Perform the isolation of limbal niche cells, or LNCs, while working under sterile conditions on an ultra-clean workbench. Retrieve the limbus tissue from the intermediate term corneal storage medium. Using a sterile surgical round blade, scrape and remove the iris and endothelium around the cornea.
Next, with the sterile surgical round blade, cut the limbus tissue into 12 equal pieces, leaving only one millimeter of tissue on both sides of the cornea and sclera. After that, transfer the 12 limbus tissue pieces to a 35 millimeter culture plate, and add 1 milliliter of collagenase A to completely immerse the tissues in the plate. Digest the tissues by incubating the culture plate in a 37 degree Celsius cell incubator For 18 hours.
Thaw the matrigel or basement membrane matrix in a refrigerator set at 4 degrees Celsius. Prepare a sterilized bag containing 200 microliter and 1 milliliter tips, a 15 milliliter centrifuge tube, and a 6 well-plate, and place the bag at minus 20 or minus 80 degrees Celsius for 20 minutes. After retrieving the tips, centrifuge tube, and 6 well-plate from the chiller, proceed to perform the next steps on an ultra-clean workbench.
Using a pre-chilled 200 microliter tip, pipette 50 microliters of the thawed basement membrane matrix into 1 milliliter of MESCM, and gently mix it. Using a pre-chilled, 1 milliliter tip fitted to a pipette, transfer the resultant 5%basement membrane matrix to a single well of the pre-chilled 6 well culture plate. Gently shake the 6 well-plate horizontally before placing it in an incubator at 37 degrees Celsius for about one hour to prepare the 5%matrigel-coated culture plate.
Following the 18-hour digestion, retrieve the collagenase A digested limbal tissue, mostly containing small visible clusters. Working under a stereo microscope, use a 200 microliter pipette tip to detach the clusters from the undigested scleral tissues. Submerge the detached clusters in 0.25%Trypsin-EDTA in a 35 millimeter culture plate, and incubate the plate for 15 minutes.
Then, add 1 milliliter of MESCM with 10%knockout serum to the plate to quench cell digestion. Gently pipette the suspension up and down with a 1 milliliter pipette tip to break the clusters into individual cells. Transfer the cell suspension to a 15 milliliter centrifuge tube, and centrifuge it at 200 G for five minutes at room temperature.
Without disturbing the cell pellet, carefully remove the supernatant, and add 1 milliliter of MESCM to resuspend the cells. Using a 1 milliliter pipette tip, pipette the content up and down two to three times to ensure the entire pellet is resuspended in MESCM. Collect 20 microliters from the suspension for cell counting.
Next, using a 1 milliliter pipette, transfer the cell suspension to the 5%basement membrane matrix coated 6 well-plate. Add MESCM to make the total volume in the well 2.5 milliliters. Gently shake the 6 well-plate horizontally to ensure a uniform distribution of cells.
After imaging the cells, transfer them to a cell culture incubator at 37 degrees Celsius. The demonstrated protocol successfully digested the cornea scleral rim tissue, generating clusters that could be visualized under the microscope. As expected, the clusters resembled the shape of a caterpillar.
The primary LNCs grew slowly and required approximately 12 days for culture. LNCs from passage 1 to passage 8 needed about three days to passage, but the growth rate of LNCs after passage 9 decreased significantly. Morphologically, LNCs were spindle-shaped and round in passage 0.
After passage 3, they were spindle-shaped with uniform sizes. The number of cell doublings or NCD represented the growth rate of the LNCs. The NCD and accumulative NCD curves revealed that the LNCs grew the fastest from passage 3 to passage 5.
