The author wishes to thank Annette Blak Rasmussen and Kristine Moeller for their technical support

The collection of skin samples from healthy adult volunteers undergoing plastic surgery requires approval from the ethical committee in the host institutions. This protocol was approved by the Regional Ethical Committee of Region Midtjylland, Denmark (M-20110027). The method described here is derived from similar studies by Maciaq et al.10 and Liu and Karasek11.
1. Isolation of Keratinocytes from Human Skin
<ol>
	<li>Start by making th...

<ol>
	<li>Barker, J. N., Mitra, R. S., Griffiths, C. E., Dixit, V. M., Nickoloff, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes+as+initiators+of+inflammation.">Keratinocytes as initiators of inflammation.</a> Lancet. 337 (8735), 211-214 (1991).</li><li>Nickoloff, B. J., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes:+key+immunocytes+of+the+integument.">Keratinocytes: key immunocytes of the integument.</a> Am J Pathol. 143 (2), 325-331 (1993).</li><li>Fuchs, E., Raghavan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+under+the+skin+of+epidermal+morphogenesis.">Getting under the skin of epidermal morphogenesis.</a> Nat Rev Genet. 3 (3), 199-209 (2002).</li><li>Bos, J. D., Kapsenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin+immune+system+Its+cellular+constituents+and+their+interactions.">The skin immune system Its cellular constituents and their interactions.</a> Immunol Today. 7 (7-8), 235-240 (1986).</li><li>Nestle, F. O., Di Meglio, P., Qin, J. Z., Nickoloff, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+immune+sentinels+in+health+and+disease.">Skin immune sentinels in health and disease.</a> Nat Rev Immunol. 9 (10), 679-691 (2009).</li><li>Albanesi, C., Scarponi, C., Giustizieri, M. L., Girolomoni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes+in+inflammatory+skin+diseases.">Keratinocytes in inflammatory skin diseases.</a> Curr Drug Targets Inflamm Allergy. 4 (3), 329-334 (2005).</li><li>Gilliet, M., Lande, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+and+self-DNA+in+autoimmune+skin+inflammation.">Antimicrobial peptides and self-DNA in autoimmune skin inflammation.</a> Curr Opin Immunol. 20 (4), 401-407 (2008).</li><li>Rohani, M. G., Pilcher, B. K., Chen, P., Parks, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+inhibits+ERK-mediated+collagenase-1+(MMP-1)+expression+in+collagen-activated+human+keratinocytes.">Cdc42 inhibits ERK-mediated collagenase-1 (MMP-1) expression in collagen-activated human keratinocytes.</a> J Invest Dermatol. 134 (5), 1230-1237 (2014).</li><li>Meephansan, J., Tsuda, H., Komine, M., Tominaga, S., Ohtsuki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+IL-33+expression+by+IFN-gamma+and+tumor+necrosis+factor-alpha+in+normal+human+epidermal+keratinocytes.">Regulation of IL-33 expression by IFN-gamma and tumor necrosis factor-alpha in normal human epidermal keratinocytes.</a> J Invest Dermatol. 132 (11), 2593-2600 (2012).</li><li>Maciag, T., Nemore, R. E., Weinstein, R., Gilchrest, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endocrine+approach+to+the+control+of+epidermal+growth:+serum-free+cultivation+of+human+keratinocytes.">An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes.</a> Science. 211 (4489), 1452-1454 (1981).</li><li>Liu, S. C., Karasek, M. <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+growth+of+adult+human+epidermal+keratinocytes+in+cell+culture.">Isolation and growth of adult human epidermal keratinocytes in cell culture.</a> J Invest Dermatol. 71 (2), 157-162 (1978).</li><li>Naaldijk, Y., Friedrich-Stockigt, A., Sethe, S., Stolzing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+cooling+rates+for+fibroblast+and+keratinocyte+cryopreservation.">Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.</a> J Tissue Eng Regen Med. 10 (10), E354-E364 (2016).</li><li>Otkjaer, K., 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=IL-20+gene+expression+is+induced+by+IL-1beta+through+mitogen-activated+protein+kinase+and+NF-kappaB-dependent+mechanisms.">IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms.</a> J Invest Dermatol. 127 (6), 1326-1336 (2007).</li><li>Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involucrin+and+other+markers+of+keratinocyte+terminal+differentiation.">Involucrin and other markers of keratinocyte terminal differentiation.</a> J Invest Dermatol. 81 (1 Suppl), 100s-103s (1983).</li><li>Boyce, S. T., Ham, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-regulated+differentiation+of+normal+human+epidermal+keratinocytes+in+chemically+defined+clonal+culture+and+serum-free+serial+culture.">Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.</a> J Invest Dermatol. 81 (1 Suppl), 33s-40s (1983).</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>Carlson, M. W., Alt-Holland, A., Egles, C., Garlick, 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=Three-dimensional+tissue+models+of+normal+and+diseased+skin.">Three-dimensional tissue models of normal and diseased skin.</a> Curr Protoc Cell Biol. , (2008).</li><li>Kamsteeg, 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=Type+2+helper+T-cell+cytokines+induce+morphologic+and+molecular+characteristics+of+atopic+dermatitis+in+human+skin+equivalent.">Type 2 helper T-cell cytokines induce morphologic and molecular characteristics of atopic dermatitis in human skin equivalent.</a> Am J Pathol. 178 (5), 2091-2099 (2011).</li><li>van den Bogaard, E. 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=Crosstalk+between+keratinocytes+and+T+cells+in+a+3D+microenvironment:+a+model+to+study+inflammatory+skin+diseases.">Crosstalk between keratinocytes and T cells in a 3D microenvironment: a model to study inflammatory skin diseases.</a> J Invest Dermatol. 134 (3), 719-727 (2014).</li><li>Raaby, 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=Langerhans+cell+markers+CD1a+and+CD207+are+the+most+rapidly+responding+genes+in+lesional+psoriatic+skin+following+adalimumab+treatment.">Langerhans cell markers CD1a and CD207 are the most rapidly responding genes in lesional psoriatic skin following adalimumab treatment.</a> Exp Dermatol. , (2017).</li></ol>

Here, we describe how to easily isolate primary human keratinocytes from adult skin, and how to culture them in vitro. This model can have a broad application for investigation of epidermal cell biology, and can be useful for researchers interested in studying cutaneous diseases.
Some of the advantages of the protocol described here is that in contrast to keratinocytes isolated from neonatal foreskin obtained from newborn males undergoing circumcision, primary human keratinocytes from...

The skin is the biggest organ of the human body and serves as a protective barrier against the external environment. The skin is composed of two main layers: the dermis and the epidermis, where the epidermis constitutes the outermost layer of the skin. The most abundant cell type in the epidermis is the keratinocytes comprising more than 95% of the cell mass1,2. The keratinocytes are maintained at various stages of differentiation in the epidermis and are organized into basal, spinous, granular, and cornified layers that correspond to specific stages of differentiation3. The primary function of keratinocytes is to provide the structural integrity of the epidermis, thereby producing an intact barrier to the outside world.
The keratinocytes also represent the first line of defense against pathogens in the skin, and therefore play an important role in the innate immune response4,5. Exposure of the keratinocytes to external stimuli leads to activation of intracellular signaling pathways and subsequently, production of a number of various inflammatory mediators including cytokines, chemokines, and antimicrobial peptides. These keratinocyte-derived proteins participate in the inflammatory response by recruiting and activating immune cells such as dendritic cells, neutrophils, and specific T cells6,7. Thus, because keratinocytes play a crucial part in numerous biological processes, the rationale behind the technique presented here was to generate an in vitro model to study skin biology. Primary keratinocyte cultures obtained from neonatal foreskin are often used to study skin biology8,9. However, with the technique described here, keratinocytes from both genders are obtained resulting in a higher biological diversity of the cells.
Here, we present a detailed protocol for the isolation and generation of primary human keratinocytes from adult skin, including maintenance and freezing of the keratinocytes. The overall goal of this method is to generate primary human keratinocytes that can be used as a model to study cutaneous biology in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">KSFM</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">17005-034</td>
			<td style="width:167px;">Cell culture medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">KSFM supplements</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">37000-015</td>
			<td style="width:167px;">Supplements for KSFM</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">DPBS</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td style="width:167px;">DPBS without Calcium and Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td style="width:167px;">Dimethyl sulfoxid</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamycin</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15710-049</td>
			<td style="width:167px;">Cell culture medium additive</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sterilization filter</td>
			<td style="width:119px;">Sartorius</td>
			<td style="width:119px;">16534</td>
			<td style="width:167px;">Syringe filter with a pore size of 0.2 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7409</td>
			<td style="width:167px;">Used to trypsinize cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7528</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">RPMI-1640</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">61870-010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">FBS</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">16000044</td>
			<td style="width:167px;">Used to inactivate trypsin</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Forceps</td>
			<td style="width:119px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Forceps from any company can be used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Scissors</td>
			<td style="width:119px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Scissors from any company can be used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Scalpel</td>
			<td style="width:119px;">Swann Morton</td>
			<td style="width:119px;">0501</td>
			<td style="width:167px;">Scalpels from any company can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% ethanol</td>
			<td style="width:119px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gauze pads</td>
			<td style="width:119px;">NOBAMED</td>
			<td style="width:119px;">875420</td>
			<td style="width:167px;">Gauze pads from any provider can be used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Foot planer</td>
			<td style="width:119px;">Credo Solingen</td>
			<td style="width:119px;">1510</td>
			<td style="width:167px;">Foot planer from any provider can be used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Petri dishes</td>
			<td style="width:119px;">TPP</td>
			<td style="width:119px;">93100</td>
			<td style="width:167px;">Petri dishes from any provider can be used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Metal filter</td>
			<td style="width:119px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">In-house 1 mm hole size metal filter</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">75 cm2 culture flasks</td>
			<td style="width:119px;">NUNC</td>
			<td style="width:119px;">156499</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">150 cm2 culture flasks</td>
			<td style="width:119px;">TTP</td>
			<td style="width:119px;">90151</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">0.05% Trypsin-EDTA solution</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25300-062</td>
			<td style="width:167px;">Used to trypsinize cells when passaging</td>
		</tr>
	</tbody>
</table>

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.

Calcium-induced Terminal Differentiation 
Human keratinocytes undergo terminal differentiation upon treatment with calcium14,15,16. Primary human keratinocytes were isolated and cultured as described in the above protocol. When approximately 50 - 60% confluent, the cells were stimulated with calcium (1.2 mM) or vehicle and pictures of the cells were taken on day 0, 1, and 2. Figure 1</s...

Watch this Scientific Journal Video about Generation and Culturing of Primary Human Keratinocytes from Adult Skin at JoVE.com

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

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. ...

The overall goal of this procedure is to generate primary human keratinocytes that can be used as a model to study cutaneous biology in vitro. This method can help answer key questions in the epidermal cell biology field, such as how human keratinocyte differentiation is regulated or as a model for studying inflammatory skin diseases. The main advantages of this technique are that primary human keratinocytes can be conventionally isolated from adult skin of both men and women and can include skin from people of any age older than 18.Demonstrating the procedure will be Annette Blak Rasmussen, a technician from my laboratory. To begin, prepare a 50 milliliter solution of 0.25%trypsin and 0.1%glucose in DPBS. Then, mix and filter sterilize the solution.Prepare 10 milliliters of RPMI 1640 plus 2%FBS, 50 milliliters of DPBS and keratinocyte serum free medium, or KSFM. Add KSFM supplements and 250 microliters of gentamicin to 500 milliliters of KSFM. Then, pre-warm the solutions to 37 degrees Celsius before use.Collect 10 centimeter by 15 centimeter skin samples from healthy adult volunteers undergoing plastic surgery. Keep the skin cool by transporting the skin samples in a styrofoam box containing a cooling element. If necessary, store the skin sample at four degrees Celsius overnight.Using sterilized scissors, scalpels and forceps, remove fat from underneath the skin section. Then, using needles, buckle out the skin section on a sterile cover on top of a plate. With a dry, sterilized gauze pad, clean the skin.Then, follow with 70%ethanol on a sterilized gauze pad. Next, using a foot planer, cut off the upper epidermal layer of the skin section and transfer it into a nine centimeter Petri dish. Then, immediately add 25 milliliters of the previously prepared DPBS trypsin glucose solution to the Petri dish.Incubate the samples at 37 degrees Celsius for 30 minutes. Using a pipette, remove the DPBS trypsin glucose solution from the Petri dish and add 10 milliliters of RPMI 1640 plus 2%FBS to inactivate the trypsin. Now, with two forceps, release the epidermal cells into the medium by gently scraping and agitating both the epidermal and the dermal compartment of the skin sections.Filter the epidermal cell suspension through a metal filter and collect the filtrate into a 50 milliliter tube. Add the remaining skin sections to a 50 milliliter tube containing 10 milliliters of RPMI 1640 without FBS and vortex for 10 seconds. Filter this suspension through the metal filter into a 50 milliliter tube containing the epidermal cell suspension.Then, add DPBS to a total volume of 50 milliliters. Centrifuge the cell suspension at 450 times G and room temperature, for 10 minutes. Then, remove the supernatant and depending on the size of the cell pellet, re-suspend the epidermal cell pellet in approximately 10 milliliters of 37 degrees Celsius KSFM.Using the trypan blue staining method, count the cells under a microscope. Then, to each 75 square centimeter culture flask, transfer eight times 10 to the sixth cells, together with 12 milliliters of 37 degrees Celsius KSFM. Gently shake the culture flasks to ensure uniform distribution of the cells.Incubate the keratinocytes in a 37 degree Celsius incubator with 100%humidity and 5%CO2. Change the medium after two days and then three times weekly. When the cells reach 70-80%confluency, keep the appropriate amount of 0.05%trypsin EDTA solution to 37 degrees Celsius in an incubator.Remove the medium from the cells and add 4.5 milliliters per 75 square centimeter culture flask of pre-warmed 0.05%trypsin EDTA solution to the cells. After placing the culture flask in the 37 degree Celsius incubator for approximately five minutes, check under the microscope if the cells have started to loosen. When approximately 50%of the cells have loosened, gently hit the culture flask against the hand to loosen the remaining cells.Then, to inactivate the trypsin, add six milliliters of 37 degree Celsius RPMI 1640 plus 2%FBS to the 75 square centimeter culture flask and transfer the cell suspension to 50 milliliter tubes. Rinse the culture flasks with three milliliters of RPMI 1640 plus 2%FBS and add the medium to the 50 milliliter tubes. Then, centrifuge the cells for 10 minutes at 450 times G and room temperature.Use 10 milliliters of 37 degree Celsius KSFM to re-suspend the pelleted cells. Then, count and transfer three times 10 to the sixth cells to a 150 square centimeter culture flask, together with 20 milliliters of 37 degree Celsius KSFM. Gently shake the culture flask to ensure uniform distribution of the cells, before returning the cells to the incubator.When the culture is 80-90%confluent, after trypsinizing and transferring the cells to 50 milliliter tubes, as demonstrated earlier in this video, rinse the culture flasks with six milliliters of RPMI 1640 plus 2%FBS and add it to the 50 milliliter tubes. Centrifuge the tubes for 10 minutes at 450 times G and four degrees Celsius. Then, prepare ice cold cell freezing medium.Re-suspend the cell pellet in KSFM plus 10%DMSO. Then, after counting the cells, freeze them at a density of six times 10 to the sixth cells per milliliter, using standard, slow-freezing cryopreservation methods. Store the keratinocytes in liquid nitrogen until ready to use.As seen here, human keratinocytes were isolated and cultured, as demonstrated in this video and then stimulated with Calcium. These images show the morphological changes due to the addition of Calcium on days one and two, compared to controlled treated cells. In addition to morphological changes, the addition of Calcium also resulted in an increase in transcription of mRNA for the differentiation marker Involucrin, 5.5-fold after 24 hours and 3.5-fold after 48 hours.In this experiment, cultured keratinocytes were stimulated with IL-1 beta for various amounts of time. As seen by Western blotting, within five minutes, IL-1 beta stimulation led to a rapid activation phosphorylation of p38 MAP kinase as IL-1 beta stimulation had no effect on p38 MAP kinase total protein levels. After one hour, IL-1 beta induced p38 MAP kinase phosphorylation returned to basal levels.After it's development, this technique paved the way for researchers in the field of human epidermal cell biology to explore the role of keratinocytes in innate immune responses, as well as in skin development. After watching this video, you should have a good understanding of how to isolate and culture primary keratinocytes from adult human skin, which can be used as a model to study cutaneous biology in vitro.

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16.0K visualizações.  Aarhus University Hospital. The overall goal of this procedure is to generate primary human keratinocytes that can be used as a model to study cutaneous biology in vitro. This method can help answer key questions in the epidermal cell biology field, such as how human keratinocyte differentiation is regulated or as a model for studying inflammatory skin diseases. The main advantages of this technique are that primary human keratinocytes can be conventionally isolated from adult skin of both men and women and can include ...