Agradecemos al Dr. Steven Austad para que nos proporciona la primera versión de este protocolo. Este trabajo fue apoyado por subvenciones del NIH y la Ellison Medical Foundation de VG y AS

1. Antes de empezar <ol><li> Esterilizar tijeras y pinzas con etanol al 70%. </li><li> Lugar pequeño agitador magnético dentro de un vaso de precipitados de 30 ml, cubierta con dos capas de papel de aluminio, y autoclave. </li><li> Prepare un 28 unidades Wunsch / ml de solución stock de Liberase Blendzyme 3 en agua estéril. Hacer alícuotas de 0,5 ml y congelar a -20 ° C. Descongelar una alícuota de nuevo antes de cada uso. La solución puede aparecer turbia después de la descongelación. Vortex la solución hasta que se aclare. </li><li> Calentar el medio de cultivo celular. </li></ol> 2. Preparación de muestras de animales Precaución: los animales salvajes pueden contener agentes patógenos tales como virus de la rabia. Que sea siempre consciente de abrir objetos punzantes. <ol><li> Sacrificar al animal y colocar el cadáver a 4 ° C. Lo mejor es utilizar el canal inmediatamente, sin embargo, si esto no es práctico, como si los animales son recogidos en el campo, los canales se pueden utilizar dentro de las 24 horas. Después de 24 horas, la reducciones de los rendimientos de la célula. </li><li> Diseccionar al animal en la sala de operaciones o en la campana química (no en la campana de cultivo de tejidos). </li><li> Área debajo del brazo es un sitio conveniente para recoger muestras de piel, como piel de las axilas es más fino, contiene menos grasa y la piel es menos densa. Limpie el sitio de la incisión con un 70% de etanol. Asegúrese de que la piel se empapa con etanol. </li><li> Afeitarse la piel alrededor del sitio de la incisión con un bisturí. Afeitarse un área más grande que el sitio de la incisión deseada. Trate de minimizar los cortes en la piel. Rocíe el área con el 70% de etanol y dejar secar. </li><li> Para cobrar una especial muestra de piel de un fragmento de aproximadamente 1 cm 2. Pellizcar la piel con pinzas de tejido y se corta con tijeras. Trate de no cortar la capa de grasa con la piel, la grasa interfiere con la digestión colagenasa. Para recoger las muestras de pulmón, lave el área del pecho con un 70% de etanol, y abrir la piel en el pecho con unas tijeras, haciendo una incisión en forma de T y separando las aletas de la piel. Lave el área del músculo abrió con un 70% de etanol y dejar secar. Cortar las costillas con las pinzas y tijeras estériles (uso de cortadores de huesos de animales de mayor tamaño). Utilice técnicas de esterilización para evitar la contaminación de los órganos internos. No toque los órganos internos con los instrumentos que tocaba el pelo de los animales. Cortar los fragmentos de pulmón de aproximadamente 1 cm 2 utilizando una pinza estéril y tijeras. </li><li> Fragmentos de tejido en lugar de tubos de 50 ml con PBS estéril para evitar la desecación. </li><li> Lavar la parte externa de la tubos de 50 ml con fragmentos de tejido con etanol, y los llevan a la capilla de cultivo de tejidos. </li><li> Como deshacerse de los cadáveres de animales. </li></ol> 3. Extraer células <ol><li> La transferencia de los fragmentos de tejido en una placa de cultivo de 10 cm de tejido utilizando bisturí estéril. No transfiera demasiado PBS con la muestra. </li><li> Cortar el tejido en trozos de 1 mm ~ con dos escalpelos. Use dos hojas con una acción de tijera a partir del centro y separando. Mantenga la bola con el tejido, no corte pieza por pieza. Cuando el corte es suficiente, la piel se asemeja a la masilla, no se separará en pedazos, pero se estrecha franja. El tejido pulmonar es más fácil de cortar, y se separará en pequeños pedazos. </li><li> El uso de un bisturí, la transferencia de los tejidos cortados en el vaso estéril de 30 ml con barra de agitación estéril. Lavar la placa utilizada para el corte de tejidos con 10 ml de DMEM/F12 los medios de comunicación, con 0,14 unidades Wunsch / mL Liberase Blendzyme 3, y 1X antibiótico / antimicótico, y la solución en el vaso de 30 mL con fragmentos de tejido. </li><li> Cubrir el vaso con una lámina estéril, y se incuba a 37 ° C, revolviendo lentamente, durante 30 a 90 minutos. La duración de la incubación depende del tipo de la especie y el tejido. Tenga cuidado de no sobre-digerir el tejido. Mejores rendimientos se obtienen cuando los fragmentos de tejidos están todavía presentes en la final de la digestión. La piel necesita más tiempo para digerir que el pulmón. La piel de animales de gran tamaño tarda más tiempo en digerir. Compruebe la digestión después de 30 minutos, y luego cada 10 min. Cuando la digestión de la piel se haya completado, los medios de comunicación se vuelve turbia y fragmentos de piel separados unos de otros, y los bordes de las piezas se convierten en &quot;confusos&quot;. La digestión de pulmón debe ser parado cuando los pulmones cambian de color fragmentos de rojo a blanco y empezar a formar las fibras pegajosas. </li><li> Pipeta la solución que contiene fragmentos de tejido de arriba abajo para romper los terrones. Transferir la solución a un tubo estéril de 50 ml. Si los fragmentos se mueven fácilmente a través del corte de una pipeta de 10 ml y la digestión se hace bien. Lavar el vaso tres veces con 10 ml de agua tibia DMEM/F12 los medios de comunicación con FBS al 15%, antibiótico / antimicótico 1X y añadir los medios de comunicación en el tubo de 50 ml con fragmentos de tejido. Cierre el tubo de 50 ml y mezclar por inversión varias veces. El FBS en los medios de comunicación se detendrá la digestión Liberase. </li><li> Giran a 524 g en una centrífuga de la cultura basculante tejido durante 5 min. Eliminar el sobrenadante. Resuspender el precipitado en 10 ml de agua tibia DMEM/F12 los medios de comunicación con FBS al 15%, 1X antibiótico / antimicótico. Pipeta con la suspensiónla máxima fuerza para romper las piezas de tejido. </li><li> Añadir otro 30 ml de DMEM/F12 los medios de comunicación con FBS al 15%, 1X antibiótico / antimicótico, mezclar y centrifugar a 524 g en una centrífuga de la cultura basculante tejido durante 5 min. Repetir una vez más para eliminar los restos de Liberase. </li><li> Resuspender el precipitado en 10 ml de DMEM/F12 los medios de comunicación con FBS al 15%, 1X antibiótico / antimicótico y la transferencia a un plato de cultivo de 10 cm de tejido y colocarlo en un cultivo de tejidos incubadora a 37 ° C, 5% de CO 2, el 3% O 2 . </li><li> Revise las placas de todos los días para los fibroblastos y el color de los medios de comunicación. Si el aislamiento se ha realizado correctamente, los fibroblastos se arrastran fuera de los fragmentos de tejido y se adhieren a la placa (Figura 1). Los fibroblastos comienzan a salir de fragmentos de tejido dentro de 2-5 días. </li><li> Si el color de los medios de comunicación cambia a amarillo, lo que indica una posible contaminación o el hacinamiento de las celdas. Examinar las placas bajo el microscopio a gran aumento. Si las bacterias, hongos o parásitos están presentes descartar las placas (esto no suele ser un problema con los animales de laboratorio, sin embargo puede ocurrir cuando las muestras se recogen de la naturaleza). Si no hay contaminación está presente, pero los medios de comunicación cambió de color, esto es causado por muchas células o fragmentos de tejido demasiados colocado en el mismo plato. Si más del 60% de la placa está cubierto por los fibroblastos adjunto, cambie los medios de comunicación en la placa y la transferencia de los fragmentos de tejido para una nueva placa con los nuevos medios. Si no los fibroblastos muchos unido a la placa, cambiar los medios de comunicación y dividir las piezas de tejido a un 2-4 placas. </li><li> Después de 7 días, si los medios de comunicación no se había modificado anteriormente, el cambio de los medios y la transferencia de los fragmentos de tejido para una nueva placa con los nuevos medios. </li><li> Se incuban las células y fragmentos de tejido durante 7 días. El día 14 todos los fibroblastos viables han salido los fragmentos de tejido. </li><li> Después de 14 días desde el inicio de la celda de aislamiento, desechar los viejos medios y fragmentos de tejido, las células de la cosecha y la placa en un plato nuevo en 5x10 5 células / EMEM placa con FBS al 15%, 1X penicilina / estreptomicina, aminoácidos no esenciales y sodio piruvato. Los medios de comunicación EMEM apoyará el crecimiento de los fibroblastos tipos de células y otros sólo se mueren o dejan de proliferar. </li><li> Después de llegar a las células del 80-90% de confluencia, congelar una parte alícuota de las células para su uso futuro. </li><li> Continuar cultivando las células de dividir a 5x10 5 células / placa cuando las células alcanzan el 80-90% de confluencia. </li></ol> 4. Resultados representante Fibroblastos normales son células grandes con protuberancias prominentes (lamellipodia) (Figura 2). Fibroblastos crecen en una monocapa. Una cultura sana de crecimiento contiene células 10.1% en la etapa M, reconocidos como detenidos células elevada sobre la superficie de la placa, pero no separado de la placa (Figura 3). Por lo general, un plato de 10 cm sembrados con 5x10 5 células confluentes se convierte en 3-4 días. El tiempo de duplicación varía mucho entre las especies, y puede ser mayor para algunas de las especies de roedores de larga duración 1. Cuando las células se llenan de una placa que detener la proliferación en la fase G1. Plato típico confluentes de fibroblastos contiene una capa apretada de las células (Figura 4). Cuando las células llegan al 90% de confluencia que están listos para partir. Si lo desea, las células se pueden mantener en una placa confluente detenidos durante largos períodos de tiempo con cambios regulares de medios (una o dos veces a la semana). <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="Figura 1" /> Figura 1. Aislamiento de fibroblastos de piel de ratón (A) y pulmón (B). El medio se cambió para eliminar las células sueltas y escombros antes de tomar las imágenes en el día 7. <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="Figura 2" /> Figura 2. Fibroblastos de ratón de pulmón. <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="Figura 3" /> Figura 3. Un campo de fibroblastos de ratón con células de M-etapa, fotografiado con un aumento de 10 veces. <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="Figura 4" /> Figura 4. Una placa confluente de fibroblastos de ratón, fotografiado con un aumento de 10 veces.

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li></ol>

Normal de los fibroblastos primarios ofrecen una excelente alternativa para establecer líneas celulares de cáncer en la investigación biológica. Una ventaja importante de los fibroblastos, es que no son portadores de mutaciones en oncogenes y los supresores de tumores y mantener intactos los puestos de control del ciclo celular. Esto hace que los fibroblastos normales de un sistema preferido para los estudios de la regulación del ciclo celular, reparación del ADN y la apoptosis. El protocolo aquí descrito proporc...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TM Research Grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05401127001</td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

establishing primary adult fibroblast cultures from rodents

La importancia del uso de células primarias, en lugar de líneas celulares de cáncer, para estudios biológicos está siendo ampliamente reconocido. Las células primarias son las preferidas en los estudios de control del ciclo celular, la apoptosis y la reparación del ADN, las células cancerosas son portadores de mutaciones en los genes implicados en estos procesos. Las células primarias no pueden ser cultivadas indefinidamente debido a la aparición de la senescencia replicativa o aneuploidization. Por lo tanto, de nuevas culturas es necesario establecer con regularidad. El procedimiento para el aislamiento de los fibroblastos embrionarios de roedores está bien establecido, pero aislando las culturas de fibroblastos adultos a menudo se presenta un desafío. Fibroblastos de roedores adultos aislados de los modelos de ratón de la enfermedad humana puede ser un control preferido cuando se los compara con los fibroblastos procedentes de pacientes humanos. Además, los fibroblastos adultos son el único material disponible cuando se trabaja con roedores salvajes donde las mujeres embarazadas no pueden ser obtenidos fácilmente. Aquí les ofrecemos un protocolo para el aislamiento y cultivo de fibroblastos de la piel de roedores adultos y los pulmones. Hemos utilizado con éxito este procedimiento para aislar los fibroblastos de más de veinte especies de roedores de los ratones y ratas de laboratorio a los roedores salvajes como los castores, puerco espín, y la ardilla.

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

En este artículo se describe un protocolo para el aislamiento y mantenimiento de cultivos primarios de fibroblastos de la piel y el tejido pulmonar de los roedores salvajes.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

establishing-primary-adult-fibroblast-cultures-from-rodents

Research

JoVE Journal

Biology

49.1K vistas.  University of Rochester. En este artículo se describe un protocolo para el aislamiento y mantenimiento de cultivos primarios de fibroblastos de la piel y el tejido pulmonar de los roedores salvajes.

Video: Establishing Primary Adult Fibroblast Cultures From Rodents

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Investigación

Biología

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

La eliminación rápida de fibroblastos de alta densidad Humanos culturas de células madre embrionarias

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparación de Primaria Myogenic precursor de célula / cultivos de mioblastos de basales Vertebrados Linajes

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

La aplicación de ácido retinoico para obtener cultivos de osteocitos principal del ratón osteoblastos

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

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 ...