Los autores agradecen a Katharina Schallmoser para proporcionar pHPL, Rohde y Eva Nicole Hofmann por sus valiosos comentarios al manuscrito, Daniela P. Thaler y Margaretha hwirth de asistencia técnica, Mónica Farrell para la edición de lingüística, Uwe Lang, Holzapfel Margit y Sandra Eppich al Departamento de Obstetricia para proporcionar las cuerdas. Este trabajo ha sido financiado por el Austrian Research Foundation (FMF, la concesión N211-NAN para DS), la Agencia de Promoción de la Investigación de Austria (FFG, otorgar N200 para DS) y por el adulto de células madre de Investigación (AR). AR es un compañero de la medicina molecular programa de doctorado de la Universidad Médica de Graz.

Parte 1: Configuración 1. Preparación del medio de cultivo celular Antes de iniciar la preparación a medio recoger todos los materiales y las herramientas que usted necesita. Descongele 2 x 56 ml alícuotas de lisado de plaquetas humanas agrupados (pHPL, hecho a sí mismo: una referencia y JoVe # 1523), 10 ml de penicilina 100x / solución de estreptomicina, 2 x 5 ml de L-glutamina (Sigma dos). Descongele las citoquinas y las alícuotas del factor de crecimiento (VEGF, bFGF, EGF, IGF, hidrocortisona, ácido ascórbico, la heparina, la anfotericina siempre como &quot;quots único&quot;), ajustado para complementar una botella (500 ml) de medio EGM-2 (Lonza). De la nevera tomar una botella de 500 ml cada uno de (i) la alfa-modificado medio mínimo esencial (a-MEM) y (ii) el medio basal endotelial (EBM). Todos los pasos siguientes se llevan a cabo en una campana de flujo laminar de cultivo de tejidos en condiciones estériles. Prepare la heparina sin conservantes (por ejemplo, Biochrom) disolviendo el polvo a una concentración final de 1000 UI / ml con agua estéril. MSC-Media: Utilizar 500 ml de una MEM, añadir 56 ml de descongelado pHPL (véase también la referencia 1 para más detalles) e IU 2 / ml (= l de solución madre 224) de la heparina sin conservantes (evita la coagulación del medio a través de la formación de grumos el fibrinógeno en el plasma) para alcanzar una concentración final del 10% pHPL. Además añade la penicilina (100U/mL) / estreptomicina (100μg/mL) y la solución 2 mM de L-glutamina (Sigma ambos). Filtrar el medio a través de 20 micras de poro del filtro de vacío de tamaño (Millipore). Etiqueta de la botella adecuadamente (contenido, la fecha). ECFC-Media: Utilice una botella (500 ml) de la MBE, añadir las alícuotas de citoquinas, 56 ml de pHPL, 10 UI / ml (= 1120μl de la solución madre) sin conservantes heparina, penicilina (100U/mL) / estreptomicina (100 g / ml) y 2 mM de L-glutamina en el medio basal y el filtro con 20 l-de tamaño de poro del filtro de vacío (Millipore). Etiqueta de la botella adecuadamente (contenido, la fecha). 2. La esterilización de instrumentos quirúrgicos Pinzas, tijeras afiladas quirúrgica y titular de bisturí debe ser el calor esterilizados desechables mineral estéril. 3. Preparación de los tubos de recogida de cable Añadir 500 UI de conservantes - heparina libre de 50 tubos de polipropileno ml (Falcon) y adaptarse a un volumen final de 20 ml con PBS. La transferencia de los tubos en la sala de partos. 4. El consentimiento informado (confirmado por el comité de ética local o IRB). Pregunte a los padres antes de la entrega si desea donar un pedazo de la cuerda y les informará adecuadamente de los efectos que va a servir. 5. Cable de donación Después de la entrega y la inspección de la placenta y el cordón cortado un pedazo de 10 cm y de inmediato se traslado al tubo precargada con heparina para evitar la coagulación de la sangre que queda dentro de los vasos del cordón umbilical. Continuar con el aislamiento de células tan pronto como sea posible. Parte 2: el aislamiento de células <ol><li> Prepare campana de flujo laminar el tejido cultural de la limpieza de superficies con INDICIN o 70% EtOH. Lugar estéril de 150 cm 2 placas de cultivo celular, 75 cm frascos de cultivo celular (tanto Corning), PBS, se esterilizan los instrumentos quirúrgicos, raspadores de células, soporte de tubo de 25 ml stripettes y jeringas de 5 ml equipado con agujas de punta roma adecuadamente en su campana limpia. </li><li> Pre-caliente el preparado α-MEM y el EGM-2 medio de cultivo celular de 37 ° C en un baño de agua. </li><li> Traiga la cuerda de la sala de partos para su laboratorio equipado con la capucha de cultivo de células. Después de transferir el cable en el flujo laminar que lavar dos veces en PBS (por transferencia a una nueva placa) para deshacerse de la contaminación de las células sanguíneas. Enjuague la vena umbilical después del canulating que por un lado con una jeringa de 5 ml estéril equipado con una aguja de punta roma con PBS hasta que el líquido que sale del otro extremo del cable está completamente transparente (rojo indica contaminación de la sangre). </li></ol> ECFCs: <ol start="4"><li> Prepare un frasco de 75 cm2 (Corning), se etiqueta y rellenar 15-20 ml de pre-calentado medio EGM-2. </li><li> Coloque el cable en una nueva placa llena de PBS estéril. Tome las tijeras quirúrgicas y cortar el cordón umbilical en dos trozos de unos 5 cm de longitud (piezas cortas son más fáciles de procesar, porque los vasos umbilicales se retuercen a lo largo de su eje). </li><li> Trate de encontrar la vena umbilical (vaso grande), insertar una hoja de las tijeras quirúrgicas y cortar la vena longitudinal. En este punto podría ser útil que un ayudante puede mantener la vena ya cortado con dos pinzas, mientras que reducir aún más. </li><li> Coloque el pastel de la médulace con el corte de vena en una nueva placa sin PBS, tomar el rascador de células y roce de la parte interna (endotelio) de superficie de la vena umbilical por frotamiento de una punta a la otra. Transferencia de las células de su frasco preparado por la limpieza de la punta de la espátula en el medio. Repita este procedimiento por lo menos 10 veces. </li><li> Cerca de su frasco y ponerlo en una incubadora (37 ° C, 5% CO2, 95% de humedad). </li></ol> MSC: <ol start="4"><li> Preparar a 150 cm 2 placas de cultivo (Corning) y la etiqueta de la misma. </li><li> Tome el trozo de cordón (después de extraer la capa endotelial) y cortar el cordón todo en trozos muy pequeños mediante el uso de tijeras estériles y bisturí estéril. El tamaño máximo de las piezas de cable debe ser de 1-2 mm. </li><li> La transferencia de sus piezas cable con la pinza estéril en su placa de cultivo de pre-etiquetados. Al dejar abierta la placa durante 5 minutos antes de llenar con el medio, la fijación de las piezas a la superficie de plástico se fortalece. </li><li> Una vez que las piezas de cable están conectados adecuadamente a las de plástico, añadir con cuidado en 30-35 ml de pre-calentado alfa modificado MEM. Trate de usar la velocidad más baja para asegurarse de que todas las piezas de permanecer unidos. </li><li> Cerrar la placa y ponerla en una incubadora (37 ° C, 5% CO2, 95% de humedad). </li></ol> Parte 3: la expansión celular y la confirmación de la inmuno-fenotipo por citometría de flujo La expansión de células EFCFs: <ol><li> Compruebe la conexión de las células endoteliales al día siguiente en un microscopio invertido y el intercambio de toda EGM-2 medio para deshacerse de todas las células no inscritos y las partículas. </li><li> Expandir las células hasta que un paso, de intercambio tercio de la media dos veces por semana. Cuando se llega a la confluencia, separar las células con el 0,05% Trypsin/0.7 mM EDTA y transferirlos a los buques de nueva cultura. Dependiendo del número de células dentro de su frasco T75, usted debe elegir el tamaño y el número de sus nuevos frascos para garantizar la densidad de siembra de baja para una mayor expansión. </li><li> Cambio de un tercio de la media dos veces por semana durante la expansión de las células. </li></ol> MSC: <ol><li> El resultado de las MSC del cordón se tomará más tiempo, así que usted puede esperar por lo menos 3-4 días hasta que compruebe en un microscopio invertido para la aparición de células fusiformes en los alrededores de las piezas de tejidos conectados. Cuando no hay suficientes células de crecimiento, las piezas tienden a separarse debido a que pierden contacto con el plástico. </li><li> Después de 10-12 días retirar las piezas de tejidos, separar las MSC del plástico con 0,05% Trypsin/0.7 mM EDTA y transferirlos a los buques de nueva cultura. Dependiendo de la cantidad de células dentro de su plato debe decidir el tamaño y el número de sus nuevos frascos para garantizar la baja densidad de siembra para una mayor expansión. </li><li> Cambio de un tercio de la media dos veces por semana durante la expansión de las células. </li></ol> Citometría de flujo Material necesario: Citómetro de flujo (BD FACSCalibur), micrónico tubos (Corning), soporte para tubos, suero de oveja (bloqueo de reactivo), Eppifuge (Eppendorf), los anticuerpos monoclonales MSC: CD45, CD14, CD19, HLA-DR, CD31, CD73, CD90, CD105, controles de isotipo ECFCs: CD45, CD14, CD19, HLA-DR, CD31, CD34, CD90, CD105, CD144, CD146, controles de isotipo <ol><li> Después de llegar a la confluencia, separar la celda con un 0,05% Trypsin/0.7 mM EDTA y se centrifuga a 300 g durante 7 min a 4 ° C. </li><li> Resuspender las células a una concentración de 1x10 6 células / ml en PBS modificado y la unión de bloques de anticuerpos inespecíficos con 10% v / v de suero de oveja durante 20 min a 4 ° C. </li><li> Marque los tubos Micronic y añadir la concentración adecuada de anticuerpos fluorescentes etiquetados. </li><li> Añadir 50 l de suspensión celular (bloqueadas) a cada tubo y se incuba durante 30 min a 4 ° C. </li><li> Lavar las células modificadas mediante la adición de PBS para eliminar los anticuerpos no son vinculantes. </li><li> Vaya con su celda marcada con el citómetro de flujo y realizar el análisis. </li></ol> Parte 4: crecimiento ECFC colonia, fijación, tinción y análisis de la jerarquía <ol><li> Semilla de la ECFCs prácticamente puro cosechado en la densidad de placas muy baja de 3 o 10 células por centímetro cuadrado en placas de cultivo celular para permitir el crecimiento de colonias individuales. </li><li> Cambio de un tercio de la media dos veces por semana. </li><li> Después de 14 días (fin de la cultura) eliminar medio, lavar dos veces con PBS y fijar las colonias con la solución de fijación fría (acetona: metanol = 3: 2) durante 15 minutos en el refrigerador. </li><li> Deseche la solución de fijación y dejar las placas abiertas a secar por 10 minutos. </li><li> Rehidratar las células con agua destilada durante 10 minutos. </li><li> Añadir Harris solución Tinción de hematoxilina las colonias fija durante 12 minutos. </li><li> Quitar H solución matoxylin y enjuague los platos con agua del grifo para eliminar la solución de tinción restantes. </li><li> Tomar fotos de cada coloniautilizando un microscopio estereoscópico y la importación *. jpg o *. tif en su ordenador. </li><li> Abra las fotos con el software ImageJ y analizar el número de células precisa de cada colonia como se describe en la animación. </li><li> Abra el software ImageJ e importar una imagen de la colonia con el &#39;Archivo \ Abrir&#39; función en el menú desplegable. </li><li> Proceder con el &quot;Proceso \ Antecedentes Reste&quot; función </li><li> Utilice la función de &quot;selección elíptica * * o cepillo&quot; en el menú de ImageJ para marcar el margen de colonia. </li><li> Borrar la imagen circundante a través del &quot;Editar \ Fuera de Clear &#39;función y recortar la imagen, seleccionando&quot; Recortar \&#39;. </li><li> Ajuste el umbral de forma manual utilizando el &#39;Imagen \ Ajuste de Umbral \&#39; función, por lo que todas las células son completamente de color rojo. </li><li> Cuente el número de células de la colonia mediante la selección de &quot;Analizar \ Analizar las partículas. Elegir la configuración adecuada (optimizado para cada tipo de célula) y seleccione &quot;Aceptar&quot;. El cuadro de resultados, incluyendo el recuento de células y una nueva imagen que contiene los lineamientos correspondientes de las células contadas aparecerá. <img src="/files/ftp_upload/1525/1525op2.jpg" alt="Figura 1" /></li></ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

Los autores no tienen conflictos de intereses que declarar.

Hay una variedad de fuentes de donde obtener MSC o ECFCs incluyendo la médula ósea, el tejido adiposo, sangre del cordón umbilical, etc Es necesario tener ambos tipos de células progenitoras de la misma fuente para comparar directamente la participación de los diferentes tipos de células en procesos como la regeneración de los tejidos y vasos o contribución a la progresión del tumor. La facilidad con que para aislar y posteriormente estudio pares autólo...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

El cordón umbilical es una fuente rica de células progenitoras con un alto potencial de proliferación de células mesenquimales del estroma incluyendo (también llamados células madre mesenquimales, MSC) y endoteliales que forman colonias células progenitoras (ECFCs). Ambos tipos de células son actores clave en el mantenimiento de la integridad de los tejidos y es probable que también participan en los procesos de regeneración y la formación de tumores. Para el estudio de su biología y su función en forma comparativa, es importante tener los dos tipos de células disponibles de un mismo donante. También puede ser beneficioso para los propósitos de regeneración para obtener CMM y ECFCs del mismo tejido. Debido a que la terapéutica celular finalmente debe encontrar su camino desde el laboratorio a la cabecera se estableció un nuevo método para aislar y expandir las células madre sin el uso de proteínas de origen animal. Lisado de plaquetas humanas agrupados (pHPL) reemplazó suero fetal bovino en todas las etapas de nuestro protocolo de evitar por completo el contacto de las células a las proteínas xenogénicas. Este vídeo muestra una metodología para el aislamiento y la expansión de las células progenitoras del cordón umbilical de uno. Todos los materiales y los procedimientos se describen.

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Este protocolo describe el aislamiento y la posterior expansión de células mesenquimales del estroma y las células endoteliales que forman colonias sin el uso de suero animal para generar pares con fines de trasplante autólogo experimental.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

isolation-animal-serum-free-expansion-human-umbilical-cord-derived

Research

JoVE Journal

Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

15.3K vistas.  Medical University of Graz, Austria. Este protocolo describe el aislamiento y la posterior expansión de células mesenquimales del estroma y las células endoteliales que forman colonias sin el uso de suero animal para generar pares con fines de trasplante autólogo experimental.

Video: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

El aislamiento de las células endoteliales de vena umbilical (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Investigación

Biología

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

Aislamiento y enriquecimiento de las células de rata madre mesenquimales (MSC) y la separación de una sola colonia Derivado MSC

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

El aislamiento de los Derechos Humanos de la arteria umbilical células musculares lisas (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Alimentador de Libre adaptación, Cultura y pases de células iPS humanas con alimentador de Libre Completa golpe de gracia suero sustitución Media

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caracterización fenotípica y funcional de las células endoteliales Formadoras de Colonias derivados de la sangre del cordón umbilical

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Aislamiento y diferenciación de las células del estroma vascular a células Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Aislamiento de las células murinas de la válvula endoteliales

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Aislamiento de CD146<sup&gt; +</sup&gt; Las células estromales mesenquimales de pulmón Residente de los pulmones de ratas

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...