Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies

Abstract

Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

Patient-derived xenograft cryopreservation and reanimation outcomes are dependent on cryoprotectant type

“…The thawing is rapid and accomplished with a water bath (25–37°C) or a running water rinse for 30–60 seconds. 37 The vial is agitated until almost all ice has thawed inside the vial itself and is then rinsed with 70% ethanol. The contents of the vial are poured onto a Petri dish and the tumor samples are thoroughly rinsed (at least twice) with PBS solution in order to remove all cryoprotectant solvents that may potentially adversely affect engraftment efficacy.…”

Simulated Night Shift Disrupts Circadian Rhythms of Immune Functions in Humans

“…For a given subject, PBMC samples for both conditions (baseline and night shift) were thawed (41)(42)(43) and placed in culture at the same time. Cryopreserved PBMCs were transferred from liquid nitrogen to a 37˚C water bath for 10 min, transferred to a 15-ml tube, and washed twice with RPMI 1640 at 200 3 g. PBMCs were resuspended in 1 ml RPMI 1640 (supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin/ streptavidin, 10 mM HEPES, 1 mM sodium pyruvate, 13 nonessential amino acids) and set at 1 3 10 6 cells/ml following their count in trypan blue using a hematocytometer.…”

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

“…Over time, several descriptive and comparative studies have approached the issue of PBMC preparation by studying isolation principles [17,18] and techniques, various isolation devices [18][19][20][21][22][23] and the effect of physical factors such as time [23][24][25], storage temperature [24,26] and cryopreservation [22,25,[27][28][29]. Commonly investigated parameters and performance indicators include cell recovery, cellular population composition, purity, viability, sterility, activation status and functionality.…”