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Rachelle Palchesko, James L Funderburgh, Adam W Feinberg; Development of a Human Bioengineered Corneal Endothelium for Lamellar Keratoplasty. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4623.
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Disease or injury to the corneal endothelium (CE) is responsible for approximately 40% of corneal transplants worldwide. Current treatments include full cornea transplant and Descemet’s Stripping Endothelial Keratoplasty (DSEK), which are successful in restoring corneal clarity; however donor corneas are limited worldwide. Here we sought to bioengineer a transplant-quality CE by culturing expanded corneal endothelial cells (CECs) on an engineered basement membrane (EBM).
Engineered sheets of collagen type IV (col4) ~100 nm thick were fabricated using a biomimetic, surface-initiated assembly process. The col4 sheets were transferred onto to a thicker collagen type I (col1) gel to form a layered scaffold termed EBM. Structure and composition was confirmed using confocal laser scanning microscopy at each stage of fabrication. Both bovine and human CECs were seeded onto the EBMs, cultured for up to 28 days and fixed and stained for the nucleus, ZO-1, col4, and F-actin.
We successfully developed an EBM with distinct col4 and col1 layers that mimics the structure of Descemet’s membrane, which is col4 and laminin rich, on top of stroma, which is col1 rich. Though clear differences exist, the EBM provides a scaffold for the CECs that they can constructively remodel. Both bovine and human CECs seeded on the EBMs formed a confluent monolayer, expressing ZO-1 at the cell borders and achieved a density of ~1600 cells/mm2 for over 14 days in culture. The engineered col4 sheet showed evidence of remodeling beginning at 14 days in culture, but the fact that it was largely maintained demonstrated the stability of the EBM. Morphologically, both types of CECs cultured on the EBMs maintained the desired cellular phenotype and monolayer structure and mimics the thickness and handling characteristics of a corneal graft prepared for a DSEK procedure.
We have demonstrated that human CECs can form a high-density monolayer on an EBM to form a bioengineered CE comparable to the portion of the cornea transplanted during a DSEK procedure. The next steps are to further evaluate the performance of the bioengineered CE to pump fluid in a modified Ussing Chamber and in vivo in animal models.
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