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

Abstract

Johansen, C. et al. Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)

This video describes the isolation of keratinocytes—the most prominent cells of human skin's epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KSFM	ThermoFisher Scientific	17005-034	Cell culture medium
KSFM supplements	ThermoFisher Scientific	37000-015	Supplements for KSFM
DPBS	ThermoFisher Scientific	14190-144	DPBS without Calcium and Magnesium
DMSO	Sigma-Aldrich	D8418	Dimethyl sulfoxide
Gentamycin	ThermoFisher Scientific	15710-049	Cell culture medium additive
Sterilization filter	Sartorius	16534	Syringe filter with a pore size of 0.2 µm
Trypsin	Sigma-Aldrich	T7409	Used to trypsinize cells
Glucose	Sigma-Aldrich	G7528	-
RPMI-1640	ThermoFisher Scientific	61870-010	-
FBS	ThermoFisher Scientific	16000044	Used to inactivate trypsin
Forceps	-	-	Forceps from any company can be used
Scissors	-	-	Scissors from any company can be used
Scalpel	Swann Morton	0501	Scalpels from any company can be used
70% ethanol	-	-	-
Gauze pads	NOBAMED	875420	Gauze pads from any provider can be used
Foot planer	Credo Solingen	1510	Foot planer from any provider can be used
Petri dishes	TPP	93100	Petri dishes from any provider can be used
Metal filter	-	-	In-house 1 mm hole size metal filter
75 cm2 culture flasks	NUNC	156499	-
150 cm2 culture flasks	TTP	90151	-
0.05% Trypsin-EDTA solution	ThermoFisher Scientific	25300-062	Used to trypsinize cells when passaging