Abstract
Purpose :
To characterize adult primary human retinal pigment epithelium (hRPE) cultured and expanded in a GMP-approved, animal material-free medium.
Methods :
The adult hRPE cells were isolated from post mortem samples using enzymatic digestion. The project adhered to the Guidelines of the Declaration of Helsinki and approved by the Regional Ethics Committee. Cells were cultured in Knockout DMEM-F12 medium supplemented with Serum replacement (20%) in the presence or absence of EGF, until reaching confluent monolayers. RT-qPCR analysis was performed to determine expression of genes related to RPE function.
Results :
The hRPE attached to the cell culture plates in the first 48 hrs and demonstrated the characteristic cobblestone morphology. The cells underwent a slow expansion phase, generating a monolayer of viable hRPE by the 3rd week, with reducing pigmentation after each duplication. Expression of RPE function-related genes was altered in vitro; RPE65 and CRALBP showed decreased, while MITF, TYR and PEDF showed increased expression compared to hRPE cells of the native retina.
Conclusions :
Preliminary data suggests hRPE obtained from aging donors can be expanded in animal material-free medium. The cells lost pigmentation during the expansion phase, however expressed genes related to RPE- function, with certain ones downregulated. Adult hRPE expanded ex vivo can probably be used similar to ESC-derived hRPE, towards cell therapy applications for treating RPE-related disorders and meet the robust quality control requirements.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.