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 undifferentiated. As basal KCs exit the cell cycle, the cells commit to differentiation and gradually migrate towards the surface of the epidermis, accompanied by the maturation of cell-cell junctions and formation of an epidermal permeability barrier (EPB). The KCs at the spinous layer express early differentiation markers such as Keratin 10 (K10); as the KCs migrate to the granular layer, the cells express late differentiation markers such as Filaggrin (FLG), Loricrin (LOR) and Involucrin (INV). At the stratum corneum, the KCs become terminally differentiated corneocytes, which are eventually shed off through desquamation as new cells replace them.

Calcium is considered the most physiological agent in the epidermis and triggers differentiation in vitro and in vivo in a similar manner. In normal skin epidermis, calcium ions form a characteristic "concentration gradient", increasing in concentration towards the skin surface^1,2,3. The calcium concentration rises from low levels in the lowest sublayers (basal and spinous layers) to a peak in the upper granular layer and then drops to negligible levels in the most superficial layer (stratum corneum). The calcium gradient also develops coincidently with the emergence of a component permeability barrier, which supports that calcium signaling plays a critical role of KC differentiation. In vitro, low calcium (0.02-0.1 mM) maintains the proliferation of basal KCs as a monolayer, whereas high calcium (>0.1 mM) induces a rapid and irreversible commitment of the cells to terminal differentiation as demonstrated by tight-junction formation and induction of LOR and INV upon high calcium treatment to the basal KCs^4,5.

In addition to barrier formation, epidermal KCs are also an important component of the skin's innate immune system. In response to pathogens or damaged-associated molecular patterns (DAMPs) released upon UVB irradiation or injury, KCs can produce large amounts of inflammatory cytokines, such as TNFα, IL6 and IFNβ, leading to immune system activation^6,7,8,9. Although proper inflammatory signaling from KCs is required for pathogen clearance, uncontrolled inflammatory response may trigger the development of auto-inflammatory skin diseases, such as psoriasis and rosacea^6,8.

Overall, KCs play a vital role in maintaining the intact skin barrier and initiating an immune response upon pathogen invasion or environmental insults. Therefore, primary culture of epidermal KCs is a useful technique to study the epithelial biology, KC differentiation, as well as KC-stimulated innate immune responses. The isolation and culture of primary mouse epidermal KCs can be a challenging process due to KC's susceptibility and sensitivity to various external stimulants. Here we describe a method to isolate and culture KCs from either neonatal mouse skin or adult mouse tail skin. For adult KC isolation, we do not use mouse dorsal skin because isolating sufficient quantities of viable KCs from this tissue can be difficult for the following reasons: First, the adult dorsal skin at the resting phase of the hair cycle (telogen) consists of a thin epidermis with only 1-2 layers of cells, leading to a low cell yield and inefficient separation of the epidermis from the dermis, which is the critical step for successful KC isolation. Second, the high hair follicle density that is present on adult dorsal skin further contributes to the difficulty in separating epidermis from the dermis. Instead, we routinely use tail skin as the source for adult mouse KCs as this epithelium is thicker with 3-5 layers of epidermal KCs. It also has a lower hair follicle density, which does not interfere with the epidermal separation, thus allowing KC isolation from any adult mouse tail skin regardless of the age and hair cycling stage of the mouse. The isolated neonatal KCs are seeded to gelatin-coated culture dishes, whereas collagen-coated dishes are used to seed isolated adult KCs due to the impaired ability of the adult cells to adhere compared to their neonatal counterparts. To culture mouse KCs, low calcium basal medium is supplemented with dGS, which contains epidermal growth factor (EGF), bovine transferrin, insulin-like growth factorc1 (IGF1), prostaglandin E2 (PGE2), bovine serum albumin (BSA) and hydrocortisone. Between 2-4 days after the initial plating, most of the differentiated KCs can be washed away during daily medium changes, and the remaining adherent cells show typical cobblestone morphology⁴, are proliferating, and do not express the early differentiation marker K10.

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 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. To peel off the whole skin, first insert sharp scissors through the hole at the tail and …

Representative 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

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