Defining Potency: A Comprehensive Guide to Mesenchymal Stromal Cell Potency Assays for Clinical Translation

Abstract

This article provides a detailed overview of the development, application, and validation of potency assays for Mesenchymal Stromal Cell (MSC) products, crucial for their success as advanced therapies. Aimed at researchers, scientists, and drug development professionals, it covers the foundational principles and regulatory requirements set by agencies like the FDA and EMA. The scope extends to established methodological approaches, including immunomodulation and bioassays, alongside emerging technologies like high-throughput microfluidic systems. Furthermore, the article addresses critical challenges such as donor variability and assay robustness, offering troubleshooting and optimization strategies. Finally, it outlines the rigorous path to analytical validation according to ICH Q2(R1) guidelines and comparative analysis of different assay formats, serving as a vital resource for ensuring MSC product quality, consistency, and eventual clinical efficacy.

The Bedrock of MSC Potency: Understanding Mechanisms and Regulatory Imperatives

In the field of Mesenchymal Stromal Cell (MSC) therapeutics, potency represents a critical quality attribute that extends far beyond a mere regulatory checkbox. It is the quantitative measure of a product's biological activity, directly linking its mechanism of action (MOA) to the intended clinical effect [1]. The development of robust potency assays has emerged as a non-negotiable requirement for advanced clinical trials because MSCs are not a simple, chemically-defined drug but a complex, living pharmaceutical with multifaceted therapeutic properties [2] [1]. The United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) classify culture-expanded MSCs as more than minimally manipulated cellular and gene therapy products, mandating potency assays as part of release criteria for advanced-phase clinical trials [2]. This requirement stems from repeated clinical trial experiences where MSC products of unproven potency contributed to suboptimal outcomes and failed phase III trials, despite encouraging preclinical data [3] [1] [4]. The recent 2024 FDA approval of remestemcel-L-rknd (Ryoncil) for pediatric acute graft-versus-host disease, preceded by earlier regulatory rejections partly due to insufficient evidence for clinically correlative MSC critical quality attributes (CQAs), underscores this imperative [5] [1].

The Scientific and Regulatory Imperative for Potency Assays

Understanding MSC Heterogeneity and Its Impact on Potency

The biological complexity of MSCs presents fundamental challenges that potency assays must address. MSCs exhibit significant donor-to-donor variability, tissue-of-origin differences, and batch-to-batch variations that directly impact their therapeutic efficacy [1] [6]. Studies have demonstrated that these variations are not captured by the minimal defining criteria established by the International Society for Cell & Gene Therapy (ISCT), which include plastic adherence, specific surface marker expression (CD73, CD90, CD105), and tri-lineage differentiation potential [3] [7] [8]. Research shows that adherence to plastic does not predict potency, and the ISCT surface marker criteria do not distinguish between MSCs with high-growth capacity versus low-growth capacity, despite their significantly different tissue-regenerating abilities [3].

Regulatory Requirements and Clinical Failures

Regulatory authorities require potency assays that are quantitative, sensitive, relevant to the mechanism of action, and validated for specific disease indications [1]. The consequences of inadequate potency assessment are evident in multiple clinical trial failures:

• COVID-19 ARDS Trials: Several phase III trials of MSCs for COVID-19-associated acute respiratory distress syndrome failed despite promising preclinical data, attributed partly to suboptimal MSC product fitness and inability to consistently deliver potent cells [1].
• Historical Rejections: The FDA's initial rejections of remestemcel-L in 2020 and 2023 cited insufficient evidence for clinically correlative MSC CQAs before ultimately approving a product with more robust characterization [1].
• Inconsistent Outcomes: The discrepancy between successful preclinical animal studies and human clinical trials highlights how MSCs of unproven potency contribute to variable outcomes [3] [6].

Table 1: Key Regulatory Milestones Highlighting Potency Assessment Imperatives

Year	Regulatory Action	Significance for Potency Assessment
2006	ISCT Minimal Criteria Established	Provided basic MSC definition but insufficient for predicting therapeutic success [7] [8]
2020	FDA Rejects Remestemcel-L	Highlighted insufficient evidence for clinically correlative CQAs [1]
2023	FDA Rejects Remestemcel-L	Reaffirmed need for robust potency assays correlating with clinical mechanism [1]
2024	FDA Approves Remestemcel-L-rknd	Demonstrated successful validation of potency measures for specific clinical indication [5]
2025	ISCT MSC Committee Statement	Emphasized immunomodulatory criteria and mechanism-aligned potency assays [5]

Comparative Analysis of Potency Assay Methodologies

Matrix Versus Single-Assay Approaches

Given the plurality of MSC biological activities—including immunomodulation, pro-angiogenic effects, anti-inflammatory properties, tissue maintenance, and repair—single potency assays cannot adequately capture their therapeutic potential [2] [1]. The ISCT consensus recommends a matrix assay approach that captures the summation of effector pathways significant to MSC immunomodulation, regeneration, and homing properties [2]. This approach recognizes that MSCs deploy multiple therapeutic mechanisms that are induced upon interaction with host microenvironmental and immune status cues [2].

Table 2: Comparison of MSC Potency Assay Methodologies

Assay Type	Measured Parameters	Strengths	Limitations
Immunomodulatory Secretome	Cytokine secretion (VEGF, GCSF, CXCL9, CCL2, CXCL10); Suppression of TNF-α, IFNγ, IL-13 [2]	Captures bidirectional MSC-immune cell interactions; Correlates with T-cell suppression [2]	Requires standardized responder PBMCs; Complex data interpretation
Transcriptomic Analysis	Gene expression of immunomodulatory and homing properties; Response to IFN-γ licensing [2]	Reveals molecular mechanisms; Can use IFN-γ as PBMC surrogate [2]	mRNA levels may not reflect protein activity or function
Functional Differentiation	Osteogenic, chondrogenic, adipogenic capacity [3] [8]	Well-established; Part of ISCT minimal criteria [7]	Poor predictor of in vivo tissue repair capability [3]
Cell Surface Marker Expansion	STRO-1, PDGFR-α, CD146 beyond standard ISCT panel [3]	Identifies subpopulations with enhanced potency [3]	Marker expression varies with culture conditions and tissue source [3]
In Vivo Bone Formation	Ectopic bone volume in animal models [3]	Direct measure of tissue regenerative capacity	Low-throughput; Ethical concerns; Not suitable for release criteria

Experimental Protocols for Key Potency Assays

Protocol 1: Immunomodulatory Secretome Profiling

This method assesses MSC potency through their secretome signature when interacting with immune cells [2].

• MSC Preparation: Culture test MSCs to 70-80% confluence in standard maintenance media. Use MSCs between passages 4-6 to avoid senescence effects [3] [2].
• PBMC Co-culture: Isolate peripheral blood mononuclear cells (PBMCs) from healthy donors. Seed activated PBMCs (using staphylococcal enterotoxin B or similar mitogen) with MSCs at escalating ratios (typically 1:8 to 1:2 MSC:PBMC) [2].
• Control Setup: Include PBMCs alone (negative control) and MSCs alone (baseline secretome reference) [2].
• Supernatant Collection: Collect conditioned media after 48-72 hours of co-culture.
• Multiplex Cytokine Analysis: Quantify 29+ biologically relevant cytokines using Luminex or ELISA, focusing on both upregulated (VEGF, GCSF, CXCL10, CCL2) and downregulated (TNF-α, IFNγ, IL-13) factors correlated with T-cell suppression [2].
• T-cell Proliferation Measurement: Assess suppression of CD3+Ki67+ T-cell proliferation using flow cytometry as functional correlation [2].

Protocol 2: Transcriptional Potency Array

This approach quantifies RNA expression of immunomodulatory genes in MSCs following inflammatory licensing.

• MSC Licensing: Treat MSCs with 10-50 ng/mL interferon-γ (IFN-γ) for 24 hours to simulate inflammatory activation [2].
• RNA Isolation: Extract total RNA using column-based purification methods.
• qRT-PCR Array: Perform quantitative PCR using customized arrays targeting key immunomodulatory genes (IDO1, TSG-6, HMOX1, PG-E2 synthesis enzymes) and homing receptors [2] [1].
• Data Analysis: Normalize expression to housekeeping genes and calculate fold-change compared to unlicensed MSCs. Use predefined thresholds for potency determination [2].

Critical Factors Influencing MSC Potency

Donor and Biological Variables

Robust potency assays must account for significant biological variables that impact MSC therapeutic efficacy:

• Donor Sex: Recent evidence demonstrates that donor sex significantly affects MSC proliferation, differentiation capabilities, gene expression, and immunomodulatory potential. Female MSCs often show enhanced proliferative capacity, while male MSCs may exhibit superior osteogenic differentiation under specific conditions [6].
• Tissue Source: Bone marrow, adipose tissue, and umbilical cord-derived MSCs demonstrate distinct differentiation preferences, proliferation rates, and gene expression profiles despite meeting standard ISCT criteria [7] [6].
• Donor Age: Potency of MSCs declines with age, with cells from younger donors exhibiting reduced senescent features and enhanced differentiation potential [6].
• Culture Expansion: Extensive expansion to meet clinical dosing requirements can diminish MSC fitness, as can cryopreservation and post-thaw heat shock [1].

Mechanism-Aligned Assay Selection

Selecting appropriate potency assays requires alignment with the proposed clinical mechanism of action:

• For Immunomodulatory Applications (GVHD, Crohn's disease): Secretome profiling and T-cell suppression assays provide the most relevant potency measures [2] [5].
• For Tissue Regeneration (bone/cartilage repair): In vivo bone formation assays or specific differentiation markers (alkaline phosphatase for osteogenesis) better predict therapeutic efficacy [3] [4].
• For Angiogenic Applications: Endothelial cell migration, proliferation, and tube formation assays correlate with vascular regenerative potential [4].

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Essential Research Reagents for MSC Potency Assessment

Reagent/Category	Specific Examples	Research Function	Application Notes
Surface Antibodies	CD73, CD90, CD105, CD34, CD45, CD11b, CD14, CD19, HLA-DR, STRO-1, PDGFR-α [3] [8]	MSC identity verification and subpopulation characterization	STRO-1 and PDGFR-α identify high-growth capacity MSCs beyond standard ISCT panel [3]
Cytokine Kits	VEGF, GCSF, CXCL9, CCL2, CXCL10, TNF-α, IFNγ, IL-13 multiplex arrays [2]	Secretome profiling for immunomodulatory potency	Measure both MSC-upregulated and PBMC-downregulated cytokines [2]
Inflammatory Licensors	Interferon-γ (IFN-γ), TNF-α, lipopolysaccharide (LPS) [2] [1]	MSC preconditioning to simulate in vivo environment	IFN-γ can serve as surrogate for activated PBMCs in potency assays [2]
Differentiation Kits	Osteogenic: Dexamethasone, β-glycerophosphate, ascorbate; Adipogenic: IBMX, indomethacin, insulin [3] [8]	Tri-lineage differentiation capacity assessment	Poor predictor of in vivo tissue repair despite ISCT requirements [3]
Cell Culture Media	DMEM-low glucose with 10% fetal calf serum, L-glutamine, penicillin/streptomycin [3]	MSC expansion and maintenance	Serum lot variations significantly impact MSC potency; standardized protocols essential [3]
qPCR Reagents	Primers for TWIST-1, DERMO-1, IDO, TSG-6, HMOX1 [3] [2]	Transcriptional potency analysis	TWIST-1 and DERMO-1 higher in high-growth capacity MSCs [3]
Estragole	Estragole (Methyl Chavicol)		Bench Chemicals
Chloroprocaine	Chloroprocaine, CAS:133-16-4, MF:C13H19ClN2O2, MW:270.75 g/mol	Chemical Reagent	Bench Chemicals

Implementation Roadmap for Robust Potency Assessment

Strategic Approach to Potency Assay Development

Developing clinically relevant potency assays requires a systematic approach:

• Define Mechanism of Action: Precisely identify how MSCs exert therapeutic effects for the specific clinical indication—whether through paracrine signaling, immunomodulation, direct differentiation, or combinations thereof [5] [1].
• Select Assay Matrix: Choose a complementary set of assays that collectively capture the key biological activities, recognizing that single assays are insufficient for complex MSC functions [2] [1].
• Establish Correlation with Clinical Outcomes: Whenever possible, link potency assay results with clinical efficacy measures from early-phase trials to validate predictive value [1] [4].
• Standardize Across Batches: Implement rigorous quality control using validated potency assays to ensure consistency between manufacturing batches and donor sources [1].

Addressing Current Challenges and Future Directions

The field continues to evolve strategies to overcome potency assessment challenges:

• Donor Heterogeneity: Pooled BM mononuclear cells from multiple donors can create more consistent MSC products with younger mitotic age and enhanced allosuppressive potential [1].
• Product Fitness Markers: Identify basal markers of "fit" MSCs such as TNFAIP6 and HMOX1 that discern inter-donor differences and correlate with therapeutic performance [1].
• Pathway-Specific Assessment: Develop assays targeting specific mechanistic pathways rather than general MSC functions to better predict clinical response [2] [1].

Defining potency through robust, mechanism-aligned assays is fundamentally non-negotiable for advancing MSC therapies from promising preclinical observations to consistent clinical success. The historical pattern of failed phase III trials using MSCs of unproven potency underscores this imperative. As the field matures with recent regulatory approvals, the focus must shift from simply meeting minimal identity criteria to comprehensively characterizing biological functionality through matrix-based potency assessment. Only by embracing this rigorous approach can MSC therapies realize their potential as reliable, effective pharmaceuticals for diverse medical conditions.

For researchers and drug development professionals working with Advanced Therapy Medicinal Products (ATMPs), particularly Mesenchymal Stromal Cell (MSC) products, developing robust potency assays represents one of the most significant translational challenges. Potency assays serve as critical quality attributes that quantitatively measure the biological activity of a cell therapy product and provide a direct link to its proposed mechanism of action (MoA) [9]. These assays are essential for demonstrating that a living therapy can achieve its intended biological effect, ensuring manufacturing consistency, evaluating product stability, and ultimately guaranteeing clinical efficacy [10] [9]. The development of these assays is complicated by the inherent variability of biological starting materials and the complex, often multi-factorial, mechanisms of action exhibited by MSC therapies [9].

The regulatory landscape for potency assays is evolving rapidly on both sides of the Atlantic. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) now emphasize that analytical confidence forms the foundation of regulatory submissions, with potency data being paramount for cell-based products [11]. This guide provides a detailed comparison of regulatory perspectives, offering experimental protocols and data presentation frameworks to help navigate requirements for MSC-based product development.

Regulatory Framework Comparison: FDA vs. EMA

Core Principles and Guidelines

The FDA and EMA share the fundamental principle that a potency assay must measure the product's biological activity relative to its mechanism of action. However, some differences in approach and emphasis exist.

• FDA Perspective: The FDA's Center for Biologics Evaluation and Research (CBER) identifies regulatory convergence as a key strategy for managing international requirements [12]. For potency testing, the FDA provides specific guidance, expecting assays to be developed early and refined throughout the product development lifecycle [9]. The agency acknowledges the need for phase-appropriate compliance, with increasing rigor required as products move toward licensure [12]. The recent approval of Ryoncil (remestemcel-L), the first MSC therapy in the U.S., underscores the FDA's focus on potency assay robustness despite complex approval pathways [13].

• EMA Perspective: The EMA's new multidisciplinary guideline for clinical-stage ATMPs, effective July 2025, consolidates information from over 40 separate guidelines and reflection papers [12]. For potency, the EMA encourages a risk-based approach when evaluating quality data and explicitly notes that immature quality development may compromise the use of clinical trial data to support a marketing authorization [12]. The EMA's framework is structured around the Common Technical Document (CTD) format, providing a roadmap for organizing information in investigational or marketing applications [12].

Key Regulatory Differences and Convergences

Table: Comparison of FDA and EMA Regulatory Approaches to Potency Assays for ATMPs

Aspect	U.S. FDA Approach	EMA Approach
Guidance Status	Specific guidance on potency tests for Cellular and Gene Therapy Products [9]	Multidisciplinary guideline on quality, non-clinical, and clinical requirements for investigational ATMPs [12]
GMP Compliance	Reliance on attestation in early phases with phase-appropriate increase; verified at pre-license inspection [12]	Mandatory self-inspections with documented results providing evidence of an effective quality system [12]
Structural Framework	Flexible, case-specific [11]	Structured, science-based, aligned with CTD format [12] [11]
Core Principle	Potency must measure biological activity linked to MoA; phase-appropriate development acceptable [9]	Risk-based approach; weak quality system could prevent clinical trial authorization [12]
Terminology	"Drug Substance" and "Drug Product" in CTD [12]	"Active Substance" and "Investigational Medicinal Product" in CTD [12]

Despite nuanced differences, significant regulatory convergence has occurred, particularly within Chemistry, Manufacturing, and Controls (CMC) disciplines [12]. Both agencies now recognize that robust analytical data, including potency, are the most sensitive indicators of product quality and consistency [11].

Analysis of Approved Products and Potency Testing Strategies

Potency Test Categories for FDA-Approved Cell Therapies

An analysis of the 31 U.S. FDA-approved Cell Therapy Products (CTPs) from 2010 through 2024 reveals the practical application of potency testing strategies. A total of 104 potency tests have been used for these products, with an average of 3.4 potency tests per product (standard deviation 2.0) [10]. After accounting for redacted information (32%), 71 non-redacted tests were categorized, demonstrating the following distribution:

Table: Categories of Potency Tests for 31 FDA-Approved Cell Therapy Products [10]

Potency Test Category	Number of Tests	Percentage of Non-Redacted Tests	Example Applications
Viability and Count	37	52%	Cell viability, total nucleated cells (TNC), viable CD34+ cell count [10]
Expression	19	27%	CAR expression by flow cytometry, CD54+ cell number [10]
Bioassays	7	7%	Interferon-γ production upon target cell stimulation, colony-forming unit (CFU) assays [10]
Genetic Modification	6	9%	Vector copy number (VCN) by qPCR, percent LVV+ cells [10]
Histology	2	3%	Tissue organization, viability & retention of important cell types [10]

This analysis indicates that multiple potency assays are typically required to fully characterize a CTP's biological activity. While "Viability and Count" and "Expression" tests are frequently used together (for 52% of CTPs), the most informative potency tests are often bioassays that functionally demonstrate the product's proposed mechanism of action [10].

Case Study: Ryoncil – The First FDA-Approved MSC Therapy

The approval timeline for Ryoncil illustrates the critical importance of addressing potency and CMC challenges in MSC therapy development:

Diagram: Ryoncil's Regulatory Journey Highlighting CMC and Potency Challenges

Ryoncil's development faced multiple regulatory hurdles, including a Complete Response Letter (CRL) specifically requesting additional data to support efficacy, and a subsequent CRL related to Chemistry, Manufacturing, and Controls (CMC) aspects [13]. The therapy was ultimately approved in December 2024 based on data from a multicenter, single-arm study in 54 pediatric patients with steroid-refractory acute graft-versus-host disease (SR-aGVHD), where 30% achieved a complete response and 41% showed partial improvement [13]. This approval highlights that despite the challenge of batch-to-batch variability in MSC products, regulators will approve therapies with variable response rates when supported by robust manufacturing controls and appropriate potency assays.

Experimental Protocols for MSC Potency Assay Development

VEGF Quantification Potency Assay for CD34+ Cell Therapy

A validated potency assay for ProtheraCytes (expanded autologous CD34+ cells) demonstrates a comprehensive approach to measuring biological activity linked to mechanism of action. Since these cells promote myocardial regeneration through angiogenesis, the assay quantifies secreted vascular endothelium growth factor (VEGF) during CD34+ cell expansion [14].

Experimental Workflow:

Diagram: Workflow for VEGF-Based Potency Assay

Detailed Methodology: [14]

• Cell Culture and Supernatant Collection: Expand CD34+ cells from patient samples or healthy donors for 9 days in a GMP-compliant, xeno-free culture medium (e.g., StemFeed medium). Collect cell culture supernatant after the expansion period.

• VEGF Quantification: Use an automated immunoassay platform (ELLA system from Bio-Techne) with a microfluidic cartridge designed for VEGF-A quantification. This system uses a sandwich-type quantitative ELISA method with specific antibodies against VEGF-A. The relative fluorescence units (RFU) measured by the fluorometer are proportional to the quantity of antibody/antigen complexes formed.

• Validation Parameters: The method was validated according to ICH Q2(R2) guidelines for:

  • Specificity: Demonstrate no interference from the culture medium.
  • Linearity: Establish a working range of 20 pg/mL to 2800 pg/mL (R² = 0.9972).
  • Precision: Achieve coefficients of variation ≤10% (repeatability) and ≤20% (intermediate precision).
  • Accuracy: Demonstrate mean recoveries between 85% and 105% for various concentrations.

Key Advantages: This potency assay is fast, reliable, quantitative, and allows for timely batch release before the cell product is shipped to the clinic for injection [14]. The validation of 38 clinical batches demonstrated consistency, making it suitable for phase III clinical trials and marketing authorization applications.

The Scientist's Toolkit: Essential Research Reagents

Table: Key Reagents and Materials for MSC Potency Assay Development

Reagent/Equipment	Function in Potency Testing	Example from Literature
Automated Immunoassay System	Quantitative protein biomarker measurement	ELLA system (Bio-Techne) for VEGF quantification [14]
Flow Cytometer	Cell surface marker expression analysis	Measurement of CD73, CD90, CD105 for MSC characterization [15]
Human Platelet Lysate	Xeno-free cell culture supplement	Supplement for GMP-compliant MSC expansion [15]
Nanoparticle Tracking Analysis	Characterization of extracellular vesicles	Size and concentration analysis of MSC-sEVs [15]
qPCR System	Genetic modification quantification	Vector copy number (VCN) analysis for gene-modified therapies [10]
4-Nitrosodiphenylamine	4-Nitrosodiphenylamine, CAS:156-10-5, MF:C12H10N2O, MW:198.22 g/mol	Chemical Reagent
Endosulfan Sulfate	Endosulfan Sulfate, CAS:1031-07-8, MF:C9H6Cl6O4S, MW:422.9 g/mol	Chemical Reagent

Strategic Recommendations for Navigating Global Regulations

Developing a Risk-Based Potency Assay Strategy

Based on the analysis of regulatory requirements and approved products, the following strategic approach is recommended for MSC potency assay development:

• Implement Parallel Development: Begin potency assay development early in product development and refine it throughout the clinical trial phases. The assay used for phase III and marketing authorization must be fully validated [9].

• Employ Multiple Assay Formats: Given that 65% of approved CTPs use measurements of gene or protein expression and 61% use cell viability or count, a combination of physical, molecular, and functional assays is often necessary to fully characterize product potency [10].

• Prioritize Mechanism of Action: Design potency assays that directly reflect the proposed biological mechanism. For example, if MSC therapy works through immunomodulation, develop assays that measure secretion of relevant cytokines (e.g., IFN-γ, IL-6, TGF-beta) [15] [10].

• Plan for Regulatory Flexibility: Acknowledge and plan for regional differences between FDA and EMA, particularly regarding GMP compliance and specific technical expectations [12]. Engage with both agencies early through scientific advice procedures.

• Leverage Emerging Technologies: Consider innovative tools such as droplet digital PCR and reporter gene assays to improve the precision and reliability of potency testing as the field evolves [9].

Addressing Common Challenges in MSC Potency Testing

MSC therapies present unique challenges for potency assay development:

• Batch-to-Batch Variability: MSCs are highly responsive to their microenvironment, leading to variations in immunomodulatory properties, viability, and functionality based on donor source, culture conditions, and expansion methods [13]. Implement rigorous donor screening and standardized culture conditions to minimize this variability.

• Multi-Factorial Mechanisms of Action: Unlike single-molecule drugs, MSCs often function through multiple parallel mechanisms. Consider a potency assay panel that captures the primary mechanisms rather than relying on a single test [9].

• Standardization Difficulties: Living cell products cannot be frozen and stored as reference standards for decades like traditional drugs. Develop well-characterized internal reference materials and cell banks to enable meaningful cross-batch comparisons [13].

The regulatory landscape for potency assays of MSC products requires a sophisticated, multi-faceted approach that addresses both FDA and EMA expectations while acknowledging the biological complexities of living therapies. The recent approval of Ryoncil demonstrates that despite the challenges, regulatory pathways exist for MSC-based therapies when supported by robust, mechanism-relevant potency data. As the field advances, success will increasingly depend on developing physiologically relevant potency assays that demonstrate a strong correlation between in vitro potency data and clinical outcomes [9]. By implementing the strategies and experimental approaches outlined in this guide, researchers and drug development professionals can navigate this complex landscape with greater confidence and efficiency, accelerating the delivery of transformative MSC therapies to patients in need.

The development of robust potency assays is a critical, yet challenging, requirement for the clinical translation of Mesenchymal Stromal Cell (MSC) therapies. These assays must reliably predict the therapeutic effectiveness of MSC products in vivo, a task complicated by several inherent biological challenges. The fundamental hurdles include significant donor-to-donor variability, a complex and multifactorial mechanism of action (MOA), and the limited stability of the live cell product post-thaw [16] [17]. These variabilities impact the consistency of MSC products, making it difficult to ensure that each batch meets the necessary quality and efficacy standards for clinical use [16]. This guide objectively compares experimental approaches and data for assessing and mitigating these challenges, providing a structured framework for researchers developing potency assays.

Challenge 1: Donor Variability

Donor variability introduces substantial heterogeneity in MSC characteristics, including their proliferation capacity, secretome profile, and consequently, their immunomodulatory and regenerative potency [16] [18]. This variability can originate from the donor's age, health status, and tissue source.

Comparative Data: Impact of Donor and Culture Conditions

The following table summarizes experimental findings on how donor characteristics and culture conditions influence MSC product quality and potency.

Table 1: Impact of Donor and Culture Conditions on MSC Product Characteristics

Variable	Experimental Comparison	Key Findings	Implication for Potency
Donor Source	Bone Marrow (BM) vs. Umbilical Cord (UC) MSCs [7]	UC-MSCs often exhibit enhanced proliferation and lower immunogenicity. BM-MSCs are the most studied with strong immunomodulation.	Potency assays must be source-specific.
Culture System	2D Monolayer vs. 3D Bioreactor Culture [19]	3D dynamic culture significantly enhanced anti-inflammatory molecule secretion (e.g., TSG-6, PGE2) and robustly inhibited TNF-α, IL-1β, IL-6, IL-8, and MIP-1.	3D culture products may require more sensitive potency assays to capture enhanced activity.
Culture Medium	DMEM vs. α-MEM [15]	Cells in α-MEM showed a trend of higher proliferative capacity and particle yield of small Extracellular Vesicles (sEVs), though not statistically significant.	Culture medium formulation can influence critical quality attributes and must be standardized.
Inter-Donor sEV Yield	sEV production from different BM-MSC donors [15]	Particle concentrations from donors 1 and 2 were significantly higher than from donors 3 and 5, despite no differences in particle size.	Potency tests for cell-free products (e.g., sEVs) must account for donor-dependent secretion capacity.

Experimental Protocol: Assessing Donor Variability in Anti-inflammatory Potency

Objective: To compare the anti-inflammatory potency of MSC-derived conditioned medium (MTF) from different donors and culture systems.

• Cell Culture: Generate MTF from multiple donors (e.g., ≥5). Use parallel 2D monolayer cultures and 3D bioreactor cultures with gelatin microcarriers in spinner flasks [19].
• Sample Collection: Conditioned medium should be collected serum-free to avoid interference, concentrated, and standardized by total protein or cell number.
• Potency Assay - Macrophage System: Differentiate THP-1 cells or primary human monocytes into macrophages. Pre-treat with LPS (e.g., 100 ng/mL) for 24 hours to activate a pro-inflammatory phenotype. Co-culture with MTF for an additional 24 hours [19].
• Readout: Quantify secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) using ELISA or a multiplex immunoassay. Calculate the percentage inhibition relative to LPS-activated controls without MTF.
• Data Analysis: Compare the coefficient of variation (CV) in inhibitory potency across different donors and culture methods to quantify variability.

Challenge 2: Complex Mechanism of Action

The therapeutic effect of MSCs is primarily attributed to their paracrine activity rather than direct cell replacement [16]. This activity is mediated by a complex secretome containing both soluble factors and extracellular vesicles (EVs), which act on diverse immune and tissue cells through multiple pathways.

Signaling Pathways in MSC Immunomodulation

The following diagram illustrates the key signaling pathways through which the MSC secretome exerts its immunomodulatory effects, highlighting the roles of different secretome components.

Comparative Data: Secretome Fraction Bioactivity

Different fractions of the MSC secretome contribute to its overall potency through distinct mechanisms, as evidenced by the following experimental data.

Table 2: Bioactivity of Different MSC Secretome Fractions

Secretome Fraction	Key Bioactive Components	Primary Experimental Effects	Proposed Potency Assay Readout
Soluble Factors (< 5 kDa)	PGE2, Kynurenine [20]	Dose-dependent inhibition of NF-κB and IRF pathways in innate immune cells (e.g., macrophages).	Inhibition of cytokine production (e.g., TNF-α) in LPS-stimulated reporter cells (e.g., THP-1 Dual).
Components > 100 kDa	Proteins, large EVs [20]	Inhibition of T-cell proliferation, even when soluble factors are removed.	Suppression of CD3/CD28-stimulated T-cell proliferation measured by CFSE dilution or BrdU incorporation.
Small EVs (sEVs, 30-150 nm)	miRNAs, CD63, CD9, TSG101 [15]	Protection of retinal pigment epithelium (ARPE-19) from Hâ‚‚Oâ‚‚-induced oxidative damage; reduction of apoptosis.	Cell viability assay (e.g., MTT) and flow cytometric analysis of apoptosis in a stressed target cell line.
Clarified Secretome	Mix of all soluble factors [20]	Strongest anti-inflammatory activity, superior to concentrated fractions, suggesting synergistic effects.	Multi-parameter assay combining innate pathway inhibition and cytokine secretion profiling.

Experimental Protocol: Fractionating the Secretome to Deconvolute MOA

Objective: To isolate and test the functional potency of different secretome fractions.

• Secretome Production: Culture MSCs in serum-free medium for 24-48 hours. Collect conditioned medium and clarify using 0.2 µm filtration (Post-Clarification (PC) sample) [20].
• Fractionation by TFF: Process the PC sample using Tangential Flow Filtration (TFF) with membranes of different molecular weight cutoffs (e.g., 5 kDa, 10 kDa, 100 kDa) to generate fractions enriched in specific size ranges [20].
• Fractionation by Ultracentrifugation: As an alternative, ultracentrifuge the PC sample at 150,000 × g for 2 hours to separate a pellet (EV-rich fraction) from the supernatant (soluble factor-rich fraction) [20].
• Parallel Potency Testing:
  • Innate Immunomodulation: Treat LPS-stimulated THP-1 Dual reporter cells with each fraction and measure inhibition of NF-κB and IRF pathway activation via secreted luciferase [20].
  • Adaptive Immunomodulation: Treat CD3/CD28-activated PBMCs with each fraction and measure suppression of T-cell proliferation via CFSE dye dilution and flow cytometry [20].
• Analysis: Correlate the activity of each fraction with its composition (e.g., via proteomics, lipidomics) to identify key potency markers.

Challenge 3: Limited Stability

MSCs are often cryopreserved as "off-the-shelf" products, but their viability and potency post-thaw can be limited, posing a significant challenge for acute treatments where administration must occur within hours of diagnosis [21].

Comparative Stability and Potency Data

The stability and functionality of MSCs after thawing are critical for their clinical application. The following table compares key metrics between freshly cultured and cryopreserved-thawed MSCs.

Table 3: Comparison of Cultured (Fresh) vs. Thawed MSC Stability and Potency

Parameter	Cultured (Fresh) MSCs	Thawed MSCs	Experimental Context
Viability at 0h	92% ± 2.7%	93% ± 2.6%	In vitro, post-preparation [21].
Viability at 6h	91% ± 2.3%	81% ± 2.5%	In vitro, post-preparation [21].
Apoptotic Cells	Lower levels	Higher levels (AV+/PI− & AV+/PI+) at 4h and 6h	In vitro, measured by Annexin V/PI staining [21].
T-cell Suppression	13% to 38% inhibition (Donor-dependent)	No significant difference from donor-matched cultured MSCs	In vitro co-culture with activated PBMCs [21].
Monocyte Phagocytosis	Significant recovery of LPS-impaired phagocytosis	Comparable recovery to donor-matched cultured MSCs	In vitro co-culture with CD14+ PBMCs [21].
Endothelial Barrier Repair	Significant decrease in LPS-induced permeability	Comparable restoration to donor-matched cultured MSCs	In vitro transwell assay with endothelial cells [21].
In vivo Efficacy (Sepsis)	Reduced plasma lactate and inflammatory cytokines	No significant difference from cultured MSCs	Murine cecal ligation and puncture (CLP) model [21].

Experimental Protocol: Assessing Post-Thaw Potency

Objective: To evaluate the immunomodulatory potency of a cryopreserved MSC product within a clinically relevant timeframe post-thaw.

• Cell Preparation: Thaw a vial of cryopreserved MSCs using a standard protocol. Compare against a log-fold cultured, donor-matched MSC sample prepared simultaneously [21].
• Short-term Stability: Measure cell viability and recovery at 0, 2, 4, and 6 hours post-thaw using Trypan Blue exclusion and Annexin V/Propidium Iodide staining by flow cytometry [21].
• Rapid Potency Assay Battery: Conduct the following assays immediately (within 2-4 hours) post-thaw:
  • T-cell Proliferation Assay: As described in Section 3.3.
  • Monocyte Phagocytosis Assay: Co-culture MSCs with CD14+ monocytes. Measure the uptake of pHrodo-labeled E. coli bioparticles by flow cytometry [21].
  • Endothelial Barrier Integrity Assay: Culture endothelial cells in a transwell system. Induce permeability with LPS and co-culture with MSCs. Measure the flux of FITC-dextran across the monolayer over time [21].
• Correlation with In Vivo Outcome: For validation, administer freshly thawed MSCs in an acute disease model (e.g., murine CLP sepsis) and assess critical outcomes such as bacterial clearance (e.g., via phagocytosis in peritoneal lavage cells) and reduction in systemic inflammatory markers (e.g., plasma IL-6, TNF-α) [21].

The Scientist's Toolkit: Essential Research Reagents and Materials

The following table lists key reagents and materials essential for conducting the potency assays and experimental protocols described in this guide.

Table 4: Key Research Reagent Solutions for MSC Potency Assays

Reagent / Material	Function / Application	Example Use Case
Human Platelet Lysate (hPL)	Serum-free, xeno-free supplement for MSC culture expansion.	GMP-compliant culture of BM-MSCs and UC-MSCs [15] [19].
Tangential Flow Filtration (TFF) System	Concentration and size-based fractionation of secretome from large-volume conditioned media.	Isolating sEVs and soluble factor fractions for MOA studies [15] [20].
THP-1 Dual Reporter Cell Line	In vitro model for innate immunomodulation; reports simultaneous activation of NF-κB and IRF pathways.	Testing inhibition of inflammatory pathways by soluble secretome fractions [20].
MACSPlex EV Kits	Multiplexed bead-based flow cytometry for high-throughput characterization of EV surface markers.	Phenotyping MSC-EVs for identity and batch-to-batch consistency [20].
Cultispher-S Microcarriers	3D substrate for the expansion of MSCs in bioreactor systems.	Scalable production of MSCs with enhanced anti-inflammatory potency [19].
Prostaglandin E2 (PGE2) ELISA Kit	Quantification of a key soluble immunomodulatory factor.	Correlating PGE2 concentration with anti-inflammatory bioactivity in potency assays [20].
Annexin V / Propidium Iodide (PI)	Flow cytometry-based detection of apoptotic and necrotic cells.	Assessing the stability and viability of MSCs post-thaw over time [21].
pHrodo E. coli BioParticles	pH-sensitive fluorescent particles for quantifying phagocytic activity.	Potency assay measuring MSC-enhanced monocyte phagocytosis [21].
Octyl Gallate	Octyl Gallate, CAS:1034-01-1, MF:C15H22O5, MW:282.33 g/mol	Chemical Reagent
2-Iminobiotin	2-Iminobiotin, CAS:13395-35-2, MF:C10H17N3O2S, MW:243.33 g/mol	Chemical Reagent

Addressing the triple challenges of donor variability, complex MOA, and limited stability is paramount for the successful development of MSC-based therapies. A one-size-fits-all approach to potency testing is insufficient. Instead, a panel of complementary assays that reflect the intended therapeutic mechanism is required. As evidenced by the experimental data, strategies such as adopting 3D bioreactor cultures can enhance and standardize product quality, while fractionation studies help deconvolute the MOA for more targeted potency measurements. Furthermore, rigorous comparability studies, like those between fresh and thawed cells, are essential to define the shelf-life and usage window of the final product. By integrating these multifaceted approaches, developers can create robust potency assays that not only ensure product quality and consistency but also pave the way for more predictable and successful clinical outcomes.

From Theory to Bench: A Practical Guide to Established and Novel Potency Assays

For developers of Mesenchymal Stromal Cell (MSC) therapies, demonstrating product potency through reliable functional assays is a critical regulatory requirement. The immunomodulatory function of MSCs represents a primary mechanism of action for many therapeutic applications, particularly for immune-related disorders such as graft-versus-host disease (GVHD), Crohn's disease, and autoimmune conditions [22]. Among the various methods available, lymphocyte suppression assays and Mixed Lymphocyte Reaction (MLR) have emerged as gold-standard functional tests for quantifying the immunomodulatory potency of MSC products. These assays provide critical data on the ability of MSCs to suppress T-cell activation, proliferation, and effector function—key therapeutic attributes that must be consistently quantified across manufacturing batches [23] [22]. While both assays measure immunomodulatory capacity, they differ significantly in their experimental design, biological context, and application in product development. This guide provides an objective comparison of these two foundational approaches, supported by experimental data and methodological details to inform strategic assay selection for MSC potency testing.

Assay Comparison: Lymphocyte Suppression vs. Mixed Lymphocyte Reaction

The table below summarizes the core characteristics, applications, and output metrics for lymphocyte suppression assays and Mixed Lymphocyte Reaction in the context of MSC potency evaluation.

Assay Characteristic	Lymphocyte Suppression Assay	Mixed Lymphocyte Reaction (MLR)
Primary Application	Potency testing for MSC products with known immunosuppressive mechanisms [22]	Preclinical immunogenicity assessment and immunomodulatory drug evaluation [24] [25]
Biological Principle	MSC suppression of mitogen- or antibody-activated T-cell proliferation [26] [23]	T-cell response to allogeneic HLA class II antigens (HLA-DR/DQ/DP) [24]
Standard Duration	3-5 days (full proliferation) or 24 hours (early activation markers) [26]	5-7 days [24] [23]
Key Readout Parameters	CD4+ T-cell proliferation suppression; IPA value; PS externalization; TNFα levels [26] [22]	Stimulation Index (SI); proliferation rates; cytokine profiles (IFN-γ, IL-6) [24] [25]
Stimulation Method	Anti-CD3/CD28 antibodies or mitogens (PHA) [23] [22]	Allogeneic monocyte-derived dendritic cells (MoDCs) [27]
Throughput Potential	Moderate to high (96-well formats) [26]	Lower (donor variability considerations) [23]
Quantitative Output	Immunopotency Assay (IPA) value: % suppression of CD4+ T-cell proliferation [22]	Stimulation Index (SI): ratio of proliferation in stimulated vs. unstimulated conditions [24]
Key Advantages	Standardized, reproducible quantitative output; direct potency measurement [22]	Physiologically relevant alloreactive response; assesses multiple immunomodulatory nodes [24]
Reported Variability	IPA values: 27% to 88% across MSC products [22]	Substantial variation unless donor pooling used [23]

Experimental Protocols and Methodologies

Lymphocyte Suppression Assay Protocol

The lymphocyte suppression immunopotency assay (IPA) utilizes anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation, measuring MSC-induced suppression of CD4+ T cells at various effector-to-target ratios compared to a reference standard MSC product [22].

Materials and Reagents:

• Cryopreserved peripheral blood mononuclear cells (PBMCs) from pooled donors (8-10 donors) [26]
• Anti-CD3/CD28 antibodies (e.g., ImmunoCult Human CD3/CD28 T Cell Activator) [22]
• Culture media: RPMI 1640 with 10% FBS, 1× GlutaMax, 10mM HEPES [26]
• 96-well flat-bottom tissue culture-treated plates [26]
• Flow cytometry reagents for proliferation analysis (CFSE, CellTrace Violet) [23]
• ATP detection reagents for luminescence-based proliferation measurement [26]

Methodology:

• PBMC Preparation: Thaw cryopreserved PBMCs and wash twice in PBMC media. Resuspend at 1.5 × 10^6 cells/mL [26].
• MSC Seeding: Seed MSCs in 96-well plates at varying densities (e.g., 5,000-60,000 cells/well) and culture for 24 hours to achieve adherence [26] [22].
• Co-culture Establishment: Add 150,000 PBMCs per well to adhered MSCs with PBMC:MSC ratios typically ranging from 2.5:1 to 202.5:1 [26] [22].
• T-cell Activation: Add anti-CD3/CD28 antibody stimulus (50 μL per well) at optimal concentration (e.g., 1-5 μg/mL) [23] [22].
• Incubation: Culture plates for 72-96 hours at 37°C, 5% COâ‚‚.
• Proliferation Measurement: Quantify CD4+ T-cell proliferation using:
  • CFSE dilution by flow cytometry [23]
  • ATP quantification via luminescence assay [26]
  • ³H-thymidine incorporation (traditional method) [24]
• Data Analysis: Calculate IPA value as percentage suppression of CD4+ T-cell proliferation compared to PBMCs cultured without MSCs [22].

Mixed Lymphocyte Reaction (MLR) Protocol

The MLR assay measures T-cell responses to allogeneic HLA class II antigens presented by monocyte-derived dendritic cells (MoDCs), quantifying a drug's ability to regulate T-cell activation, proliferation, and effector molecule secretion [24] [27].

Materials and Reagents:

• CD14+ monocytes from healthy donors for MoDC generation [27]
• CD4+ or CD8+ T cells from unrelated healthy donors [27]
• Dendritic cell differentiation cytokines: IL-4 (50 ng/mL) and GM-CSF (100 ng/mL) [27]
• Culture media: RPMI 1640 GlutaMAX supplemented with 10% heat-inactivated serum [27]
• Ultra-low attachment 96-well microplates [27]
• Proliferation detection: ³H-thymidine, EdU incorporation, or CFSE dilution [24] [28]
• Cytokine analysis: HTRF, LEGENDplex, or AlphaLISA platforms [25]

Methodology:

• MoDC Generation: Isolate CD14+ monocytes from PBMCs using magnetic bead separation. Culture monocytes (10^6/mL) for 7 days in complete medium with IL-4 (50 ng/mL) and GM-CSF (100 ng/mL) to generate monocyte-derived dendritic cells (MoDCs) [27].
• T-cell Isolation: Purify CD4+ or CD8+ T cells from PBMCs of unrelated donors using negative selection kits [27].
• Co-culture Setup: Culture freshly isolated CD4+ or CD8+ T cells (10^5 cells) with allogeneic MoDCs (10^4 cells) in ultra-low attachment 96-well plates using a 10:1 responder:stimulator ratio [27].
• Experimental Conditions: Include controls with T cells alone and T cells with anti-CD3/CD28 antibodies as positive proliferation control [27].
• Incubation: Culture for 5-7 days at 37°C, 5% COâ‚‚ [24] [23].
• Proliferation Measurement: Assess T-cell proliferation using:
  • ³H-thymidine incorporation during the final 8-18 hours of culture [24]
  • EdU incorporation with flow cytometric detection [28]
  • CFSE dilution by flow cytometry [23]
• Cytokine Analysis: Quantify cytokine secretion (IFN-γ, IL-2, IL-6, TNF-α) in supernatant using multiplex immunoassays [25] [27].
• Data Analysis: Calculate Stimulation Index (SI) as ratio of proliferation in co-culture to T cells alone [24].

Signaling Pathways in Lymphocyte Suppression and MLR

MLR Immunological Synapse and MSC Intervention Points

The MLR cascade offers multiple intervention nodes for immunomodulatory drugs and MSC products, targeting key signaling pathways in T-cell activation and proliferation [24].

Research Reagent Solutions for MLR and Lymphocyte Suppression Assays

The table below details essential reagents and their functions for implementing these immunomodulatory potency assays.

Reagent Category	Specific Examples	Function in Assay
Cell Separation	CD14 Microbeads (Miltenyi); CD4/CD8 T Cell Isolation Kits (Miltenyi) [27]	Isolation of specific immune cell populations for assay setup
Dendritic Cell Generation	IL-4 (50 ng/mL); GM-CSF (100 ng/mL) [27]	Differentiation of monocytes into monocyte-derived dendritic cells (MoDCs)
T-cell Activation	Anti-CD3/CD28 Antibodies (ImmunoCult); PHA-P/PHA-L [23] [22]	Polyclonal T-cell activation for proliferation induction
Proliferation Detection	CFSE/CellTrace Violet; ³H-thymidine; EdU; ATP Luminescence Kits [24] [26] [28]	Quantification of cell division and proliferation rates
Cytokine Analysis	HTRF; LEGENDplex; AlphaLISA; Multiplex ELISA [25] [27]	Measurement of cytokine secretion profiles
Cell Culture Media	RPMI 1640 with GlutaMAX; MSC-qualified FBS; Human Serum Albumin [26] [27]	Maintenance of cell viability and function during culture
Cryopreservation	CryoStor CS10; DMSO; Human Serum Albumin [23]	Preservation of cell viability for consistent assay performance

Comparative Performance Data and Research Applications

Quantitative Performance Metrics

Lymphocyte Suppression Assay Performance:

• In standardized immunopotency assays (IPAs), MSC products from multiple manufacturing centers demonstrated IPA values ranging from 27% to 88% suppression of CD4+ T-cell proliferation, highlighting significant product-to-product variability [22].
• For rapid potency screening, phosphatidyl serine externalization (PS+) on live CD3+ T cells at 24 hours serves as a robust surrogate marker, showing dose-dependent and reproducible suppression by MSCs [26].
• TNFα release by mitogen-activated PBMCs provides a sensitive early readout for MSC immunomodulation, with reliable detection of suppression after 24 hours of co-culture [26].

MLR Assay Performance:

• In MLR models, JAK inhibitors like Baricitinib (10 nM) reduced CD4+ T-cell proliferation by 62% (p<0.001) and IFN-γ secretion by 85%, demonstrating precise quantitation of immunomodulatory effects [24].
• CTLA-4 fusion proteins (e.g., Abatacept) significantly lower MLR stimulation indices (SI) from 5.3 to 1.8 (p=0.003), establishing robust detection of costimulation blockade [24].
• MLR assays effectively identify TCR-knockout UCAR-T cells with 10-fold lower alloreactivity (SI<1.5) compared to conventional CAR-T products [24].

Strategic Application in MSC Product Development

The selection between lymphocyte suppression assays and MLR depends on specific development phase requirements and therapeutic targets:

Lymphocyte Suppression Assays are optimal for:

• Quality Control Testing: Routine potency assessment for MSC product batch release [22]
• Rapid Screening: Early-stage screening of multiple MSC donors or manufacturing conditions [26]
• Standardized Potency Metrics: Quantitative comparison across MSC products using standardized IPA values [22]

MLR Assays are preferred for:

• Mechanistic Studies: Investigating specific immunomodulatory pathways and intervention points [24] [27]
• Physiological Relevance: Modeling allogeneic immune responses more closely mimicking in vivo conditions [23]
• Comprehensive Profiling: Multi-parameter assessment of immunomodulation including proliferation, cytokine secretion, and checkpoint expression [27]

For comprehensive MSC product characterization, many developers implement both assays at different development stages—using lymphocyte suppression assays for lot-release testing and MLR for deeper mechanistic profiling during preclinical development.

Within the development of mesenchymal stromal cell (MSC) therapies, establishing robust and predictive potency assays is a critical regulatory and manufacturing challenge. The immunosuppressive function of MSCs is not driven by a single mechanism but is the result of overlapping and cumulative effector pathways [29] [30]. This complexity necessitates an "assay matrix" strategy, where multiple critical quality attributes are measured to form a unified potency score [29]. Among the many candidate biomarkers, Indoleamine 2,3-dioxygenase (IDO) activity, Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6), and Programmed Death-Ligand 1 (PD-L1) have emerged as particularly promising surrogate markers. This guide provides an objective comparison of these three key biomarkers, detailing their quantification methods, correlation with immunosuppressive function, and application in the context of MSC potency assays for researchers and drug development professionals.

The table below provides a high-level comparison of the three surrogate markers, highlighting their primary functions and their specific roles in the mechanism of action of MSCs.

Table 1: Core Characteristics of Key MSC Potency Markers

Marker	Primary Function	Role in MSC Immunosuppression
IDO Activity	Catalyzes the conversion of tryptophan to kynurenine, a rate-limiting step in the kynurenine pathway [31] [32].	Depletes local tryptophan and accumulates kynurenine, suppressing T-cell proliferation and promoting regulatory T-cell (Treg) differentiation [32] [30].
TSG-6	A secreted anti-inflammatory protein that inhibits neutrophil migration and degranulation, and modulates TLR2 signaling [30].	Mediates MSC-mediated suppression of sterile inflammation (e.g., in corneal injury, peritonitis, and lung injury models); its transcript levels correlate with in vivo efficacy [30].
PD-L1	An immunoinhibitory ligand that binds to PD-1 on T cells, suppressing their activation and cytokine secretion [33] [34].	Provides a contact-dependent immunosuppressive signal from MSCs to T cells, inhibiting T-cell proliferation and effector function [30] [35].

Quantitative Data and Correlation with Potency

The expression levels and functional activity of these markers are inducible and show a direct correlation with the immunosuppressive potency of MSCs. Key quantitative data and correlations are summarized below.

Table 2: Quantitative Correlations and Experimental Support for MSC Potency Markers

Marker	Inducing Stimuli	Correlation with Functional Potency	Key Supporting Evidence
IDO Activity	IFN-γ, TNF-α [30]	Strong positive correlation with suppression of T-cell proliferation ((R^2 = 0.90)) [30].	IFN-γ-licensed MSCs from different donors showed variable suppression (10-80% inhibition), which was strongly predicted by IDO-1 protein levels [30].
TSG-6	TNF-α [30]	Transcript levels correlate with efficacy in mouse models of sterile inflammation [30].	In a panel of MSCs from 13 donors, TSG-6 transcript levels were identified as a key correlate of in vivo efficacy [30].
PD-L1	IFN-γ [30]	Positive correlation with suppression of T-cell proliferation [30].	Protein expression of PD-L1 on licensed MSCs correlates with their ability to suppress T-cell responses [30].

Detailed Experimental Protocols for Marker Quantification

IDO Activity Measurement

The gold standard for assessing functional IDO enzyme activity is the quantification of tryptophan degradation and kynurenine production, typically using High-Performance Liquid Chromatography (HPLC) or Liquid Chromatography-Mass Spectrometry (LC-MS).

Protocol: IDO Activity via HPLC [31] [32] [36]

• Sample Preparation: Incubate MSCs (licensed with IFN-γ or other stimuli) in a tryptophan-containing medium. After a set period (e.g., 24-48 hours), collect the cell culture supernatant.
• Deproteinization: Precipitate proteins from the supernatant by adding 30% trichloroacetic acid, vortexing, and centrifuging (e.g., 10,000 × g for 10 minutes).
• Chromatographic Separation:
  • Column: A reverse-phase C18 column.
  • Mobile Phase: A mixture of buffers, such as 0.1 M potassium phosphate buffer (pH 4.0) and 0.1 M potassium phosphate buffer (pH 4.0) mixed with acetonitrile in a specific ratio (e.g., 94:6 v/v).
  • Flow Rate: 1.0 mL/min.
  • Detection: Use a UV/Vis or diode array detector. Kynurenine is detected at 365 nm, and tryptophan can be monitored at 280 nm.
• Calculation of Activity: Calculate the Kynurenine/Tryptophan (Kyn/Trp) ratio from their respective concentrations. This ratio is a direct indicator of IDO activity in the sample [32].

TSG-6 Quantification

As a secreted protein, TSG-6 is typically measured in the cell culture supernatant using an Enzyme-Linked Immunosorbent Assay (ELISA).

Protocol: TSG-6 via ELISA [30]

• Sample Collection: Culture MSCs under appropriate stimulatory conditions (e.g., with TNF-α). Collect the conditioned medium and centrifuge to remove any cells or debris.
• Antibody Coating: Coat a 96-well plate with a capture antibody specific for human TSG-6. Incubate overnight at 4°C, then block the plate with a protein-based blocking buffer.
• Sample & Standard Incubation: Add the conditioned medium samples and a series of known concentrations of recombinant TSG-6 protein (for the standard curve) to the wells. Incubate to allow TSG-6 to bind to the capture antibody.
• Detection Antibody: After washing, add a biotinylated detection antibody specific for TSG-6.
• Signal Development: Incubate with a streptavidin-Horseradish Peroxidase (HRP) conjugate. Finally, add a chromogenic HRP substrate (e.g., TMB) and measure the absorbance using a microplate reader.
• Quantification: Interpolate the concentration of TSG-6 in the samples from the standard curve.

PD-L1 Expression Analysis

PD-L1 is a cell surface protein, and its expression is most commonly and reliably quantified using flow cytometry.

Protocol: PD-L1 via Flow Cytometry [30]

• Cell Harvesting: Harvest MSCs, typically after licensing with IFN-γ, using a non-enzymatic cell dissociation solution to preserve surface protein integrity.
• Staining: Resuspend the cell pellet in a cold flow cytometry buffer. Aliquot cells and stain with a fluorescently conjugated anti-human CD274 (PD-L1) antibody. Include an isotype control antibody to account for non-specific binding.
• Incubation & Washing: Incubate the cells with antibodies for 30-60 minutes on ice or in the dark. Wash the cells twice with buffer to remove unbound antibody.
• Data Acquisition: Resuspend the cells in buffer and analyze them on a flow cytometer. Acquire a sufficient number of events (e.g., 10,000) for the MSC population, gated based on forward and side scatter.
• Data Analysis: The level of PD-L1 expression can be reported as the Geometric Mean Fluorescence Intensity (Geo MFI) or the percentage of PD-L1 positive cells.

Signaling Pathways and Biological Context

The immunosuppressive function of MSCs is often licensed by inflammatory signals. The following diagram illustrates how key stimuli induce the expression of the surrogate markers, which in turn mediate immunosuppressive effects on immune cells.

Diagram 1: MSC Immunosuppressive Pathway

The Scientist's Toolkit: Essential Research Reagents

The table below lists key reagents required for the quantification of these three potency markers, based on the described experimental protocols.

Table 3: Essential Research Reagents for Potency Marker Analysis

Reagent Category	Specific Example	Critical Function in the Assay
Cell Licensing Agents	Recombinant Human IFN-γ, Recombinant Human TNF-α	Induces the expression of IDO-1, PD-L1, and TSG-6 in MSCs, mimicking an inflammatory environment [30].
Chromatography Standards	L-Tryptophan, L-Kynurenine	Used as pure standards to create calibration curves for the accurate quantification of these metabolites via HPLC/LC-MS [31] [32].
Antibodies for Immunoassays	Anti-human CD274 (PD-L1) Antibody (conjugated), Anti-human TSG-6 Capture & Detection Antibodies	Enable specific detection and quantification of PD-L1 (via flow cytometry) and TSG-6 (via ELISA) [30].
Assay Kits	Commercial Human TSG-6 ELISA Kit	Provides a standardized, off-the-shelf solution for the sensitive and specific quantification of TSG-6 protein in conditioned media, streamlining the workflow.
Enzymatic Assay Components	L-Tryptophan, Ascorbic Acid, Methylene Blue	Key components of a colorimetric IDO activity assay; tryptophan is the substrate, while ascorbic acid and methylene blue help maintain the enzyme's active, reduced state [32].
Ftivazide	Ftivazide	Ftivazide is a thiosemicarbazone for research on multi-drug resistant tuberculosis (MDR-TB). This product is For Research Use Only. Not for human or veterinary use.
1-Bromo-5-methoxypentane	1-Bromo-5-methoxypentane, CAS:14155-86-3, MF:C6H13BrO, MW:181.07 g/mol	Chemical Reagent

While the immunomodulatory properties of Mesenchymal Stromal Cells (MSCs) have long been a research focus, their angiogenic potential represents an equally crucial therapeutic mechanism, particularly for applications in tissue regeneration and wound healing [17] [2]. The endothelial tube formation assay has emerged as a fundamental in vitro tool for quantifying this aspect of MSC potency, providing critical data for pre-clinical assessment of cellular therapeutic products [17] [37]. For MSC researchers transitioning from immunomodulation studies to vascular biology applications, understanding the capabilities, quantification methods, and advancements in tube formation assays is essential for comprehensive potency assessment. This guide objectively compares the spectrum of available assay methodologies, from traditional 2D approaches to advanced 3D microphysiological systems, providing experimental frameworks for their implementation in MSC research.

Methodological Spectrum: From Conventional to Advanced Assay Platforms

Conventional 2D Tube Formation Assay

The traditional 2D tube formation assay represents the most widely established approach for initial angiogenic potential assessment. This method involves plating endothelial cells (ECs) or endothelial progenitors on a basement membrane matrix such as Matrigel or Geltrex, where they spontaneously form capillary-like tubular structures within hours [37]. The assay captures the later stages of angiogenesis, specifically the reorganization and morphogenesis phase where endothelial cells align into nascent vascular networks.

Key Protocol Steps: Coating wells with extracellular matrix (50-100 μL/cm²), incubating for 30 minutes at 37°C for gel solidification, seeding cells at optimized density (3.5-4.5 × 10⁴ cells/200 μL for HUVECs), and incubating for 4-18 hours before imaging [37]. For MSC applications, this typically involves testing MSC-conditioned media or co-culture systems to evaluate paracrine effects on endothelial tubulogenesis.

Advantages and Limitations: This platform offers simplicity, cost-effectiveness, short culture periods, and amenability to higher-throughput screening [37]. However, significant limitations include large variation among different lots of endothelial cells and matrices, absence of heterospecific cell interactions, and the fact that tube formation may not represent true endothelial differentiation [37]. Additionally, conventional 2D systems lack physiological fluid flow and three-dimensional microenvironment cues present in vivo [38].

Advanced 3D and Microfluidic Assay Systems

Advanced model systems have emerged to address the physiological limitations of conventional 2D assays, offering enhanced predictive validity for clinical outcomes [39] [38].

Microfluidic 3D Angiogenesis Platforms: These systems incorporate endothelial cells within three-dimensional hydrogels (fibrin, collagen, or synthetic matrices) under perfused conditions, enabling application of physiological shear stress and spatial-temporal control over biomolecular gradients [38]. The OrganoPlate platform (Mimetas), for instance, comprises 40-96 microfluidic chips patterned underneath a microtiter plate, allowing formation of perfused microvessels against a patterned extracellular matrix [38]. These systems demonstrate key angiogenic behaviors including tip cell formation with characteristic filopodia, directional sprouting, and lumen formation [38].

On-Chip 3D Potency Assays: Recently developed microfluidic on-chip 3D systems culture cells in synthetic hydrogels (e.g., PEG-4MAL presenting RGD peptide) with continuous media perfusion [39]. These platforms have demonstrated elevated levels of immunomodulatory and trophic proteins compared to 2D culture, with improved correlation to clinical outcomes in a phase 3 trial for osteoarthritis [39].

Table 1: Comparison of Tube Formation Assay Platforms

Parameter	Conventional 2D Assay	3D Microfluidic Systems	On-Chip 3D Potency Assay
Physiological Relevance	Low; lacks flow, 3D environment	High; includes flow, 3D matrix, gradients	High; includes perfusion, 3D microenvironment
Throughput	High; amenable to 96-well format	Medium; 40-96 chips/plate	Medium; limited by device complexity
Assay Duration	Short (4-18 hours)	Extended (days to weeks)	24-hour perfusion typical
Cell Types	Primarily endothelial cells	Endothelial cells with supporting cells	Complex cocultures (e.g., BMAC samples)
Data Complexity	Basic morphometric parameters	Advanced parameters (sprouting distance, tip cells)	Secretory profile + morphological data
Clinical Correlation	Limited translation	Improved predictive value	Demonstrated correlation with clinical outcomes [39]
Cost & Accessibility	Low cost, widely accessible	Higher cost, specialized equipment	Highest cost, specialized expertise required

Quantitative Analytical Frameworks for Tube Formation Assessment

Traditional Morphometric Analysis

Conventional quantification of tube formation assays typically involves measuring basic parameters from 2D microscope images, including total tubule length, number of nodes (branching points), number of meshes, and mesh area [40]. These parameters provide gross morphological assessment but treat the vascular network as disconnected features rather than an integrated system [41]. The Angiogenesis Analyzer for ImageJ represents a widely used tool for this level of analysis, extracting characteristic points and elements of endothelial networks through an integrated program that avoids requirement for image pretreatment with additional plugins [40].

Graph-Theoretic Quantitative Framework

A recently developed graph-theoretic framework significantly advances the quantitative analysis of tube formation assays by transforming skeletonized images of endothelial networks into mathematical graphs [41]. This approach captures both morphological differences and temporal progression through computation of 11 graph-based metrics with biological relevance:

Table 2: Graph Theory Metrics for Tube Formation Quantification [41]

Graph Metric	Description	Biological Significance
Number of Nodes	Count of all distinct pixels forming part of the vascular structure	Indicates complexity of vascular structure; higher count suggests increased branching
Number of Edges	Total number of direct connections between nodes	Describes how well-connected each vessel junction is
Average Node Degree	Average number of connections per node	Higher values indicate better network interconnection
Clustering Coefficient	Measures degree of interconnection between a node's neighbors	Reflects network robustness and redundancy
Tortuosity	Ratio of actual path length to straight-line distance	Higher values indicate more curved, less efficient vessels
Global Efficiency	Inverse of the average shortest path length	Measures how efficiently the network transports materials
Connectivity Index	Quantifies how well-connected the network is overall	Reflects overall network quality and complexity
Largest Component Size	Number of nodes in the largest connected subgraph	Indicates degree of network integration versus fragmentation

This framework has demonstrated sensitivity in distinguishing different network morphologies, with sparse networks exhibiting significantly higher average node degree (p = 0.00079), clustering coefficient (p = 0.00109), and tortuosity, while dense networks showed greater node and edge counts (p = 0.00109) [41]. Temporal progression was effectively captured, with networks evolving from fragmented forms at 2 hours to integrated structures at 18 hours, reflected by increased largest component size (p = 0.00216), connectivity index (p = 0.00216), and efficiency (p = 0.0152) [41].

Experimental Design and Protocol Implementation

Standard Endothelial Tube Formation Assay Protocol

For researchers establishing tube formation assays to assess MSC angiogenic potential, the following protocol provides a robust foundation:

Day 0: Cell Preparation

• Prepare supplemented Medium 200PRF by adding Low Serum Growth Supplement (LSGS) [37]
• Seed cryopreserved human umbilical vein endothelial cells (HUVECs) at 2 × 10⁵ viable cells per 75-cm² tissue culture flask using LSGS-supplemented Medium 200PRF (15 mL total volume) [37]
• Change culture medium 24-36 hours after seeding and every other day thereafter until approximately 80% confluent (5-6 days) [37]

Day 5: Matrix Preparation

• Thaw extracellular matrix (Geltrex) at 4°C overnight [37]

Day 6: Assay Setup

• Add 50-100 μL of matrix per cm² to growth surface and incubate for 30 minutes at 37°C to allow gel solidification [37]
• Harvest HUVECs using trypsin/EDTA treatment and neutralization [37]
• Centrifuge cells at 180 × g for 7 minutes, remove supernatant, and resuspend in non-supplemented Medium 200PRF [37]
• Determine cell concentration and dilute to 3.5-4.5 × 10⁴ cells per 200 μL in non-supplemented medium (for negative control) or medium containing MSC-conditioned factors (for experimental conditions) [37]
• Gently add cells to matrix-coated wells at final media volume of ~200 μL/cm² [37]
• Incubate plate at 37°C, 5% COâ‚‚ for 4-18 hours [37]

Imaging and Analysis

• Image tubular structures using phase contrast or fluorescence microscopy (if using calcein-AM staining) [37]
• Analyze images using Angiogenesis Analyzer for ImageJ or custom graph-theoretic algorithms [41] [40]

Signaling Pathways in Endothelial Tube Formation

The tube formation process involves coordinated signaling events that can be modulated by MSC-derived paracrine factors. The following diagram illustrates key signaling pathways involved in angiogenesis that can be assessed through tube formation assays:

Essential Research Reagent Solutions

Successful implementation of tube formation assays requires specific reagent systems with careful quality control:

Table 3: Essential Research Reagents for Tube Formation Assays

Reagent Category	Specific Examples	Function & Importance	Quality Considerations
Extracellular Matrices	Geltrex, Matrigel, Collagen-I, Fibrin gels, PEG-4MAL hydrogel	Provides scaffolding for tube formation; biochemical and mechanical cues	High lot-to-lot variability requires batch testing; protein concentration affects results [37] [39]
Endothelial Cells	HUVECs, ECFCs, iPSC-ECs	Primary cellular component forming tubes	Donor variation affects reproducibility; iPSC-ECs offer unlimited expansion [42] [38]
Culture Media	Medium 200PRF with LSGS, EGM-2, endothelial basal media	Provides nutrients and growth factors	Serum content affects tube stability; growth factor composition crucial [42] [37]
Pro-Angiogenic Factors	VEGF, FGF-2, S1P, PMA	Induce and enhance tube formation	Concentration optimization required; VEGF typically 10-50 ng/mL [42] [38]
Inhibitors (Controls)	Sunitinib, 3PO, Suramin	Negative control compounds for assay validation	Sunitinib IC~50~ ~20-66 nM depending on cell type [38]
Detection Reagents	Calcein-AM, Calcein Blue, CellTrace dyes	Fluorescent visualization of tubular structures	Cell-permeable dyes must not disrupt tube architecture [37]

Tube formation assays represent a crucial component in the comprehensive potency assessment of mesenchymal stromal cell products, particularly for applications where vascularization is therapeutic. The methodological spectrum ranges from cost-effective conventional 2D assays for initial screening to advanced 3D microphysiological systems with demonstrated clinical correlation [39]. The emerging graph-theoretic analytical framework provides sophisticated quantitative tools that capture topological and organizational properties beyond basic morphometrics [41], while standardized protocols ensure reproducibility across research settings.

For MSC researchers, integrating tube formation assays with secretome analysis [2] and immunomodulatory potency assessments [17] creates a multidimensional potency matrix that better predicts in vivo performance. As regulatory requirements for cell therapy products evolve [2] [39], these biologically relevant angiogenesis assays will play increasingly important roles in product characterization and lot-release criteria, bridging the gap between in vitro assessment and clinical outcomes in regenerative medicine applications.

The development of cell therapies, particularly those using human Mesenchymal Stromal Cells (hMSCs), has been stymied by a significant translational challenge: the inability to accurately predict clinical efficacy, or potency, before product administration [43]. Current standard potency metrics for hMSCs often rely on co-culture systems with third-party immune cells, which face substantial challenges including donor variability and poor scalability, ultimately hindering their predictive power [43]. Furthermore, regulatory agencies like the US FDA require quantitative measures of biologic function for late-stage clinical investigations and commercial applications, a requirement that has led to rejected applications due to inadequate potency attributes [39]. This gap underscores the urgent need for innovative assay platforms that can reliably forecast clinical outcomes.

Microfluidic technology represents a paradigm shift in potency assay development. By enabling the miniaturization of laboratory processes into chip-based systems, microfluidics allows for the precise control of fluidic environments, high-throughput screening of cellular functions, and perfused 3D culture that better mimics the in vivo microenvironment [44] [45]. These systems require tiny sample volumes, reduce reagent consumption, and provide a more cost-effective platform for assessing the therapeutic potential of cell products [44]. The implementation of high-throughput on-chip microfluidic technologies is therefore poised to transform the development and quality control of cell-based therapies by providing clinically predictive potency data.

Core Microfluidic Technology Platforms for Potency Assessment

3D On-Chip Potency Assay for Clinical Outcome Prediction

A groundbreaking microfluidic on-chip 3D system has been developed specifically for predicting clinical outcomes of cell therapy candidates. This platform addresses the limitations of traditional 2D planar culture by providing cells with 3D structural, mechanical, and biochemical cues within a perfused microenvironment [39]. In a landmark study involving patient-derived bone marrow aspirate concentrate (BMAC) samples from a phase 3 multicenter trial for knee osteoarthritis (NCT03818737), this on-chip 3D system demonstrated superior predictive validity compared to conventional 2D assays [39].

The system utilizes a poly(dimethylsiloxane) (PDMS) microfluidic device where cells are encapsulated in a synthetic hydrogel—specifically, a 4-arm maleimide-functionalized poly(ethylene-glycol) (PEG-4MAL) hydrogel presenting cell-adhesive RGD peptide and cross-linked with protease-degradable peptides [39]. This engineered hydrogel supports cell viability and secretory activity while allowing media perfusion. The platform incorporates a specially formulated osteoarthritis simulated synovial fluid (simSF) that mimics the protein composition and viscous properties of native synovial fluid, creating a more physiologically relevant environment for testing [39].

When BMAC samples were cultured in this system, they exhibited elevated levels of immunomodulatory and trophic proteins compared to 2D culture, including cytokines, chemokines, cell adhesive proteins, and MMPs [39]. Most importantly, linear regression prediction models built using the secreted analyte data from the on-chip 3D system showed improved clinical prediction accuracy for patient pain scores compared to models using 2D culture data, establishing this platform as a powerful decision tool for cell therapy development [39].

High-Throughput On-Chip hMSC Potency Prediction

Another significant advancement comes from a specialized high-throughput, scalable, low-cost on-chip microfluidic potency assay designed specifically for hMSCs [43]. This platform addresses the critical limitation of current surrogate potency markers by offering improved functional predictive power and better recapitulation of in vivo secretory responses [43].

The assay compares hMSC secretory responses to functional hMSC-mediated immune cell suppression, identifying specific on-chip microfluidic potency markers with enhanced predictive capability [43]. Research demonstrates that hMSCs cultured in this on-chip system achieve secretory performance with improved similarity to in vivo models compared to traditional planar methods, highlighting the platform's ability to better mimic physiological conditions [43]. This technology enables researchers to overcome the shortcomings of current culture practices and provides a novel system with improved functional predictive power for assessing hMSC therapeutic potential.

Microfluidic Assay for Quantification of Cell Invasion (MAqCI)

For predicting metastatic potential in cancer research—a valuable corollary to potency assessment—the Microfluidic Assay for quantification of Cell Invasion (MAqCI) platform demonstrates how microfluidic technology can isolate and characterize rare cell subpopulations with enhanced functional capacity [46].

MAqCI features a unique design with parallel seeding and collection channels connected by Y-shaped microchannels that mimic the cross-sectional areas of in vivo pores and tracks (ranging from 10–300μm²) through which cells migrate during metastasis [46]. The device includes a relatively large feeder channel (20μm width × 10μm height) that branches into narrower channels (10μm or 3μm width), forcing cells to undergo mechanical constraints similar to those encountered during in vivo invasion [46].

This platform requires only 50,000 cells to accurately detect rare subpopulations constituting less than 0.1% of the total population, achieving 96% accuracy, 89% sensitivity, and 100% specificity in predicting metastatic potential when optimized for 12-14 hours with a threshold of 7-9% migratory cells [46]. In comparison, traditional transwell migration assays showed only 72% accuracy with significantly reduced sensitivity (22-33%) [46]. The ability to both quantify migratory behavior and isolate viable cells for subsequent molecular characterization makes this platform particularly valuable for comprehensive potency assessment.

Comparative Performance Analysis of Microfluidic Platforms

Table 1: Comparison of Key Microfluidic Potency Assay Platforms

Platform Feature	3D On-Chip Potency Assay	High-Throughput hMSC Potency Assay	MAqCI (Cell Invasion)
Primary Application	BMAC potency for osteoarthritis [39]	hMSC potency prediction [43]	Metastatic potential assessment [46]
Key Measured Parameters	Secreted immunomodulatory & trophic proteins [39]	Secretory responses, immune cell suppression [43]	Migratory cell abundance, proliferative index [46]
Throughput Capability	Multiple donors/conditions in parallel [39]	High-throughput, scalable design [43]	50,000 cells screened in <24h [46]
Clinical Predictive Power	Improved prediction of patient pain scores vs. 2D [39]	Improved functional predictive power vs. planar methods [43]	96% accuracy for metastatic potential [46]
Sample Volume/Cell Number	Not specified	Low sample volume [44]	50,000 cells [46]
Culture Dimension	3D with perfusion [39]	On-chip 3D microenvironment [43]	2D confinement channels [46]

Table 2: Analytical Performance Comparison with Traditional Methods

Performance Metric	Traditional 2D/Transwell Assays	Microfluidic On-Chip Platforms
Predictive Accuracy	Limited clinical predictive value [39]	Improved correlation with clinical outcomes [39]
Sensitivity	22-33% (transwell for metastasis) [46]	89% (MAqCI for metastasis) [46]
Specificity	94-100% (transwell for metastasis) [46]	100% (MAqCI for metastasis) [46]
Physiological Relevance	Low; lacks 3D structure and perfusion [39]	High; perfused 3D microenvironment [39]
Scalability	Limited by donor variability [43]	High-throughput capability [43]
Sample Consumption	Higher reagent/sample volumes [45]	Minimal reagent consumption [45]

Experimental Protocols for Implementation

Protocol for 3D On-Chip Potency Assay

The following detailed methodology outlines the implementation of the 3D on-chip potency assay for BMAC samples, as used in the phase 3 clinical trial analysis [39]:

• Device Preparation: Fabricate PDMS microfluidic devices using soft lithography techniques. The device design incorporates a central chamber for hydrogel containment with fluidic inlets and outlets for perfusion.

• Hydrogel Preparation and Cell Encapsulation:

  • Prepare the 4-arm maleimide-functionalized poly(ethylene-glycol) (PEG-4MAL) hydrogel solution.
  • Functionalize with cell-adhesive RGD peptide at a concentration of 2.0 mM.
  • Cross-link with a combination of protease-degradable peptide (1.0 mM) and non-degradable dithiothreitol (2.0 mM).
  • Mix BMAC cells with the hydrogel precursor solution at a concentration of 10-20 million cells/mL.
  • Pipette the cell-hydrogel mixture into the device chamber and allow cross-linking to complete (approximately 15-20 minutes at room temperature).

• Perfusion Culture:

  • Connect the device to a perfusion system with precise flow control.
  • Perfuse with appropriate media (control media or media supplemented with 10% simulated synovial fluid) at a rate of 1.0 μL/min for 24 hours.
  • Maintain temperature at 37°C with 5% COâ‚‚ supplementation.

• Sample Collection and Analysis:

  • Collect effluent media for secreted protein analysis.
  • Analyze samples using multiplexed immunoassays (Luminex or similar) for 24 immunomodulatory and trophic proteins relevant to MSC function.
  • Process data using hierarchical clustering and linear regression models to predict clinical outcomes.

Protocol for High-Throughput hMSC Potency Assessment

While specific methodological details are more limited in the available sources, the general approach for hMSC on-chip potency assessment involves [43]:

• Chip Design and Fabrication: Utilize high-throughput microfluidic designs capable of parallel processing of multiple hMSC donors and conditions.

• On-Chip Culture: Culture hMSCs in a microfluidic environment that promotes 3D organization and secretory function, potentially using micro-patterned surfaces or encapsulated 3D cultures.

• Stimulation and Secretion Analysis: Expose hMSCs to pro-inflammatory stimuli (such as IFN-γ) to activate immunomodulatory functions, then measure secretion of key potency markers (e.g., IDO, PGE2, TSG-6).

• Functional Correlation: Correlate secretory profiles with functional outcomes in immune cell suppression assays to validate predictive power.

Protocol for MAqCI Implementation

For cell invasion and migration assessment using MAqCI [46]:

• Device Priming: Pre-treat microfluidic channels with appropriate extracellular matrix proteins to facilitate cell adhesion and migration.

• Cell Loading: Introduce 50,000 cells in suspension into the seeding channel of the MAqCI device.

• Time-Lapse Imaging: Monitor cell movement through the Y-shaped microchannels using time-lapse microscopy over 12-14 hours.

• Data Analysis:

  • Classify cells as non-migratory if movement is limited to the feeder channel.
  • Classify as migratory if cells reach the bifurcation region and enter branch channels.
  • Calculate the percentage of migratory cells and establish correlation with functional potency.

Experimental Workflow and Signaling Pathways

Figure 1: Workflow for 3D On-Chip Potency Assay. This diagram illustrates the sequential process from sample preparation through clinical correlation, highlighting key steps in microfluidic potency assessment.

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Essential Research Reagents for Microfluidic Potency Assays

Reagent/Material	Function	Application Example
PEG-4MAL Hydrogel	Synthetic ECM for 3D cell encapsulation	Provides tunable 3D microenvironment for BMAC/hMSC culture [39]
RGD Peptide	Cell-adhesive ligand	Promotes integrin-mediated cell adhesion in synthetic hydrogels [39]
Protease-degradable Peptide	Matrix remodeling capability	Allows cell-mediated degradation and migration in 3D [39]
Simulated Synovial Fluid (simSF)	Physiological culture medium	Mimics in vivo joint environment for OA-relevant potency testing [39]
Lyo-Ready Direct RNA/DNA LAMP	Nucleic acid amplification	Detection of pathogen nucleic acids in diagnostic microfluidics [47]
Eva Green Fluorescent Dye	DNA intercalating dye	Real-time monitoring of amplification in microfluidic LAMP [47]
Tetraamminepalladium(2+) dinitrate	Tetraamminepalladium(2+) Dinitrate|CAS 13601-08-6
Isothiazole-5-carboxylic acid	Isothiazole-5-carboxylic acid, CAS:10271-85-9, MF:C4H3NO2S, MW:129.14 g/mol	Chemical Reagent

The implementation of high-throughput on-chip microfluidic technologies represents a transformative advancement in potency assessment for cell-based therapies. Platforms such as the 3D on-chip potency assay and high-throughput hMSC prediction system demonstrate superior clinical predictive power compared to traditional 2D methods, enabling more accurate forecasting of patient outcomes [43] [39]. The ability of these systems to recapitulate critical aspects of the in vivo microenvironment through perfused 3D culture, while simultaneously enabling scalable, cost-effective screening, positions them as essential tools for overcoming the translational challenges that have plagued the cell therapy field.

As microfluidic technology continues to evolve, emerging trends including AI-driven design, biodegradable chip materials, and increased integration with electronic and optical sensing will further enhance their capabilities [45]. For researchers and drug development professionals, adopting these innovative assay platforms now provides a strategic advantage in developing more effective, reliably potent cell therapies with a greater likelihood of clinical and regulatory success. The future of potency assessment lies in these integrated, physiologically relevant systems that bridge the gap between in vitro characterization and in vivo performance.

Overcoming Hurdles: Strategies for Robust and Reproducible Potency Testing

In the development of Mesenchymal Stromal Cell (MSC)-based therapies, demonstrating product potency through robust and reproducible in vitro assays is a critical regulatory requirement. Among various approaches, co-culture systems using Peripheral Blood Mononuclear Cells (PBMCs) have emerged as a gold standard for evaluating the immunomodulatory capacity of MSCs, a key attribute of their therapeutic function [48] [7]. These assays measure the ability of MSCs to suppress immune cell proliferation, reflecting their potential efficacy in treating autoimmune diseases, graft-versus-host disease (GVHD), and other inflammatory conditions [49] [7]. However, the inherent biological variability of PBMC donors, combined with differences in experimental design, can significantly impact assay results, leading to challenges in interpreting data and comparing results across studies and laboratories [48]. This guide objectively compares critical parameters in PBMC-based MSC potency assays—including PBMC sourcing, activation stimuli, and co-culture ratios—based on current experimental data, providing researchers with evidence-based strategies to enhance assay robustness and predictive value.

PBMC Sourcing and Donor Variability

The biological source of PBMCs constitutes a fundamental variable that can dramatically influence the outcome of MSC potency assays. Research indicates that different PBMC donors exhibit significant variation in their proliferative response to stimulation and their subsequent sensitivity to MSC-mediated immunosuppression [48].

Impact of Donor-Specific Cytokine Profiles

A comprehensive study investigating the correlation between cytokine production from PBMCs and the immunosuppressive function of MSCs revealed several key inflammatory mediators that significantly influence assay outcomes. The research identified that specific cytokines released by PBMCs upon stimulation showed a strong correlation with the degree of MSC-mediated inhibition of PBMC proliferation [48].

Table 1: Cytokines Correlated with MSC Immunosuppressive Potency in MLR Assays

Cytokine/Chemokine	Correlation with MSC Suppressive Function	Potential Role in Assay Outcome
IFN-γ	Significant positive correlation	Priming of MSC immunosuppressive pathways [49] [48]
TNF-α	Significant positive correlation	Synergizes with IFN-γ to enhance MSC function [49] [48]
CXCL10	Significant positive correlation	Chemoattractant for immune cells [48]
CCL5	Significant positive correlation	Regulates T-cell proliferation and activation [48]
HGF	Significant positive correlation	Paracrine immunomodulatory factor [48]

Strategies for Robust PBMC Sourcing

To mitigate donor-related variability and enhance assay consistency, researchers can adopt the following strategies based on experimental findings:

• Donor Pre-screening: Select PBMC donors demonstrating high proliferative capacity and production of key cytokines (e.g., IFN-γ, TNF-α) upon phytohemagglutinin (PHA) stimulation [48]. Using pre-qualified donors ensures a consistent and robust response platform for evaluating MSC potency.
• Pooled PBMCs: Consider creating small pools (e.g., 2-3 donors) from pre-screened donors to average out individual variations while maintaining a strong signal-to-noise ratio. This approach can enhance reproducibility compared to single-donor sources.
• Quality Control Metrics: Implement routine quality control for incoming PBMC lots, including viability assessment (>95% recommended) and baseline proliferation assays. This practice effectively increases the robustness of the in vitro assay system [48].

Stimuli Selection for PBMC Activation

The choice of stimulus to activate PBMCs and initiate an immune response is a critical experimental parameter that directly influences the mechanistic pathways involved and the subsequent immunomodulatory response of MSCs.

Comparison of Activation Methods

Table 2: Comparison of PBMC Activation Methods for MSC Potency Assays

Activation Method	Mechanism of Action	Advantages	Disadvantages
Phytohemagglutinin (PHA)	Mitogen; non-specifically stimulates T-cell proliferation via lectin-mediated receptor cross-linking.	- Strong, reproducible proliferative response- Well-established protocol- Suitable for standardization [48]	- Non-physiological activation- May not engage all relevant immune pathways
Anti-CD3/CD28 Beads	Mimics antigen-specific T-cell receptor activation and co-stimulation.	- More physiologically relevant- Consistent stimulation	- Higher cost- Requires bead removal from culture
Mixed Lymphocyte Reaction (MLR)	One-way reaction where PBMCs respond to allogeneic antigens from irradiated or mitomycin-C-treated feeder cells.	- Models allogeneic immune responses- Clinically relevant for transplantation	- Higher variability- More complex setup- Requires donor matching

Experimental Protocol: Standardized MLR Assay

A robust protocol for assessing MSC immunosuppressive function using PHA-activated PBMCs involves the following key steps [48]:

• PBMC Preparation: Thaw frozen PBMCs from qualified donors and rest overnight in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
• MSC Seeding: Seed MSCs at a predetermined density (e.g., 10,000-20,000 cells/well) in a 96-well plate and allow to adhere overnight in complete MSC medium.
• Co-culture Establishment: Label PBMCs with CFSE (5-10 μM) or similar cell proliferation dye according to manufacturer's instructions. Activate labeled PBMCs with PHA at an optimized concentration (typically 5-10 μg/mL). Add activated PBMCs to MSC monolayers at the selected ratio (e.g., 10:1 PBMC:MSC).
• Incubation and Analysis: Co-culture cells for 3-5 days in a humidified incubator at 37°C with 5% COâ‚‚. Analyze PBMC proliferation by flow cytometry using CFSE dilution or alternative methods like BrdU incorporation.

Co-culture Ratios and Conditioning Effects

The ratio of PBMCs to MSCs in co-culture systems directly influences the degree of immune activation and the subsequent immunomodulatory response. Furthermore, these interactions can "condition" or "prime" MSCs, enhancing their therapeutic properties.

Impact of Cell Ratios on Assay Outcomes

The PBMC:MSC ratio must be carefully optimized to balance sufficient immune activation for a measurable response with the immunosuppressive capacity of the MSCs being tested. Research indicates that ratios ranging from 5:1 to 10:1 (PBMC:MSC) often provide a suitable dynamic range for detecting differences in MSC potency [49]. Using ratios that are too high may overwhelm the MSC immunosuppressive capacity, while very low ratios may not provide adequate activation signals.

PBMC Conditioning of MSCs

Beyond their role in potency assays, PBMCs can actively enhance MSC function through a process called conditioning. Studies show that MSC conditioned by PBMC (cMSC) exhibit significantly enhanced immunomodulatory properties compared to resting MSCs (rMSC) [49].

• Molecular Mechanisms of Conditioning: Co-culture with PBMCs triggers significant changes in MSC gene expression, with 244 differentially expressed genes identified in cMSC compared to rMSC [49]. Key upregulated immune mediators include CCL2, CCL11, DPP4 (CD26), ICAM1 (CD54), IL6, PDCD1LG2 (CD273), and TNFRSF11B [49].
• Phenotypic Changes: Flow and mass cytometry analyses reveal that cMSC upregulate surface markers including CD26, CD54, and CD273 and intracellular molecules IDO1 and PTGS2 (COX-2), all associated with enhanced immunomodulation [49].
• Functional Consequences: This conditioning leads to functional improvements, as cMSC demonstrate a greater capacity to suppress activated T-cell proliferation in vitro and significantly reduce disease severity in a humanized mouse model of Myasthenia Gravis, halving severity scores from 2 weeks post-injection [49].

Diagram 1: PBMC Conditioning Enhances MSC Therapeutic Potential. Co-culture with PBMCs triggers molecular, phenotypic, and functional enhancements in MSCs [49].

Integrated Experimental Workflow for Assay Optimization

Implementing a systematic approach to PBMC-based potency assay development ensures reliable and reproducible results. The following workflow integrates the critical parameters discussed in this guide.

Diagram 2: Integrated Workflow for PBMC-Based MSC Potency Assay Development. A systematic, multi-step approach for optimizing critical assay parameters [49] [48].

The Scientist's Toolkit: Essential Research Reagents

Successful implementation of PBMC-based MSC potency assays requires specific reagents and materials. The following table details key solutions used in the featured experiments and their functional roles.

Table 3: Essential Research Reagents for PBMC-MSC Co-culture Studies

Reagent / Material	Function in Assay	Examples from Literature
PBMC Isolation Media	Density gradient separation of mononuclear cells from whole blood.	Lymphoprep (Axis-Shield) or equivalent polysucrose-based media [49].
PBMC Cryopreservation Medium	Long-term storage of PBMC lots for assay consistency.	FBS supplemented with 10% DMSO [49].
MSC Culture Medium	Expansion and maintenance of mesenchymal stromal cells.	αMEM or DMEM supplemented with 5-10% human platelet lysate (hPL) or FBS [49] [15].
PBMC Activation Stimuli	Induction of immune cell proliferation for potency measurement.	Phytohemagglutinin (PHA) at 5-10 μg/mL [48] or anti-CD3/CD28 activation beads.
Cell Proliferation Tracking	Quantification of PBMC division in response to MSC suppression.	CFSE dye dilution assay [49] or BrdU/EdU incorporation.
Cytokine Analysis Kits	Measurement of cytokine secretion correlated with MSC potency.	Multiplex immunoassays (e.g., Luminex) or ELISA for IFN-γ, TNF-α, CXCL10, etc. [48].
Menoctone	Menoctone, CAS:14561-42-3, MF:C24H32O3, MW:368.5 g/mol	Chemical Reagent

Optimizing PBMC sourcing, activation stimuli, and co-culture ratios is fundamental to developing robust MSC potency assays that yield meaningful, reproducible data for regulatory submissions and product characterization. Key takeaways include the importance of PBMC donor pre-screening to control for biological variability, the strategic selection of activation methods aligned with the mechanism of action of the MSC product, and the careful titration of cell ratios to ensure a measurable dynamic range. Furthermore, recognizing that PBMCs are not merely passive indicators but active participants that can condition MSCs to enhance their immunomodulatory function provides a more comprehensive physiological context for interpreting assay outcomes. By systematically applying these evidence-based parameters, researchers can significantly enhance the reliability and predictive power of their MSC potency assays, thereby accelerating the development of effective cell-based therapies.

For decades, two-dimensional (2D) cell culture on rigid plastic surfaces has been the standard tool for biological research and drug development. However, this method presents a starkly artificial environment that fails to replicate the complex three-dimensional architecture of human tissues. The growing emphasis on physiological relevance in preclinical studies, particularly for advanced therapies involving mesenchymal stromal cells (MSCs), has accelerated the adoption of three-dimensional (3D) culture systems. Within the context of potency assay development for MSC products, the choice of culture platform is not merely a matter of convenience but fundamentally influences cellular phenotype, secretome composition, and ultimately, therapeutic efficacy. This guide provides an objective comparison between 2D and 3D culture methodologies, supported by experimental data, to inform strategic decisions in therapeutic development.

Key Differences Between 2D and 3D Microenvironments

The transition from 2D to 3D culture represents more than just a geometrical change; it introduces critical biochemical and biophysical cues that closely mimic the in vivo niche. The table below summarizes the core distinctions that impact cell behavior.

Table 1: Fundamental Characteristics of 2D vs. 3D Culture Systems

Feature	2D Culture	3D Culture
Cell-Matrix Interactions	Primarily focal adhesions on a flat, rigid surface [50]	Multidirectional engagement with a soft, pliable matrix [50] [51]
Cell Morphology	Flattened, stretched cytoskeleton [50]	Condensed, volumetrically compact shape [52] [51]
Nutrient/Gradient Formation	Uniform exposure to nutrients and gases [50]	Diffusion-limited, creating oxygen/nutrient/metabolite gradients [50] [51]
Physiological Relevance	Low; oversimplifies tissue architecture [50] [53]	High; mimics key aspects of native tissue microenvironment [50] [53] [51]
Mechanical Cues	Excessively stiff substrates (~100,000 kPa) [52]	Tunable stiffness resembling soft native tissues [52]

Comparative Analysis: Functional Outcomes in MSC Research

Extensive research has quantified how these microenvironmental differences translate to variations in MSC functionality, which is critical for designing potency assays and manufacturing therapeutic products. The following data, compiled from recent studies, highlights these disparities.

Table 2: Experimental Data Comparison of MSC Behavior in 2D vs. 3D Culture

Parameter	2D Culture Findings	3D Culture Findings	Experimental Context
Cell Size	Significant enlargement over passages [52]	Reduced cell size; ~64% smaller volume in spheroids [52] [51]	Placenta-derived MSCs; alternated 2D/3D culture [52]
Immunomodulatory Function	Standard anti-inflammatory activity [19]	Enhanced potency; significantly higher inhibition of TNF-α, IL-1β, IL-6, IL-8, and MIP-1α [19]	UC-MSC conditioned medium on LPS-activated macrophages [19]
EV Yield & Cargo	Baseline EV secretion [50]	Significantly higher EV yield; enrichment of specific miRNAs (e.g., miR-1246, miR-21) and proteins (e.g., GPC-1) [50]	PANC-1 pancreatic cancer cell spheroids in ULA plates [50]
Therapeutic Efficacy (In Vivo)	Improved lung function in a bleomycin-induced fibrosis model [54]	Worsened outcomes; increased collagen deposition, leukocyte infiltration, and failed to improve lung function [54]	MSC-EVs administered in a murine model of lung fibrosis [54]
Production Cost/Efficiency	Higher cost per unit of anti-inflammatory effect [19]	11-40% lower cost for 1% inhibition of TNF-α [19]	Cost analysis of anti-inflammatory MTF production [19]

Detailed Experimental Protocols for 3D Culture

To ensure reproducibility in potency assay development, detailed methodologies for establishing 3D cultures are essential. Below are protocols for two common 3D systems cited in the comparative data.

Protocol 1: Generating MSC Spheroids Using Low-Attachment Plates

This scaffold-free method relies on cell-to-cell adhesion to form 3D aggregates [54] [52].

• Cell Seeding: Harvest MSCs from 2D monolayer culture and prepare a single-cell suspension. Seed cells into a non-adherent (ultra-low attachment) 96-well plate, typically at a density of 2.5 x 10^4 cells per well [54]. The well geometry promotes aggregation.
• Centrifugation: Centrifuge the plate at a low speed (e.g., 300-400 x g) for a few minutes to gather cells at the bottom of the wells.
• Culture: Incubate the plate for 24-72 hours at 37°C and 5% COâ‚‚. To prevent aggregate dispersal, cultures can be placed on a rotary shaker [50] [54].
• Media Formulation: Use serum-free media or media supplemented with exosome-depleted FBS for EV production studies [50]. To enhance spheroid viability and compactness, media can be supplemented with 0.25% methylcellulose [54] or defined extracellular matrix components [52].
• Harvesting: After incubation, compact spheroids are ready for downstream analysis. Conditioned media can be collected for EV isolation or analysis of secreted factors [50] [54].

Protocol 2: 3D Dynamic Culture in a Spinner Flask Bioreactor

This scalable, scaffold-based system uses microcarriers for large-scale production [19].

• Microcarrier Preparation: Hydrate and sterilize gelatin-based microcarriers (e.g., Cultispher-S) according to the manufacturer's instructions [19].
• System Inoculation: Inoculate a 125 mL disposable spinner flask with 1.33 x 10^6 MSCs and 0.35 g of prepared microcarriers in a medium volume of 100 mL.
• Cell Attachment: Apply intermittent agitation for 8 hours (e.g., 3 minutes at 80 rpm followed by 27 minutes at rest) to facilitate cell attachment to the microcarriers.
• Continuous Culture: After attachment, set the agitator to a constant 60 rpm. Maintain the culture for 5-6 days, monitoring glucose levels twice daily.
• Media Management: Perform partial media changes (25-50%) to maintain glucose levels above 60 mg/dL. On day 5, adjust to the final working volume of 125 mL for conditioned medium collection [19].

Signaling Pathways and Cellular Adaptations in 3D

The improved physiological relevance of 3D environments triggers distinct signaling cascades and cellular adaptations. The diagram below illustrates the key pathways and outcomes influenced by the 3D microenvironment, particularly in MSC spheroids.

Diagram: MSC Signaling Adaptations in 3D. The 3D microenvironment triggers adaptations through three primary mechanisms: altered adhesion leading to morphological changes and improved function; a metabolic shift towards glycolysis and enhanced mitochondrial activity that supports a robust secretome; and an activated stress response that promotes survival and pro-angiogenic signaling.

Essential Research Reagent Solutions

Transitioning to 3D culture requires specific reagents and tools. The following table lists key materials essential for establishing and analyzing 3D culture models, based on the protocols and studies cited.

Table 3: Key Research Reagents for 3D Cell Culture Applications

Reagent / Material	Function in 3D Culture	Example Application
Ultra-Low Attachment (ULA) Plates	Prevents cell adhesion, forcing cell-cell contact and spheroid self-assembly [50] [54].	Formation of scaffold-free MSC or cancer cell spheroids for potency or EV studies [50] [52].
Gelatin Microcarriers (e.g., Cultispher-S)	Provides a biodegradable 3D scaffold for cell attachment in dynamic bioreactor systems [19].	Large-scale expansion of MSCs in spinner flasks for production of conditioned medium [19].
RGD-Functionalized Alginate	Synthetic hydrogel incorporating RGD peptides to mimic integrin-binding sites of the ECM, enhancing cell adhesion [52].	Creating engineered 3D microenvironments (e.g., AlgTubes) for scalable cell aggregation [52].
Exosome-Depleted FBS	Provides essential growth factors while eliminating contaminating bovine EVs that could interfere with downstream analysis [50].	Production of cell-derived EVs in serum-containing conditioned media [50].
Methylcellulose	Increases medium viscosity to prevent agglomerate dispersal and promote formation of single, compact spheroids [54].	Enhancing the uniformity and integrity of MSC spheroids in ULA plates [54].
TrypLE Express	Enzyme-free cell dissociation reagent gentle enough to dissociate cells from sensitive 3D scaffolds and microcarriers [19].	Harvesting MSCs from 3D microcarrier cultures in bioreactors [19].

The evidence demonstrates that 3D culture systems are not simply an alternative but a necessary evolution for enhancing the physiological relevance of in vitro models, particularly in MSC research and potency assay development. While 2D culture remains useful for high-throughput screening, its limitations in predicting in vivo outcomes are clear. The 3D microenvironment fundamentally alters cell phenotype, improves secretome potency, and can offer more cost-effective manufacturing. However, the choice of 3D model is critical, as the functional outcomes—including paradoxical pro-fibrotic effects in certain disease models—are highly specific to the culture method. Researchers must therefore carefully select the 3D platform that best aligns with their therapeutic targets and potency metrics to successfully translate laboratory findings into clinical applications.

For developers of mesenchymal stromal cell (MSC) products, demonstrating biological activity through potency assays is a critical regulatory requirement. These assays must not only be scientifically valid but also feasible for the commercial lifecycle of a therapeutic. The journey from research-grade methods to Good Manufacturing Practice (GMP)-compliant quantitative assays presents significant challenges in scalability, cost-effectiveness, and seamless integration into controlled manufacturing workflows. This guide objectively compares alternative approaches for establishing potency assays, focusing on practical implementation within a GMP environment for MSC-based products, including emerging applications like MSC-derived small extracellular vesicles (sEVs).

Experimental Protocols for Potency Assay Evaluation

Cell-Based Bioassays for MSC-sEV Potency

Objective: To evaluate the therapeutic potential and lot-to-lot consistency of bone marrow MSC-derived sEVs (BM-MSC-sEVs) using a relevant cell-based assay.

Methodology: The protocol involves inducing oxidative stress in a retinal pigment epithelium cell line (ARPE-19) to model retinal degeneration, a target for MSC-sEV therapies [15].

• Cell Culture: ARPE-19 cells are maintained in standard culture conditions.
• Oxidative Stress Induction: Cell damage is induced by exposure to hydrogen peroxide (Hâ‚‚Oâ‚‚).
• Therapeutic Intervention: BM-MSC-sEVs are applied to the damaged ARPE-19 cells at a concentration of 50 µg/mL. Two treatment regimens are evaluated: application 24 hours before Hâ‚‚Oâ‚‚ exposure (preventive) and 24 hours after exposure (therapeutic) [15].
• Viability Assessment: Cell viability is quantified post-treatment. Viability of ARPE-19 cells after Hâ‚‚Oâ‚‚ exposure alone was 37.86 ± 0.61% [15].
• Apoptosis Analysis: Flow cytometry is used to measure the percentage of total apoptotic cells, providing a mechanistic insight into the sEVs' protective effect [15].

Key Findings: Application of BM-MSC-sEVs significantly increased ARPE-19 cell viability to 54.60 ± 3.59% (pre-treatment) and 52.68 ± 0.49% (post-treatment), and demonstrated a significant reduction in total apoptotic cells [15].

Flow Cytometry for MSC and sEV Characterization

Objective: To ensure the identity and purity of the starting MSC population and characterize sEV surface markers, which is a prerequisite for a meaningful potency assay.

Methodology: This multi-parameter technique is used for immunophenotyping.

• Cell Staining: MSCs or sEVs are stained with fluorescently-labeled antibodies against specific markers [55].
• Data Acquisition: Samples are run through a flow cytometer, which measures fluorescence intensity and light scatter properties for each event (cell or particle) [55].
• Gating Strategy:
  • Viable Cell Selection: A gate is set based on forward and side scatter to eliminate dead cells and debris from the analysis [55].
  • Single Cells: A gate on forward scatter area versus height is used to exclude doublets or cell aggregates, ensuring analysis of single cells [55].
  • Population Identification: Subsequent gates are used to identify and quantify specific cell populations based on their marker expression (e.g., MSC positive markers: CD73, CD90, CD105; negative markers: CD34, CD45) [15] [55].
• Quantification: The percentage of cells expressing a particular marker is calculated from the gated populations [55].

Comparative Analysis of Quantitative Data

The following tables summarize key experimental data from the evaluation of MSC-sEV production and potency, providing a basis for comparing methodological choices.

Table 1: Comparison of sEV Isolation Methods and Functional Outcomes

Data derived from studies on Bone Marrow MSC-derived sEVs (BM-MSC-sEVs) [15].

Parameter	Ultracentrifugation (UC)	Tangential Flow Filtration (TFF)	Functional Outcome of sEVs on ARPE-19 cells
Particle Yield	Lower	Statistically higher than UC	Cell Viability after H₂O₂ exposure: 37.86% ± 0.61% (untreated control)
Scalability	Low; suited for research scale	High; suited for larger-scale production	Viability with sEV pre-treatment: 54.60% ± 3.59%
Process Integration	Manual, open-process risk	Amenable to automation and closed systems	Viability with sEV post-treatment: 52.68% ± 0.49%
Key Advantage	Considered a classical, widely published method	Increased yield and improved scalability	Significant reduction in total apoptotic cells demonstrated by flow cytometry

Table 2: Impact of Culture Media on MSC Growth and sEV Production

Data showing the effect of basal culture media on BM-MSCs and their subsequent sEV characteristics [15].

Parameter	DMEM-based Medium	α-MEM-based Medium	sEV Characterization (Both Media)
Cell Population Doubling Time (Passage 3-6)	1.90 to 2.25 days	1.85 to 1.99 days	Particle Morphology: Cup-shaped, confirmed by TEM
Expansion Ratio	Lower	Higher	Size Range: 107 - 114 nm, confirmed by NTA
Particle Yield per Cell	3,751 ± 2,059 particles/cell	4,319 ± 2,110 particles/cell	Positive Markers: CD9, CD63, TSG101, confirmed by Western Blot
Statistical Significance	Not significant (p-value > 0.05)	Not significant (p-value > 0.05)	Inter-donor Variability: Significant differences in particle concentration observed between donors

Visualizing Experimental Workflows

The following diagrams illustrate the core experimental and data analysis processes described in this guide.

Diagram 1: sEV Potency Bioassay Workflow

Diagram 2: Flow Cytometry Gating Strategy

The Scientist's Toolkit: Key Research Reagent Solutions

A robust potency assay requires high-quality, well-characterized reagents. The table below details essential materials and their functions in the context of MSC and MSC-sEV potency evaluation.

Reagent/Material	Function in Assay Workflow	Key Considerations for GMP Integration
Human Platelet Lysate (hPL)	Serum supplement for xeno-free MSC culture expansion; supports cell growth and sEV production [15].	Sourced from GMP-compliant vendors with full traceability and rigorous pathogen testing.
Characterized Antibody Panels	Immunophenotyping of MSCs (CD73, CD90, CD105) and characterization of sEV surface markers (CD9, CD63) [15] [55].	Validation for intended use is critical. Certificates of Analysis (CoA) detailing specificity, cross-reactivity, and conjugation efficiency are required.
Cell Culture Media (α-MEM/DMEM)	Basal medium formulation impacting MSC proliferative capacity and subsequent sEV yield [15].	Raw material identity testing and adherence to compendial standards (e.g., USP) ensure consistency and reduce process variability.
Apoptosis/Necrosis Assay Kits	Quantification of cell death mechanisms (e.g., in ARPE-19 cells) as a measure of sEV therapeutic potency [15].	Kits should be qualified for performance in the specific assay format. Component-level CoAs support regulatory filings.
Magnetic Beads & Cell Sorting Reagents	Isolation and purification of specific cell populations (e.g., Tregs, MSCs) as a starting material for therapy and assay development [56].	GMP-grade, clinical-grade reagents are essential for manufacturing. Closed-system separation technologies are preferred for scalability [56].

The journey from a research-grade assay to a GMP-compliant potency method requires strategic decisions based on scalability, cost, and integration potential. The data demonstrates that isolation methods like Tangential Flow Filtration (TFF) offer a clear advantage over Ultracentrifugation (UC) for scalable sEV production, a critical consideration for commercial therapeutics [15]. Furthermore, the foundational choices of cell culture media, while not always yielding statistically significant differences in a research setting, can impact cell growth and particle yield, influencing long-term process robustness [15].

Integrating "quality-by-design" principles from the outset, using standardized and automatable methods like flow cytometry and scalable filtration, and selecting GMP-suitable reagents are not just best practices—they are essential for ensuring that the potency assays for innovative MSC products are not only feasible but also robust and compliant throughout the product lifecycle [57]. This structured approach, which balances scientific rigor with manufacturing practicality, is key to successfully advancing mesenchymal stromal cell products from the research bench to the patient.

Ensuring Quality and Compliance: Validation and Comparative Analysis of Potency Assays

For researchers and drug development professionals working with Mesenchymal Stromal Cell (MSC) products, the development of robust potency assays represents one of the most significant challenges in translational science. Potency is defined by regulatory agencies as the "specific ability or capacity of the product to effect a given result" and is recognized as a critical quality attribute (CQA) for biologics [58] [59]. The International Council for Harmonisation (ICH) provides the harmonized framework for analytical procedure validation through its ICH Q2(R1) guideline, which has become the global gold standard for ensuring the reliability of analytical data [60] [61]. For MSC-based therapies, this validation process is particularly complex due to the multifactorial mechanism of action (MoA) and inherent biological variability of living cell products [62] [59].

The path to validation requires a methodical approach to demonstrating that a potency assay is accurate, precise, and robust enough to serve as a reliable measure of biological activity throughout the product lifecycle. This process begins early in development and evolves through clinical stages, culminating in a fully validated assay required for Biologics License Application (BLA) submission [63] [59]. This guide examines the experimental approaches and quantitative criteria necessary to meet ICH Q2(R1) standards for three fundamental validation parameters—accuracy, precision, and robustness—within the context of MSC potency assay development, providing directly applicable methodologies and acceptance criteria for scientific teams.

Core Validation Parameters: Experimental Designs and Acceptance Criteria

Accuracy: Demonstrating Closeness to True Value

Accuracy represents the closeness of agreement between the measured value and a reference value accepted as either a conventional true value or an accepted reference value [60] [61]. For MSC potency assays, which typically measure relative potency (%RP) rather than absolute values, accuracy validation requires careful experimental design using well-characterized reference materials.

Experimental Protocol for Accuracy Determination:

• Sample Preparation: Prepare a minimum of nine determinations across at least three concentration levels covering the specified range of the assay (e.g., 50%, 100%, 150% of target potency) [60]. For MSC immunomodulatory potency assays, this may involve spiking known quantities of reference standard into matrices such as culture media or patient samples.
• Reference Standard: Use a qualified MSC reference standard with assigned potency units. The reference material should be prepared using the same manufacturing process as the test product and fully characterized [59].
• Calculation: Express accuracy as percentage recovery of the known, spiked amount. For relative potency assays, compare the measured potency to the expected potency of the reference standard [60].

Table 1: Accuracy Acceptance Criteria for MSC Potency Assays

Sample Type	Concentration Level	Target Recovery	Acceptance Criteria
MSC Secretome	50%	Theoretical potency	80-120%
Cell-Based Therapy	100%	Reference standard	90-110%
Immunomodulatory	150%	Theoretical potency	80-120%

Data from implementation of an IL-1RA secretion potency assay for MSC batches demonstrated accuracy with percentage recoveries within 90-110% across the validation range, successfully meeting ICH Q2(R1) criteria [64].

Precision: Quantifying Method Variability

Precision validation encompasses three tiers: repeatability (intra-assay precision), intermediate precision (inter-assay precision), and reproducibility (inter-laboratory precision) [60] [61]. For MSC potency assays, higher variability is expected due to biological systems, making precision evaluation particularly critical.

Experimental Protocol for Precision Determination:

• Repeatability: Have a single analyst perform the assay on six independent preparations of the same homogeneous MSC sample under identical conditions within the same day [60]. Calculate the mean, standard deviation, and percentage coefficient of variation (%CV).
• Intermediate Precision: Introduce intentional variations including different analysts, different days, and different equipment [60]. Maintain the same MSC reference standard and critical reagents. A full experimental design should include a minimum of two analysts testing the same sample on three different days.
• Reproducibility: Conduct collaborative studies between two or more laboratories using the same standardized protocol and reference materials [60].

Table 2: Precision Acceptance Criteria for MSC Potency Assays

Precision Level	Experimental Design	Acceptable CV	Application Phase
Repeatability	6 replicates, same analyst/day	≤15-20%	Early development
Intermediate Precision	2 analysts, 3 days, same lab	≤20-25%	Phase I-II trials
Reproducibility	2 labs, standardized protocol	≤25-30%	Phase III validation

Statistical approaches for potency assay variability estimation utilize linear mixed models to differentiate between various sources of variability, including inter-run, intra-run, and dilution series variability [63]. Reportable results for MSC products often average multiple %RP values from different assay runs to control precision, with the number of runs selected based on the assay's inherent variability and the desired out-of-specification (OOS) rate [63].

Robustness: Evaluating Method Resilience

Robustness measures the capacity of an assay to remain unaffected by small, deliberate variations in method parameters, providing indication of its reliability during normal usage [60] [61]. For cell-based potency assays, this parameter is particularly important due to the complexity of biological systems.

Experimental Protocol for Robustness Determination:

• Parameter Selection: Identify critical method parameters that may vary during routine testing. For MSC immunomodulatory assays, this may include incubation time (±2 hours), temperature (±1°C), cell passage number (±2 passages), reagent lots (at least 3 different lots), and analyst proficiency [23] [59].
• Experimental Design: Systematically vary one parameter at a time while keeping others constant. For each condition, analyze the same MSC reference standard and calculate the percentage difference from the nominal value obtained under standard conditions.
• Acceptance Criteria: Establish predefined acceptance criteria, typically requiring that all variations produce results within ±15% of the nominal value [59].

Implementation examples from optimized immunomodulatory potency assays demonstrate how variations in critical parameters such as mitogen concentration (e.g., PHA, CD3/CD28 antibodies), PBMC donor variability, and culture conditions can be systematically evaluated to establish robustness ranges [23]. The use of cryopreserved PBMCs from characterized donors, standardized cryomedium formulations, and predefined system suitability criteria all contribute to enhanced assay robustness [23].

Advanced Methodologies: 3D Platforms and Orthogonal Approaches

Traditional 2D culture systems for MSC potency assessment often lack the physiological relevance needed for clinical predictivity. Recent advances include the development of 3D microfluidic platforms that better recapitulate the in vivo microenvironment [39]. These systems demonstrate enhanced secretory profiles of immunomodulatory and trophic factors from MSC samples compared to 2D cultures, potentially offering improved correlation with clinical outcomes [39].

For complex MSC products with pleiotropic mechanisms of action, a matrix of potency assays rather than a single test may be necessary to fully capture the biological activity [62] [59]. This approach aligns with regulatory expectations for a potency assay or matrix of assays to capture the complete MoA when scientifically feasible [63]. Orthogonal methods with different scientific principles can strengthen the overall potency assurance strategy.

The following workflow visualization outlines the complete validation pathway for MSC potency assays, integrating all critical parameters and decision points:

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful implementation of validated MSC potency assays requires carefully selected reagents and materials. The following table details key research reagent solutions and their critical functions in assay development and validation:

Table 3: Essential Research Reagent Solutions for MSC Potency Assays

Reagent/Material	Function	Application Examples	Validation Considerations
Qualified PBMCs	Respondent cells in immunomodulatory assays	Lymphocyte proliferation inhibition [23]	Donor characterization, cryopreservation protocol standardization
Reference Standard	Well-characterized MSC material of known potency	System suitability testing, relative potency calculation [63]	Extensive characterization, stability monitoring, establishment of biological activity units
Critical Mitogens/Stimuli	Induce lymphocyte proliferation for inhibition measurements	PHA, CD3/CD28 antibodies, MLR [23]	Titration to optimal concentration, qualification of multiple lots
GMP-Compliant Cryomedium	Maintain cell viability and function after thawing	CryoStor CS10, HSA-based formulations [23]	Validation of post-thaw recovery and functionality
Detection Antibodies/Analytes	Quantify secreted factors or surface markers	IL-1RA ELISA, multiplex cytokine panels [64] [39]	Specificity demonstration, cross-reactivity profiling
Cell Culture Matrix	3D scaffold for microphysiological systems	PEG-4MAL hydrogel, synthetic scaffolds [39]	Characterization of mechanical and biochemical properties
Culture Media/Supplements	Support MSC function and viability	Simulated synovial fluid, specialized media formulations [39]	Composition consistency, growth factor qualification

Statistical Analysis and Data Interpretation

The statistical framework for analyzing potency data relies heavily on relative potency (RP) calculation derived from dose-response curves, most commonly using parallel-line analysis or 4-parameter logistic (4PL) models [63] [58]. The fundamental assumption of parallelism must be met for meaningful derivation of %RP, ensuring that the test sample and reference standard have similar curve shapes [63].

For precision estimation, linear mixed models are employed to differentiate between various sources of variability, including inter-run, intra-run, and dilution series variability [63]. The variability of reportable results can be optimized by adjusting the number of independent assay runs, with the relationship between run numbers and out-of-specification (OOS) rates informing the testing strategy [63].

The following diagram illustrates the statistical relationships and decision process in potency assay validation:

The path to successful potency assay validation for MSC products requires a phase-appropriate approach that begins early in development and evolves with increasing product understanding [63] [59]. During early-phase clinical trials, the focus should be on demonstrating accuracy, precision, and robustness sufficient to support initial safety assessments and early efficacy signals. As the product advances toward commercialization, the validation package must expand to include full method validation per ICH Q2(R1) requirements [60] [61].

The most successful validation strategies incorporate risk-based principles aligned with ICH Q9 and ICH Q14, focusing resources on the most critical assay parameters that impact product quality and patient safety [61]. By systematically addressing accuracy, precision, and robustness through well-designed experimental protocols and appropriate statistical analysis, development teams can establish potency assays that not only meet regulatory expectations but also provide meaningful insights into product quality and consistency throughout the product lifecycle.

Mesenchymal stromal cells (MSCs) have emerged as a promising cellular therapy for immune-mediated disorders, including graft-versus-host disease (GvHD) and autoimmune conditions, due to their potent immunomodulatory properties [65] [66]. The transition of MSC products from research to advanced-phase clinical trials necessitates the development of robust potency assays that can quantitatively measure their biological activity [2]. Regulatory authorities, including the U.S. Food and Drug Administration (FDA) and European Medicines Agency, mandate potency assays as part of product release criteria for cellular therapy products [2] [67]. These assays must demonstrate accuracy, precision, linearity, and robustness according to International Council for Harmonisation (ICH) Q2 (R1) guidelines [65] [66]. The mixed lymphocyte reaction (MLR) has emerged as a leading platform for assessing MSC potency by measuring their capacity to suppress T-cell proliferation, a key mechanism of action relevant to many clinical applications [66] [68]. This case study examines the full validation of two distinct MLR potency assay formats and provides a comparative analysis of their performance characteristics, methodologies, and applications in the context of MSC product development.

MLR Potency Assay Methodologies and Experimental Protocols

Core MLR Experimental Design and Principle

The fundamental principle of the MLR potency assay involves co-culturing MSCs with allogeneic peripheral blood mononuclear cells (PBMCs) and quantifying the inhibition of T-cell proliferation [65] [66]. The percentage of inhibition of lymphocyte proliferation is calculated using the formula: Ratio × percentage of inhibition = (control percentage of division — ratio × percentage of division)/control percentage of division [65]. The assay can be configured in different formats, including one-way MLR (with inactivated stimulator cells) and two-way MLR (where both populations can respond) [68]. The inhibition percentage of lymphocyte proliferation as a function of co-culture ratios is typically represented in a line graph, with the corresponding area under the curve (AUC) representing MSC's ability to inhibit lymphocyte proliferation [65].

Detailed Cell Culture and Co-culture Protocols

PBMC Isolation and Preparation: PBMCs are isolated from buffy coats or whole blood using density gradient centrifugation with Lymphocyte Separation Medium or Ficoll-Hypaque [65] [66]. Cells are washed twice with phosphate-buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) and pipetted through a 70 μm cell strainer [66]. PBMC purity and cell counts are determined using an automated hematology analyzer [66]. For proliferation tracking, PBMCs are labeled with fluorescent dyes such as CellTrace Violet (CTV) or Violet Proliferation Dye 450 (VPD450) at concentrations of 2.5 μM for 20 minutes at 37°C [65] [66]. The staining reaction is stopped by adding an equal volume of complete medium supplemented with human serum.

MSC Culture and Preparation: MSCs are typically isolated from bone marrow and cultured in minimum essential medium α (MEM α) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml fibroblast growth factor (FGF) [65]. For clinical-grade MSCs, culture may use human thrombocyte lysate instead of FBS [66]. MSCs at passages 2-5 are used, characterized by flow cytometry for surface markers (CD45-, CD73+, CD90+, CD105+) according to International Society for Cell & Gene Therapy (ISCT) recommendations [65]. Before co-culture, MSCs are mitotically inactivated using gamma irradiation (30 Gy) and seeded at 0.3 × 10^6 cells/cm² [66].

Co-culture Setup: Various MSC to PBMC ratios are tested, typically ranging from 1:1 to 1:300 [65]. PBMC numbers per well remain constant (e.g., 3 × 10^5), while MSC numbers vary according to the ratios [65]. T-cell stimulants such as anti-CD3 and anti-CD28 antibodies are added at final concentrations of 0.4 μg/mL each [66]. Co-cultures are maintained in complete medium (RPMI-1640 with GlutaMAX supplemented with 10% FBS or human serum) for 4-7 days at 37°C in a 5% CO₂ humidified atmosphere [65] [66].

Analytical Validation of MLR Potency Assays

Comparative Performance of Validated MLR Assay Formats

Two comprehensive studies have validated MLR potency assays according to ICH Q2 (R1) guidelines, with key analytical performance parameters summarized in the table below.

Table 1: Comparative Analytical Validation of MLR Potency Assays for MSCs

Validation Parameter	Flow Cytometry-Based MLR [66]	CTV-Based MLR with AUC [65]
Precision (Repeatability)	<10% variation	6.1% standard deviation
Intermediate Precision	<15% variation	4.6% standard deviation
Linearity Range	PBMC:MSC ratios 1:1 to 1:0.01	Linear across tested ratios (1:1 to 1:300)
Robustness	Unaffected by PBMC inter-donor variability	Robust to PBMC concentration variations
Specificity	Specific inhibition by viable MSCs	Differentiates MSC with varying inhibition activities
Measurement Range	Distinguishes subpotent batches	AUC calculation provides quantitative measure
Reference Method Correlation	r = 0.9021 with BrdU assay	Not specified

Critical Methodological Variations and Their Impact

Stimulation Methods: MLR assays can utilize different stimulation methods. The allogeneic response relies on T-cell receptor recognition of non-self MHC molecules on stimulator cells, engaging 1-10% of the T-cell repertoire [68]. Alternatively, antibody-based stimulation using anti-CD3 and anti-CD28 antibodies provides a more standardized and reproducible activation signal, specifically triggering T-cell proliferation through TCR and co-stimulatory pathway engagement [66].

Detection Methods: Flow cytometry-based proliferation tracking using dye dilution (CTV, VPD450, or CFSE) enables precise quantification of cell division at single-cell resolution and allows for immunophenotyping of responding cells [65] [66]. Traditional methods include [3H]-thymidine incorporation, which measures DNA synthesis, and metabolic assays, though these lack cellular resolution [68].

Assay Readouts: The percentage of inhibition can be calculated based on division indices of T cells, or the area under the curve (AUC) can be determined from inhibition curves across multiple MSC:PBMC ratios, providing a comprehensive potency measure [65]. Additional readouts include cytokine profiling (IFN-γ, TNF-α, IL-2) and activation marker expression (CD25, CD69) [68].

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagent Solutions for MLR Potency Assays

Reagent Category	Specific Examples	Function in MLR Assay
Cell Isolation Media	Lymphocyte Separation Medium (Eurobio), Ficoll-Hypaque	PBMC isolation via density gradient centrifugation
Proliferation Tracking Dyes	CellTrace Violet, VPD450, CFSE	Fluorescent cell labeling to track division history
Cell Culture Media	RPMI-1640 with GlutaMAX, MEM α with GlutaMAX	Base medium for cell co-culture
Serum Supplements	Fetal bovine serum (FBS), human AB serum	Provide essential growth factors and nutrients
T-cell Stimulants	Anti-CD3/CD28 antibodies, staphylococcal enterotoxin B (SEB)	Activate T-cell proliferation pathways
Flow Cytometry Antibodies	Anti-CD4, CD8, CD25, CD69, Ki67	Immunophenotyping of responding T cells
Cell Viability Markers	7-AAD, trypan blue	Distinguish viable from non-viable cells
Cytokine Detection	ELISA kits, multiplex bead arrays	Quantify cytokine secretion profiles

T-cell Activation Signaling Pathways in MLR

The MLR assay recapitulates key T-cell activation signaling events that occur in response to allogeneic stimulation. Understanding these molecular mechanisms is essential for proper assay design and interpretation.

Applications in Drug Discovery and MSC Product Development

Assessing Immunomodulatory Drug Effects

The MLR potency assay serves as a robust platform for evaluating the effects of various therapeutic agents on T-cell function [68]. Immune checkpoint inhibitors (anti-PD-1, anti-PD-L1, anti-CTLA-4 antibodies) demonstrate enhanced T-cell responses in MLR by blocking inhibitory pathways [68]. Conversely, immunosuppressive agents (calcineurin inhibitors like tacrolimus and cyclosporine, mTOR inhibitors like sirolimus, corticosteroids) show dose-dependent suppression of T-cell proliferation [68]. Immunomodulatory drugs (lenalidomide, TGF-β receptor inhibitors) exhibit complex modulation of T-cell responses, which can be quantified using MLR readouts [68].

Quality Control for MSC-Based Therapeutics

For MSC manufacturers, validated MLR potency assays are essential for batch release testing and ensuring consistent product quality [65] [66]. The assays can differentiate between MSC batches with varying immunosuppressive capacities, preventing the release of subpotent products [66]. MSC potency can be correlated with clinical outcomes in conditions like GvHD, where the mechanism of action directly involves T-cell suppression [66]. The use of cryopreserved PBMC banks from multiple donors (e.g., 10 donors) provides standardized responder cells, reducing inter-assay variability and enabling long-term stability of testing reagents [65].

The full validation of MLR potency assays according to ICH Q2 guidelines represents a critical advancement in the field of MSC therapeutics. The two assay formats examined demonstrate that properly validated MLR methods exhibit excellent precision, linearity, and robustness suitable for quality control testing of clinical-grade MSC products [65] [66]. The flow cytometry-based approach offers the advantage of multiparametric analysis and correlation with reference methods [66], while the CTV-based method with AUC calculation provides a comprehensive measure of inhibition across multiple cell ratios [65]. As the field progresses, MLR potency assays will likely be incorporated into matrix testing approaches that capture the plurality of MSC effector pathways [2], potentially combined with secretome analysis [2] and advanced in vitro models [67] to better predict clinical performance. The standardization of such potency assays across manufacturing facilities will be essential for comparing MSC products from different sources and advancing the entire field of cellular therapy.

Within the development of cell-based medicinal products, such as mesenchymal stromal cell (MSC) therapies, potency assays are mandatory release criteria required by regulatory authorities for advanced-phase clinical trials [2]. These assays measure the biological activity of a product and its ability to elicit a specific therapeutic effect. The central challenge lies in selecting an assay format that accurately predicts clinical efficacy while being feasible for routine quality control. This guide provides an objective comparison between two fundamental approaches: functional assays, which measure a product's biological effect on a living system, and surrogate assays, which quantify a specific physical or chemical attribute that correlates with the biological activity [67]. The analysis is framed within the context of potency testing for MSC products and their derivatives, such as extracellular vesicles (MSC-EVs).

Core Concepts and Definitions

Functional Potency Assays

Functional, or biological, potency assays use a living system (in vitro, in vivo, or ex vivo) to measure a product's functional capacity. These assays are designed to model the pathophysiological environment and directly measure a therapeutic response linked to the mechanism of action (MoA) [64] [67]. For MSCs, a key MoA is immunomodulation, and a classic functional assay involves co-culturing MSCs with immune cells, such as activated peripheral blood mononuclear cells (PBMCs) or polarized macrophages, and measuring the suppression of T-cell proliferation or the secretion of anti-inflammatory factors [2] [64].

Surrogate Potency Assays

Surrogate potency assays are non-biological tests that measure a specific critical quality attribute (CQA), such as the concentration of a key biomarker, that has been demonstrated to correlate with the product's biological function [69] [67]. These assays are often physicochemical, such as an enzyme-linked immunosorbent assay (ELISA) to quantify a specific protein, or a Surface Plasmon Resonance (SPR) assay to characterize binding kinetics [69] [70]. The surrogate biomarker must be rigorously validated against a functional readout to ensure it faithfully represents therapeutic potency.

Direct Comparison of Assay Formats

The following table summarizes the key characteristics of functional and surrogate assay formats, highlighting their comparative merits and challenges.

Table 1: Direct Comparison of Functional and Surrogate Potency Assays

Feature	Functional Assays	Surrogate Assays
Fundamental Principle	Measures a biological response in a living system [2] [64]	Measures a physicochemical attribute that correlates with function [69] [67]
Therapeutic Relevance	High; directly models biological mechanism of action [64]	Variable; depends entirely on the validated link between the biomarker and function [67]
Complexity & Duration	High; can involve cell culture, co-cultures, and multi-day protocols [2]	Low; typically faster, simpler, and more straightforward [67]
Robustness & Reproducibility	Variable; susceptible to biological variability of responder cells [2]	High; more easily standardized and validated [67]
Throughput	Low; difficult to automate and run in high-throughput formats	High; amenable to automation and high-throughput screening [67]
Cost	Higher due to reagents, cell culture, and labor	Generally lower per sample
Regulatory Acceptance	Considered the gold standard for reflecting biological activity [17]	Accepted when a robust correlation to function is proven [69]
Primary Challenge	Capturing the complexity of MSC multifunctionality with a single assay [2]	Identifying a single biomarker that faithfully represents a complex mechanism of action [67]

Experimental Data and Performance

Case Study: Functional Assay for MSC Immunomodulation

A robust functional assay was developed to measure the anti-inflammatory capacity of MSCs in an M1 macrophage-driven environment [64].

• Experimental Protocol: Skin-derived MSCs were co-cultured with THP-1 monocyte-derived M1-polarized macrophages. Successful macrophage differentiation and polarization were confirmed by flow cytometry for markers CD36 and CD80, and by the release of proinflammatory tumor necrosis factor α (TNF-α). The key readout was the quantification of interleukin-1 receptor antagonist (IL-1RA) secreted by the MSCs into the co-culture supernatant using a validated ELISA.
• Key Findings: The assay demonstrated guideline-concordant selectivity, accuracy, and precision. Testing a range of MSC/macrophage ratios identified an optimal ratio for near-maximal IL-1RA stimulation. This assay was successfully used for batch release testing of 71 consecutively manufactured MSC batches, showing a low failure rate and high comparability between donors [64].

Case Study: Surrogate Assay for MSC-EV Immunomodulation

Research on MSC-EVs has identified transforming growth factor-beta 1 (TGF-β1) as a surrogate biomarker for immunomodulatory potency [69].

• Experimental Protocol: MSC-EVs were isolated from monolayer and microcarrier cultures. The TGF-β1 and let-7b-5p content in the EVs was quantified. The surrogate assay data was correlated with:
  • An in vitro bioassay: suppression of IFN-γ secretion in stimulated splenocytes.
  • An in vivo functional model: efficacy in halting disease progression in murine experimental autoimmune uveoretinitis (EAU).
• Key Findings: A strong correlation was established between TGF-β1 levels in MSC-EVs and their capacity to suppress IFN-γ in splenocytes. In vivo, MSC-EVs with high TGF-β1 levels (MC-EVs) exhibited greater efficacy than those with lower levels (ML-EVs) in the EAU model, thereby validating TGF-β1 as a predictive surrogate biomarker for immunomodulatory potency [69].

The Assay Matrix and Integrated Workflow

Given that MSCs deploy multiple overlapping effector mechanisms, a single assay is often insufficient. Regulatory consensus recommends an assay matrix approach, which combines multiple assays to capture the summation of critical effector pathways [2]. The workflow below illustrates how functional and surrogate assays can be integrated to form a comprehensive potency testing strategy.

The Scientist's Toolkit: Essential Research Reagents

The following table details key reagents and their functions in the experimental protocols cited in this guide.

Table 2: Key Reagent Solutions for Featured Potency Assays

Research Reagent / Assay	Function in Potency Analysis
THP-1 Monocyte Cell Line	Differentiated into M1-polarized macrophages to create a standardized, pro-inflammatory in vitro environment for testing MSC anti-inflammatory capacity [64].
IL-1RA ELISA	Quantifies secretion of interleukin-1 receptor antagonist, a key anti-inflammatory mediator, serving as a direct functional readout of MSC potency [64].
TGF-β1 ELISA	Quantifies TGF-β1 levels in MSC-EV preparations, serving as a validated surrogate biomarker for predicting immunomodulatory potency in vivo [69].
Recombinant Human ACE-2 Protein	A critical component in surrogate virus neutralization tests (sVNTs); used to measure neutralizing antibodies against the SARS-CoV-2 RBD in a competitive ELISA format [71].
Surface Plasmon Resonance (SPR)	A biosensor technique used for surrogate potency assays of biologics; characterizes critical binding kinetics (e.g., antigen and Fc-receptor binding) without biological systems [70].
PBMCs (Peripheral Blood Mononuclear Cells)	Used as responder cells in functional immunomodulation assays to measure the suppressive effect of MSCs on T-cell proliferation and cytokine secretion [2].

The choice between functional and surrogate assay formats is not a matter of selecting a superior option, but of applying the right tool for the specific stage of product development and the critical quality attribute being measured. Functional assays provide the highest biological relevance and are indispensable for initial MoA studies and as a gold standard for correlation. Surrogate assays offer the practicality, robustness, and throughput required for routine quality control and product release. For complex therapies like MSCs, the most powerful strategy is an integrated "assay matrix" that leverages the strengths of both formats. This approach combines the physiological depth of functional assays with the precision and efficiency of validated surrogate biomarkers, ultimately ensuring that cellular products are both clinically potent and consistently manufactured.

Mesenchymal stromal cells (MSCs) represent a promising therapeutic tool for various inflammatory, immune-mediated, and degenerative diseases due to their regenerative properties, anti-inflammatory effects, and immunomodulatory capabilities [72] [7]. However, the transition from promising preclinical results to consistent clinical success has been challenging. A significant hurdle in this translation is the inherent complexity and variability of MSC products, which stems from multiple factors including tissue source (bone marrow, adipose tissue, umbilical cord), donor heterogeneity, culture conditions, and manufacturing processes [72] [73].

The United States Food and Drug Administration and European Medicines Agency classify culture-expanded MSCs as more than minimally manipulated cellular and gene therapy products, and for advanced-phase clinical trials, regulatory authorities mandate the development of potency assays as part of release criteria [2]. Potency assays are essential for measuring the biological activity of MSC products and ensuring batch-to-batch consistency. However, MSCs deploy multiple effector pathways through diverse mechanisms including secretion of bioactive molecules, extracellular vesicles, and direct cell-cell contacts [7]. This complexity means that analyzing a single effector pathway as a surrogate measure of potency provides an incomplete picture and can be misleading [2].

The Scientific Rationale for a Matrix Approach

Multifaceted Mechanisms of Action

MSCs exert their therapeutic effects through a diverse array of mechanisms that cannot be captured by any single assay. They modulate immune responses by suppressing T-cell proliferation, inhibiting natural killer cell activation, preventing dendritic cell maturation, promoting macrophage polarization toward an anti-inflammatory phenotype, and generating regulatory T cells [72]. Simultaneously, they secrete trophic factors that promote tissue repair and angiogenesis [7]. The specific mechanisms deployed vary depending on environmental cues and inflammatory signals in the recipient's tissue [2].

Dynamic Response to Inflammatory Cues

MSCs are not constitutively immunosuppressive but require "licensing" or activation by inflammatory signals to achieve their full immunomodulatory potential [72]. This bidirectional interaction between MSCs and inflammation means their functional state is highly dynamic. Under strong inflammation, MSCs become immunosuppressive, while weak inflammation may enhance their immune responses [72]. This dynamic responsiveness cannot be adequately captured by measuring a single parameter at a single time point.

Addressing Donor and Source Variability

Significant functional heterogeneity exists between MSCs from different donors and tissue sources [72]. Research has shown that cytokine priming can reduce this inter-donor variability, but inherent differences remain that must be accounted for in potency assessment [72]. Furthermore, donor age impacts MSC function, with MSCs from pediatric donors exhibiting higher RAB27B expression and secreting more small extracellular vesicles with immunomodulatory cargo compared to those from adult donors [74].

Established Matrix Assay Platforms and Their Components

Researchers have developed several complementary analytical systems to capture the multifunctional responses of MSCs. The International Society for Cell Therapy (ISCT) has recommended an assay matrix approach that best captures the summation of effector pathways significant to MSC immunomodulation, regeneration, and homing properties [2] [75].

Table 1: Key Components of MSC Potency Assay Matrix

Assay Platform	Measured Parameters	Functional Correlates
Secretome Analysis	Upregulated factors: VEGF, GCSF, CXCL9, CXCL10, CCL2, IL-7 [2]	Correlated with T-cell suppression [2]
	Downregulated factors: TNF-α, IFNγ, IL-13, IL-5, IL-2R, CCL3, CCL4 [2]	Reflect MSC modulation of immune cell responses [2]
Transcriptome Analysis	IDO-1, TSG-6, A20, other immunomodulatory genes [72] [74]	Predictive of immunosuppressive capacity [76]
Phospho-STAT Signaling	STAT1 and STAT3 phosphorylation patterns [75]	Correlates with and predicts allogeneic T-cell suppression [75]
Metabolomic Profiling	Intracellular metabolite levels (e.g., UDP-glucuronate, phosphocreatine) [77]	Predicts immunomodulatory capacity pre-stimulation [77]
Extracellular Vesicle Analysis	Quantity, size distribution, cargo (A20, TSG-6) [74]	Mediates communication with immune cells [74]

Secretome-Based Matrix

The secretome-based assay matrix defines MSC and peripheral blood mononuclear cell (PBMC) responses to each other upon reciprocal interactions [2]. This approach identifies both upregulated and downregulated cytokines and chemokines that inform MSC functionality. Dose dependency analysis has identified at least eight PBMC-derived cytokines that display significantly correlated downregulation upon interaction with MSCs, including IL-13, TNF-α, CCL3, IL-5, IL-2R, IFNγ, CCL4, and IL-12 [2]. Simultaneously, factors such as VEGF, IFNα, GCSF, CXCL9, CCL2, IL-7, FGF-basic, and CXCL10 are upregulated in a dose-dependent manner with MSC presence [2].

Phospho-STAT Matrix Approach

A innovative "loop analytical" phospho-matrix approach captures STAT protein phosphorylation signals in MSCs when they are exposed to secretome arising from co-culture with activated PBMCs [75]. This method uses MSCs as both generators and sensors of the secretome, with heat-inactivated MSCs serving as reference controls. Research demonstrates that the secretome of live MSCs co-cultured with activated PBMCs reduces STAT1 and STAT3 phosphorylation in sensor MSCs, while secretome from heat-inactivated MSCs does not [75]. This phosphorylation signature correlates with and predicts T-cell suppression capacity across bone marrow, adipose, and umbilical cord-derived MSCs.

Metabolic Profiling Matrix

Metabolomics has emerged as a promising approach for predicting MSC immunomodulatory capacity. Studies employing both NMR spectroscopy and UPLC-MS have identified intracellular metabolites that correlate with MSC immunosuppressive function [77]. Notably, metabolites including UDP-glucuronate, hydroxyproline, phosphocreatine, FAD, sn-glycero-3-phosphocholine, and cysteine-glutathione disulfide have shown strong predictive value for T-cell suppression capacity, potentially serving as non-destructive, in-process markers during manufacturing [77].

Experimental Protocols for Key Matrix Assays

Secretome Analysis Protocol

Principle: Measure cytokine expression changes when MSCs interact with immune cells, quantifying both MSC-derived factors and MSC-mediated modulation of immune cell-derived factors [2].

Method Details:

• Co-culture Setup: Culture MSCs with SEB-activated PBMCs at varying MSC:PBMC ratios (e.g., 1:8 to 1:2) [2]. Maintain PBMC numbers constant while escalating MSC numbers for dose/effect analysis.
• Control Cultures: Include PBMCs alone (positive control), MSCs alone, and non-activated PBMCs (negative control).
• Supernatant Collection: Collect culture supernatants after 24-72 hours of co-culture.
• Cytokine Measurement: Analyze 29+ biologically relevant cytokines and morphogens using multiplex immunoassays (Luminex) or ELISA.
• Data Analysis: Identify significantly correlated (R² ≥ 0.5) upregulation or downregulation of cytokines compared to PBMC-only controls. Calculate fold changes and correlate with T-cell suppression data.

Phospho-STAT Signaling Assay Protocol

Principle: Assess STAT phosphorylation patterns in MSCs exposed to secretome from MSC-PBMC co-cultures, using MSCs as sensors of their own immunomodulatory capacity [75].

Method Details:

• Secretome Generation: Generate conditioned media from: (1) live MSCs + activated PBMCs, (2) heat-inactivated MSCs + activated PBMCs, (3) activated PBMCs alone, (4) live MSCs alone, and (5) heat-inactivated MSCs alone.
• Sensor MSC Stimulation: Expose naive "sensor" MSCs from the same donor to these conditioned media for 15 minutes.
• Cell Fixation and Staining: Fix cells immediately, permeabilize, and stain with fluorescently labeled antibodies against phosphorylated STAT1, STAT3, STAT4, STAT5, and STAT6.
• Flow Cytometry Analysis: Analyze using phosflow technology to quantify phosphorylation levels.
• Data Interpretation: Compare phosphorylation patterns induced by different secretome conditions. Potent MSCs generate secretome that reduces STAT1 and STAT3 phosphorylation in sensor MSCs.

IDO-1 Functional Potency Assay

Principle: Measure indoleamine 2,3-dioxygenase activity, a key MSC immunomodulatory mechanism, through kynurenine production [76].

Method Details:

• MSC Stimulation: Activate MSCs with IFN-γ (50-100 ng/mL) for 24-48 hours to induce IDO-1 expression.
• Tryptophan-Kynurenine Conversion: Supplement cultures with L-tryptophan and measure kynurenine production in supernatants.
• Detection: React supernatant with Ehrlich's reagent and measure absorbance at 490nm, comparing to kynurenine standards.
• Quantification: Calculate IDO-1 activity as kynurenine production rate normalized to cell number.
• Threshold Setting: Establish minimum activity thresholds (e.g., ≥75% IDO-1 positive cells) correlating with T-cell suppression [76].

Visualization of Matrix Assay Relationships

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagents for MSC Potency Matrix Implementation

Reagent/Category	Specific Examples	Application in Potency Assessment
Cytokine Priming Cocktails	IFN-γ (20ng/ml), TNF-α (10ng/ml), IL-1β (20ng/ml) [72]	Enhance immunomodulatory capacity; reduce donor variability
Immune Cell Activation Reagents	Staphylococcal Enterotoxin B (SEB), Concanavalin A (ConA), anti-CD3/CD28 beads [2] [74]	Activate PBMCs for functional co-culture assays
Flow Cytometry Antibodies	CD73, CD90, CD105, CD45, CD34, HLA-DR; pSTAT1, pSTAT3 [75] [77]	Phenotypic characterization & signaling pathway analysis
Secretome Analysis Tools	Multiplex cytokine arrays (29+ plex), ELISA for IDO, PGE2, TSG-6 [2] [76]	Quantify soluble immunomodulatory factors
Extracellular Vesicle Characterization	Nanoparticle Tracking Analysis, CD63/CD81 antibodies, TSG-6/A20 detection [74]	EV quantification, sizing, and cargo analysis
Metabolomics Platforms	NMR spectroscopy, UPLC-MS [77]	Non-destructive prediction of immunomodulatory capacity

Comparative Performance of MSC Products in Matrix Assays

Different MSC sources exhibit distinct profiles in matrix assays, informing their appropriate therapeutic application.

Table 3: Matrix Assay Performance Across MSC Sources

MSC Source	Secretome Signature	Phospho-STAT Response	IDO-1 Inducibility	EV Production	Therapeutic Advantages
Bone Marrow	Strong IFNγ-dependent IDO-1 induction [76]	Robust STAT1/3 modulation [75]	High (10-90% range) [76]	Moderate	Gold standard, well-characterized [7]
Adipose Tissue	Balanced immunomodulatory profile [2]	STAT1/3 modulation with STAT5/6 elements [75]	Moderate	High, age-dependent [74]	Abundant source, rapid proliferation [78]
Umbilical Cord	Enhanced immunomodulatory factor secretion [78]	Similar STAT1/3 to BM-MSCs [75]	Variable	High, less age-dependent [78]	High proliferation, low immunogenicity [7]
Placental	Enhanced anti-inflammatory factors [78]	Data limited	Data limited	High	High proliferative capacity [78]

The field of MSC therapeutics is increasingly recognizing that a single assay approach is insufficient to capture the complex and multifaceted nature of these living pharmaceutical products. The matrix approach represents a paradigm shift in potency assessment, moving from reductionist single-parameter measurements to comprehensive profiling that better predicts clinical performance.

As MSC research advances, the integration of high-throughput technologies, multi-omics data integration, and artificial intelligence for pattern recognition in matrix data will further enhance our ability to ensure consistent product quality. Additionally, the development of standardized reference materials and harmonized protocols across laboratories will facilitate comparability between studies and products [76].

For researchers and drug development professionals, implementing a well-designed matrix potency approach is no longer optional but essential for advancing MSC-based therapies through regulatory approval and into clinical practice. By embracing this comprehensive assessment strategy, the field can overcome the challenges of product variability and mechanism complexity, ultimately delivering on the promising therapeutic potential of mesenchymal stromal cells.

Conclusion

The development of robust, predictive, and validated potency assays is the cornerstone for the successful clinical translation of MSC therapies. As synthesized from the four core intents, this requires a deep understanding of the complex mechanisms of action, a practical mastery of diverse methodological tools, proactive strategies to troubleshoot inherent variabilities, and rigorous adherence to validation standards. The future of the field lies in moving beyond simple phenotypic characterization to embrace matrix approaches that combine functional bioassays with physiologically relevant surrogate markers. Emerging technologies, particularly high-throughput microfluidic systems that better recapitulate in vivo conditions, hold great promise for improving the predictive power of potency measurements. Ultimately, by systematically addressing these areas, researchers and manufacturers can ensure the production of consistent, high-quality, and clinically effective MSC products, thereby fulfilling their immense therapeutic potential.

References

• 1. Hallmarks of MSCs: Key quality attributes for pharmacology ... [https://www.sciencedirect.com/science/article/abs/pii/S1934590925001882]
• 2. Potency Analysis of Mesenchymal Stromal Cells Using a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5855117/]
• 3. Establishing Criteria for Human Mesenchymal Stem Cell ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5363381/]
• 4. Potency Assays for Mesenchymal Stromal Cell Secretome ... [https://www.mdpi.com/1422-0067/24/11/9379]
• 5. From “stem” to “stromal”: Reframing the nomenclature of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12553919/]
• 6. The potency of mesenchymal stem/stromal cells: does donor ... [https://stemcellres.biomedcentral.com/articles/10.1186/s13287-024-03722-3]
• 7. Mesenchymal stem cells in treating human diseases [https://www.nature.com/articles/s41392-025-02313-9]
• 8. Methods to Validate Mesenchymal Stem Cell Quality [https://www.rndsystems.com/resources/articles/markers-and-methods-verify-mesenchymal-stem-cell-identity-potency-and-quality]
• 9. Advancing Potency Assay Development for Advanced Therapy Medicinal Products: A Comprehensive Approach and Regulatory Insights - PubMed [https://pubmed.ncbi.nlm.nih.gov/40257954/]
• 10. Analysis of the measurements used as potency tests for the 31 US FDA-approved cell therapy products - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC11877884/]
• 11. Biosimilar Development in 2025: From Comparative Trials to ... [https://clinpharmdevsolutions.com/biosimilar-development-in-2025/]
• 12. EMA Guideline On Clinical-Stage ATMPs Comes Into Effect: On The Verge Of Convergence? [https://www.cellandgene.com/doc/ema-guideline-on-clinical-stage-atmps-comes-into-effect-on-the-verge-of-convergence-0001]
• 13. February 2025 - FDA's Approval of Ryoncil [https://www.isctglobal.org/telegrafthub/blogs/ken-ip1/2025/02/12/na-lra-watchdog-update-february-2025]
• 14. Validation of a potency assay for CD34 + cell based therapy [https://www.nature.com/articles/s41598-025-14828-w]
• 15. Comparison of production methods for mesenchymal stem ... [https://www.nature.com/articles/s41598-025-21218-9]
• 16. Mesenchymal stromal cell therapy: Progress to date and ... [https://www.sciencedirect.com/science/article/pii/S1525001625000930]
• 17. Human Mesenchymal Stromal Cells: Identifying Assays to Predict... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3659751/]
• 18. Investigation of MSC potency metrics via integration ... [https://www.sciencedirect.com/science/article/pii/S2211124724009082]
• 19. Comparison of Cost and Potency of Human Mesenchymal ... [https://www.mdpi.com/2306-5354/10/8/930]
• 20. Size-dependent immunomodulation by MSC secretome: soluble factors... [https://stemcellres.biomedcentral.com/articles/10.1186/s13287-025-04670-2]
• 21. Thawed Mesenchymal Stem Cell Product Shows Comparable... [https://www.nature.com/articles/s41598-019-54462-x?error=cookies_not_supported&code=b5e8a407-0658-4419-aa87-4ac77ecd6647]
• 22. A Reproducible Immunopotency Assay to Measure ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4297551/]
• 23. Optimizing an immunomodulatory potency assay for ... [https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.1085312/full]
• 24. Mixed Lymphocyte Reaction (MLR): A Cornerstone of ... [https://www.milecell-bio.com/news1/254.html]
• 25. The mixed lymphocyte reaction (MLR): a classic assay for ... [https://www.revvity.com/blog/mixed-lymphocyte-reaction-mlr-classic-assay-modern-drug-immune-assessment]
• 26. Short-term assays for mesenchymal stromal cell ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10562633/]
• 27. Characterization of CD4 + and CD8 + T cells responses in ... [https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1320481/full]
• 28. Evaluation of equine xenogeneic mixed lymphocyte ... [https://www.sciencedirect.com/science/article/pii/S0165242722000502]
• 29. Opportunities with artificial intelligence in assessing the ... [https://pubmed.ncbi.nlm.nih.gov/41159402/]
• 30. Mesenchymal Stromal Cells Inducible indoleamine 2,3- ... [https://www.sciencedirect.com/science/article/abs/pii/S1465324918300380]
• 31. Quantification of IDO1 enzyme activity in normal and malignant tissues - PMC [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347075/]
• 32. Indoleamine 2,3-Dioxygenase (IDO) Activity: A Perspective Biomarker for Laboratory Determination in Tumor Immunotherapy - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC10377333/]
• 33. PD-L1 expression in human cancers and its association ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4990391/]
• 34. Regulatory mechanisms of PD-1/PD-L1 in cancers [https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-024-02023-w]
• 35. The expression mechanism of programmed cell death 1 ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10859298/]
• 36. Quantification of IDO1 enzyme activity in normal and malignant tissues - PubMed [https://pubmed.ncbi.nlm.nih.gov/31727243/]
• 37. Endothelial Cell Tube Formation Assay [https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/cell-profilteration-assay-protocols/angiogenesis-protocols/endothelial-cell-tube-formation-assay.html]
• 38. Robust and Scalable Angiogenesis Assay of Perfused 3D ... [https://www.mdpi.com/1422-0067/21/13/4804]
• 39. On-chip 3D potency assay for prediction of clinical ... [https://www.nature.com/articles/s41467-025-60158-w]
• 40. Angiogenesis Analyzer for ImageJ — A comparative ... [https://www.nature.com/articles/s41598-020-67289-8]
• 41. A graph-theoretic framework for quantitative analysis of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12492523/]
• 42. Advanced tube formation assay using human endothelial ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6205943/]
• 43. High-Throughput On-Chip Human Mesenchymal Stromal Cell ... [https://pubmed.ncbi.nlm.nih.gov/34725948/]
• 44. High-Throughput Screening Using Microfluidics [https://microfluidics.insightconferences.com/events-list/high-throughput-screening-using-microfluidics]
• 45. Microfluidics in 2025: Applications, Trends & How It Work [https://eden-microfluidics.com/news-events/microfluidics-overview/]
• 46. A microfluidic assay for the quantification of the metastatic ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6563615/]
• 47. A fully automated rotary microfluidic platform for high ... [https://www.nature.com/articles/s41378-025-01044-9]
• 48. Original Article Increasing robustness of in vitro assay for ... [https://www.sciencedirect.com/science/article/pii/S2352320424002360]
• 49. Mesenchymal stromal cells conditioned by peripheral blood ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12333171/]
• 50. improving physiological relevance and yield with 3D cell culture [https://pubs.rsc.org/en-ca/content/articlehtml/2025/nr/d5nr00707k]
• 51. Specificity of 3D MSC Spheroids Microenvironment [https://pubmed.ncbi.nlm.nih.gov/32681232/]
• 52. Alternating 2D and 3D culture reduces cell size and ... [https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2025.1632810/full]
• 53. Imaging and Analysis of Three-Dimensional Cell Culture ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3699323/]
• 54. Effect of 2D and 3D Culture Microenvironments on ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8882622/]
• 55. Data analysis in flow cytometry [https://www.abcam.com/en-us/technical-resources/guides/flow-cytometry-guide/data-analysis-in-flow-cytometry]
• 56. Treg cell therapy manufacturability: current state of the art ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12141221/]
• 57. Smarter Ways to Scale in Cell and Gene Therapy ... [https://www.patheon.com/us/en/insights-resources/blog/smarter-pathways-to-scale-in-cell-and-gene-therapy-manufacturing.html]
• 58. Potency Assays for Cell and Gene Therapy [https://insights.forgebiologics.com/how-potency-assays-support-gene-therapy-success]
• 59. A guide for potency assay development of cell-based ... [https://www.drugdiscoverynews.com/a-guide-for-potency-assay-development-of-cell-based-product-candidates-7802]
• 60. Validation of analytical procedures according to the ICH ... [https://efor-group.com/en/validation-of-analytical-procedures-according-to-the-ich-guidelines/]
• 61. ICH and FDA Guidelines for Analytical Method Validation [https://www.labmanager.com/ich-and-fda-guidelines-for-analytical-method-validation-34252]
• 62. Potency Assays for Mesenchymal Stromal Cell Secretome- ... [https://pubmed.ncbi.nlm.nih.gov/37298329/]
• 63. Potency Assay Variability Estimation in Practice - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC11788244/]
• 64. Potency assay to predict the anti-inflammatory capacity of a ... [https://www.sciencedirect.com/science/article/pii/S1465324924000501]
• 65. Mesenchymal stem/stromal cell quality control: validation of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7531151/]
• 66. Validation of an ICH Q2 Compliant Flow Cytometry-Based ... [https://www.mdpi.com/2073-4409/12/6/850]
• 67. Functional and potency assays for mesenchymal stromal ... [https://www.sciencedirect.com/science/article/abs/pii/S2468867324000129]
• 68. Mixed Lymphocyte Reaction for Drug Discovery: Principles ... [https://www.marinbio.com/mixed-lymphocyte-reaction-for-drug-discovery-principles-methods-and-applications/]
• 69. Biopotency and surrogate assays to validate the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11322862/]
• 70. Surrogate potency assays: Comparison of binding profiles ... [https://www.sciencedirect.com/science/article/pii/S2095177917301417]
• 71. Performance evaluation of newly developed surrogate ... [https://www.nature.com/articles/s41598-023-31114-9]
• 72. Improving the therapeutic profile of MSCs: Cytokine ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11872697/]
• 73. Mesenchymal Stem Cell-Based Therapies: Challenges ... [https://link.springer.com/article/10.1007/s12013-025-01895-z]
• 74. Human multipotent mesenchymal stromal cells cytokine ... [https://stemcellres.biomedcentral.com/articles/10.1186/s13287-020-02050-6]
• 75. Potency analysis of Mesenchymal Stromal Cells using a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6729138/]
• 76. 221 A Potency Assay Matrix for Establishing Consistency of ... [https://www.sciencedirect.com/science/article/pii/S2666636725002854]
• 77. Metabolomics and cytokine profiling of mesenchymal ... [https://www.sciencedirect.com/science/article/abs/pii/S1465324921007751]
• 78. Mesenchymal stem cells: opening a new chapter in the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12465317/]