Tissue Culture of Retinal Pigment Epithelium Following Isolation with a Gelatin Matrix Technique

Abstract

Prior to transplantation of the retinal pigment epithelium, it is necessary to develop techniques to harvest viable retinal pigment epithelium as an organized monolayer. Unfortunately, current techniques result in contraction of the harvested monolayer and coiling of the cell sheets, which hinders successful transplantation. The purpose of this study was to develop a method for harvesting retinal pigment epithelium from eye cups and from tissue culture prior to transplantation. Passage 1 porcine retinal pigment epithelium and native retinal pigment epithelium from fresh porcine eye cups were pretreated with 0.25% edetic acid for 12 minutes and coated with a 100 micron layer of 12£ gelatin. Patches of the retinal pigment epithelial cell monolayer were harvested and transferred to culture plates, and cell viability and the ability of the transferred cells to proliferate in culture was determined. Our results demonstrated that retinal pigment epithelium can be harvested from tissue culture and eye cups as an organized monolayer with high efficiency (94.7±3.5% and 99.7±0.3% harvesting rates, respectively) and high cell viability (91.3±2.9% and 89.4±4.3%, respectively). Cells harvested from tissue culture plates divided and became confluent within 10 to 14 days. Cells harvested from eye cups maintained a differentiated phenotype and migrated outward from the margin of the exoplant. There was no contraction of the retinal pigment epithelial monolayer isolated from either substrate. Thus, we were able to harvest retinal pigment epithelium as an organized monolayer from tissue culture plates and freshly enucleated eyes with edetic acid and gelatin. The harvested cells were viable and proliferated without contraction of the monolayerin vitro.