Research paper
Maximizing the retention of antigen specific lymphocyte function after cryopreservation

https://doi.org/10.1016/j.jim.2005.09.011Get rights and content

Abstract

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 °C prior to washing, the mean viability was 69.7 ± 12.5%, at 25 °C 92.55 ± 3.1%, and at 37 °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.

Introduction

The ability to analyze cryopreserved peripheral blood mononuclear cells (PBMC) for antigen specific T cell immunity is needed in evaluating response to immune based therapies. Extensive studies of cryopreservation of PBMC in subjects with HIV infections have demonstrated that freezing cells can significantly impact function (Betensky et al., 2000, Weinberg et al., 2000). We questioned whether an optimized cryopreservation protocol could be developed that would retain antigen specific T cell function after freezing to the same level as fresh PBMC. Furthermore, we aimed to develop a protocol that would not include additives, such as cytokines, that could have the potential to artificially augment antigen specific immunity. Previous studies had demonstrated that retention of T cell function after cryopreservation was associated with cell viability greater than or equal to 70% (Betensky et al., 2000). We used cell viability measures to evaluate the effect of altering several key steps in the cryopreservation process such as the volume of washes, number of cells frozen per tube, media additives, and temperature during the thawing process.

Data presented here demonstrates that altering two key parameters resulted in an optimized cryopreservation method that preserved both cell viability and antigen specific T cell function as assessed by lymphoproliferation. Human serum albumin (HSA) was an optimal media additive for decreasing cryodamage and diluting thawed the cells in a 37 °C media bath maximized cell survival. PBMC preserved using the optimized cryopreservation protocol described here retained antigen specific function against tetanus toxoid, a moderately immunogenic antigen, as compared to freshly isolated PBMC. The ability to cyropreserve PBMC and retain antigen specific T cell function at similar levels as freshly isolated PBMC will greatly facilitate the conduction of clinical studies of immune based therapies as well as the development of new technologies to measure T cell immunity.

Section snippets

Subjects and sample preparation

Volunteer donors signed informed consent and donated 100 ml of whole blood. Peripheral blood mononuclear cells (PBMC) were purified from whole blood by aliquoting 20 ml of heparinized blood into 50 ml conical tubes (BD Falcon, Franklin Lakes, NJ). Hanks' Balanced Salts Solution (HBSS) (Mediatech Inc., Herndon,VA) was added to a total volume of 50 ml. After centrifugation at 300 RCF for 10 min at room temperature, the top 25 ml was aspirated and volume was brought up to 40 ml with HBSS. The

Several factors do not affect cell viability after cryopreservation

Our initial studies focused on evaluating which steps in the cryopreservation and thawing of PBMC most affect the viability of the cells at reconstitution. To simulate shipping on dry ice, ten cryopreserved PBMC samples were stored in liquid nitrogen, or on dry ice for 24, 48, and 72 h. The mean viability of the samples that remained in liquid nitrogen prior to thawing was 98.2 ± 1.0. Those stored on dry ice demonstrated over 95% viability; at 24 h 98.4 ± 1.6, at 48 h 96 ± 1.4, and at 72 h 97.3 ± 1.0.

Discussion

We performed an analysis of multiple parameters involved in the cyropreservation process that would have the potential to adversely impact the function of antigen specific lymphocytes at thawing. We determined that two factors were critical in preserving viability and antigen specific function of T cells after cryopreservation; the type of protein additive in the cryopreservation media and the temperature of the media at the time of thawing the cells. Our investigations were facilitated by

Acknowledgements

This work was supported for all authors by NIH NCI U54 CA090818. Subject specimens were collected, in part, at the General Clinical Research Center Facility at the University of Washington (NIH MO1-RR-00037). We would like to thank all members of the Immunologic Monitoring Consortium as well as Mr. Robert Schroeder for assistance in manuscript preparation.

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