Der Autor bedankt sich bei Annette Blak Rasmussen und Kristine Moeller für ihre technische Unterstützung

Die Sammlung von Hautproben von gesunden Erwachsenen Probanden einer plastischen Operation erfordert Genehmigung durch die Ethik-Kommission in den Einrichtungen. Dieses Protokoll wurde von der regionalen ethischen Ausschuss der Region Midtjylland, Dänemark (M-20110027) genehmigt. Die hier beschriebene Methode leitet sich von ähnlichen Studien von Maciaq Et al. 10 und Liu und Karasek11.
1. Isolation von Keratinozyten aus der menschlichen Haut
<ol>
	<li>Beginnen, indem Sie die folgenden Lösungen: 50 mL Lösung von 0,25 % Trypsin und 0,1 % Glukose in DPBS. Mischung und Filter zu sterilisieren (0,2 µm) die Lösung. 10 mL RPMI-1640 + 2 % FBS Lösung, 50 mL des DPBS und Keratinozyten serumfreien Medium (KSFM) vorzubereiten. 500 mL KSFM KSFM Ergänzungen und 250 µL Gentamycin hinzufügen. Vorwärmen der Lösungen auf 37 ° C vor dem Gebrauch.</li>
	<li>Haut von gesunden Erwachsenen Probanden einer plastischen Operation zu sammeln. Die Hautproben verwendet sind ca. 10 cm x 15 cm, aber können in der Größe variieren. Halten Sie die Haut kühl unter Transport durch den Transport der Hautproben in einer Styropor-Box mit einem Kühlelement. Bei Bedarf kann die Hautprobe über Nacht bei 4 ° C gespeichert werden.</li>
	<li>Entfernen Sie Fett unter der Haut-Sektion mit sterilisierten Scheren, Skalpelle und Pinzetten.</li>
	<li>Schnalle aus Abschnitt Haut (ca. 10 cm x 15 cm) auf eine sterile Abdeckung oben auf einen Teller mit Nadeln. Zuerst reinigen Sie die Haut mit einem trockenen steriler Gaze-Pad. Dann reinigen Sie die Haut mit einem sterilen Tupfer mit 70 % Ethanol.</li>
	<li>Mit einem Fuß-Hobel, der obersten Hautschicht (Epidermis) des Abschnitts Haut schneiden Sie und legen Sie es in einem 9 cm Petrischale. Fügen Sie dann sofort 25 mL der DPBS/Trypsin/Glucose-Lösung (vorbereitet in Schritt 1.1) in der Petrischale. 30 min bei 37 ° c inkubieren</li>
	<li>Mit einer Pipette entfernen Sie die DPBS/Trypsin/Glukose-Lösung aus der Petrischale und 10 mL RPMI-1640 + 2 % FBS, die Trypsin zu inaktivieren.</li>
	<li>Mit zwei Pinzetten, lassen Sie jetzt die Epidermiszellen in das Medium durch sanft kratzen und rühren die epidermalen und dermalen Abteil des die Hautpartien.</li>
	<li>Filtern der epidermalen Zellsuspension durch einen Metallfilter (1 mm Lochgröße), und sammeln Sie in einem 50 mL-Tube. Hinzu kommt der übrigen Haut eine 50 mL-Tube mit 10 mL RPMI-1640 ohne FBS und Vortex für 10 S. Filter filtern die Aussetzung durch das Metall in ein 50 mL-Tube mit der epidermalen Zellsuspension. Einem Gesamtvolumen von 50 mL DPBS hinzufügen.</li>
	<li>Zentrifugieren der Zellsuspension für 10 min bei 450 X g bei Raumtemperatur.</li>
	<li>Entfernen Sie den Überstand und Aufschwemmen der epidermalen Zelle Pellet in ca. 10 mL von 37 ° C KSFM abhängig von der Größe des Pellet-Zelle. Zählen Sie die Zellen mit dem Trypan blau Färbung Methode unter Mikroskop. Die Anzahl der Zellen aus einen 10 x 15 cm-Haut-Abschnitt ist ca. 50-100 x 106 Zellen. Übertragen Sie an jedem 75 cm2 Kultur Kolben, 8 x 106 Zellen zusammen mit 12 mL von 37 ° C KSFM. Schütteln Sie die Kulturflaschen um gleichmäßige Verteilung der Zellen zu gewährleisten.</li>
	<li>Inkubieren Sie die Keratinozyten in einem Inkubator 37 ° C mit 100 % Luftfeuchtigkeit und 5 % CO2. Ändern Sie das Medium nach 2 Tagen und dreimal wöchentlich. Durchgang-Zellen, wenn die Kultur Zusammenfluss von 70-80 %, was ungefähr 2 Wochen je nach die Proliferationsrate der Keratinozyten dauert.</li>
</ol>
(2) Passagierung der Keratinozyten
<ol>
	<li>Erhitzen Sie die entsprechende Menge an Trypsin-EDTA-Lösung 0,05 % auf 37 ° C im Brutschrank. Verwenden Sie 4,5 mL 0,05 % Trypsin-EDTA-Lösung für eine 75 cm2 Kultur Kolben.</li>
	<li>Entfernen Sie Mittel aus den Zellen zu, und fügen Sie 4,5 mL/75 cm2 Kultur Flasche 0,05 % Trypsin-EDTA-Lösung zu den Zellen vorgewärmt. Legen Sie den Kultur-Kolben in den Inkubator (37 ° C) und nach ca. 5 min unter dem Mikroskop zu prüfen Sie, ob die Zellen lösen gestartet haben.</li>
	<li>Bei etwa 50 % der Zellen gelockert haben, schlagen Sie sanft den Kultur-Kolben gegen die Hand, die restlichen Zellen zu lösen. Fügen Sie dann um die Trypsin zu inaktivieren, 6 mL RPMI-1640 + 2 % FBS (37 ° C) in den 75 cm2 Kultur Kolben und übertragen Sie die Zellsuspension auf 50 mL Röhrchen.</li>
	<li>Spülen Sie die Kulturflaschen mit 3 mL RPMI 1640 + 2 % FBS und Hinzufügen der 50 mL-Tuben. Zentrifugieren Sie Zellen für 10 min bei 450 X g bei Raumtemperatur.
	<ol>
		<li>Aufschwemmen der gebeizte Zellen in 10 mL KSFM (37 ° C). Zählen der Zellen und 3 x 106 Zellen auf ein 150 cm2 Kultur Fläschchen mit 20 mL KSFM (37 ° C) übertragen. Schütteln Sie den Kultur-Kolben um gleichmäßige Verteilung der Zellen zu gewährleisten. Einfrieren der Zellen, wenn die Kultur ist 80-90 % konfluierende, die dauert ca. 4-6 Tage je nach die Proliferationsrate der Keratinozyten (siehe Abschnitt 3, Einfrieren Protokoll unten). Erweitern Sie die Keratinozyten um entsprechende gefrorenen Vorräte zu generieren.</li>
	</ol>
	</li>
</ol>
(3) Einfrieren von Keratinozyten
<ol>
	<li>Erhitzen Sie die entsprechende Menge an Trypsin-EDTA-Lösung 0,05 % auf 37 ° C im Brutschrank. Verwenden Sie 9 mL 0,05 % Trypsin-EDTA-Lösung für ein 150 cm2 Kultur Kolben.</li>
	<li>Entfernen Sie Mittel aus den Zellen zu, und fügen Sie 9 mL/150 cm2 Kultur Flasche 0,05 % Trypsin-EDTA-Lösung zu den Zellen vorgewärmt. Legen Sie den Kultur-Kolben in den Inkubator (37 ° C) und nach ca. 5 min unter dem Mikroskop zu prüfen Sie, ob die Zellen lösen gestartet haben.</li>
	<li>Bei etwa 50 % der Zellen gelockert haben, schlagen Sie sanft den Kultur-Kolben gegen die Hand um die verbleibenden Zellen zu lösen. Fügen Sie dann um die Trypsin zu inaktivieren, 12 mL RPMI-1640 + 2 % FBS (37 ° C) in den 150 cm2 Kultur Kolben und aspirieren der Zellsuspension auf 50 mL Röhrchen.</li>
	<li>Spülen Sie die Kulturflaschen mit 6 mL RPMI 1640 + 2 % FBS und Hinzufügen der 50 mL-Tuben. Zentrifugieren Sie die Rohre für 10 min bei 450 X g bei 4 ° C. Bereiten Sie eiskalte Zelle Medium (KSFM + 10 % DMSO) Einfrieren.</li>
	<li>Aufschwemmen der Zelle Pellet in KSFM + 10 % DMSO, zählen die Zellen und Zellen bei einer Dichte von 6 x 106 Zellen/mL mit standard langsam-Einfrieren Kryokonservierung Methoden12einfrieren.</li>
	<li>Die Keratinozyten in flüssigem Stickstoff (Dampfphase) erst unmittelbar vor Gebrauch zu speichern.</li>
</ol>
(4) Auftauen und Kultivierung gefrorenen Keratinozyten
<ol>
	<li>Tauen Sie schnell die entsprechenden gefrorenen Fläschchen in einem 37 ° C-Wasserbad. Verwenden Sie 70 % Ethanol, reinigen Sie die Außenseite der Cryoröhrchen. Die entsprechende Anzahl von Zellen (ca. 15.000 Zellen/cm2) auf einem Kultur-Kolben übertragen und sofort den Kultur-Kolben vorgewärmt Wachstumsmedium (KSFM) hinzufügen. Jede Größe von Kulturflaschen kann je nach Aufbau des Experiments verwendet werden.</li>
	<li>Verwenden Sie 5 mL Kulturmedium für eine 25 cm2 Kultur Kolben.</li></ol>Die Anzahl der Zellen hinzugefügt, dass die Kultur-Flasche variieren kann, da das Wachstum der Zellen vom Spender, Spender variiert. Inkubieren Sie Zellen in einem Inkubator 37 ° C mit 100 % Luftfeuchtigkeit und 5 % CO2.
	<li>Änderung des Mediums nach 2 Tagen und dreimal Verwendung wöchentlich frisch vorgewärmte KSFM. Hinweis: Unsere Daten haben gezeigt, dass durchschnittlich 95 % der Zellen isoliert von dem hier beschriebenen Protokoll positiv für die Keratinozyten-Marker-Cytokeratine-14-13 sind.</li>

<ol>
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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>

. Der Autor hat keinen Interessenskonflikt.

Hier beschreiben wir, wie primären menschlichen Keratinozyten von Erwachsenen Haut leicht zu isolieren und sie in-vitro-Kultur. Dieses Modell kann nützlich sein für Forscher, die Interesse an einem Studium Haut-Krankheiten kann haben eine breite Anwendung für die Untersuchung der epidermalen Zelle Biologie
Einige der Vorteile von dem hier beschriebenen Protokoll ist, dass im Gegensatz zu Keratinozyten isoliert von Neugeborenen Vorhaut erhalten von Neugeborenen männlichen Beschnei...

Die Haut ist das größte Organ des menschlichen Körpers und dient als eine schützende Barriere gegen die Außenwelt. Die Haut besteht aus zwei Schichten: die Dermis und Epidermis, wo die Epidermis die äußerste Schicht der Haut bildet. Die am häufigsten vorkommenden Zelltyp in der Epidermis ist die Keratinozyten, bestehend aus mehr als 95 % der Zelle Masse1,2. Die Keratinozyten werden in verschiedenen Stadien der Differenzierung in der Epidermis und gliedern sich in basalen, abstehenden, körnige und verhornten Schichten, die bestimmte Phasen der Differenzierung3entsprechen. Die primäre Funktion der Keratinozyten soll die strukturelle Integrität der Epidermis, wodurch eine intakte Barriere nach außen zur Verfügung zu stellen.
Die Keratinozyten auch die erste Linie der Verteidigung gegen Krankheitserreger in der Haut darstellen und daher eine wichtige Rolle in der angeborenen Immunantwort4,5. Exposition der Keratinozyten auf äußere Reize führt zu einer Aktivierung der intrazellulären Signalwege und anschließend Produktion einer Reihe von verschiedenen Entzündungsmediatoren wie Zytokine, Chemokine und antimikrobielle Peptide. Diese Keratinozyten gewonnenen Proteinen beteiligt die entzündliche Reaktion durch die Rekrutierung und Aktivierung von Immunzellen wie dendritische Zellen, Neutrophilen und spezifische T-Zellen6,7. So weil Keratinozyten in vielen biologischen Prozessen eine entscheidende Rolle spielen, war das Grundprinzip hinter der hier vorgestellten Technik erzeugen eine in-Vitro -Modell, um die Haut Biologie zu studieren. Primären Keratinozyten Kulturen gewonnenen neonatale Vorhaut werden häufig verwendet, um Haut Biologie8,9zu studieren. Jedoch mit der hier beschriebenen Technik Keratinozyten aus beiden Geschlechtern ergeben sich wiederum eine höhere biologische Vielfalt der Zellen.
Hier präsentieren wir Ihnen ein detailliertes Protokoll zur Isolierung und Generierung von primären menschlichen Keratinozyten aus Erwachsenen Haut, einschließlich Wartung und das Einfrieren der Keratinozyten. Das übergeordnete Ziel dieser Methode ist es, die primären menschlichen Keratinozyten zu generieren, die als Modell kann, zur kutanen Biologie in-vitro-Studie verwendet werden.

<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

Die Hauptfunktion der Keratinozyten soll die strukturelle Integrität der Epidermis, eine mechanische Barriere für die Außenwelt erhalten bleibt. Darüber hinaus spielen die Keratinozyten eine wesentliche Rolle bei der Initiierung, Wartung und Regelung der epidermalen Immunantworten als Teil des angeborenen Immunsystems Reaktion auf Antigene Reize schnell und unspezifisch. Wir beschreiben hier, ein Protokoll für die Isolierung der primären menschlichen Keratinozyten von Erwachsenen Haut und zeigen, dass diese Zellen auf Kalzium-induzierte terminal Differenzierung, gemessen durch eine erhöhte Expression von Differenzierung Marker Involucrin reagieren. Darüber hinaus zeigen wir, dass die isolierte Keratinozyten, IL-1β-induzierte Aktivierung des intrazellulären Signalwege reagieren, wie durch die Aktivierung der p38 MAPK Weg gemessen. Zusammengenommen, beschreiben wir eine Methode zur Isolierung und Kultivierung von primären menschlichen Keratinozyten von Erwachsenen. Da die Keratinozyten die vorherrschenden Zelltyp in der Epidermis sind, eignet sich diese Methode zu molekulare Mechanismen in kutane Biologie in-vitro-Studie.

Kalzium-induzierte Terminal Differenzierung Menschlichen Keratinozyten unterziehen terminal Differenzierung bei der Behandlung mit Kalzium14,15,16. Primären menschlichen Keratinozyten waren isoliert und kultiviert wie im obigen Protokoll beschrieben. Bei ca. 50-60 % Zusammenfluss, die Zellen wurden mit Kalzium (1,2 mM) stimuliert oder Fahrzeug und Bilder der Zellen wurden am Tag 0, 1 und 2. <strong class="xf...

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

Die menschliche Haut wirkt als eine erste Verteidigungslinie gegen die Außenumgebung. Wir präsentieren Ihnen eine Methode zur Isolierung von primären menschlichen Keratinozyten von Erwachsenen. Diese isolierten Keratinozyten eignen sich zahlreiche Versuchsaufbauten und sind ein sehr geeignetes Modell für die Untersuchung der molekularer Mechanismen in kutane Biologie in-vitro-.

Generation und Kultivierung der primären menschlichen Keratinozyten von Erwachsenen

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

generation-culturing-primary-human-keratinocytes-from-adult

Research

JoVE Journal

Developmental Biology

16.2K Aufrufe.  Aarhus University Hospital. Die menschliche Haut wirkt als eine erste Verteidigungslinie gegen die Außenumgebung. Wir präsentieren Ihnen eine Methode zur Isolierung von primären menschlichen Keratinozyten von Erwachsenen. Diese isolierten Keratinozyten eignen sich zahlreiche Versuchsaufbauten und sind ein sehr geeignetes Modell für die Untersuchung der molekularer Mechanismen in kutane Biologie in-vitro-.

Serie: Generation and Culturing of Primary Human Keratinocytes from Adult Skin

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolierung und Charakterisierung von Quantitative Immunzytochemische Primäre myogenen Zellen und Fibroblasten aus menschlichen Skelettmuskel

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Forschung

Entwicklungsbiologie

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Transfektion, Selektion und Colony-Picking für Menschen induzierten pluripotenten Stammzellen mit einem GFP-Gen in die AAVS1 Safe Harbor TALEN ziel

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generation der Integration freie Menschen induzierten pluripotenten Stammzellen Mit Haarstamm Keratinozyten

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Herstellung von genetisch veränderten Organotypische Hautkulturen Mit devitalisierten Menschen Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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.

Isolieren von Haarfollikelstammzellen und epidermalen Keratinozyten von Dorsal Mäusehaut

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 ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Herstellung und Kultur von Myogenic Vorläuferzellen / Primär-Myoblasten aus Skelettmuskel von adulten und im Alter von Menschen

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definierte und Scalable Generierung von Leber-ähnlichen Zellen aus humanen pluripotenten Stammzellen

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Die Isolation und die Kultur der primären Epicardial Zellen aus menschlichen Erwachsenen und fetalen Herzens Exemplare

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

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.

Eine vereinfachte und effiziente Methode zur primären menschlichen Keratinozyten aus Erwachsenen Hautgewebe zu isolieren

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

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generation von 3D Haut organoide aus Cord Blood-abgeleitete induzierte pluripotente Stammzellen

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...