Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques.

Cryopreservation of peripheral blood mononuclear cells (PBMCs) is common for large clinical trials for which phenotypical characterization of lymphocytes is retrospectively performed in specialized core laboratories. It is therefore essential to assess the comparability between fresh and frozen samples. No side-by-side comparison of B and plasma cells of rhesus macaques (RM), which serve as useful models for several human diseases has been conducted until now. Hence, we performed an extensive comparative analysis between fresh and thawed mononuclear cells (MNCs) from blood and various tissues of healthy RM to analyze for the possible effects of cryopreservation on phenotype and functionality. Our data demonstrate that -80°C cryopreservation induces profound changes compared to fresh ex vivo-derived material. Percentages of B cells were stable in PBMCs, but were increased in all organs analyzed. The expression of CD27, a marker for differentiation between naïve and memory B cells, was massively reduced in PBMCs and MNC from organs with the most severe changes observed in cells from bone marrow (BM). Additionally, similar low percentages of CD27(+) memory B cells were detected in PBMCs and BM samples stored in liquid nitrogen. Therefore, cryopreservation is not suitable for the phenotypical and functional characterization of B cells. Further optimization of cryoconservation protocols monitoring the surface expression of CD27, which was identified as a marker for the quality of cryopreserved material of RM, will be essential.