Research paperMaximizing the retention of antigen specific lymphocyte function after cryopreservation
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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