Effect of freezing/thawing conditions and their optimization for quality control of PBMC viability and functional assays
Boston, MA, May 12-16, 2005
Raddassi K, Bourcier K, Estevam J, Hafler DA, Seyfert-Margolis V.
Brigham and Women's Hospital, Harvard University, Boston MA Immune Tolerance Network, Bethesda, MD
Objective: To compare and optimize freezing protocols for PBMCs taken from whole blood for detection of autorective T cells.
Background: To detect the effects of potentially immunomodulatory drugs on autoreactive T cells, clinical trials performed by the Immune Tolerance Network employ assays that quantify T cell responses to autoantigens. Centralized assay facilities are used to reduce the problem of inter-site variability. However, some inter-site variability can occur during the preparation of PBMC samples at various geographic locations. These steps require freezing of PBMCs prior to shipping, as well as thawing of samples prior to assay. W e have therefore examined the effects of our freezing and thawing procedures to minimize inter-site variability of the results of T cell assays.
Methods: PBMCs from healthy donors and MS patients were isolated by ficoll separation according to published techniques. Parts of the samples were exposed to different doses of tetanus toxoid, myelin antigens, PHA or no antigen, while the other part was frozen for three weeks using various freezing media and temperatures prior to stimulation in T cell assays. Proliferation ([ 3 H]-Thymidine incorporation), cytokine production (ELISA, flow cytometry and elispot) and cell death/apoptosis (flow cytometry using Guava viacount) were assayed in parallel in both fresh and frozen samples. Subpopulations of PBMCs were phenotyped by flow cytometric analysis.
Results: Cell viability and recovery decreased after a cycle of freezing/thawing (by 2 to 10% and 20 to 60%, respectively). The viability as well as PBMC response to antigens were significantly improved when human AB serum (+10% DMSO) was used to freeze the cells. The viability of PBMCs using fetal bovine serum (+10% DMSO) to freeze the cells resulted in a fair viability (91%) but high background for proliferation and cytokine production. Using cold (4ºC) freezing media decreased both the viability (by 4%) and the response of T cells to antigens (by 20%). PBMCs were better preserved (numerically and functionally) and retained a greater response to antigens when the temperature of freezing medium used was kept at 25ºC than 4º. Using these optimal conditions we did not find any significant differences in the cell population percentage between the fresh and frozen regarding CD3, CD4, CD8, CD14, CD19, activated or memory T cells.
Conclusions : We have found as expected that freezing and thawing of PBMCs decrease cell viability by [2 to 10%] and T cell response (by 50%). This decrease in PBMC count and viability did not disproportionately affect a specific cell population among those examined. The best combination of freezing conditions was obtained by using human AB serum+10% DMSO at 25ºC. Thus, we have identified procedures optimal for PBMC viability and T cell responses to PHA and a panel of nominal and self-antigens. This protocol has been adopted as standard operating procedure for all studies conducted by theITN and thereforeexpand the ITN's capability to evaluate mechanisms of disease and treatment response in multicenter trials.