En bloc transfer of extracellular matrix in vitro
Journal
Investigative Ophthalmology and Visual Science
Journal Volume
37
Journal Issue
3
Date Issued
1996-02-15
Author(s)
Abstract
Purpose. Extracellular (ECM) plays a major role in the development and regeneration of retinal pigment epithelium (RPE), and in the reattachment of transplanted RPE to Bruch's membrane. We have developed an effective technique for the en bloc transfer of ECM secreted by cultured cells. Methods. ECM was prepared from bovine corneal endothelium, porcine corneal endothelium, porcine RPE, and human RPE by treating a confluent monolayer cells of the particular cell type with 0.02 N ammonium hydroxide. The ECM was coated with a 100 μ layer of 12% gelatin. A trephine was used to harvest patches of the ECM, and the patches were transferred to another culture plate at 4°C. Immunohistochemistry was used to determine the ECM components transferred. The functional integrity of the ECM was determined by measuring the ability of human RPE cells reattachment rate to the transferred ECM. Results. ECM can be transferred en bloc with this technique, and fibronectin, laminin, and type IV collagen are present in the transferred ECM. Reattachment to transferred ECM is higher (87.3 ± 6.5%) than reattachment to bare plastic of culture plate (57.6 ± 9.8) Conclusions. ECM produced by various cell lines in tissue culture plates can be isolated and transferred en bloc. The transferred ECM contains laminin, fibronectin, and collagen IV, and is able to promote cell reattachment. Simultaneous transplantation of harvested ECM and RPE cells may facilitate the reattachment of transplanted RPE to Bruch's membrane in vivo.
Type
journal article