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

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 grasp the skin, and gently peel the whole skin off the body and over the leg stumps with one continuous motion.
   1. Peel off the skin as one intact piece and be careful not to break the skin into pieces as this will result in cell loss during the dispase step.
3. Rinse the peeled skin with 15 mL of sterile PBS in a 10 cm Petri dish, then place the skin from each pup into a 2 mL tube filled with ice cold dispase digestion buffer, which contains 4 mg/mL dispase in KC growth medium (KC basal medium has 0.06 mM CaCl₂, dGS, and antibiotics/antimycotics).
   NOTE: Multiple skin isolates can be combined and incubated in one 15 mL tube (up to 5 skins per tube) containing 12 mL of dispase solution. Incubate the skin tube overnight in a 4 °C refrigerator on a rotator.
   1. Make sure that the skin is not folded in the tube as this will result in insufficient exposure of the folded region to the dispase solution.
4. On the next day (after 12-18 h in dispase), transfer the skin together with the dispase solution into a Petri dish. Transfer each tissue piece to a new Petri dish with 15 mL sterile PBS to wash away excess dispase.
5. Using two pairs of forceps, grasp the skin from PBS, lift the skin, and transfer to a new Petri dish with the epidermal side down and the dermis side up. Carefully stretch the skin folds so that it is fully extended on the Petri dish.
6. Place 500 µL of a trypsin-based digestion solution for each skin in a new Petri dish. Using forceps, slowly lift the dermis up and away from the epidermis, while holding the epidermis down. Dispose the dermis as biohazardous material. Transfer the separated epidermis and float on each drop of trypsin solution with the basal layer downward.
   1. If the epidermis is folded on the trypsin solution, remove any folds using forceps to ensure efficient digestion of basal KCs from the epithelium.
7. Incubate the skin on a Petri dish in trypsin solution at room temperature (with the lid covered) on a horizontal shaker with gentle agitation for 20 min.
8. Add 2 mL of supplemented KC growth medium per epidermis (about 1 square inch in size per epidermis) to the Petri dish. Grasp the epidermis using forceps, and vigorously rub the epidermis back and forth to release single cells from the epidermal sheet.
   1. Watch the medium turn increasingly turbid as cells are detached from the epidermis. Tilt the Petri dish to collect and transfer the cell suspension to a new tube leaving the remaining epidermal sheet on the dish.
9. Repeat two more times the rubbing of the epidermal sheet after adding 2 mL KC growth medium, and combine the cell suspensions in the same tube.
10. Pipet the cell solution up-and-down gently a few times to break any cell clumps using the appropriate serological pipette, then pass it through a 100 µm filter to a new 50 mL centrifuge tube.
11. Centrifuge the filtered cells for 5 min at 180 x g.
12. Aspirate off the supernatant and gently resuspend the cell pellet in 1 mL cold KC growth medium/epidermis.
13. Count the cells by a hemocytometer.
    NOTE: The KCs can spontaneously differentiate in suspension, so the cells should be plated as soon as possible after isolation to ensure the optimal performance of the cells. We recommend placing the cells on ice while counting as this will reduce the rate of KC spontaneous differentiation.
14. Seed the cells at a density of 5 x 10⁴/cm² in KC growth medium in culture dishes pre-coated with an extracellular matrix (ECM) product that promotes cell attachment.
    NOTE: We use a commercial available (e.g. attachment factor, which is a gelatin-based coating material (see the Table of Materials for details). Cells were cultured in humidified incubator with 5% CO₂ at 37 °C, and fresh medium were replenished every other day.
    1. For coating, coat the culture dishes with the cell attachment coating material warmed to room temperature for 10 min to 2 h prior to the cell seeding. Remove the attachment factor just before adding the cell suspension.
15. Change the medium 24 h after the initial plating to remove unattached cells, and change the medium daily until the cells reach the desired confluency prior to the experiment.

2. Primary Mouse KC Isolation from Adult Tail Skin

1. Sacrifice an adult C57BL/6 mouse (preferably between 6-15 weeks of age; either male or female) according to the animal facility regulations. Cut off the tail off the body from the tail base, and cut off ~2 mm of the tail tip so that there is a visible hole at the tip.
2. To peel the tail skin off, first use a sharp blade to cut along tail skin from the base to the tail tip. Next, use one forceps to grasp the exposed tail bone and the other forceps to grasp the skin, and gently peel the tail skin off the bone with one continuous motion. Cut the peeled skin from the mid-line so that each piece is less than 2-3 cm long.
3. Rinse the peeled skin with 15 mL of sterile PBS in a 10 cm Petri dish, then place the skin from each tail into a 2 mL tube filled with ice cold dispase digestion buffer (4 mg/mL dispase in KC growth medium).
   NOTE: Multiple skin pieces can also be combined and incubated in one 15-mL tube (up to 5 tails per tube) containing 12 mL of dispase solution.
   1. Incubate the skin tube overnight in a 4 °C refrigerator on a rotator.
4. Perform steps 1.4 to 1.13 to isolate the single cell suspension from the tail epidermis.
5. Seed the adult cells at a density of 10 x 10⁴/cm² (counted by a hematocytometer) in KC growth medium in culture dishes pre-coated with collagen.
   NOTE: We use a commercially available collagen-based coating kit (see the Table of Materials for details). Cells were cultured in humidified incubator with 5% CO₂ at 37 °C, and fresh medium were replenished every other day. In general, the culture dishes should be coated with collagen for 30 min to 1 h prior to the cell seeding. We observe that there is a significant enhancement of adult KC attachment to the dish when coated with collagen compared to the attachment factor, which is a gelatin-based coating material.
6. Change the medium 24 h after the initial plating to remove unattached cells, and change the medium daily until the cells reach the desired confluency prior to the experiment.

3. In Vitro Differentiation of KCs by High Calcium

1. To induce terminal differentiation, add CaCl₂ to 0.2 mM in the culture medium.
2. Alternatively, starve the cultured KCs overnight without added growth supplements in low calcium KC basal medium prior to the addition of high calcium.
   NOTE: Growth factor starvation will enhance cell commitment to early differentiation and the induction of early differentiation markers, such as K10⁵.

4. UVB Irradiation Mediated Cell Death and TNFα Release from KCs

1. Culture the mouse KCs in KC growth medium (in humidified incubator with 5% CO₂ at 37 °C) with daily medium change until the cells reach confluency. Immediately before the UVB irradiation, remove the growth medium and replace with 500 µL of PBS. Then subject the cells to 25 mJ/cm² UVB or mock control (no UVB).
   NOTE: UVB irradiation is performed using handheld UVB lamps with two 8 W bulbs (312 nm) as previously described¹⁰. After UVB irradiation, PBS is aspirated and fresh medium is added.
2. Measure and quantify the cell viability by the CCK-8 cell viability assay at 6, 8, and 24 h post-UVB treatment following the manufacturer's instruction¹¹.
3. Collect the conditioned medium from the KCs treated with UVB at desired time points and measure the TNFα released in the conditioned medium by the Mouse TNF (Mono/Mono) ELISA kit following the manufacturer's instruction^7,12.

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

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