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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Keratinocytes from Human Skin

1. Start by making the following solutions: 50 mL solution of 0.25% trypsin and 0.1% glucose in DPBS. Mix and filter sterilize (0.2 µm) the solution. Prepare 10 mL of RPMI-1640 + 2% FBS solution, 50 mL of DPBS and keratinocyte serum-free medium (KSFM). Add KSFM supplements and 250 µL of gentamycin to 500 mL of KSFM. Pre-warm the solutions to 37 °C before use.
2. Collect skin from healthy adult volunteers undergoing plastic surgery. The skin samples used are approximately 10 cm x 15 cm but can vary in size. Keep the skin cool under transportation by transporting the skin samples in a Styrofoam box containing a cooling element. If necessary, the skin sample can be stored at 4 °C overnight.
3. Remove fat from underneath the skin section using sterilized scissors, scalpels, and forceps.
4. Buckle out the skin section (approximately 10 cm x 15 cm) on a sterile cover on top of a plate using needles. First, clean the skin with a dry sterilized gauze pad. Then, clean the skin using a sterilized gauze pad with 70% ethanol.
5. Using a foot planer, cut off the upper layer (epidermis) of the skin section and put it in a 9 cm Petri dish. Then, immediately add 25 mL of the DPBS/trypsin/glucose solution (prepared in step 1.1) to the Petri dish. Incubate for 30 min at 37 °C.
6. Using a pipette, remove the DPBS/trypsin/glucose solution from the Petri dish and add 10 mL of RPMI-1640 + 2% FBS to inactivate the trypsin.
7. Using two forceps, now release the epidermal cells into the medium by gently scraping and agitating both the epidermal and the dermal compartment of the skin sections.
8. Filter the epidermal cell suspension through a metal filter (1 mm hole size), and collect in a 50 mL tube. Add the remaining skin sections to a 50 mL tube containing 10 mL of RPMI-1640 without FBS and vortex for 10 s. Filter the suspension through the metal filter into a 50 mL tube containing the epidermal cell suspension. Add DPBS to a total volume of 50 mL.
9. Centrifuge the cell suspension for 10 min at 450 x g at room temperature.
10. Remove the supernatant and resuspend the epidermal cell pellet in approximately 10 mL of 37 °C KSFM depending on the size of the cell pellet. Count the cells using the Trypan Blue staining method under a microscope. The number of cells obtained from a 10 cm x 15 cm skin section is approximately 50-100 x 10⁶ cells. To each 75 cm² culture flask, transfer 8 x 10⁶ cells together with 12 mL of 37 °C KSFM. Gently shake the culture flasks to ensure uniform distribution of the cells.
11. Incubate the keratinocytes in a 37 °C incubator with 100% humidity and 5% CO₂. Change the medium after 2 days and three times weekly. Passage cells when the culture is 70-80% confluent, which takes approximately 2 weeks depending on the proliferation rate of the keratinocytes.