The Retinal Pigment Epithelial Cell Differentiation and Cell Marker Expression Following Cryopreservation at -80°C and under Liquid Nitrogen at -196°C

Abstract

Cryopreserved retinal pigment epithelial (RPE) cells at -80°C can be used for transplantation studies. Long-term viability at -196°C has been used for many other cell lines. The origin and purity of human RPE cell lines were assessed by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) using RPE cell markers [cytokeratin, cellular retinaldehyde binding protein (CRALBP), tyrosinase, tyrosinase-related proteins I and II (TRP-I and II), Na,K-ATPase α₁ and β₂]. Cultured human RPE cell lines were cryopreserved at -80°C and -196°C. After cryopreservation, human RPE cells were re-cultured on laminin-coated polystyrene dishes and laminin-coated collagen sheets. Differentiation was evaluated by the expression level of RPE marker genes using RT-PCR. It was found that cryopreserved human RPE cells on laminin-coated collagen sheets disclosed relatively strong re-expression of their marker genes if compared to human RPE cells cultured on laminin-coated polystyrene dishes. Cryopreservation of RPE cells at both temperatures, thawing and subsequent culturing on laminin-coated collagen sheets can enhance the feasibility of using cryopreserved RPE cells for transplantation.