Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

- Keratinocytes are the most prominent cells present in the epidermis, or the outermost layer of the skin. They arrange themselves in multiple layers over the dermis, associated with various other cell types such as melanocytes, dendritic cells, and merkel cells.

To isolate keratinocytes, begin by taking the epidermal tissue from human skin. Next, treat with an enzymatic digestion buffer. Enzymes in the buffer degrade the extracellular matrix proteins present in the epidermal tissue, thereby initiating cell dissociation. Now, add a suitable media and mechanically disrupt the tissue.

To release the dissociated cells into suspension, filter the epidermal cell suspension through a strainer to remove any cell clumps or debris. Centrifuge to pelletize the epidermal cells and separate them from the enzyme-containing supernatant. Remove the supernatant and resuspend the cells using a preferred keratinocyte growth media. Culture the cells for the desired duration.

During culture, the optimized media enrich the expansion of keratinocytes by promoting their selective growth over the other epidermal cell types. In the following protocol, we will show the isolation of keratinocytes from adult human skin tissue.

- Collect 10 centimeter by 15 centimeter skin samples from healthy adult volunteers undergoing plastic surgery. Keep the skin cool by transporting the skin samples in a Styrofoam box containing a cooling element. If necessary, store the skin sample at 4 degrees Celsius overnight. Using sterilized scissors, scalpels, and forceps, remove fat from underneath the skin section.

Then, using needles, buckle out the skin section on a sterile cover on top of a plate. With a dry, sterilized gauze pad, clean the skin. Then, follow with 70% ethanol on a sterilized gauze pad. Next, using a foot planer, cut off the upper epidermal layer of the skin section, and transfer it into a 9 centimeter petri dish.

Then, immediately add 25 milliliters of the previously prepared DPBS/trypsin/glucose solution to the petri dish. Incubate the samples at 37 degrees Celsius for 30 minutes. Using a pipette, remove the DPBS/trypsin/glucose solution from the petri dish and add 10 milliliters of RPMI-1640 plus 2% FBS to inactivate the trypsin.

Now, with two forceps, release the epidermal cells into the medium by gently scraping and agitating both the epidermal and the dermal compartment of the skin sections. Filter the epidermal cell suspension through a metal filter and collect the filtrate into a 50 milliliter tube. Add the remaining skin sections to a 50 milliliter tube containing 10 milliliters of RPMI-1640 without FBS and vortex for 10 seconds.

Filter this suspension through the metal filter into a 50 milliliter tube containing the epidermal cell suspension. Then, add DPBS to a total volume of 50 milliliters. Centrifuge the cell suspension at 450 x g at room temperature for 10 minutes. Then, remove the supernatant, and depending on the size of the cell pellet, resuspend the epidermal cell pellet in approximately 10 milliliters of 37 degrees Celsius KSFM.

Using the Trypan Blue staining method, count the cells under a microscope. Then, to each 75 square centimeter culture flask, transfer 8 times 10 to the sixth cells together with 12 milliliters of 37 degrees Celsius KSFM. Gently shake the culture flasks to ensure uniform distribution of the cells. Incubate the keratinocytes in a 37 degrees Celsius incubator with 100% humidity and 5% CO₂. Change the medium after two days, and then, 3 times weekly.