Related Experiment Video
Updated: May 28, 2026
Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells
Published on: October 20, 2023
Standardized Serum-Free Cryomedia Maintain Peripheral Blood Mononuclear Cell Viability, Recovery, and
Insights
New serum-free cryomedia improve peripheral blood mononuclear cell (PBMC) recovery and T-cell functionality. These optimized freezing methods are essential for reliable analysis of immune responses in biobanked samples.
Area of Science:
- Immunology
- Cell Biology
- Cryobiology
Background:
- Cryopreserved peripheral blood mononuclear cells (PBMCs) are crucial for evaluating immune-based therapies.
- Cell viability and functionality after cryopreservation significantly impact assay outcomes.
- Traditional cryomedia additives like fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) present challenges, including lot variability, import restrictions, and cytotoxicity.
Purpose of the Study:
- To develop novel, standardized, serum-free cryomedia formulations for preserving PBMC viability and T-cell functionality.
- To identify suitable alternatives to FCS and optimize DMSO concentration for reduced cytotoxicity.
- To ensure GMP-compliant cryopreservation methods for reliable analysis of immune responses.
Main Methods:
- Comparison of various cryomedia additives, including bovine serum albumin fraction V as an FCS substitute.
- Optimization of DMSO concentration in conjunction with hydroxyethyl starch.
- Assessment of PBMC recovery, viability, and T-cell functionality (via ELISpot) after cryopreservation.
Main Results:
- Developed two distinct serum-free cryomedia formulations manufactured under GMP conditions.
- Achieved over 83% PBMC recovery and 98% PBMC viability using the new cryomedia.
- Demonstrated optimal T-cell functionality post-cryopreservation, as measured by ELISpot assays.
Conclusions:
- The developed serum-free cryomedia effectively minimize cryoprotectant cytotoxicity while maintaining PBMC viability and T-cell function.
- These standardized, GMP-compliant formulations offer a reliable solution for analyzing cryopreserved PBMCs from biobanks.
- The new methods support robust evaluation of immune responses crucial for advancing immune-based therapies.
Abstract:
The ability to analyze cryopreserved peripheral blood mononuclear cells (PBMCs) from biobanks for antigen-specific T-cell immunity is necessary to evaluate responses to immune-based therapies. Comprehensive studies have demonstrated that the quality of frozen PBMCs is critical and the maintenance of cell viability and functionality by using appropriate cryopreservation techniques is a key to the successful outcome of assays using PBMCs. Different cryomedia additives affect cell viability. The most common additive is fetal calf serum (FCS), although it is widely known that each FCS lot has to be tested before usage to prevent nonspecific stimulation of T-cells. Also, shipping of samples containing FCS is critical because of many import restrictions. Often, dimethyl sulfoxide (DMSO) is added as a cryoprotectant. However, DMSO concentration has to be reduced significantly because of its toxic effect on cells at room temperature. Therefore, we have developed freezing approaches to minimize cytotoxicity of cryoprotectants and maintain T-cell functionality. We compared different additives to the widely used FCS and found bovine serum albumin fraction V to be an appropriate substitute for the potentially immune-modulating FCS. We also found that DMSO concentration can be reduced by the addition of hydroxyethyl starch. Using our serum-free cryomedia, the PBMC recovery was more than 83% and the PBMC viability was more than 98%. Also, the T-cell functionality measured by enzyme-linked immunospot (ELISpot) was optimal after cryopreservation with our new cryomedia. On the basis of our experimental results, we could finally design 2 different, fully working cryomedia that are standardized, serum free, and manufactured under GMP conditions.
