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

Samenvatting

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....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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