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

Published: April 30, 2023

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 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…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Citar este artículo
Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples. J. Vis. Exp. (Pending Publication), e20354, doi: (2023).

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