L'autore desidera ringraziare Annette Blak Rasmussen e Kristine Moeller per il loro supporto tecnico

La raccolta di campioni di pelle da volontari sani adulti sottoposti a chirurgia plastica richiede l'approvazione del comitato etico nelle istituzioni dell'host. Questo protocollo è stato approvato dal regionale Comitato etico della regione Midtjylland, Danimarca (M-20110027). Il metodo qui descritto è derivato dagli studi simili da Maciaq et al. 10 e Liu e Karasek11.
1. isolamento dei cheratinociti da pelle umana
<ol>
	<li>Iniziare facendo le seguenti soluzioni: 50 mL di soluzione di glucosio di tripsina e 0,1% 0,25% in DPBS. Mix e filtro sterilizzare (0,2 µm) la soluzione. Preparare 10 mL di RPMI-1640 + soluzione di 2% FBS, 50 mL di medium senza siero DPBS e cheratinociti (KSFM). Aggiungere 500 mL di KSFM KSFM integratori e 250 µ l di gentamicina. Pre-riscaldare le soluzioni a 37 ° C prima dell'uso.</li>
	<li>Raccogliere pelle da volontari sani adulti sottoposti a chirurgia plastica. I campioni di pelle utilizzati sono circa 10 cm x 15 cm, ma possono variare in dimensione. Mantenere la pelle fresca sotto trasporto trasportando i campioni di pelle in una scatola di polistirolo contenente un elemento di raffreddamento. Se necessario, il campione di pelle possa essere conservato a 4 ° C durante la notte.</li>
	<li>Rimuovere il grasso da sotto la sezione di pelle utilizzando pinze, bisturi e forbici sterilizzate.</li>
	<li>Fibbia la sezione della pelle (circa 10 cm x 15 cm) su un telo sterile sopra una piastra usando gli aghi. In primo luogo, pulire la cute con un tampone di garza sterile asciutta. Quindi pulire la pelle usando un tampone di garza sterile con etanolo al 70%.</li>
	<li>Utilizzando una pialla di piede, taglio di livello superiore della sezione della pelle (epidermide) e metterlo in una capsula di Petri di 9 cm. Quindi, immediatamente aggiungere 25 mL della soluzione di glucosio/DPBS/tripsina (preparata al punto 1.1) per la capsula di Petri. Incubare per 30 min a 37 ° C.</li>
	<li>Utilizzando una pipetta rimuovere la soluzione DPBS/tripsina/glucosio da di Petri e aggiungere 10 mL di RPMI-1640 + 2% FBS per inattivare la tripsina.</li>
	<li>Utilizzando due pinze, ora rilasciare cellule epidermiche nel medium delicatamente raschiando e agitazione sia epidermico e dermico vano delle sezioni della pelle.</li>
	<li>Filtrare la sospensione cellulare epidermica attraverso un filtro metallico (dimensione del foro di 1 mm) e raccogliere in un tubo da 50 mL. Aggiungere le restanti sezioni di pelle per un tubo da 50 mL contenente 10 mL di RPMI-1640 senza FBS e vortexare per 10 s. filtro filtrare la sospensione attraverso il metallo in una provetta da 50 mL contenente la sospensione delle cellule epidermiche. Aggiungere DPBS per un volume totale di 50 mL.</li>
	<li>Centrifugare la sospensione cellulare per 10 min a 450 x g e a temperatura ambiente.</li>
	<li>Rimuovere il supernatante e risospendere il pellet cellulare epidermico in circa 10 mL di 37 ° C KSFM a seconda della dimensione del pellet cellulare. Contare le celle utilizzando il blu di Trypan che macchia il metodo sotto il microscopio. Il numero di cellule ottenute da una sezione di pelle 10 cm x 15 cm è di circa 50-100 x 106 celle. A ciascun matraccio di cultura2 75cm, trasferimento 8 x 106 cellule insieme a 12 mL di 37 ° C KSFM. Agitare delicatamente i matracci di cultura per assicurare una uniforme distribuzione delle cellule.</li>
	<li>Incubare i cheratinociti in un incubatore a 37 ° C con 100% umidità e 5% CO2. Modificare il mezzo dopo 2 giorni e tre volte alla settimana. Cellule di passaggio quando la cultura è 70-80% confluenti, che prende circa 2 settimane a seconda del tasso di proliferazione dei keratinocytes.</li>
</ol>
2. passaggio dei cheratinociti
<ol>
	<li>Riscaldare la giusta quantità di soluzione di tripsina-EDTA 0,05% a 37 ° C in un incubatore. Utilizzare 4,5 mL di soluzione di tripsina-EDTA 0,05% per un matraccio di cultura2 75 cm.</li>
	<li>Rimuovere il mezzo dalle cellule e aggiungere matraccio di cultura di 4,5 mL/75 cm2 pre-riscaldati soluzione di tripsina-EDTA 0,05% per le celle. Posizionare il matraccio di cultura nell'incubatore (37 ° C) e dopo circa 5 minuti, controllare al microscopio se le cellule hanno iniziato allentamento.</li>
	<li>Quando circa il 50% delle cellule hanno allentato, delicatamente colpire il matraccio di cultura contro la mano per allentare le cellule rimanenti. Poi, per inattivare la tripsina, aggiungere 6 mL di RPMI-1640 + 2% FBS (37 ° C) nel matraccio di cultura di 75 cm2 e trasferire la sospensione cellulare per provette da 50 mL.</li>
	<li>Sciacquare i matracci di cultura con 3 mL di RPMI 1640 + 2% FBS e aggiungere le provette da 50 mL. Centrifugare le cellule per 10 min a 450 x g e a temperatura ambiente.
	<ol>
		<li>Risospendere le cellule pellettate in 10 mL di KSFM (37 ° C). Contare le celle e trasferire 3 x 106 cellule in un pallone di cultura di2 cm 150 con 20 mL di KSFM (37 ° C). Agitare delicatamente il matraccio di cultura per assicurare una uniforme distribuzione delle cellule. Congelare le cellule quando la cultura è 80-90% confluenti, che prende circa 4-6 giorni a seconda del tasso di proliferazione dei keratinocytes (Vedi sezione 3, protocollo sottostante di congelamento). Espandere i cheratinociti per generare gli stock congelati appropriati.</li>
	</ol>
	</li>
</ol>
3. congelamento dei cheratinociti
<ol>
	<li>Riscaldare la giusta quantità di soluzione di tripsina-EDTA 0,05% a 37 ° C in un incubatore. Utilizzare 9 mL di soluzione di tripsina-EDTA 0,05% per un matraccio di cultura2 150 cm.</li>
	<li>Rimuovere il mezzo dalle cellule e aggiungere matraccio di cultura di 9 mL/150 cm2 pre-riscaldati soluzione di tripsina-EDTA 0,05% per le celle. Posizionare il matraccio di cultura nell'incubatore (37 ° C) e dopo circa 5 minuti, controllare al microscopio se le cellule hanno iniziato allentamento.</li>
	<li>Quando circa il 50% delle cellule hanno allentato, colpire delicatamente il matraccio di cultura contro la mano al fine di allentare le cellule rimanenti. Quindi, per inattivare la tripsina, aggiungere 12 mL di RPMI-1640 + 2% FBS (37 ° C) nel matraccio di cultura2 cm 150 e aspirare la sospensione cellulare per provette da 50 mL.</li>
	<li>Sciacquare i matracci di cultura con 6 mL di RPMI 1640 + 2% FBS e aggiungere le provette da 50 mL. Centrifugare le provette per 10 min a 450 x g a 4 ° C. Preparare il medium (KSFM + 10% DMSO) di congelamento delle cellule ghiacciata.</li>
	<li>Risospendere il pellet cellulare in KSFM + 10% DMSO, contare le celle e congelare le cellule ad una densità di 6 x 106 cellule/mL utilizzando metodi standard crioconservazione di congelamento lento12.</li>
	<li>Memorizzare i cheratinociti in azoto liquido (fase vapore) fino all'uso.</li>
</ol>
4. lo scongelamento e coltura di cheratinociti congelati
<ol>
	<li>Scongelare rapidamente i flaconcini congelati appropriati in bagnomaria a 37 ° C. Utilizzare etanolo al 70% per pulire l'esterno del cryovial. Trasferire il numero appropriato di cellule (circa 15.000 cellule/cm2) in un pallone di cultura e immediatamente aggiungere mezzo di crescita pre-riscaldato (KSFM) nel matraccio di cultura. Qualsiasi dimensione di matracci di cultura può essere utilizzato a seconda della configurazione dell'esperimento.</li>
	<li>Utilizzare 5 mL terreno di coltura per un matraccio di cultura 25 cm2 .</li></ol>Aggiunto il numero delle cellule, che il matraccio di cultura potrebbe variare perché la crescita delle cellule varia da donatore a donatore. Incubare le cellule in un incubatore a 37 ° C con 100% umidità e 5% CO2.
	<li>Cambia il mezzo dopo 2 giorni e tre volte settimanalmente utilizzando freschi KSFM pre-riscaldato. Nota: I nostri dati hanno dimostrato che in media il 95% delle cellule isolate tramite il protocollo descritto qui sono positivo per il marcatore del keratinocyte citocheratine-1413.</li>

<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>

. L'autore non ha alcun conflitto di interessi.

Qui, descriviamo come facilmente isolare keratinocytes umani primari da pelle adulta e come loro coltura in vitro. Questo modello può avere una vasta applicazione per indagini di biologia delle cellule epidermiche e può essere utile per i ricercatori interessati a studiare le malattie cutanee.
Alcuni dei vantaggi del protocollo descritto qui è che, contrariamente ai cheratinociti isolati da prepuzio neonatale ottenuto da neonati maschi sottoposti a circoncisione, keratinocytes uman...

La pelle è l'organo più grande del corpo umano e serve come una barriera protettiva contro l'ambiente esterno. La pelle è composta di due strati principali: il derma e l'epidermide, dove l'epidermide costituisce lo strato più esterno della pelle. Il tipo più abbondante di cellule nell'epidermide è i keratinocytes che comprende oltre il 95% delle cellule massa1,2. I cheratinociti sono tenuti nelle varie fasi di differenziazione nell'epidermide e sono organizzati in livelli basali, spinosi, granulari e cornified che corrispondono a specifiche fasi di differenziazione3. La funzione primaria dei cheratinociti è per fornire l'integrità strutturale dell'epidermide, producendo così una barriera intatta al mondo esterno.
I cheratinociti rappresentano anche la prima linea di difesa contro gli agenti patogeni della pelle e quindi giocano un ruolo importante nella risposta immunitaria innata4,5. L'esposizione dei keratinocytes agli stimoli esterni conduce all'attivazione di vie di segnalazione intracellulare e, successivamente, produzione di un numero di vari mediatori infiammatori, tra cui citochine, chemochine e peptidi antimicrobici. Queste proteine derivate da cheratinociti partecipano nella risposta infiammatoria di reclutamento e l'attivazione di cellule immunitarie come specifico T cellule6,7, neutrofili e cellule dendritiche. Così, perché keratinocytes gioca una parte cruciale in numerosi processi biologici, la spiegazione razionale dietro la tecnica presentata qui era per generare un modello in vitro per studiare la biologia della pelle. Primario del keratinocyte colture ottenute da prepuzio neonatale sono spesso utilizzati per studiare biologia pelle8,9. Tuttavia, con la tecnica qui descritta, cheratinociti da entrambi i sessi sono ottenuti con conseguente una maggiore diversità biologica delle cellule.
Qui, presentiamo un protocollo dettagliato per l'isolamento e la generazione di keratinocytes umani primari da pelle adulta, compresa la manutenzione e il congelamento dei keratinocytes. L'obiettivo generale di questo metodo è quello di generare keratinocytes umani primari che può essere utilizzato come modello per studiare biologia cutanea 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

La funzione principale dei cheratinociti è per fornire l'integrità strutturale dell'epidermide, mantenendo quindi una barriera meccanica al mondo esterno. Inoltre, cheratinociti svolgono un ruolo essenziale nell'iniziazione, la manutenzione e la regolazione delle risposte immunitarie epidermiche facendo parte del sistema immunitario innato risponde agli stimoli antigenici in modo veloce, non specifico. Qui, descriviamo un protocollo per l'isolamento dei keratinocytes umani primari da pelle adulta e dimostrare che queste cellule rispondono a differenziazione terminale calcio-indotta, come misurato da un'aumentata espressione del involucrin indicatore di differenziazione. Inoltre, ci mostra che i keratinocytes isolati sono reattivi al-1 β-induced attivazione delle vie di segnalazione intracellulare come misurato tramite l'attivazione del pathway MAPK p38. Presi insieme, descriviamo un metodo per l'isolamento e coltura dei keratinocytes umani primari da pelle adulta. Poiché i cheratinociti sono il tipo predominante delle cellule nell'epidermide, questo metodo è utile per studiare i meccanismi molecolari in biologia cutanea in vitro.

Calcio-indotto differenziazione terminale Cheratinociti umani subiscono differenziazione terminale previo trattamento con calcio14,15,16. Keratinocytes umani primari è stato isolato e coltivato come descritto nel protocollo di cui sopra. Quando circa 50-60% confluenti, le cellule sono state stimolate con calcio (1,2 mM) o veicolo e immagini delle cellule sono state scattate il giorno 0, 1 e 2. <strong class=...

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

La pelle umana agisce come una prima linea di difesa contro l'ambiente esterno. Presentiamo un metodo per isolare i keratinocytes umani primari da pelle adulta. Questi keratinocytes isolato sono utili in numerose configurazioni sperimentali e sono un modello altamente adatto per studiare i meccanismi molecolari in biologia cutanea in vitro.

Generazione e la coltura dei Keratinocytes umani primari da pelle adulta

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 Views.  Aarhus University Hospital. La pelle umana agisce come una prima linea di difesa contro l'ambiente esterno. Presentiamo un metodo per isolare i keratinocytes umani primari da pelle adulta. Questi keratinocytes isolato sono utili in numerose configurazioni sperimentali e sono un modello altamente adatto per studiare i meccanismi molecolari in biologia cutanea in vitro.

Video: 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.

Isolamento e quantitativa immunocitochimica caratterizzazione di cellule miogeniche primarie e fibroblasti da muscolo scheletrico umano

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

Ricerca

Biologia dello sviluppo

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.

Transfection, selezione, e Colonia-picking di cellule staminali umane pluripotenti indotte TALEN-mirati con Gene GFP nel AAVS1 Safe Harbor

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.

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

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.

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

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.

Isolare le cellule staminali del follicolo pilifero e epidermico cheratinociti da pelle dorsale del mouse

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.

Preparazione e la cultura di cellule miogeniche Precursore / mioblasti primari dal muscolo scheletrico di adulti e esseri umani invecchiati

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.

Definito e Generazione scalabile di cellule epatociti-come da cellule staminali umane pluripotenti

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.

L'isolamento e coltura di cellule primarie dell'epicardio ricavate da campioni di cuore umano adulto e fetale

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.

Un metodo semplificato ed efficiente per isolare i Keratinocytes umani primari dal tessuto pelle adulta

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.

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

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 ...