Agradecemos ao Dr. Steven Austad para fornecer-nos com a primeira versão do presente protocolo. Este trabalho foi financiado por concessões do NIH e da Ellison Medical Foundation para VG e AS

1. Antes de Começar <ol><li> Esterilizar a tesoura e uma pinça com etanol 70%. </li><li> Lugar agitador magnético pequeno dentro de um copo 30 mL, cobrir com duas camadas de papel alumínio, e autoclave. </li><li> Prepare a 28 unidades Wunsch / mL solução estoque de Liberase Blendzyme 3 em água estéril. Faça alíquotas 0,5 mL e congelar a -20 ° C. Descongelar uma alíquota de novo antes de cada utilização. A solução pode parecer turvo após o descongelamento. Vortex a solução até que fique claro. </li><li> Aqueça o meio de cultura celular. </li></ol> 2. Preparação de amostras de animais Cuidado: animais selvagens podem conter agentes patogênicos, como um vírus da raiva. Sempre estar ciente de abrir farelos. <ol><li> Eutanásia do animal e colocar a carcaça a 4 ° C. O melhor é usar a carcaça de imediato, no entanto, se isso não é prático, como se os animais são recolhidos no campo, as carcaças podem ser usados ​​dentro de 24 horas. Após 24 horas a queda de produção celular. </li><li> Dissecar o animal no centro cirúrgico ou na capa químicos (não na capa da cultura de tecidos). </li><li> Axilas área é um local conveniente para coletar amostras de pele como a pele das axilas é mais fino, contém menos gordura, ea pele é menos denso. Limpar o local da incisão com etanol 70%. Verifique se o pêlo é embebido com álcool. </li><li> Raspar os pêlos ao redor do local da incisão com um bisturi afiado. Raspar uma área maior do que o local da incisão desejada. Tentar minimizar cortes na pele. Pulverizar a área com álcool 70% e deixe secar. </li><li> Para coletar uma amostra de pele especial sobre o consumo de um fragmento de aproximadamente 1 cm 2. Beliscar a pele com uma pinça, e cortar com tesoura. Tente não cortar a camada de gordura com a pele, como gordura interfere com a digestão da colagenase. Para coletar as amostras de pulmão, lave a área do peito com etanol 70%, e abra a pele do peito com uma tesoura, fazendo uma incisão em forma de T e puxar as abas para além da pele. Lave a área do músculo abriu com etanol 70% e deixe secar. Cortar a caixa torácica com a pinça estéril e tesoura (use cortadores de osso para animais de maior porte). Uso de técnicas estéreis para evitar a contaminação dos órgãos internos. Não toque os órgãos internos com instrumentos que tocou a pele de animal. Cortar fragmentos de pulmão de aproximadamente 1 cm 2 usando uma pinça e tesouras estéreis. </li><li> Fragmentos de tecido lugar em tubos de 50 ml com PBS estéril, para evitar a secagem. </li><li> Lavar a parte externa do tubos de 50 ml com fragmentos de tecido com etanol, e levá-los para a capa de cultura de tecidos. </li><li> Devidamente dispor de carcaça animal. </li></ol> 3. Células extrair <ol><li> Transferir os fragmentos de tecido em um prato de 10 centímetros cultura de tecidos utilizando bisturi estéril. Não transferir PBS muito com a amostra. </li><li> Corte o tecido em pedaços ~ 1 mm com dois bisturis. Use duas lâminas usando uma tesoura ação a partir do centro e afastamento. Manter o tecido enrolado, não corte pedaço por pedaço. Quando o corte é suficiente a pele se assemelha putty, ele não vai separar em pedaços, mas vai esticar fina. O tecido pulmonar é mais fácil de cortar, e ele irá separar-se em pequenos pedaços. </li><li> Usando um bisturi, transferir o tecido cortado em copo mL estéril 30 com barra de agitação estéril. Lave a placa usada para cortar tecido com 10 mL de DMEM/F12 mídia com 0,14 unidades Wunsch / mL Liberase Blendzyme 3, e 1X antibiótico / antimicótico e adicionar a solução para o copo 30 mL com fragmentos de tecido. </li><li> Cobrir o copo com papel alumínio estéril e incubar a 37 ° C, mexendo lentamente, por 30 a 90 minutos. A duração da incubação depende do tipo de espécies e tecidos. Tome cuidado para não sobre-digerir o tecido. Melhores rendimentos são obtidos quando fragmentos de tecido ainda estão presentes no final da digestão. Pele demora mais tempo para digerir do que o pulmão. Pele de animais de grande porte leva mais tempo para digerir. Verifique a digestão depois de 30 min, e depois a cada 10 min. Quando a digestão da pele é concluída, a mídia torna-se turva e fragmentos de pele separados uns dos outros, e as bordas das peças tornam-se &quot;fuzzy&quot;. Digestão pulmonar deve ser interrompido quando mudam de cor fragmentos de pulmão de vermelho para branco e começar a formar fibras pegajosas. </li><li> Solução de pipeta contendo o fragmento de tecido para cima e para baixo para quebrar os aglomerados. Transferir a solução para tubo de 50 mL estéril. Se os fragmentos de mover-se facilmente através do corte da pipeta 10 mL e digestão foi bem feito. Lavar o copo 3 vezes com 10 mL de morno DMEM/F12 media com 15% FBS, 1X antibiótico / antimicótico e adicione os meios de comunicação para o tubo de 50 mL com fragmentos de tecido. Fechar o tubo de 50 mL e misturar por inversão várias vezes. A FBS na mídia vai parar a digestão Liberase. </li><li> Giram em 524 g em uma centrífuga balançando cultura balde do tecido por 5 min. Remover o sobrenadante. Ressuspender o sedimento em 10 mL de morno DMEM/F12 media com 15% FBS, 1X antibiótico / antimicótico. Pipeta com a suspensãoforça máxima para quebrar os pedaços de tecido. </li><li> Adicionar outro, 30 mL de DMEM/F12 media com 15% FBS, 1X antibiótico / antimicótico mix, e centrifugar a 524 g em uma centrífuga balançando cultura balde do tecido por 5 min. Repita mais uma vez para remover os vestígios do Liberase. </li><li> Ressuspender o sedimento em 10 mL de DMEM/F12 mídia com FBS 15%, 1X antibiótico / antimicótico e transferir para um prato de 10 centímetros cultura de tecidos e coloque em uma cultura de tecidos incubadora a 37 ° C, 5% CO 2, 3% O 2 . </li><li> Verifique as placas todos os dias para fibroblastos e cor media. Se o isolamento foi bem-sucedida, fibroblastos rastejar para fora dos fragmentos de tecido e fixe a placa (Figura 1). Os fibroblastos começam a sair fragmentos de tecido dentro de 2-5 dias. </li><li> Se a mídia muda de cor para amarelo, indica contaminação em potencial ou a superlotação de celas. Examine as placas sob o microscópio em grandes ampliações. Se as bactérias, fungos ou vermes estão presentes descartar as placas (isso raramente é um problema com animais de laboratório, no entanto pode ocorrer quando as amostras são coletadas na natureza). Se não houver contaminação está presente, mas a mídia mudou de cor, isso é causado por um excesso de células ou fragmentos de tecido demais colocados no mesmo prato. Se mais de 60% da placa é coberta por fibroblastos anexa, alterar a mídia sobre a placa e transferir os fragmentos de tecido para um novo prato com a nova mídia. Se não fibroblastos muitos ligados à placa, alterar a mídia e dividir os pedaços de tecido para um 2-4 pratos. </li><li> Após 7 dias, se a mídia não tivesse sido alterado anteriormente, alterar a mídia e transferir os fragmentos de tecido para um novo prato com as novas mídias. </li><li> Incubar as células e fragmentos de tecido para um adicional de 7 dias. Por 14 dias todos os fibroblastos viáveis ​​tenham saído os fragmentos de tecido. </li><li> Após 14 dias desde o início do isolamento de células, descartar a velha mídia e fragmentos de tecido, a colheita das células e placa-los em um novo prato de 5x10 5 células / EMEM placa com FBS 15%, 1X Penicilina / Estreptomicina, não aminoácidos essenciais , sódio e piruvato. EMEM mídia vai apoiar o crescimento de fibroblastos tipos de células e outros só vai morrer ou parar de proliferar. </li><li> Depois que as células chegar confluência de 80-90%, congelar uma alíquota de células para utilização futura. </li><li> Continue cultivando as células, dividindo-os em 5x10 5 células / placa quando as células atingem confluência de 80-90%. </li></ol> 4. Resultados representante Fibroblastos normais são células grandes com saliências proeminentes (lamellipodia) (Figura 2). Fibroblastos crescer em uma monocamada. Uma cultura saudável e em crescimento contém células 1-10% na fase M, reconhecido como arredondado células elevados sobre a superfície da placa, mas não retirado da placa (Figura 3). Tipicamente, um prato de 10 cm semeados com 5x10 5 células se torna confluente em 3-4 dias. O tempo de duplicação varia muito entre as espécies, e pode ser maior para algumas das espécies de vida longa de roedores 1. Quando as células encher um prato eles prendem a proliferação na fase G1. Prato típico confluentes de fibroblastos contém uma camada de células hermeticamente embalados (Figura 4). Quando as células atingem confluência de 90% que eles estão prontos para dividir. Se desejado, as células podem ser mantidos em uma placa presa confluentes por longos períodos de tempo com mudanças regulares de mídia (uma ou duas vezes por semana). <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento de fibroblastos da pele do rato (A) e pulmão (B). A mídia foi mudado para remover as células solto e detritos antes de tirar as fotos no dia 7. <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="Figura 2" /> Figura 2. Fibroblastos de pulmão de rato. <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="Figura 3" /> Figura 3. Um campo de fibroblastos de rato com células em estágios M, fotografadas com ampliação de 10x. <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="Figura 4" /> Figura 4. Uma placa confluentes de fibroblastos de rato, fotografadas com ampliação de 10x.

<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>

Não há conflitos de interesse declarados.

Fibroblastos normais primárias oferecem uma excelente alternativa para as linhas de células de câncer estabelecido na pesquisa biológica. Uma vantagem importante de fibroblastos é que eles não carregam mutações em oncogenes e supressores de tumor e manter postos de controle do ciclo celular intacto. Isso faz com que fibroblastos normais de um sistema preferido para os estudos de regulação do ciclo celular, reparo do DNA e apoptose. O protocolo aqui descrito fornece uma receita simples para o isolamento e manut...

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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

A importância de usar pilhas, ao invés de linhas celulares de cancro, para estudos biológicos está se tornando amplamente reconhecida. Pilhas são preferidas em estudos de células a apoptose ciclo de controle, e reparo do DNA, como as células cancerosas carregam mutações em genes envolvidos nestes processos. Pilhas não podem ser cultivadas indefinidamente devido ao início da senescência replicativa ou aneuploidization. Assim, novas culturas devem ser estabelecidas regularmente. O procedimento para o isolamento de fibroblastos de roedores embrionárias está bem estabelecida, mas isolar culturas de fibroblastos adultos, muitas vezes representa um desafio. Fibroblastos de roedores adultos isolados a partir de modelos do rato da doença humana pode ser um controle preferidos quando comparando-os com fibroblastos de pacientes humanos. Além disso, os fibroblastos adultos são o único material disponível quando se trabalha com roedores silvestres onde as fêmeas grávidas não podem ser facilmente obtidos. Aqui nós fornecemos um protocolo para isolamento e cultura de fibroblastos da pele de roedores adultos e os pulmões. Nós usamos este procedimento com sucesso para isolar os fibroblastos de mais de vinte espécies de roedores a partir de ratos de laboratório e em ratos de roedores silvestres como o castor, porco-espinho, e esquilo.

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

Establishing Primary Adult Fibroblast Cultures From Rodents

Este artigo descreve um protocolo para isolamento e manutenção de culturas de fibroblastos primários da pele e do tecido pulmonar dos roedores silvestres.

Estabelecer culturas de fibroblastos adultos primária de 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

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Biology

49.2K visualizações.  University of Rochester. Este artigo descreve um protocolo para isolamento e manutenção de culturas de fibroblastos primários da pele e do tecido pulmonar dos roedores silvestres.

Vídeo: 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.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

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

Biologia

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.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Cultura primária de Miócitos Coração Adulto Rat

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.

Culturas de células primárias de Drosophila Os embriões Gástrula

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.

A remoção rápida de fibroblastos de Alta Densidade Humanos culturas de células estaminais embrionárias

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&#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 isolamento de culturas primárias 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.

Preparação de Primário Miogênica de Células Precursoras / Culturas de mioblastos de Vertebrados basais Linhagens

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;

A aplicação de ácido retinóico para obter culturas de osteócitos principal do mouse 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.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e 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 ...