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>

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	<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>

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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><tbody><tr><td>KSFM</td><td>ThermoFisher Scientific</td><td>17005-034</td><td>Cell culture medium</td></tr><tr><td>KSFM supplements</td><td>ThermoFisher Scientific</td><td>37000-015</td><td>Supplements for KSFM</td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14190-144</td><td>DPBS without Calcium and Magnesium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>Dimethyl sulfoxid</td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15710-049</td><td>Cell culture medium additive</td></tr><tr><td>Sterilization filter</td><td>Sartorius</td><td>16534</td><td>Syringe filter with a pore size of 0.2 &amp;micro;m</td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T7409</td><td>Used to trypsinize cells</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>-</td></tr><tr><td>RPMI-1640</td><td>ThermoFisher Scientific</td><td>61870-010</td><td>-</td></tr><tr><td>FBS</td><td>ThermoFisher Scientific</td><td>16000044</td><td>Used to inactivate trypsin</td></tr><tr><td>Forceps</td><td>-</td><td>-</td><td>Forceps from any company can be used</td></tr><tr><td>Scissors</td><td>-</td><td>-</td><td>Scissors from any company can be used</td></tr><tr><td>Scalpel</td><td>Swann Morton</td><td>0501</td><td>Scalpels from any company can be used</td></tr><tr><td>70% ethanol</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Gauze pads</td><td>NOBAMED</td><td>875420</td><td>Gauze pads from any provider can be used</td></tr><tr><td>Foot planer</td><td>Credo Solingen</td><td>1510</td><td>Foot planer from any provider can be used</td></tr><tr><td>Petri dishes</td><td>TPP</td><td>93100</td><td>Petri dishes from any provider can be used</td></tr><tr><td>Metal filter</td><td>-</td><td>-</td><td>In-house 1 mm hole size metal filter</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>NUNC</td><td>156499</td><td>-</td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>TTP</td><td>90151</td><td>-</td></tr><tr><td>0.05% Trypsin-EDTA solution</td><td>ThermoFisher Scientific</td><td>25300-062</td><td>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. ...

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16.3K 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

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 ...

Ricerca

Biologia dello sviluppo

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 ...

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

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

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

Creazione di un sistema di coltura sferoide epidermica ad alto rendimento per modellare la plasticità delle cellule staminali dei cheratinociti

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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

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

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

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

Biologia

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

Coltivare un'epidermide umana ricostruita tridimensionale su larga scala

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Bioingegneria

Generating Primary Cultures for Keratinocyte Live Cell Imaging

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

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

Generazione di colture primarie per l'imaging di cellule vive di cheratinociti

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

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Isolamento, coltura e caratterizzazione di cellule primarie di Schwann, cheratinociti e fibroblasti dal prepuzio umano

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Neuroscienze

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

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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

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

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

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

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

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

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

Isolamento dei cheratinociti della pelle umana: un metodo per coltivare i cheratinociti primari da campioni di pelle

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