Name: isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin
Uploaded: 2017-07-14T14:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

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 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C refrigerator on a rotator.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place 500 &#181;L 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.
	<ol>
		<li>If the epidermis is folded on the trypsin solution, remove any folds using forceps to ensure efficient digestion of basal KCs from the epithelium.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#181;m filter to a new 50 mL centrifuge tube.</li>
	<li>Centrifuge the filtered cells for 5 min at 180 x g.</li>
	<li>Aspirate off the supernatant and gently resuspend the cell pellet in 1 mL cold KC growth medium/epidermis.</li>
	<li>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.</li>
	<li>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 &#176;C, and fresh medium were replenished every other day.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Primary Mouse KC Isolation from Adult Tail Skin
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Incubate the skin tube overnight in a 4 &#176;C refrigerator on a rotator.</li>
	</ol>
	</li>
	<li>Perform steps 1.4 to 1.13 to isolate the single cell suspension from the tail epidermis.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
</ol>
3. In Vitro Differentiation of KCs by High Calcium
<ol>
	<li>To induce terminal differentiation, add CaCl2 to 0.2 mM in the culture medium.</li>
	<li>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.</li>
</ol>
4. UVB Irradiation Mediated Cell Death and TNF&#945; Release from KCs
<ol>
	<li>Culture the mouse KCs in KC growth medium (in humidified incubator with 5% CO2 at 37 &#176;C) with daily medium change until the cells reach confluency. Immediately before the UVB irradiation, remove the growth medium and replace with 500 &#181;L 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.</li>
	<li>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&#39;s instruction11.</li>
	<li>Collect the conditioned medium from the KCs treated with UVB at desired time points and measure the TNF&#945; released in the conditioned medium by the Mouse TNF (Mono/Mono) ELISA kit following the manufacturer&#39;s instruction7,12.</li>
</ol>

<ol>
	<li>Mauro, T., 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=Acute+barrier+perturbation+abolishes+the+Ca2++and+K++gradients+in+murine+epidermis:+quantitative+measurement+using+PIXE.">Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.</a> J Invest Dermatol. 111 (6), 1198-1201 (1998).</li><li>Menon, G. K., Grayson, S., Elias, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+calcium+reservoirs+in+mammalian+epidermis:+ultrastructural+localization+by+ion-capture+cytochemistry.">Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry.</a> J Invest Dermatol. 84 (6), 508-512 (1985).</li><li>Elias, P. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip2+is+a+dynamic+regulator+of+epidermal+proliferation+and+differentiation+by+integrating+EGFR+and+Notch+signaling.">Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling.</a> J Cell Sci. 125 (Pt 23), 5733-5744 (2012).</li><li>Lai, Y. P., 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=Commensal+bacteria+regulate+Toll-like+receptor+3-dependent+inflammation+after+skin+injury.">Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury.</a> Nat Med. 15 (12), 1377-1382 (2009).</li><li>Bernard, J. J., 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=Ultraviolet+radiation+damages+self+noncoding+RNA+and+is+detected+by+TLR3.">Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.</a> Nat Med. 18 (8), (2012).</li><li>Zhang, L. J., 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=Antimicrobial+Peptide+LL37+and+MAVS+Signaling+Drive+Interferon-beta+Production+by+Epidermal+Keratinocytes+during+Skin+Injury.">Antimicrobial Peptide LL37 and MAVS Signaling Drive Interferon-beta Production by Epidermal Keratinocytes during Skin Injury.</a> Immunity. 45 (1), 119-130 (2016).</li><li>Borkowski, A. W., 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=Toll-Like+Receptor+3+Activation+Is+Required+for+Normal+Skin+Barrier+Repair+Following+UV+Damage.">Toll-Like Receptor 3 Activation Is Required for Normal Skin Barrier Repair Following UV Damage.</a> J Invest Dermatol. 135 (2), 569-578 (2015).</li><li>Borkowski, A. W., 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=Toll-like+receptor+3+activation+is+required+for+normal+skin+barrier+repair+following+UV+damage.">Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.</a> J Invest Dermatol. 135 (2), 569-578 (2015).</li><li>Zillessen, P., 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=Metabolic+role+of+dipeptidyl+peptidase+4+(DPP4)+in+primary+human+(pre)adipocytes.">Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes.</a> Sci Rep. 6, 23074 (2016).</li><li>Wells, C. A., 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=The+macrophage-inducible+C-type+lectin,+mincle,+is+an+essential+component+of+the+innate+immune+response+to+Candida+albicans.">The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.</a> J Immunol. 180 (11), 7404-7413 (2008).</li><li>Vasioukhin, V., Bauer, C., Yin, M., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+actin+polymerization+is+the+driving+force+for+epithelial+cell-cell+adhesion.">Directed actin polymerization is the driving force for epithelial cell-cell adhesion.</a> Cell. 100 (2), 209-219 (2000).</li><li>Zhuang, L., 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=TNF+receptor+p55+plays+a+pivotal+role+in+murine+keratinocyte+apoptosis+induced+by+ultraviolet+B+irradiation.">TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation.</a> J Immunol. 162 (3), 1440-1447 (1999).</li><li>Lichti, U., Anders, J., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+short-term+culture+of+primary+keratinocytes,+hair+follicle+populations+and+dermal+cells+from+newborn+mice+and+keratinocytes+from+adult+mice+for+in+vitro+analysis+and+for+grafting+to+immunodeficient+mice.">Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.</a> Nat Protoc. 3 (5), 799-810 (2008).</li><li>Dlugosz, A. A., Glick, A. B., Tennenbaum, T., Weinberg, W. C., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+utilization+of+epidermal+keratinocytes+for+oncogene+research.">Isolation and utilization of epidermal keratinocytes for oncogene research.</a> Methods Enzymol. 254, 3-20 (1995).</li><li>Jones, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+keratinocytes.">Isolation and culture of mouse keratinocytes.</a> CSH Protoc. 2008, (2008).</li><li>Pirrone, A., Hager, B., Fleckman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+keratinocyte+culture.">Primary mouse keratinocyte culture.</a> Methods Mol Biol. 289, 3-14 (2005).</li><li>Yuspa, S. H., Kilkenny, A. E., Steinert, P. M., Roop, D. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+cell+cycle+exit,+and+onset+of+terminal+differentiation+in+cultured+keratinocytes:+pre-programmed+pathways+in+control+of+C-Myc+and+Notch1+prevail+over+extracellular+calcium+signals.">Proliferation, cell cycle exit, and onset of terminal differentiation in cultured keratinocytes: pre-programmed pathways in control of C-Myc and Notch1 prevail over extracellular calcium signals.</a> J Invest Dermatol. 124 (5), 1014-1025 (2005).</li><li>Marcelo, C. L., Gold, R. C., Fairley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+1.2+mmol/l+calcium,+triamcinolone+acetonide,+and+retinoids+on+low-calcium+regulated+keratinocyte+differentiation.">Effect of 1.2 mmol/l calcium, triamcinolone acetonide, and retinoids on low-calcium regulated keratinocyte differentiation.</a> Br J Dermatol. 111, 64-72 (1984).</li></ol>

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><tbody><tr><td>C57BL/6 neonates or adult wildtype mice</td><td>Jackson Laboratory</td><td>000664</td><td>Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td></tr><tr><td>KC basal medium (EpiLife)</td><td>Invitrogen, Carlsbad, CA</td><td>MEPICF500</td><td>basal medium for keratinocyte culture with 0.06 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Defined Growth Supplement (dGS)</td><td>Invitrogen, Carlsbad, CA</td><td>S0125</td><td>defined growth supplements for culture medium</td></tr><tr><td>Dispase powder</td><td>Invitrogen, Carlsbad, CA</td><td>17105041</td><td>enzyme to dissociate the epidermis from dermis</td></tr><tr><td>Attachment Factor</td><td>Invitrogen, Carlsbad, CA</td><td>S006100</td><td>gelatin-based coating material</td></tr><tr><td>Coating Matrix</td><td>Invitrogen, Carlsbad, CA</td><td>R011K</td><td>Collagen-based coating material</td></tr><tr><td>TrypLE</td><td>Invitrogen, Carlsbad, CA</td><td>12604-013</td><td>A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td></tr><tr><td>100 &amp;mu;m Cell Strainer Nylon mesh</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>CCK-8 cell viability Kit</td><td>Dojindo Molecular Technologies, Rockville, MD</td><td>CK04-11</td><td></td></tr><tr><td>Mouse TNF (Mono/Mono) ELISA Set II</td><td>BD Biosciences, San Jose, CA</td><td>555268</td><td></td></tr><tr><td>Corded Hand-Held UV Lamps</td><td>Spectronics, Westbury, NY</td><td>EB-280C</td><td></td></tr><tr><td>8-watt UV tubes</td><td>Spectronics, Westbury, NY</td><td>BLE-8T312</td><td></td></tr><tr><td>Light Inverted Microscope for cell culture</td><td>ZEISS, Jena, Germany</td><td>Axio Observer</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Olympus</td><td>BX41</td><td></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 ...

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Research

JoVE Journal

Biology

30.7K Views.  UC San Diego. 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.

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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Developmental Biology

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

Bioengineering

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

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

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;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.

Izolacja keratynocytów ludzkiej skóry: metoda hodowli pierwotnych keratynocytów z próbek skóry

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...