Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media

Abstract

Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using BuOF.

Fig 1. Cell viability and cell recovery in fresh and frozen–thawed cells.

(A) Cell viability of fresh and frozen-thawed cells as determined by trypan blue dye exclusion immediately after thawing. (B) Percentage of cell recovery of frozen–thawed cells after 24 h (MEF, C18-4, CHO, HEK, and mES cells) and 72 h (hPBMCs and mBMCs) of culture. (C) Cell viability as determined by trypan blue dye exclusion after 24 h (MEF, C18-4, CHO, HEK, and mES cells) and 72h (hPBMCs and mBMCs) of culture. Data represent mean ± SEM off our trials for each cell type in each group. Bars with different letters are significantly different at P < 0.05.

Fig 2. Expression of proliferation-, pro-apoptosis-, anti-apoptosis-, and early apoptosis-specific proteins in frozen-thawed cells.

Expression of cell PCNA, BAX, BCL2, and Annexin V respectively in fresh and frozen–thawed cells after 24 h (MEF, C18-4, CHO, HEK, and mES cells) and 72 h (hPBMCs and mBMCs) of culture. Thirty-microgram aliquots of each cell extract were subjected to SDS-PAGE and western blot analysis. (A) Representative western blotting and densitometry analysis of (B) BAX/BCL2 ratio, (C) PCNA, and (D) Annexin V expression relative to GAPDH. Data represent mean ± SEM off our trials for a cell type in each group. Bars with different letters are significantly different at P < 0.05.