Cellulose acetate membranes as an artificial support in subretinal RPE and IPE transplantation

Abstract

Abstract: : Purpose: To explore the use of porous cellulose acetate membranes as an artificial support for the subretinal transplantation of retinal pigment epithelium (RPE) or iris pigment epithelium (IPE). These cells and membrane would serve as autologous replacements for damaged RPE and Bruch’s membrane in age–related macular degeneration (AMD) or other degenerative diseases of the retina. Methods: Cellulose acetate membrane with a 100 kD molecular weight permeability cutoff was sterilized by immersion in 70% ethanol for 2 minutes and then rinsed with a sterile buffered saline solution three times. Human RPE (ARPE–19) were cultured on the membranes in RPE media (DMEM/F–12 with 1% FBS and 1% penicillin/streptomycin) at 37º C. IPE cells were harvested from New Zealand red rabbits though enzymatic digestion of iridectomy samples in 0.25 % trypsin and 200 U/mL collagenase at 37º C. The cells were subsequently cultured on membranes in standard IPE media (Ham F–12 with 20% FBS and 50 µg/mL gentamycin) at 37º C. Analysis of the growth characteristics of RPE and IPE were performed after culturing for 1, 2, 3, 5, 7, 8, 12, 14, 21, and 28 days. Analytical methods included: (1) light and electron microscopy for morphology, and (2) immunofluroescence stains for ZO–1 (tight junctions) and phalloidin (actin) for function. Cells were cultured on mylar plastic as a control. Results: Both RPE and IPE were successfully cultured on cellulose acetate membranes for several months. Cultured cells formed a confluent and uniform monolayer by the second day of culture with adhesion to the membrane and with intact organelles (as visualized by electron microscopy). Cell cytoskeletons were seen to be functional (via phalloidin immunostaining) and fully formed tight junctions (via ZO–1 immunostaining) were seen in the cells within 12 days of culture. These results were comparable to the behavior of cells grown on plastic. Conclusions: Cellulose acetate membranes are stable, porous (unlike plastic substrates), and compatible with RPE and IPE growth on its surface. Furthermore, the cells cultured on cellulose acetate demonstrated properties essential for proper epithelial cell function (e.g. monolayer formation and tight junction formation). Thus, these membranes have the potential to serve as artificial substrates for RPE and IPE transplantation into the subretinal space.

Keywords: retinal pigment epithelium • transplantation • age–related macular degeneration