Related Experiment Video
Updated: Dec 7, 2025
Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs
Published on: December 24, 2015
Mesenchymal stem/stromal cell quality control: validation of mixed lymphocyte reaction assay using flow cytometry
Tess Nicotra1, Aurélie Desnos1, Justine Halimi1
1AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, 75010, Paris, France.
Insights
This study validates a method to quantify the immunomodulatory activity of mesenchymal stem/stromal cells (MSC) by measuring their ability to inhibit lymphocyte proliferation. The validated assay shows low variability and can be used for qualifying MSC batches in clinical trials.
Area of Science:
- Immunology
- Cell Biology
- Stem Cell Therapy
Background:
- Mesenchymal stem/stromal cells (MSC) possess immunomodulatory properties relevant to various diseases.
- Clinical trials require validated quality controls to confirm MSC immunomodulatory activity.
- This study focuses on validating a method to assess MSC's lymphocyte proliferation inhibition capacity.
Purpose of the Study:
- To validate a quantitative method for assessing MSC immunomodulatory activity.
- To ensure the reliability and reproducibility of MSC immunomodulatory assays for clinical applications.
- To establish a standardized assay for qualifying MSC batches based on their lymphocyte proliferation inhibition.
Main Methods:
- Co-culture of MSC with CellTrace™ Violet-labeled Peripheral Blood Mononuclear Cells (PBMC) from ten donors at seven ratios for seven days.
- Flow cytometry analysis to determine T cell division percentage.
- Calculation of MSC's lymphocyte proliferation inhibition percentage and area under the curve (AUC) to quantify immunomodulatory capacity.
Main Results:
- The validated PBMC bank demonstrated stability up to 509 days and showed no inter-bank variability.
- The assay exhibited low repeatability (6.1% SD) and reproducibility (4.6% SD) with good linearity and robustness.
- The method effectively differentiated MSC batches with varying inhibitory potentials, with MSC quantity identified as a factor influencing results.
Conclusions:
- The validated quantification method demonstrates low analytical and no inter-bank PBMC variability.
- Pre-culture MSC quantification is recommended to minimize variability.
- The assay is suitable for qualifying the immunomodulatory activity of MSC batches for clinical use.
Background:
Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method's validation, assessing MSC's ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard.
Methods:
MSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division-ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC's ability to inhibit lymphocyte proliferation.
Results:
Two cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities.
Conclusion:
This quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches' immunomodulatory activity.
More Related Videos
12:05Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation
Published on: November 3, 2018
09:57Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia
Published on: March 5, 2018