Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.
The general goal of this video is to introduce a new simple method to isolate and culture primary human epidermal cells from an adult skin tissue. This advanced method is more suitable for producing a large number of high-potential epidermal cells for both laboratory and clinical applications. Wash the tissue with PBS before weighing.
Weigh the skin tissue by an electronic scale. Rinse the skin tissue with 70%ethanol for 30 seconds. Incubate the tissue in PBS with antibiotics twice for five minutes each time.
Transfer the tissue to another sterile dish and homogenize thoroughly by using scalpel blades. Add 200 microliters of PBS every five minutes to keep the tissue wet. Transfer the homogenized tissue into a 50 milliliter tube.
Add 10 milliliters of enzyme mixture for every one gram of skin tissue. Mix the enzymes thoroughly with the homogenized tissues. Incubate the mixture in a 37 degree Celsius water bath with shaking.
After that, add 1/5 the volume of 0.25%trypsin. Continue the incubation for 30 minutes in a 37 degree Celsius water bath. Add Dnase I solution to the mixture at a one to 100 ratio.
Then incubate for another five minutes. Stop the digestion process by adding the same volume of neutralization medium. Pipette the solution up and down for about 20 times.
Filter the dissociated cells through a 100 micrometer cell strainer to remove tissue debris. Centrifuge at 200 g for five minutes. Observe the pellet at the bottom of the tube.
Remove the supernatants and wash the cell pellet with 10 milliliters of neutralization medium. Then centrifuge at 200 g for five minutes. Resuspend the cell pellet in the inoculation medium with 10 micromolar ROCK inhibitor.
Plate two times 10 to the sixth cells in a 100 millimeter culture dish. After three days, replace the inoculation medium with serum-free keratinocyte medium. Change the medium every two days.
When the cells have reached about 80%of the density, passage the cells. Wash the cells with PBS. If necessary, trypsinize for two minutes and wash the cells with PBS to remove contaminated dermal cells.
Add the same volume of neutralization medium. Centrifuge at 200 g for five minutes. Finally, remove the supernatants and resuspend the cell pellet.
The isolated keratinocytes from human skin tissues were incubated separately by two methods. The conventional method is a two-step digestion which involves a two-day procedure. By contrast, the new method is a one-step digestion which takes around three hours to perform.
On day three, we can easily find many cell clusters cultured by the new method while this phenomenon does not occur when using the conventional method. On the fifth day, it was observed that the new method produced a larger amount of primary cells. To test the differentiation status of cultured keratinocytes, we analyzed the basal cell marker K5 and terminal differentiation marker Loricrin.
We found that 90%of the total cells were K5 positive, but less than 10%of the cells expressed Loricrin at passage three, indicating that they are undifferentiated keratinocytes. We checked the expression of vimentin which is expressed in the dermal fibroblast to examine the contamination of the dermal cells during isolation. We found that around 3%of the dermal cells appeared in the initial passage, but only around 0.02%of the dermal cells were detected at passage three, indicating a high purity of the epidermal cells after passage.
