Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization

Abstract

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4(+) and CD8(+) T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at -70 degrees C for < or =3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at -70 degrees C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

FIG. 1.

Viability and viable cell recovery of cryopreserved PBMC after variable periods of storage in liquid nitrogen. The graphs show the data from the laboratory with typical performance among the six participating laboratories. Data were generated using multiple aliquots of cryopreserved PBMC from a single donor tested after 1 to 15 months of storage in liquid nitrogen. The lines depict the regression of the outcome measure as a function of time.

FIG. 2.

Reproducibility of LPA results for cryopreserved PBMC after variable periods of storage in liquid nitrogen. The data were derived from the laboratory with typical performance. Multiple aliquots from a single donor were tested after 1 to 15 months of storage in liquid nitrogen. The line represents the log SI regression as a function of time.

FIG. 3.

Reproducibility of CD4⁺ and CD8⁺ T-cell enumeration with cryopreserved PBMC after variable periods of storage in liquid nitrogen. Multiple aliquots from a single donor were tested after 1 to 15 months of storage in liquid nitrogen. The graphs show the results of the laboratory with typical performance. The lines represent the regression analysis of results over time.

FIG. 4.

Reproducibility of the enumeration of the percentages of CD4⁺ CD45RA⁺ CD95⁻, CD4⁺ CD38⁺, and CD8⁺ CD38⁺ in cryopreserved PBMC after variable periods of storage in liquid nitrogen. The graphs show the results of the laboratory with typical performance. Multiple aliquots of PBMC from a single donor cryopreserved on a single occasion were tested after 1 to 15 months of storage in liquid nitrogen. The lines represent regression analyses.