Recently, oral keratinocytes have attracted attention because of their unique property. We provide a protocol for culturing mouse oral keratinocytes in a stem cell-like state suitable for downstream applications. The culture of mouse oral keratinocyte and different characteristic from skin keratinocyte.
In this protocol, we have successfully isolated primary mouse oral keratinocyte and developed a long-term culture technique. This culture system can be employed in molecular and biochemical assays to further understand the feature of oral basilar stem cell and their related diseases. After sacrificing an adult C57BLK6J mouse, remove the hair around the mouth with a shaver and using scissors, cut from the cheeks toward the jaw on both sides.
Next, use forceps to open the mouth wide and absorb any blood using a cotton swab, then disinfect the palette by wiping the inside of the mouth with a cotton swab containing 10%povidone iodine. To harvest the mouse palette, first using a surgical scalpel blade, make a full thickness marginal incision along the palette side of the maxillary teeth. Then using a raspatorium, carefully dissect the entire palette.
Quickly transfer the palette tissue to a 15 milliliter tube containing four milliliters of complete medium supplemented with antibiotics and antimycotics. Keep the tissues on ice until ready for incubation. In a laminar flow hood, transfer the tissues to a 60 millimeter dish containing four milliliters of complete medium with antibiotics and antimycotics.
Then using short blunt forceps and a scalpel blade, gently remove any blood from the tissues and wash the tissues 10 times with the complete medium. After the last wash, transfer the tissues to a 35 millimeter dish containing four milliliters of 0.025%trypsin supplemented with antibiotics and antimycotics. Place the tissues with the epithelial surface facing down and incubate them for 16 hours at room temperature in the culture hood.
After overnight trypsin digestion, use one pair of blunt forceps to remove the tissue from the trypsin solution and transfer it to a 60 millimeter dish containing a trypsin inhibitor solution with the epithelial surface facing up. Next, holding onto the edge of the palette with forceps, use the scalpel blade to gently scrape the epithelial layer off the underlying lamina propria. To collect the maximum amount of epithelial cells from the tissues, transfer the tissue into another 60 millimeter dish with four milliliters of complete medium and repeat scraping as demonstrated previously.
Next, place a sterile 100 micron cell strainer on top of a 50 milliliter conical tube. And using a sterile pipette, transfer two milliliters of trypsin solution into the strainer to wet its surface. Then using a pipette, mix the cell suspension in the 60 millimeter dish a few times and filter the cells through the 100 micron cell strainer.
To count the number of cells, add 15 microliters of the cell suspension to 50 microliters of trypan blue solution, then transfer 10 microliters of the cell trypan blue mix to a hemocytometer and count the number of cells. Next, centrifuge the cell suspension, remove the supernatant and add two milliliters of complete medium containing Chelex FBS to the tube. Resuspend the cell pellet by titrating several times using a five milliliter pipette.
Then plate two to five times 10 to the fifth cells from one mouse into one well of 24-well plate pre-coated with collagen type one and incubate the cells at 37 degrees Celsius for two days without changing the medium. Primary oral keratinocytes grew as a monolayer and displayed a cobblestone morphology. Small keratinocyte colonies were visible after three and five days.
After one week of incubation, these colonies grew larger and formed tight colonies. The first passage was performed two weeks from the initial plating and at later passages, the keratinocytes exhibited stable growth with a shorter period of culture. The keratinocytes stopped growing if significant fibroblast contamination occurred during the isolation process.
After culturing, the keratinocytes expressed keratin 14 and alpha6-integrin. Early and late passage cells showed uniform expression of P63 confirming their stemness. However, keratinocytes treated with high calcium exhibited decreased P63 expression indicating that high-calcium treatment suppresses stem cell-related genes.
The differentiation marker keratin 13 showed rare or no expression in early and late passages, but significant expression in high-calcium treatment. The fibroblast marker PDGFR alpha was not expressed in the keratinocyte culture indicating that this protocol could successfully isolate basal keratinocytes and maintain these cells in the undifferentiated state. Trapping should start from the epidural side and 10 minute per tissue sample.
Also generally, pipetting of cells is important for better cell viability. After two or three passes, oral keratinocyte becomes stable for further functional experiments. Importantly, this protocol can be combined with transgenic mouse life for cellular and molecular assay in vitro.
Recent study have reported the dynamics and heterogeneity of oral basilar stem cell. This method will facilitate the study of oral stem cell biology and leads to better understanding of oral diseases.
