MINIMUM NUMBER OF ADULT HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS REQUIRED TO ESTABLISH A CONFLUENT MONOLAYER IN-VITRO

Purpose. To determine the minimum number of cells required to establis h a confluent monolayer of retinal pigment epithelium (RPE) with an ep itheloid morphology in vitro. Methods. Primary or passaged human RPE w ere harvested by trypsinization from 6 donors and plated onto bovine c orneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable ce lls/well (0.03-2062 viable cells/mm(2)) for primary cells or 1 to 100, 000 viable cells/well (0.03-3112 viable cells/mm(2)) for passaged cell s. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and c ircularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. Results. Prima ry RPE plated at densities above 10 viable cells/mm(2) (320 cells/well ) and passaged RPE plated above 2 viable cells/mm(2) (64 cells/well) r eached confluence on every occasion, There was a negative correlation between the plating density and time required to reach confluence. Pla ting densities above 3 viable cells/mm(2) (96 cells/well) and 50 viabl e cells/mm(2) (1600 cells/well) yielded smaller, rounder cells at conf luence for primary and passaged RPE, respectively. Conclusions. As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggest s that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.