Isolation and Identification of Limbal Niche Cells

Our studies focus on the reconstruction of the limbal needs to treat limbal stem cell deficiency and to restore the patient's vision. We are trying to answer whether or not limbal stem cells are the key cells supporting the function of limbal stem cells and if so, how it works. Recent studies reported that subjunctive transplantation of mesenchymal stem cells, from bone marrow, prevented the limbal stem cell deficiency of live burned eyes.

One of the technologies currently used to advance our tech research is single cell sequencing, which can analyze the key signaling pathway activities at a single cell level for limbal stem cell characterization. Although RNC have the potential to become an important clinical tool in the future, their growth characteristics have not been explored in much details. Considering their promising scientific and clinical applications, it is necessary to summarize the isolation, purification, identification, and characteristics of RNC.

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 one 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 matrix gel or basement membrane matrix in a refrigerator set at four degrees Celsius. Prepare a sterilized bag containing 200 microliter and one milliliter tips, a 15 milliliter centrifuge tube and a six well plate and place the bag at minus 20 or minus 80 degrees Celsius for 20 minutes. After retrieving the tips, centrifuge tube, and six 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 one milliliter of MESCM and gently mix it. Using a pre chilled one milliliter tip fitted to a pipette, transfer the resultant 5%basement membrane matrix to a single well of the pre chilled six well culture plate. Gently shake the six 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 one milliliter of MESCM with 10%knockout serum to the plate to quench cell digestion. Gently pipette the suspension up and down with a one milliliter pipette tip to break the clusters into individual cells. Transfer the cell suspension to a 15 milliliter centrifuge tube and centrifuge it at 200G for five minutes at room temperature.

Without disturbing the cell pellet, carefully remove the supernatant and add one milliliter of MESCM to resuspend the cells. Using a one 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 one milliliter pipette, transfer the cell suspension to the 5%basement membrane matrix coated six well plate, add MESCM to make the total volume in the well 2.5 milliliters, gently shake the six 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 one to passage eight needed about three days to passage but the growth rate of LNCs after passage nine decreased significantly. Morphologically, LNCs were spindle shaped and round in passage zero.

After passage three, they were spindle shaped with uniform sizes. The number of cell doublings, or NCD, represented the growth rate of the LNCs. The NCD and acumulative NCD curves revealed that the LNCs grew the fastest from passage three to passage five.

To identify the limbal niche cells, or LNCs, by immunofluorescence staining, add one milliliter of 0.25%trypsin EDTA per well to the LNCs cultured in a 5%matrigel coated six well plate, digest the cells by incubating the plate for five minutes at 37 degrees Celsius. Quench the digestion by adding one milliliter of knockout serum containing MESCM to the cell suspension. Transfer the cell suspension to a centrifuge tube and centrifuge it at 200G for five minutes before carefully aspirating the supernatant, resuspend the pellet in one milliliter of MESCM.

Next, using a cytofuge, deposit 80 microliters of the cell suspension equally on four microscope slides per the manufacturer's instruction. Fix the cells with 4%paraformaldehyde for approximately 10 minutes. Then, permeabilize the cells on the slides using 0.2%Triton X 100 and PBS for 15 minutes.

Block the cells with 2%bovine serum albumin for one hour before incubating them with primary antibodies on a shaker overnight at four degrees Celsius. Post incubation, remove unbound antibodies by washing the slides with PBST three times for five minutes each. Next, incubate the sample with suitable secondary antibodies at 37 degrees Celsius for one hour before washing any unbound antibodies as demonstrated previously.

Counterstain the nuclei with DAPI, having a concentration of five micrograms per milliliter. After sealing the slides, perform imaging using a fluorescence microscope. Using an RNA isolation kit, extract total RNA from the LNCs per the manufacturer's instructions.

Then, using a high capacity complimentary DNA, or CDNA transcription kit, reverse transcribe one to two micrograms of the RNA. Prepare a 20 microliter solution containing 2.5 microliters of the CDNA, 0.8 microliters of corresponding genetic primer, 10 microliters of a universal PCR master mix, and 6.7 microliters of double distilled water. Then, perform a quantitative polymerase chain reaction using the appropriate conditions.

Finally, employ the comparative CT technique to examine the relative gene expression. Double immuno staining of the fourth passage LNCs clearly revealed that these cells were consistently panCK negative, Vim positive, CD90 positive, CD105 positive, SCF positive, and PDGFR positive.