This work was supported by NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES grant (R01AR069653 to Zhang LJ), and National Institutes of Health Support (5T32AR062496-03 to CA).

All animal experiments are approved by the UCSD Institutional Animal Care and Use Committee.
1. Primary Mouse KC Isolation and Culture from Neonatal Skin
<ol>
	<li>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.</li>
	<li>To peel off the whole skin, first insert sharp scissors through the hole at the tail and ...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+epidermal+calcium+gradient+coincides+with+key+milestones+of+barrier+ontogenesis+in+the+rodent.">Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent.</a> J Invest Dermatol. 110 (4), 399-404 (1998).</li><li>Hennings, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+regulation+of+growth+and+differentiation+of+mouse+epidermal+cells+in+culture.">Calcium regulation of growth and differentiation of mouse epidermal cells in culture.</a> Cell. 19 (1), 245-254 (1980).</li><li>Zhang, L. J., Bhattacharya, S., Leid, M., Ganguli-Indra, G., Indra, A. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+murine+epidermal+differentiation+markers+is+tightly+regulated+by+restricted+extracellular+calcium+concentrations+in+vitro.">Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.</a> J Cell Biol. 109 (3), 1207-1217 (1989).</li><li>Yuspa, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+for+proliferation+and+differentiation+in+keratinocytes.">Signal transduction for proliferation and differentiation in keratinocytes.</a> Ann N Y Acad Sci. 548, 191-196 (1988).</li><li>Fang, N. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+enhances+mouse+keratinocyte+differentiation+in+vitro+to+differentially+regulate+expression+of+papillomavirus+authentic+and+codon+modified+L1+genes.">Calcium enhances mouse keratinocyte differentiation in vitro to differentially regulate expression of papillomavirus authentic and codon modified L1 genes.</a> Virology. 365 (1), 187-197 (2007).</li><li>Kolly, C., Suter, M. M., Muller, E. 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The authors have nothing to disclose.

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 ...

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 &#34;concentration gradient&#34;, increasing in concentration towards the skin surface1,2,3. 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 (&#62;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 KCs4,5.
In addition to barrier formation, epidermal KCs are also an important component of the skin&#39;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 TNF&#945;, IL6 and IFN&#946;, leading to immune system activation6,7,8,9. 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 rosacea6,8.
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&#39;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.

<table width="633">
	<tbody>
		<tr height="100">
			<td height="100" style="width:171px;height:100px;">C57BL/6 neonates or adult wildtype mice</td>
			<td style="width:177px;">Jackson Laboratory</td>
			<td style="width:119px;">000664</td>
			<td style="width:167px;">Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">KC basal medium (EpiLife)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">MEPICF500</td>
			<td style="width:167px;">basal medium for keratinocyte culture with 0.06 mM CaCl2</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">Defined Growth Supplement (dGS)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S0125</td>
			<td style="width:167px;">defined growth supplements for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Dispase powder</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">17105041</td>
			<td style="width:167px;">enzyme to dissociate the epidermis from dermis</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Attachment Factor</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S006100</td>
			<td style="width:167px;">gelatin-based coating material</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Coating Matrix</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">R011K</td>
			<td style="width:167px;">Collagen-based coating material</td>
		</tr>
		<tr height="80">
			<td height="80" style="width:171px;height:80px;">TrypLE</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">12604-013</td>
			<td style="width:167px;">A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">100 &#956;m Cell Strainer Nylon mesh</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">352360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">CCK-8 cell viability Kit</td>
			<td style="width:177px;">Dojindo Molecular Technologies, Rockville, MD</td>
			<td style="width:119px;">CK04-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Mouse TNF (Mono/Mono) ELISA Set II</td>
			<td style="width:177px;">BD Biosciences, San Jose, CA</td>
			<td style="width:119px;">555268</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Corded Hand-Held UV Lamps</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">EB-280C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">8-watt UV tubes</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">BLE-8T312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Light Inverted Microscope for cell culture</td>
			<td style="width:177px;">ZEISS, Jena, Germany</td>
			<td style="width:119px;">Axio Observer</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">Fluorescent Microscope</td>
			<td style="width:177px;">Olympus</td>
			<td style="width:119px;">BX41</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

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.

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...

Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

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

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.

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 ...

The overall goal of this procedure is to isolate primary mouse keratinocytes from neonatal or adult mouse skin from their in-vitro differentiation or to study their innate immune responses to UVb exposure. This method can help answer key questions in the epidermal biology field about how keratinocyte differentiation is regulated or how epidermal keratinocytes respond to external stimuli. The main advantages of this technique are that the keratinocytes can be conventionally isolated from both mouse, neonatal and adult mouse skin and that they can be cultured be with our serum.Demonstrating the procedure with me will be, Fengwu Li, a staff research associate from my laboratory. Begin by removing the limbs of a wild type C57 BL6 postnatal day zero to two mouse neonatal, just above the wrist entangled joints and cut off the tail completely leaving a small ball. Insert sharp scissors through the tail hole and cut the skin along the dorsal midline of the body to the opening on the neck.Using one forceps, grasp the exposed body and use another forceps to grasp the skin. Then gently peel the whole skin off of the body and over the leg stumps in one continuous motion, taking care not to break the skin into pieces. Rinse the peeled skin with 50 millimeters of sterile PBS in a 10 centimeter petri dish and place the skin into a two milliliter tube containing ice cold, dispase digestion buffer, taking care that the skin is not folded.When all of the neonatal skin sheets have been collected, place the tube on a rotator at four degree celsius overnight. The next morning, dispense the tube contents into a new petri dish. Then, transfer each piece of tissue into a new petri dish containing 50 milliliters of sterile PBS to remove any excess dispase.Use two forceps, place the skin samples into individual petri dishes, epidermal side down and carefully stretch the skin folds so that the pieces are fully extended on the dish bottoms. Add 500 microliters of a trypsin based digestion solution to each dish and to use one forceps to slowly lift each dermis up and away from the epidermis, while holding the epidermis in place with a second forceps. Float the isolated epidermises in the trypsin solution with the basal levels facing down and cover the dishes.Then incubate the skin pieces on a horizontal shaker at room temperature, with gentle agitation for 20 minutes. At the end of the incubation, add two milliliters of supplemented keratinocyte growth medium per epidermis to each petri dish and use forceps to vigorously rub the epidermal sheets releasing the single cells from the skin tissue. When the medium becomes turbid, tilt the dishes to allow the collection and transfer of the cell suspensions into a new tube.Leaving the remaining epidermal tissue in the dish. After the third round of cell collection, gently triturate the pooled cell suspension a few times to break up any cell clumps, and filter the cells through a 100 micron strainer into a new 50 milliliter conical tube. Collect the cells by centrifugation and gently re-suspend the pellet in one milliliter of cold keratinocyte growth medium on ice, for counting.Seed the cells at a 5x10 to the fourth centimeter squared density in keratinocyte growth medium in cultured dishes, pre-coated with an extra-cellular matrix product that promotes cell attachment. Then incubate the cells at 37 degrees celsius and 5%carbon dioxide, replacing the supernatant daily with fresh medium until the cells have reached the appropriate confluency. To isolate keratinocytes from an adult mouse, first remove the tail at its base.Followed by removal of approximately two millimeters of the tail tip so there is visible hole. Use a sharp blade to cut along the tail skin from the base to the tip. Then, using one forceps to grasp the exposed tail bone and another forceps to grasp the skin, gently peel the skin tissue from the bone in one continuous motion.Cut the peeled skin at the midline, so that each piece is less than two to three centimeters long, and rinse the pieces in 15 milliliters of sterile PBS in a 10 centimeter petri dish. Place the skins from up to five tails into a 15 milliliter tube containing up to 12 milliliters of ice cold, dispase digestion buffer and incubate the skins overnight at four degree celsius, with rotation. The next morning, isolate the epidermal cells from the partially digested skin sheets, as just demonstrated for the neonatal skin samples.Then seed the isolated adult keratinocyte cells at 1x10 to the fifth power centimeter square density in kerationcyte growth medium in culture dishes pre-coated with collagen. Changing the supernatant daily, until the cells reach the appropriate confluency. To induce the terminal differentiation of either type of keratinocyte culture, add calcium chloride for the cells at 2 nanomolar concentrations.For UVb irradiation induced death, replace the growth medium from a confluent keratinocyte culture, with 500 microliters of PBS and briefly expose the cells to 15 millijoules per square centimeter of UVb. Replace the PBS with fresh growth medium and return the cells to the cell culture incubator for six hours. After collecting the conditioned medium at each experimental time point, quantify the cell viability by the cell counting kit aid viability assay, according to the manufacturer's instructions.Then measure the TNL for release by the irradiated keratinocytes into conditioned medium according to standard protocols. Primary mode's epidermal keratinocytes plated and maintained at 06 nanomolar calcium chloride, grew as a monolayer with the individual cells exhibiting a polygonal shape with distinct inter-cellular spaces and demonstrating an overall cobblestone appearance when confluent. Elevating the calcium chloride to 2 millimolar, induces a rapid morphology change in the cells.With the cells becoming flattened and the distinct inter-cellular space becoming less apparent within eight hours. And the cell to cell adhesion between the tight junctions becoming more striking within 24 hours. By 48 to 72 hours, the formation of the cornified cell envelope and the vertical cell stratification, become apparent.Phalloidin staining of high calcium treated keratinocytes, reveals the formation of actin fiber-rich filopodial projections between adjacent cells, as early as three hours, post calcium switch. Between six and 24 hours, further actin fiber remodeling becomes evident. With the prominent amount of cell stratification apparent at 48 hours post high calcium exposure.UVb irradiation triggers the time dependent cell death of keratinocyte cultures as evidenced by both phase contrast imaging and cell viability assay. Indeed, by 24 hours, nearly all of the cells become rounded and detached, and are measured as unviable by cell viability assay. Most likely due to the high levels of TNF alpha, relieved by the keratinocytes, in response to UVb exposure.While attempting this procedure, it's important to remember that to keep the dissociated keratinocyte on ice at all times and to plate the cells as soon as possible to avoid terminal differentiation of the isolated cells. After its development, this technique paved the way for researchers in the field of epidermal biology to explore the roles of keratinocytes in skin development and in an innate immune defense of the skin. After watching this video, you should have a good understanding of how to isolate and culture primary epidermal keratinocytes from neonatal or adult mouse skin.

isolation-culture-primary-mouse-keratinocytes-from-neonatal-adult

Research

JoVE Journal

Biology

29.9K visualizações.  UC San Diego. The overall goal of this procedure is to isolate primary mouse keratinocytes from neonatal or adult mouse skin from their in-vitro differentiation or to study their innate immune responses to UVb exposure. This method can help answer key questions in the epidermal biology field about how keratinocyte differentiation is regulated or how epidermal keratinocytes respond to external stimuli. The main advantages of this technique are that the keratinocytes can be conventionally isolated from both ...