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

Hari Ramachandran,Richard Caspell,Ramu A Subbramanian,Paul V Lehmann,Ioana Moldovan,Jessica Laux

doi:10.3390/cells1030313

Hari Ramachandran, Richard Caspell + Show 4 more

Open Access

https://doi.org/10.3390/cells1030313

Copy DOI

Journal: Cells	Publication Date: Jul 25, 2012
Citations: 111	License type: CC BY 3.0

Affiliation: Shaker Heights Public Library

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.

Highlights

Many established immune assays in wide use today require timely and coordinated access to assay instrumentation and highly trained technical personnel to handle sample runs and data acquisition
Lab-specific variations are common in how cryopreserved peripheral blood mononuclear cells (PBMC) are thawed and such variations have the potential to affect the performance of lymphocytes in downstream immune assays [1,13,14,17]
PBMC derived from 7 donors were subjected in parallel to these thawing conditions and were evaluated for viability in initial experiments

Summary

Introduction

Many established immune assays in wide use today require timely and coordinated access to assay instrumentation and highly trained technical personnel to handle sample runs and data acquisition. In the context of complex clinical studies that entail immune monitoring, coupling the use of freshly isolated peripheral blood mononuclear cells (PBMC) with downstream bioassays and sample runs presents significant logistical challenges. The osmotic, temperature, and solute changes that the PBMC undergo during cryopreservation and thawing could significantly affect the viability and functionality of the recovered cells. We have previously shown that the use of pre-chilled freezing media used in traditional cryopreservation methods [10,11] significantly impairs the viability and functionality of frozen cells while use of room temperature freezing results in optimal viability and functionality of PBMC [1]. While general consensus suggests gradual freezing leads to optimal viability of cryopreserved cells by minimizing the formation of ice crystals both within and outside the cells undergoing cryopreservation [12], systematic comparison of thawing conditions of cryopreserved PBMC is currently missing. The study addresses if CD4 and CD8 cell-driven responses are differentially affected by the different conditions during the thawing procedure

Results

Discussion

Thawing and Handling of Cryopreserved PBMC and Antigens

Conclusions

Methods