En bloc transfer of extracellular matrix in vitro

Extracellular matrix (ECM) plays a major role in the development and regeneration of various epithelial cells including retinal pigment epithelium (RPE), and attachment to ECM inhibits RPE apoptosis. Transplantation of ECM prior to the transplantation of RPE may modulate the survival and subsequent proliferation of transplanted RPE. Thus, we have developed a technique to harvest and transfer native ECM produced by bovine, porcine and human cell lines.

ECM was prepared by treating a confluent monolayer of cells with 0.02 N ammonium hydroxide. The ECM was then coated with a thin 100 mu layer of 12% gelatin and cooled to 4 degrees C. Patches of the ECM were isolated and transferred to another culture plate. The transferred ECM was characterized by immunohistochemistry. We determined the ability of cultured RPE to reattach to the harvested ECM, and the ability of the harvested ECM to inhibit RPE apoptosis.

Native ECM can be transferred to another location en bloc with this technique. Immunohistochemistry demonstrates that the transferred ECM contains fibronectin, laminin and collagen IV. The reattachment rate of human RPE to each type of transferred ECM is higher (83.6 +/- 2.8%) than RPE reattachment to bare tissue culture plastic (57.6 +/- 9.8%). The apoptotic rate of attached RPE cells on transferred bovine corneal endothelial ECM (4.3 +/- 1.4%) is lower than their apoptotic rate on bare plastic (69.3 +/- 4.1%). The apoptotic rates of unattached cells are 80.3 +/- 4.4% on transferred bovine corneal endothelial ECM and 79.2 +/- 3.4% on bare plastic.

We conclude that ECM produced by various cell lines can be harvested and transferred by this technique. The transferred ECM promotes cell reattachment and inhibits RPE cell apoptosis. Harvesting and transfer of ECM at the time of RPE transplantation may inhibit apoptosis and promote survival of the transplant.