Effects of different cryoprotectants on the viability and biological characteristics of porcine preadipocyte

Abstract

Effective techniques for the cryopreservation of porcine preadipocytes could increase the usefulness of these cells as a model in obesity studies. The objective of this study was to test the effects of the following cryoprotective agents (CPAs) on the cytotoxicity, post-thaw survival, proliferation and differentiation capacity of porcine preadipocytes: ethylene glycol (EG), dimethyl sulphoxide (Me 2SO), polyvinylpyrrolidone (PVP), Me 2SO+PVP, and no-CPA. In addition to the CPAs, the CPA medium contained 80% DMEM/F12 plus 10% FBS. Trypan blue exclusion tests showed that among the CPA treatments in this study, only EG was toxic to porcine preadipocytes. The highest survival rate (94.96%) and cell viability were obtained when preadipocytes were cryopreserved with 10% PVP. Morphologically, PVP cryopreserved preadipocytes resembled fibroblasts and most underwent attachment, proliferation, and growth arrest with subsequent accumulation of intracellular lipid droplets before becoming mature adipocytes. There were no significant differences in the GPDH activity between adipocytes in the PVP treatment and primary cells from days 3 to 10 of the culture. Analysis of RT-PCR confirmed that there was no significant difference of PPARγ2 mRNA levels between the cells in the 10% PVP treatment and primary cells. In summary, porcine preadipocytes cryopreserved with DMEM/F12 medium containing 10% PVP and 10% FBS have high survival rate and proliferation potential. Furthermore, the cryopreserved cells synthesize a range of markers that are consistent with this cell type. We conclude that 10% PVP is a suitable CPA for porcine preadipocytes.