This cell culture protocol is designed to prevent corneal endothelial cells from undergoing epithelial to mesenchymal transition during isolation, expansion, and subculture. Cell cultures are established from corneal tissue explants, which can be reused to ensure an ongoing supply of primary cells from one donor. Begin by coating the wells of a six-well tissue culture plate with attachment factor using the manufacturer's instructions.
Then add one milliliter of culture medium to each coded well. Place the cornea endothelium side up into a sterile Petri dish on the stage of a dissecting microscope. Adjust the illumination so that the corneal surface is well lit with sufficient contrast to highlight the endothelial layer, and adjust the zoom so that the edge and some central corneal endothelium is in view.
Use sterilized watchmaker forceps to gently lift and tear Descemet's membrane away from the underlying stroma. Place the membrane strip into one of the prepared wells of the culture plate, making sure to immediately replace the lid of the plate to reduce the risk of contamination. Remove the strips of Descemet's membrane from the extreme periphery of the endothelium first, and from the central regions later, placing all strips from one cornea into a single well.
When finished, incubate the explants in a humidified cell culture incubator set to 37 degrees Celsius and 5%carbon dioxide. Assemble the upper chamber of the micro Boyden chamber by placing a red O ring into its center. Use an 18 millimeter diameter trephine to punch out a disk from a biomaterial sheet on a PTFE cutting board.
Then, place the disk over the red O ring in the micro Boyden chamber's upper chamber. Screw the lower chamber onto the upper chamber, securing the bio material's disk in between. And soak the assembled micro Boyden chamber in 70%ethanol for one hour to sterilize it.
After the soak, completely immerse the chamber in sterile HBSS for 10 minutes to remove the ethanol. Then wash the chamber in culture medium for 10 minutes. And transfer to culture medium inside a well of a six-well plate, making sure that the upper chamber is oriented upwards.
Adjust the level of the medium so that it contacts the lower surface of the biomaterial membrane, but does not flow into the upper chamber. Pipette 100 microliters of cell suspension onto the membrane into the micro Boyden chamber, and incubate it in the tissue culture incubator for four hours, before adding medium to completely submerge the chamber. Most explants that were derived from the corneas of one to two-year-old sheep or human donors of less than 30 years of age attached to attachment factor coded tissue culture plates within a week.
After six weeks, many small, tightly-packed cells were present next to the explant. Small, tightly-packed cells within the corneal endothelial cell cultures are resistant to digestion with triple, while the larger fibroblastic cells are more sensitive to it. This difference was exploited to selectively remove large cells from the cultures before transferring the smaller cells to new plates.
Immunal flourescence analyses were conducted on corneal endothelial cell cultures to locate ZO-1, a tight junction protein, and N-cadherin, an adherence junction protein. The same antibodies were used for both sheep and human cells, and both proteins were detected in the plasma membranes of cells from both species. The custom-made micro Boyden chamber allowed both sides of a membrane to be used as cell culture surfaces simultaneously.
A cross-section view of a tissue engineered cell construct shows a single layer of corneal endothelial cells on one surface, and a multilayered culture of corneal stromal cells on the other. When attempting this protocol, it's important to avoid scraping the endothelium with your forceps when you're peeling Descemet's membrane away from the cornea. Following this procedure, the integrity of a corneal endothelial cell layer on a membrane can be checked by measuring its electrical resistance.
