El autor desea agradecer a Annette Blak Rasmussen y Kristine Moeller por su soporte técnico

La recogida de muestras de piel de voluntarios sanos adultos sometidos a cirugía plástica requiere la aprobación del Comité de ética en las instituciones de acogida. Este protocolo fue aprobado por la Regional ética Comité de región Midtjylland, Dinamarca (M-20110027). El método aquí descrito se deriva de estudios similares por Maciaq et al. 10 y11del Liu y Karasek.
1. aislamiento de queratinocitos de la piel humana
<ol>
	<li>Empezar haciendo las siguientes soluciones: solución de 50 mL de 0.25% tripsina y 0.1% glucosa en DPBS. Mix y el filtro de esterilización (0,2 μm) la solución. Preparar 10 mL de RPMI 1640 + 2% solución FBS, 50 mL de medio sin suero DPBS y queratinocitos (KSFM). Añadir suplementos KSFM y 250 μl de gentamicina a 500 mL de KSFM. Caliente previamente la solución a 37 ° C antes de usar.</li>
	<li>Recoger la piel de voluntarios sanos adultos sometidos a cirugía plástica. Las muestras de piel utilizadas son aproximadamente 10 cm x 15 cm, pero pueden variar de tamaño. Enfriar la piel bajo transporte de transporte de las muestras de piel en una caja de espuma de poliestireno que contienen un elemento de enfriamiento. Si es necesario, la muestra de piel puede almacenarse a 4 ° C durante la noche.</li>
	<li>Quitar la grasa de debajo de la sección de la piel con pinzas, bisturís y tijeras esterilizadas.</li>
	<li>La hebilla de la sección de la piel (aproximadamente 10 cm x 15 cm) en una cubierta estéril sobre una placa con agujas. Primero, limpie la piel con una gasa esterilizada seca. Después, limpie la piel con una gasa esterilizada con etanol al 70%.</li>
	<li>Usando un cepillo de pies, corte de la capa superior (epidermis) de la sección de la piel y ponerla en un plato de Petri de 9 cm. Entonces, inmediatamente agregar 25 mL de la solución de tripsina/DPBS/glucosa (preparada en el paso 1.1) a la caja Petri. Incubar durante 30 min a 37 ° C.</li>
	<li>Usando una pipeta retirar la solución de tripsina/glucosa/DPBS de la caja Petri y añadir 10 mL de RPMI 1640 + 2% FBS para inactivar la tripsina.</li>
	<li>Utilizando dos pinzas, ahora liberan las células epidérmicas en el medio suavemente raspando y agitar el epidérmico y el compartimiento cutáneo de las secciones de la piel.</li>
	<li>Filtrar la suspensión de células epidérmicas a través de un filtro de metal (tamaño de orificio de 1 mm) y recoger en un tubo de 50 mL. Añadir las secciones restantes de la piel a un tubo de 50 mL que contiene 10 mL de RPMI-1640 sin FBS y vortex para filtro de 10 s. la suspensión a través del metal del filtro en un tubo de 50 mL que contiene la suspensión de células epidérmicas. Añadir DPBS hasta un volumen total de 50 mL.</li>
	<li>Centrifugue la suspensión celular por 10 min a 450 x g a temperatura ambiente.</li>
	<li>Quite el sobrenadante y resuspender el precipitado de células epidérmicas en aproximadamente 10 mL de 37 ° C KSFM dependiendo del tamaño de la pelotilla de la célula. Contar las células con el azul de tripano tinción bajo microscopio. El número de células obtenidas de una sección de piel de 10 cm x 15 cm es aproximadamente 50-100 x 106 células. A cada frasco de cultivo de 75 cm2 , 8 x 106 células junto con 12 mL de 37 ° C KSFM de transferencia. Sacuda suavemente los frascos de cultivo para asegurar una distribución uniforme de las células.</li>
	<li>Incubar los queratinocitos en una incubadora de 37 ° C con 100% humedad y 5% CO2. Cambiar el medio después de 2 días y tres veces semanalmente. Células de paso cuando la cultura es 70-80% confluente, que tarda aproximadamente 2 semanas dependiendo de la tasa de proliferación de los queratinocitos.</li>
</ol>
2. pases de queratinocitos
<ol>
	<li>Calentar la cantidad apropiada de solución de tripsina-EDTA 0,05% a 37 ° C en una incubadora. 4,5 mL de solución al 0.05% tripsina-EDTA se utiliza para un frasco de cultivo de 75 cm2 .</li>
	<li>Retire el medio de las células y añadir frasco de 4,5 mL/75 cm2 cultura previamente calentado 0.05% solución de tripsina-EDTA a las células. Coloque el frasco de cultivo en la incubadora (37 ° C) y después de aproximadamente 5 minutos, comprobar al microscopio si las células han comenzado a aflojar.</li>
	<li>Cuando aproximadamente el 50% de las células se hayan aflojado, golpear suavemente el frasco de cultura contra la mano para aflojar las células restantes. Luego, para inactivar la tripsina, añadir 6 mL de RPMI 1640 + 2% FBS (37 ° C) al matraz de cultivo 75 cm2 y transferir la suspensión a tubos de 50 mL.</li>
	<li>Enjuague los frascos de cultivo con 3 mL de RPMI 1640 + 2% FBS y añadir a los tubos de 50 mL. Centrifugar las células durante 10 min a 450 x g a temperatura ambiente.
	<ol>
		<li>Resuspender las células concentradas en 10 mL de KSFM (37 ° C). Contar las células y la transferencia de 3 x 106 células a un matraz de cultivo de 150 cm2 con 20 mL de KSFM (37 ° C). Agite suavemente el frasco de cultivo para asegurar una distribución uniforme de las células. Congelar las células cuando la cultura es 80-90% confluente, que toma aproximadamente 4 a 6 días dependiendo de la tasa de proliferación de los queratinocitos (ver sección 3, a continuación del Protocolo de congelación). Ampliar los queratinocitos para generar adecuadas acciones congeladas.</li>
	</ol>
	</li>
</ol>
3. congelación de los queratinocitos
<ol>
	<li>Calentar la cantidad apropiada de solución de tripsina-EDTA 0,05% a 37 ° C en una incubadora. 9 mL de solución al 0.05% tripsina-EDTA se utiliza para un frasco de cultivo de 150 cm2 .</li>
	<li>Retire el medio de las células y añadir frasco de 9 mL/150 cm2 cultura previamente calentado 0.05% solución de tripsina-EDTA a las células. Coloque el frasco de cultivo en la incubadora (37 ° C) y después de aproximadamente 5 minutos, comprobar al microscopio si las células han comenzado a aflojar.</li>
	<li>Cuando aproximadamente el 50% de las células se hayan aflojado, golpear suavemente el frasco de cultura contra la mano para aflojar las células restantes. Entonces, para inactivar la tripsina, añadir 12 mL de RPMI 1640 + 2% FBS (37 ° C) en el matraz de 150 cm2 cultura y aspirar la suspensión celular en tubos de 50 mL.</li>
	<li>Enjuague los frascos de cultivo con 6 mL de RPMI 1640 + 2% FBS y añadir a los tubos de 50 mL. Centrifugar los tubos durante 10 min a 450 x g a 4 ° C. Preparan celda helada congelación medio (KSFM + 10% DMSO).</li>
	<li>Resuspender el precipitado de células en KSFM + 10% DMSO, contar las células y congelar las células a una densidad de 6 x 106 células/mL con estándar de métodos de criopreservación congelación lenta12.</li>
	<li>Almacenar los keratinocytes en nitrógeno líquido (fase vapor) hasta que esté listo para usar.</li>
</ol>
4. descongelación y cultivo de queratinocitos congelados
<ol>
	<li>Rápidamente descongelar los viales congelados apropiados en un baño de agua de 37 ° C. Utilizar el etanol del 70% para limpiar el exterior de la cryovial. Transferir el número apropiado de células (aproximadamente 15.000 células/cm2) a un matraz de cultivo y añadir medio precalentado (KSFM) al matraz de cultivo. Cualquier tamaño de frascos de cultivo se puede utilizar dependiendo de la configuración del experimento.</li>
	<li>Utilizar medio de cultivo 5 mL para un frasco de cultivo de 25 cm2 .</li></ol>El número de células ha añadido que al matraz de cultivo puede variar ya que el crecimiento de las células varía de un donante a donante. Incubar las células en una incubadora de 37 ° C con 100% humedad y 5% CO2.
	<li>Cambio el medio después de 2 días y tres veces semanalmente usando fresco KSFM precalentado. Nota: Los datos han demostrado que en promedio el 95% de las células aisladas por el protocolo descrito aquí son positivo para el marcador del keratinocyte citoqueratina 1413.</li>

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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. 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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. 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. El autor no tiene conflicto de interés.

Aquí, describimos cómo fácilmente aislar queratinocitos humano primario de piel adulto y su cultivo en vitro. Este modelo puede tener un amplio uso para la investigación de Biología de la célula epidérmica y puede ser útil para los investigadores interesados en el estudio de enfermedades cutáneas.
Algunas de las ventajas del protocolo descrito aquí es que en contraste con queratinocitos de prepucio neonatal de los varones recién nacidos sometidos a la circuncisión, keratin...

La piel es el órgano más grande del cuerpo humano y sirve como una barrera protectora contra el medio ambiente externo. La piel se compone de dos capas principales: la dermis y la epidermis, donde la epidermis constituye la capa más externa de la piel. El tipo de célula más abundante en la epidermis es keratinocytes que comprende más del 95% de la masa1,de célula2. Los queratinocitos se mantienen en distintas etapas de diferenciación de la epidermis y se organizan en estratos basals, espinosas, granuloso y cornified que corresponden a etapas específicas de diferenciación3. La función principal de los queratinocitos es proporcionar la integridad estructural de la epidermis, produciendo una barrera intacta al mundo exterior.
Los queratinocitos también representan la primera línea de defensa contra patógenos en la piel y por lo tanto juegan un papel importante en la respuesta inmune innata4,5. Exposición de los queratinocitos a los estímulos externos conduce a la activación de vías de señalización intracelular y posteriormente, producción de un número de varios mediadores inflamatorios como citoquinas, quimiocinas y péptidos antimicrobianos. Estas proteínas derivados de queratinocitos participan en la respuesta inflamatoria por reclutar y activar las células inmunes tales como células dendríticas, neutrófilos y específicos6,de las células de T7. Así, porque los queratinocitos desempeñan un papel crucial en numerosos procesos biológicos, el fundamento de la técnica presentada aquí fue generar un modelo en vitro para estudiar la biología de la piel. Cultivos de queratinocitos primarios obtenidos de prepucio neonatal se utilizan a menudo para estudiar la piel biología8,9. Sin embargo, con la técnica descrita aquí, queratinocitos de ambos sexos se obtienen dando por resultado una mayor diversidad biológica de las células.
Aquí, presentamos un protocolo detallado para el aislamiento y la generación de keratinocytes humanos primarios de piel de adultos, incluyendo el mantenimiento y la congelación de los queratinocitos. El objetivo general de este método es generar keratinocytes humanos primarios que pueden utilizarse como modelo para estudiar biología cutánea 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 función principal de los queratinocitos es proporcionar la integridad estructural de la epidermis, manteniendo una barrera mecánica al mundo exterior. Además, queratinocitos juegan un papel esencial en la iniciación, mantenimiento y regulación de la respuesta inmune epidérmica por ser parte del sistema inmune innato responde a los estímulos antigénicos de una manera rápida y no específica. Aquí, describimos un protocolo para el aislamiento de keratinocytes humanos primarios de la piel adulta y demostrar que estas células responden a la diferenciación terminal inducida por calcio, medida por un aumento de expresión de la diferenciación marcador involucrin. Además, mostramos que los queratinocitos aislados responden a la activación de vías de señalización intracelular inducida por IL-1β como es medido por la activación de la vía de p38 MAPK. Tomados en conjunto, se describe un método para el aislamiento y cultivo de keratinocytes humanos primarios de la piel adulta. Porque los queratinocitos son el tipo celular predominante en la epidermis, este método es útil para el estudio de mecanismos moleculares en biología cutánea in vitro.

Diferenciación Terminal inducida por calcio Keratinocytes humanos experimentan diferenciación terminal sobre el tratamiento con calcio14,15,16. Queratinocitos humanos primarios fueron aisladas y cultivadas como se describe en el protocolo anterior. Cuando aproximadamente 50-60% de confluencia, las células fueron estimuladas con calcio (1,2 mM) o vehículo y fotografías de las células fueron tomadas en el...

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 piel humana actúa como primera línea de defensa contra el medio ambiente externo. Presentamos un método para aislar queratinocitos humano primario de piel adulto. Estos queratinocitos aislados son útiles en numerosas configuraciones experimentales y son un modelo muy adecuado para el estudio de mecanismos moleculares en biología cutánea en vitro.

Generación y cultivo de Keratinocytes humanos primarios de piel adulto

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 vistas.  Aarhus University Hospital. La piel humana actúa como primera línea de defensa contra el medio ambiente externo. Presentamos un método para aislar queratinocitos humano primario de piel adulto. Estos queratinocitos aislados son útiles en numerosas configuraciones experimentales y son un modelo muy adecuado para el estudio de mecanismos moleculares en biología cutánea en 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.

Generación de Genéticamente Modificados cultivos de piel organotípicos Uso Desvitalizada Dermis Humana

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

Investigación

Biología del Desarrollo

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 método simplificado y eficiente para aislar Keratinocytes humanos primarios de tejido adulto de la piel

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.

Establecimiento de un sistema de cultivo de esferoides epidérmicos de alto rendimiento para modelar la plasticidad de las células madre de queratinocitos

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.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

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

Biología

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.

El cultivo de una epidermis humana reconstruida tridimensional a gran escala

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

Bioingeniería

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.

Generación de cultivos primarios para la obtención de imágenes de células vivas de queratinocitos

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.

Aislamiento, cultivo y caracterización de células primarias de Schwann, queratinocitos y fibroblastos del prepucio humano

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

Neurociencias

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.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

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.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón 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.

Aislamiento de queratinocitos de piel humana: un método para cultivar queratinocitos primarios a partir de muestras de piel

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