Probably the most common way to cryopreserve cells, including human peripheral blood mononuclear cells (PBMC) is using a mixture of 90% serum with 10% DMSO. However, serum is very expensive, and every new lot must first be tested for its effects on the background and performance of the various cellular assays performed. A recent article in Cancer, Immunology, Immunotherapy, by Filbert et. al, reports on the results of an effort led by the Cancer Immunotherapy Immunoguiding Program to compare the viability, recovery, and performance in IFN-gamma ELISPOT assays of PBMCs cryopreserved in serum-containing versus various serum-free mediums.
This was a large-scale study which engaged 31 labs across ten countries. This study is part of a larger concerted effort by the Immunoguiding Program of the Cancer Immunotherapy Association and the Cancer Research Institute’s Cancer Immunotherapy Consortium to assess the importance of harmonizing the most commonly utilized immunological assays across institutions, such that standardized results can be obtained. The major inertia driving this effort is to establish a platform for standardized evaluation of patient immune responses to support the growing field of clinical immunotherapeutics.
In this study, three different freezing media were compared in 31 labs and seven freezing media were compared in a single center. Human PBMCs from HLA-A*0201 donors were cryopreserved in these various freezing mediums and sent to the different labs for evaluation of viability, recovery, and performance in IFN-gamma ELISPOT protocols against several HLA-A*0201-restricted epitopes from HCMV, Influenza, and EBV viruses. Each lab used its own established ELISPOT protocol.
All 31 labs compared PBMCs cryopreserved in (1) 90 % heat-inactivated human AB serum + 10 % DMSO, (2) CryoMaxx II, and (3) 10 % human serum albumin (HSA) + 10 % DMSO + 80 % RPMI. Interestingly, the viability of cells after thawing as well as the number of cells recovered after thawing and after a 1-24 hour rest, were found to be significantly higher in both serum-free mediums compared to the human AB serum-containing media. The overall cell loss from the number of cells initially cryopreserved ended up being an average of 35.2 % for PBMCs cryopreserved in the human AB serum-containing media, and roughly 22% for both of the serum-free mediums. Thus, these assays suggest that these serum-free mediums provide more optimal freezing conditions compared with the human AB serum-containing media. The performance in ELISPOT assays however, was not found to be significantly different for cells frozen in these different mediums.
In addition to those three mediums, a single laboratory made the same assessments for PBMCs cryopreserved in an additional four mediums: (4) CryoKit ABC, (5) 90 % heat-inactivated FCS + 10 % DMSO, (6) 12.5 % BSA + 77.5 % RPMI + 10 % DMSO, and (7) 12.5 % BSA + 77.5 % RPMI + 5 % DMSO + 5 % hydroxyethyl starch. In this comparison however, serum-free and serum-containing mediums had similar effects on viability, cell recovery, and in the ELISPOT assay, although the BSA-containing mediums had the worst performance overall.
In conclusion, although commonly used FBS and FCS-containing mediums were not compared in the multi-lab test, the strong performance of cells cryopreserved in serum-free media regarding subsequent viability, recovery, and in ELISPOT assays recommends that further consideration be given to cryopreservation in such serum-free media. Long term storage quality of cells frozen in various serum-free media is still an issue to be addressed as well as the comparative performance of PBMCs in the many other immunological assays. Using defined serum-free media as opposed to lot-variant serum-containing media may allow for more robust standardization of immunological assays.
Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols. Filbert H, Attig S, Bidmon N, Renard BY, Janetzki S, Sahin U, Welters MJ, Ottensmeier C, van der Burg SH, Gouttefangeas C, Britten CM. Cancer Immunol Immunother. 2013 Apr;62(4):615-27. doi: 10.1007/s00262-012-1359-5. Epub 2012 Nov 9.
The impact of harmonization on ELISPOT assay performance. Janetzki S, Britten CM. Methods Mol Biol. 2012;792:25-36. doi: 10.1007/978-1-61779-325-7_2.
Harmonization of immune biomarker assays for clinical studies. van der Burg SH, Kalos M, Gouttefangeas C, Janetzki S, Ottensmeier C, Welters MJ, Romero P, Britten CM, Hoos A. Sci Transl Med. 2011 Nov 9;3(108):108ps44. doi: 10.1126/scitranslmed.3002785.
Standardized Serum-Free Cryomedia Maintain Peripheral Blood Mononuclear Cell Viability, Recovery, and Antigen-Specific T-Cell Response Compared to Fetal Calf Serum-Based Medium. Germann A, Schulz JC, Kemp-Kamke B, Zimmermann H, von Briesen H. Biopreserv Biobank. 2011 Sep;9(3):229-236.