Maximizing the retention of antigen specific lymphocyte function after cryopreservation.
The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.
Disis
Mary L
ML
Tumor Vaccine Group, Center for Translational Medicine in Women's Health, 815 Mercer Street, 2nd Floor, Box 358050, University of Washington, Seattle, WA 98195-8050, USA. ndisis@u.washington.edu
dela Rosa
Corazon
C
Goodell
Vivian
V
Kuan
Ling-Yu
LY
Chang
Jennie C C
JC
Kuus-Reichel
Kristina
K
Clay
Timothy M
TM
Kim Lyerly
H
H
Bhatia
Sonny
S
Ghanekar
Smita A
SA
Maino
Vernon C
VC
Maecker
Holden T
HT
D000941:Antigens / Q000008:administration & dosage; D049109:Cell Proliferation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D003470:Culture Media; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology; D013745:Tetanus Toxoid / Q000008:administration & dosage / Q000276:immunology
D000941:Antigens; D003451:Cryoprotective Agents; D003470:Culture Media; D013745:Tetanus Toxoid