T H Tezel1, L V Del Priore. 1. Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Abstract
BACKGROUND: Transplantation of the retinal pigment epithelium (RPE) involves delivering donor RPE onto a bare area of host Bruch's membrane that is surrounded by a confluent monolayer of RPE. We investigated the effects of different plating densities and the presence of an adjacent confluent monolayer on the growth characteristics and final morphology of pig RPE in vitro. METHODS: In single-well experiments, porcine RPE were plated in 24-well plates at densities varying from 1 to 75 cells/mm2. Triplicate plates were counted on the 3rd and 10th days after plating and at confluence. A multiwell chamber was built to allow cells plated at different densities to be bathed with conditioned media from adjoining wells. RESULTS: In single-well experiments, plating at low densities increased the time to reach confluence and resulted in fewer, larger and more fusiform RPE at confluence. In multiwell experiments, the growth rate of cells plated at low density decreased as the amount of high-density medium increased in communicating wells and led to smaller, rounder cells at confluence. The presence of low-density RPE in adjoining wells increased the growth rate of RPE plated at high density and produced fewer, larger and more fusiform cells at confluence. Newly plated RPE grew more slowly when confluent monolayers of RPE were present in adjoining wells. CONCLUSIONS: Plating density is a critical factor in determining the growth rate and the final morphology of RPE in tissue culture. The presence of a neighboring confluent monolayer of RPE inhibits the growth rate of newly plated RPE in vitro.
BACKGROUND: Transplantation of the retinal pigment epithelium (RPE) involves delivering donor RPE onto a bare area of host Bruch's membrane that is surrounded by a confluent monolayer of RPE. We investigated the effects of different plating densities and the presence of an adjacent confluent monolayer on the growth characteristics and final morphology of pig RPE in vitro. METHODS: In single-well experiments, porcine RPE were plated in 24-well plates at densities varying from 1 to 75 cells/mm2. Triplicate plates were counted on the 3rd and 10th days after plating and at confluence. A multiwell chamber was built to allow cells plated at different densities to be bathed with conditioned media from adjoining wells. RESULTS: In single-well experiments, plating at low densities increased the time to reach confluence and resulted in fewer, larger and more fusiform RPE at confluence. In multiwell experiments, the growth rate of cells plated at low density decreased as the amount of high-density medium increased in communicating wells and led to smaller, rounder cells at confluence. The presence of low-density RPE in adjoining wells increased the growth rate of RPE plated at high density and produced fewer, larger and more fusiform cells at confluence. Newly plated RPE grew more slowly when confluent monolayers of RPE were present in adjoining wells. CONCLUSIONS: Plating density is a critical factor in determining the growth rate and the final morphology of RPE in tissue culture. The presence of a neighboring confluent monolayer of RPE inhibits the growth rate of newly plated RPE in vitro.
Authors: G A Mitchell; L C Brody; I Sipila; J E Looney; C Wong; J F Engelhardt; A S Patel; G Steel; C Obie; M Kaiser-Kupfer Journal: Proc Natl Acad Sci U S A Date: 1989-01 Impact factor: 11.205