Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

Summary

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Abstract

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Introduction

The skin is the largest organ in the body with the epidermis as the outer most layer. The epidermis plays a critical role in forming an intact epidermal barrier to separate the body from the environment, and thus prevents water loss and provides protection from environmental insults, such as allergens, pathogens and UVB exposure. The epidermis develops from a single layer of undifferentiated basal keratinocytes (KCs) into a multi-layered stratified epithelium consisting of a basal layer, followed by a spinous layer, granular layer, and stratum corneum. Basal KCs, consisting of both epidermal stem cells and transit-amplifying cells, are proliferative and undifferentiat....

Protocol

All animal experiments are approved by the UCSD Institutional Animal Care and Use Committee.

1. Primary Mouse KC Isolation and Culture from Neonatal Skin

1. Sacrifice the post-natal day 0-2 neonates from the C57B/6 wildtype mouse strain by decapitation using scissors. Cut off the limbs just above the wrist and ankle joints, then cut off the tail completely, leaving a small hole.
2. To peel off the whole skin, first insert sharp scissors through the hole at the tail and cut the skin along the dorsal midline of the body to the opening on the neck. Next, use one forceps to grasp the exposed body and the other forceps to....

Results

High calcium induced terminal differentiation of neonatal and adult KCs. The primary mouse epidermal KCs plated and maintained at 0.06 mM CaCl₂ grew as a monolayer, and individual cells had a polygonal shape with distinct intercellular space, showing a cobblestone appearance when confluent (Figure 1A and Figure 2A). Elevating the CaCl₂ to 0.2 mM induced a rapid morphology change of the cells.......

Discussion

The skin epidermis functions as a critical barrier to separate and protect the body from the outside environment and damage from water loss, pathogens, heat and UV irradiation. The KCs are the predominant cell lineage of the epidermis, and primary culture of epidermal KCs is a useful tool to study and understand the biological processes of barrier formation and the response of KCs to environmental insults in vitro.

Here we describe methods to isolate and culture primary epidermal KCs .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 neonates or adult wildtype mice	Jackson Laboratory	000664	Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.
KC basal medium (EpiLife)	Invitrogen, Carlsbad, CA	MEPICF500	basal medium for keratinocyte culture with 0.06 mM CaCl2
Defined Growth Supplement (dGS)	Invitrogen, Carlsbad, CA	S0125	defined growth supplements for culture medium
Dispase powder	Invitrogen, Carlsbad, CA	17105041	enzyme to dissociate the epidermis from dermis
Attachment Factor	Invitrogen, Carlsbad, CA	S006100	gelatin-based coating material
Coating Matrix	Invitrogen, Carlsbad, CA	R011K	Collagen-based coating material
TrypLE	Invitrogen, Carlsbad, CA	12604-013	A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet
100 μm Cell Strainer Nylon mesh	Corning	352360
CCK-8 cell viability Kit	Dojindo Molecular Technologies, Rockville, MD	CK04-11
Mouse TNF (Mono/Mono) ELISA Set II	BD Biosciences, San Jose, CA	555268
Corded Hand-Held UV Lamps	Spectronics, Westbury, NY	EB-280C
8-watt UV tubes	Spectronics, Westbury, NY	BLE-8T312
Light Inverted Microscope for cell culture	ZEISS, Jena, Germany	Axio Observer
Fluorescent Microscope	Olympus	BX41