Standardized serum-free cryomedia maintain peripheral blood mononuclear cell viability, recovery, and antigen-specific T-cell response compared to fetal calf serum-based medium
| Autor: | Hagen von Briesen, Heiko Zimmermann, Beatrice Kemp-Kamke, Julia C. Schulz, Anja Germann |
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| Přispěvatelé: | Publica |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Cryoprotectant
biology Dimethyl sulfoxide business.industry ELISPOT Medicine (miscellaneous) Original Articles Cell Biology General Medicine Peripheral blood mononuclear cell General Biochemistry Genetics and Molecular Biology Cryopreservation Andrology chemistry.chemical_compound Immune system chemistry Immunology biology.protein Medicine Viability assay Bovine serum albumin business |
| Popis: | 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 approa ches 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. |
| Databáze: | OpenAIRE |
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