biopsias de piel de pacientes con psoriasis fueron amablemente proporcionados por el Dr. Andreas Koerber (departamento de Dermatología de la Universidad de Essen, Alemania) tras el consentimiento informado por escrito o por vía oral para uso científico. XH también es apoyado por SNCF 61263039 y 11101321 SNCF.

NOTA: Las biopsias de piel de individuos sanos se obtuvieron a partir de restos de piel abdominal de los individuos sometidos a cirugía plástica electiva tras el consentimiento informado por escrito o por vía oral para uso científico. El uso de piel humana fue aprobado y de acuerdo con las normas establecidas por los comités de ética médica para la investigación humana del centro médico de la Universidad Radboud, Nijmegen, Países Bajos y la Universidad de Essen, Alemania. 1. Preparación de suspensiones de células individuales de la piel humana (Trabajo estéril en un gabinete de flujo si el cultivo celular subsiguiente es obligatorio) <ol><li> Preparar medio de cultivo celular: RPMI 1640 + penicilina / estreptomicina (concentraciones finales de 100 unidades / ml y 100 mg / ml, respectivamente) + piruvato (0,02 mM) y glutamax (0,02 mM), sin suero añadido. </li><li> Para preparar el medio de cultivo completo: medio de cultivo preparado en el paso 1.1 + 10% de suero humano agrupado (HPS); almacenar a 4 ° C. Llevar medio a 20 ° C ± 2 frente a nosotrosEn g. </li><li> Obtener la biopsia de la piel utilizando un instrumento de biopsia por punción redonda de 4 mm y mantenerlo en medio de cultivo completo RPMI 1640 a 20 ° C ± 2 para un máximo de 4 horas oa 4 ° C ON. Procesar la biopsia tan pronto como sea posible a su llegada al laboratorio. NOTA: más largo de almacenamiento de la piel influirá en el rendimiento de células y la viabilidad celular. </li><li> Etiquetar un tubo de disociación con tapa azul y añadir 5 ml de medio de cultivo completo en el tubo etiquetado. </li><li> Añadir 2 ml de medio de cultivo completo en cada pocillo (un total de 3 pocillos) de una placa de cultivo estéril de 6 pocillos. Utilice herramientas estériles para colocar la biopsia en un solo pozo, enjuague, moverlo hacia un segundo pozo y repetir este paso una vez más, logrando así un total de tres enjuagues. </li><li> Transferir la biopsia de la piel bien enjuagado a una placa de Petri estéril, añadir 100 l de medio de cultivo completo en la parte superior de la biopsia, y cuidadosamente raspar el tejido graso subcutáneo utilizando un acero inoxidable desechable bisturí estéril. NOTA: Jes que es un paso crítico. </li><li> Cortar cada biopsia de piel en 4 trozos más pequeños en una placa de Petri estéril. Transferir las muestras (hasta cuatro de las biopsias de 4 mm por tubo) en el tubo de disociación preparado que contiene 5 ml de medio de cultivo completo. </li><li> Cerrar herméticamente los tubos con la tapa y coloque boca abajo a la camisa del disociador automatizado de tejidos. Asegúrese de que todo el material de muestra se encuentra en la zona del rotor. </li><li> Iniciar el proceso de disociación mediante la ejecución del &quot;_01 m_spleen programa&quot; (un programa predefinido proporcionada por la memoria interna del instrumento o de la tarjeta de programa acompañada) para disociar la biopsia a la velocidad de rotación apropiada para 56 seg. </li><li> Después del procesamiento, separar el tubo de la disociación del disociador y asegurarse de que todo el material disociado se recoge en la parte inferior del tubo. </li><li> Añadir 150 l de colagenasa IA (80 mg / ml) en el tubo de disociación e incubar la muestra en un baño de agua con agitación a 37° C durante 60 min. Añadir 100 l de DNasa I (5 MU / ml) en tubo de thedissociation, mezclar bien. NOTA: Mayor concentración de colagenasa o el tiempo de incubación más largo va a alterar la viabilidad celular. </li><li> Una el tubo de disociación para la manga de la disociador tejido automatizado y ejecutar el &quot;m_ programa bazo _01&quot; para disociar la biopsia una vez más. </li><li> Colocar un filtro de células de nylon 70 M en la parte superior de un tubo Falcon 50 ml. Aplicar materiales de muestra disociadas a este filtro de células para eliminar grupos de células / restos de tejido. </li><li> Lavar filtro de células una vez con 5 ml de medio de cultivo completo. Centrifugar a 20 ° C ± 2, 450 xg durante 10 min y el sobrenadante aspirado. </li><li> Repita el paso de lavado una vez más. Volver a suspender los sedimentos celulares en 300 l de medio de cultivo completo. una sola célula suspensiones están listos para su posterior análisis; continuar con protocolos para el cultivo de células ex vivo o citometría de flujo análisis. </li><li> En caso de un nuevo itinción de citocinas ntracellular, estimular las células con PMA (12,5 ng / ml), ionomicina (500 ng / ml) y Brefeldine A (5 g / ml) durante 4 horas a 37 ° C, 5% de CO 2 incubadora durante 4 h antes de la realización de flujo El análisis de citometría. </li></ol> 2. policlonal activación de células T cutáneas Residentes (Ex Vivo Cultura de la célula) <ol><li> Alícuota de 100 l suspensiones unicelulares (preparado en la etapa 1.15) en una placa de fondo redondo de 96 pocillos. NOTA: Para cada uno de 4 mm biopsia de la piel, las suspensiones de una sola célula adquiridos se pueden dividir en al menos dos pocillos de una placa de 96 pocillos. </li><li> Añadir anti-CD3 / anti-CD28 microperlas recubiertas con mAb (25.000 cuentas por pocillo), recombinante humana de citoquinas rIL-2 (concentración final 25 U / ml) y rIL-1ß (concentración final 50 ng / ml). </li><li> Cubrir la placa de cultivo con una tapa de placa de marcado, se incuban en 5% de CO 2, el 100% de humedad, 37 ° C incubadora. Cambie medio cuando el color medio se pone en amarillo. <br/&gt; NOTA: Una colonia de células claras se puede observar a partir de día 8 de cultivo celular. </li></ol> 3. Análisis de citometría de flujo de células de la piel primaria / Residentes T cultivadas <ol><li> Preparar tampón FACS: PBS + BSA al 0,2%. </li><li> Transferencia de células de interés en una placa de 96 pocillos de fondo en v. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Resuspender el precipitado en 100 l de PBS. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Las células se tiñen con 100 l de eFluorescence780 preparados conjugados colorante de viabilidad corregible (1: 1000 dilución utilizando PBS) a 4 ° C durante 30 min. Añadir 100 l de tampón de FACS, se centrifuga a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Seleccionar mAbs requeridos marcador de superficie celular, por ejemplo: CD45-BV421 (HI30, 01:20), CD3-ECD (UCHT1, 1:50), CD4-PC5.5 (1388,2, 1: 200), CD8-APC-AlexFluo700 (B9.11, 1: 400), CD14-FITC (TUK4, 01:50), CD19-APC-AlexFluo750 (13-119, 1:50), CD56-PE (MEM-188, 1:50), CD25-PeCy7 (FC96, 1:50), CD11c-PeCy7 (BU15, 1:10), y CD1c-APC (AD5-8E7, 01:10), preparar los mAbs mezcla usando tampón de FACS en función de cada factor de dilución mAb probado. Definir ajustes de la compuerta utilizando control de isotipo de anticuerpo junto con la muestra no mancha. </li><li> Añadir 25 l de preparado mAbs-mezcla en cada pocillo. Incubar 20 minutos a 20 ° C ± 2, protegerlo de la luz. </li><li> Añadir 100 l de tampón de FACS, se centrifuga a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Para las muestras que sólo requieren tinción de la superficie celular, continúe con el paso 3.16. </li><li> En el caso de la tinción de Foxp3 intra-celular: <ol><li> Preparar la fijación y permeabilización de amortiguación mediante la mezcla de una parte de concentrado con 3 partes de diluyentes. </li><li> Preparar tampón de permeabilización 1x: 1 parte de tampón de permeabilización 10x + 9 partes de H 2 O. esterilizada </li></ol></li><li> pellets de resuspender en 100 l de fijación y permeabilición tampón, mezclar bien e incubar a 4 ° C durante 30 min. </li><li> Añadir 100 l de tampón de permeabilización a cada pocillo, se centrifuga a 450 xg a TA durante 2 min, y el sobrenadante aspirado. </li><li> Lavar las células con tampón de permeabilización una vez más. </li><li> Seleccionar mAbs intracelulares necesarias, por ejemplo, Foxp3-eFluo450 (PCH101, 01:50), IL-17A-AlexFluo488 (eBio64DEC1, 01:50) y IFN-PeCy7 (4S.B3, 1: 400). Preparar la mezcla de anticuerpos monoclonales usando tampón de permeabilización que contenía suero de rata normal 2% de acuerdo a cada factor de dilución del AcMo probado. Definir ajustes de la compuerta utilizando control de isotipo de anticuerpo junto con la muestra no mancha. </li><li> Añadir 20 l de preparado mAb-mezcla en cada pocillo. Incubar a 4 ° C durante 30 minutos, protegerlo de la luz. </li><li> Después de 30 min de incubación, añadir 100 l de tampón de permeabilización en cada pocillo. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Lavar las células con buffe permeabilizaciónr una vez más. sedimento se vuelve a suspender en 110 l de tampón FACS, y las suspensiones de células en un tubo de transferencia de microFACS. NOTA: La muestra está lista para medición mediante citometría de flujo 10-color. </li><li> En el caso del número de células exacta requerida, añadir 10 l de suspensión uniforme bien mezclada de fluoroesferas en cada muestra inmediatamente antes de realizar las mediciones de citometría de flujo. </li></ol>

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The authors have nothing to disclose.

A continuación, se presenta un protocolo para aislar eficazmente las células T residentes de la piel a partir de biopsias de piel humana. La ventaja de este protocolo es el aislamiento de un número relativamente alto de linfocitos viables, y la expresión de marcadores de superficie correspondientes. Los subconjuntos de células identificadas fueron:, células CD4 + y linfocitos T CD8 + y CD25 + Foxp3 + CD11c + DCS. Es importante destacar que la cultura ex vi...

La piel, como la interfaz principal entre el cuerpo y el medio ambiente, proporciona la primera línea de defensa contra la física externa, químicas y biológicas insultos tales como heridas, la radiación ultravioleta y los microorganismos. Piel comprende dos compartimientos principales, la epidermis y la dermis, y contiene una variedad de células inmunes incluyendo las células de Langerhans, macrófagos, células dendríticas (DC), y cerca de 20 millones de células T de memoria, casi dos veces el número presente en todo el volumen de sangre 1 , 2. Una creciente cantidad de datos apoyan la noción de que la piel tiene funciones inmunológicas esenciales, tanto durante la homeostasis del tejido y en diversas condiciones patológicas. Se cree que las células inmunes residentes en la piel normal para llevar a cabo la inmunovigilancia 3 y se ha demostrado que desempeñan un papel en el desarrollo de trastornos inflamatorios tales como psoriasis 4. En la piel lesional psoriásica, tanto CD4 + y CD8 + se infiltraron las células T eran observed y se demostró que la relación de la CD4 y CD8 varía en función del estado de la enfermedad 5. Sin embargo, estas poblaciones de células son difíciles de estudiar porque las técnicas actuales permiten el aislamiento de sólo unas pocas células. Las técnicas actualmente ampliamente utilizados para el aislamiento de células T de la piel humana se combinan disociación mecánica de la piel con tratamiento enzimático. Las biopsias de piel humana son ampliamente picada y se incubaron con enzimas como la tripsina, colagenasa y / o EDTA 6-8. Teniendo en cuenta que la piel es un tejido de barrera que es altamente resistente a las fuerzas de tracción y desagregación mecánica, los métodos establecidos de aislamiento de células T producen muy pocas células, e incluso un menor número de células viables, lo que hace ex vivo de cultivo de células de estas poblaciones de células difícil y desafiante. Aquí, se presenta un método modificado para aislar los linfocitos de la piel humana psoriásica saludable y participar mediante la combinación de mechandisociación ical de la piel usando un disociador de tejido automatizado en lugar del método establecido de picado ampliamente, junto con la digestión enzimática usando colagenasa. Se observaron varios subconjuntos de células inmunes viables, incluyendo países en desarrollo y las células T después de la preparación de una suspensión de una sola célula. Es importante destacar que la expresión de los marcadores de superficie CD3, CD4 y CD8 se conservó bien. Las células preparadas de este modo, están listos para su uso en cultivos de células ex vivo o análisis de citometría de flujo. Este protocolo se ha empleado con éxito para el análisis de biopsias de piel individuales (4 mm) derivadas de la piel lesional de pacientes con psoriasis. Los resultados mostraron que las células T de la piel del paciente residente producen citoquinas inflamatorias como la más IL-17 y IFN en comparación con voluntarios sanos 9.

<table><tbody><tr><td>Disposable Biopsy Punch</td><td>Kai Europe</td><td>BP-40F</td><td>4 mm</td></tr><tr><td>Disposable sterile scalpels</td><td>Dalhausen</td><td>1100000510</td><td></td></tr><tr><td>gentleMACS C tube</td><td>Miltenyi Biotech</td><td>130-093-237</td><td>Blue-capped, used as the dissociation tube.</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Automated tissue dissociator. Using the &quot;program spleen_01&quot; for dissociation of the skin biopsy.</td></tr><tr><td>Cell strainer&amp;nbsp;</td><td>BD</td><td>352350</td><td>70 &amp;micro;m nylon</td></tr><tr><td>96-well U-bottom plate</td><td>Greiner Bio-One</td><td>650180</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>22409-015</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Human Pooled Serum (HPS)</td><td>in house prepared</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Sigma-Aldrich</td><td>C2674</td><td>Type 1-A, suitable for cell culture</td></tr><tr><td>DNase I</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>PBS</td><td>B Braun</td><td>3623140</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503-500G</td><td></td></tr><tr><td>Fixable Viability Dye (FVD) APC-eFluo780</td><td>eBioscience</td><td>65-0865-18</td><td>Stain dead cells prior to cell fixation; dilute with PBS at 1:1,000</td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td></td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Permeabilization Buffer (10x)</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>BV421 Mouse anti-human CD45</td><td>BD</td><td>563879</td><td>Clone: HI30; dilution factor 1:50</td></tr><tr><td>FITC Mouse anti-human CD14</td><td>Dako</td><td>T0844</td><td>Clone: TUK4; dilution factor 1:50</td></tr><tr><td>PE Mouse anti-human CD56</td><td>Dako</td><td>R7127</td><td>Clone: MOC-1; dilution factor 1:50</td></tr><tr><td>ECD Mouse anti-human CD3</td><td>Beckman - Coulter</td><td>A07748</td><td>Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td></tr><tr><td>PC5.5 Mouse anti-human CD4</td><td>Beckman - Coulter</td><td>B16491</td><td>Clone: 13B8.2; dilution factor 1:200</td></tr><tr><td>PeCy7 Mouse anti-human CD11c</td><td>Beckman - Coulter</td><td>A80249</td><td>Clone: BU15; dilution factor 1:50</td></tr><tr><td>APC Mouse anti-human CD1c</td><td>Miltenyi Biotech</td><td>130-090-903</td><td>Clone: AD5-8E7; dilution factor 1:10</td></tr><tr><td>APC-AlexFluo700 Mouse anti-human CD8</td><td>Beckman - Coulter</td><td>A66332</td><td>Clone: B9.11; dilution factor 1:400</td></tr><tr><td>APC-AlexFluo750 Mouse anti-human CD19</td><td>Beckman - Coulter</td><td>A94681</td><td>Clone: J3-119; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human CD25</td><td>eBioscience</td><td>AD5-8E7</td><td>Clone: BC96; dilution factor 1:50</td></tr><tr><td>&amp;nbsp;PE Rat anti-human CLA</td><td>Miltenyi Biotech</td><td>130-091-635</td><td>clone: HECA-452; dilution factor 1:</td></tr><tr><td>eFluo450 Rat anti-human Foxp3</td><td>eBIoscience</td><td>48-4776-42</td><td>Clone: PCH101; intracellular staining; dilution factor 1:50</td></tr><tr><td>AlexFluo488 Mouse anti-human IL-17A</td><td>eBioscience</td><td>53-7179-42</td><td>Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human IFNg</td><td>eBioscience</td><td>25-7319-41</td><td>Clone: 4S.B3; intracellular staining; dilution factor 1:400</td></tr><tr><td>Flow-Count Fluorospheres</td><td>Beckman - Coulter</td><td>7547053</td><td>Counting beads, for flow cytometry</td></tr></tbody></table>

lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture

La piel humana tiene una función de barrera importante y contiene diversas células inmunes que contribuyen a la homeostasis del tejido y la protección contra los agentes patógenos. A medida que la piel es relativamente fácil acceso, que proporciona una plataforma ideal para estudiar los mecanismos de regulación inmune periférica. células residentes inmunes en sano conducta piel inmunovigilancia, pero también juegan un papel importante en el desarrollo de trastornos inflamatorios de la piel, tales como psoriasis. A pesar de ideas emergentes, nuestra comprensión de la biología subyacente de diversas enfermedades inflamatorias de la piel es aún limitada. Hay una necesidad de una buena calidad de las poblaciones (individual) de células aisladas a partir de muestras de piel biopsia. Hasta ahora, los procedimientos de aislamiento han sido seriamente obstaculizados por la falta de la obtención de un número suficiente de células viables. El aislamiento y el análisis posterior también se han visto afectados por la pérdida de marcadores de linaje de células inmunes, debido a la tensión mecánica y química causada por los procedimientos de disociación actuales para obtenersuspensión de células individuales. A continuación, describimos un método modificado para aislar las células T de la piel humana psoriásica saludable y participar mediante la combinación de disociación mecánica de la piel utilizando un disociador automatizado de tejidos y el tratamiento de colagenasa. Esta metodología conserva la expresión de la mayoría de los marcadores de linaje inmunes tales como CD4, CD8, Foxp3 y CD11c en la preparación de suspensiones de células individuales. Se muestran ejemplos de éxito de aislamiento de células T CD4 + y la posterior fenotípica y análisis funcional.

El protocolo que aquí se presenta producirá entre 2.200 ± 615 (media ± SEM, piel de voluntarios sanos) hasta 178.000 ± 760 (media ± SEM, piel lesionada de pacientes con psoriasis) linfocitos viables de la piel humana cuando se utiliza una sola biopsia de piel de 4 mm. Se identificaron diferentes tipos de células CD45 + en una sola célula suspensiones derivadas de la piel de individuos sanos, incluyendo las células T CD4 + (~ 45%), las células T CD8 +</sup...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

14.5K vistas.  Radboud University Medical Centre. We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Video: Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Investigación

Inmunología e Infección

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Aislamiento de fibroblastos papilares y reticulares de la piel humana mediante la clasificación celular activada por fluorescencia

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genética

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Aislamiento de linfocitos de ratón del tracto genital Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Medicina

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

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Aislar los linfocitos desde el sistema de inmune Intestinal pequeño ratón

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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

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

Johansen, C. et al. <a href="https://jove.unicartagenaproxy.elogim.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 ...

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica

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Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro