This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

O protocolo a seguir é projetado para o isolamento dos cardiomiócitos de uma ninhada de filhotes de ratos neonatos (10-14 filhotes). Se o tamanho da leitegada é significativamente diferente, o procedimento pode ter de ser escalado para compensar. Os filhotes devem ser em torno de 48 ± 6 horas de idade. Todos os procedimentos aqui foram aprovados pela North Carolina State University Animal Care Institucional e Comitê de Uso (IACUC). 1. Preparação cela de isolamento NOTA: Execute as seguintes etapas no dia anterior isolamento. <ol><li> Autoclave os seguintes: grandes tesouras, tesouras pequenas, pequenas pinças afiadas, grandes fórceps. Use um ciclo de gravidade durante 60 minutos a um mínimo de 121 ° C e um tempo de secagem de 30 minutos. </li><li> Colocar 40 ml de solução de tripsina a 0,1% em frigorífico para descongelar S / N. Sais Equilibrados Solução de Refrigerar Hank (HBSS) se já não estiver frio. Prepare um soro de bovino (FBS) solução de mídia estoque fetal 20%. <ol><li> Em 400 ml de Iscmeios Dulbecco modificado por ove (IMDM) adicionar 100 ml de FBS, 5 mL de L-glutamina (200 mM), 2,5 ml de gentamicina (10 mg mL -1), e 0,9 ml de 2-mercaptoetanol. Esterilizar usando filtro de vácuo. Meios Armazenar a 4 ° C durante até 3 semanas e esta é escalável para volumes maiores ou menores. Dilui-se 20% de FBS meios estoque 1: 1 com IMDM para produzir 10% de meio de FBS NRCM. Apenas fazer em pequenos lotes para evitar um aquecimento e resfriamento de mídia repetido. </li></ol></li></ol> 2. cela de isolamento Dia 1 <ol><li> Execute o seguinte trabalho em uma capa de cultura para evitar a contaminação das amostras. Para melhor momento iniciar este trabalho no período da tarde uma vez que existe um O / N incubação. </li><li> Prepare espaço de trabalho dentro de uma capa de cultura estéril. Coloque pad banco na capa de cultura. Encha dois copos de 250 ml com 70% de etanol e coloque os instrumentos cirúrgicos por eles. Coloque pequeno balde de gelo no capô. Certifique-se de que é grande o suficiente para armazenar vários tubos e um prato de 150 milímetros. UV esterilizar o capô for 10 min. Colocar 40 ml de tripsina a 0,1% em gelo. </li><li> Recuperar filhotes de ratos recém-nascidos da mãe. Para manter os animais quente até uso, coloque-os em um recipiente isolado, como um balde de gelo envolto em um bloco de banco para mantê-los aquecidos. </li><li> Encha uma 150 milímetros prato de cultura com 25 ml de HBSS e colocar no gelo. Unwrap 2-3 compressas de gaze e coloque na área de trabalho. Segurando pup pela pele na parte de trás do pescoço, pulverizá-lo com etanol e 70%, em lugar do exaustor. NOTA: Para melhor manter a esterilidade, isto é pode ser feito por uma outra pessoa que, em seguida, coloca o filhote limpo no capô. </li><li> Segurar o cachorro pela pele na parte de trás do pescoço. Em seguida, usando as grandes tesouras, em um rápido corte forte, eutanásia por decapitação. Blot o sangue com gaze. NOTA: Devido a variações nas exigências IACUC, pode ser mais apropriado para limpar o filhote com um etanol em vez de limpar aspersão. Certifique-se de verificar com a universidade IACUC como às suas diretrizes para essa etapa. </li><li> Continuar a cortar face anterior da chest através da caixa torácica. Apertar a pele no pescoço para ajudar a abrir a caixa torácica até que o coração é visível. Usando as pequenas tesouras, remover o coração e o lugar em HBSS prato de cultura em gelo. Repita o procedimento para os outros filhotes. </li><li> Remova qualquer tecido não-cardíaca a partir das amostras e agite a HBSS suavemente para lavar os corações. Transferir o tecido do coração para outro prato de cultura contendo HBSS e lavar o tecido. Corte o tecido mais até que as peças são 1-3 mm 3, e depois transferir para a solução de tripsina. Coloque solução de tripsina no balancim em 4 ° C geladeira O / N. </li></ol> Isolamento celular Dia 2 NOTA: Enquanto a aspiração nas etapas seguintes, utilizar uma pipeta, em vez de uma linha de vácuo. O tecido se move com facilidade, e uma linha de vácuo vai entupir ou remover o tecido ainda contendo células. É melhor usar uma pipeta de aspiração em seu lugar. Se o tecido entra na pipeta, pipeta de volta para removê-lo. <ol><li> Faça três alíquotas de 10% NRCM Mídia: um com 25 ml e dois com 15 ml. Coloque um tubo de 25 ml de meios de comunicação em 37 ° C banho de água. Adicionar 4 ml HBSS para 5 diferentes 15 ml tubos cônicos e coloque tudo no gelo. Escreva em cada 15 ml tubo # 1-5. </li><li> Adicionar 40 ml de HBSS para um tubo cónico de 50 ml. Adicionar 10 ml de HBSS a colagenase, pipeta para suspender, e combinar as soluções para criar 50 ml de 1 mg ml -1 solução de colagenase. Esterilizar usando 0,22 um filtro de vácuo, e armazenar à temperatura ambiente. </li><li> Recuperar coração tubo de tecido / tripsina. Certifique-se de que a tripsina é clara, e o tecido aparece fofo. Permitir que o tecido para liquidar a esquina do tubo e aspirar o máximo de líquido possível. </li><li> Adicionar 25 ml quente 10% mídia FBS NRCM ao tubo e agite com a mão. Cap e rodar em 37 ° C num banho de água durante 2 min. Aspirar líquido novamente. O tecido deve flutuar nesta etapa; em caso afirmativo, aspirado a partir do fundo do tubo. </li><li> Adicionar 10 ml de colagenase. Gire em banho-maria por 2 min ou até que a solução está turva. Quickly aspirar o líquido. Adicionar 10 ml de colagenase fresco ao tubo contendo o tecido e rodar em banho de água durante 2 min. Pipeta para misturar, solução de transferência de tubo # 1, e colocar no gelo. Pode não ser possível retirar todo o líquido. Repita os passos de 3,2-3,6 para os outros três tubos de 15 ml cônicas. Transferir o líquido remanescente para tubo # 5. </li><li> Centrifugar tubos de 15 ml a 410 rcf durante 8 min. Enquanto centrifugação, coloque 15 ml de 10% tubo de mídia FBS NRCM em banho-maria. Colocar 40 um filtro de células no topo de um tubo cónico de 50 ml e molhado com 2 ml de HBSS frio. </li><li> Aspirar o sobrenadante a partir de células centrifugadas. Adicione 6 ml frio HBSS a cada tubo e ressuspender. As suspensões de células pipeta através do filtro em 50 ml cónico para remover qualquer tecido. Lavar os lados de cada tubo de 15 ml com 3 ml de HBSS frio e adicionar ao filtro. Centrifugar células a 410 rcf durante 10 min. Aspirar o sobrenadante. Ressuspender pellet celular em 10 ml 10% de media FBS NRCM e transferência para T-75 frasco. Enxágüe cônico com media de 5 ml e adicionar à flperguntar. </li><li> Incubar durante 1 h a 37 ° C e 5% de CO 2. Coloque últimos 15 ml 10% tubo de mídia FBS NRCM em banho-maria. Conteúdo de T-75 para um novo frasco T-175 Transferir, enxaguar T-75 com 15 ml de meio e adicionar a T-175. Incubar durante 1 h. </li></ol> 4. Criopreservação <ol><li> Coloque mídia congelamento no gelo. Transferência de mídia a partir de células T-175 a 50 ml tubo cônico, e coletar 10 ml de suspensão celular para celular contagem utilizando Trypan azul. Centrifugar a 410 rcf durante 5 min. Enquanto a centrifugação, contagem de células usando um contador de células. </li><li> Aspirar meios de cultura de células, e ressuspender em meio de congelação a uma concentração de 2-4.000.000 células por ml. Coloque 1 ml de suspensão de células em criotubo, então lugar frascos uma taxa controlada de refrigeração recipiente de congelamento, e coloque no freezer -80 ° C O / N. Dentro de 1-2 dias, transferem frascos em azoto líquido. </li></ol> 5. descongelamento de células <ol><li> Prato Brasão em fibronectina dissolvendo 250 ul60; de fibronectina (concentração de 1 mg ml-1) em 9,75 ml de água (concentração final de 0,025 mg mL -1). Adicionar solução suficiente para cobrir as placas de cultura e incuba-se a 37 ° C durante pelo menos 30 min. <ol><li> Em quatro de 15 ml tubos cônicos, fazer as misturas DMSO / mídia da seguinte forma adicionando DMSO a 20% de Mídia. Adicionar (5%) 9,5 ml de meio com 500 ul de DMSO. Adicionar (2,5%) 9,75 ml de meio com 250 ul de DMSO. Adicionar 10 ml de mídia (fazer duas delas). </li></ol></li><li> Aqueça as misturas de mídia acima mencionadas em 37 ° C banho de água. Remover as células a partir de azoto líquido e colocar em gelo seco. Quando misturas de mídia são quentes, descongelar as células em 37 ° C banho de água até pouco descongelado. Não deixe as células em banho-maria por muito tempo, pois isso pode causar danos às células. </li><li> Transferir conteúdo do frasco criogênico a 5% tubo de DMSO / media. Centrifugar a 410 rcf durante 8 min. Aspirar o sobrenadante deixando sedimento celular. Adicione o conteúdo do tubo de 2,5% DMSO / media para a célula pellet e resuspend. Centrifugar a 410 rcf durante 8 min. Aspirar o sobrenadante deixando sedimento celular. </li><li> Adicionar conteúdo de um tubo de 0% DMSO / media para a célula pellet e ressuspender. Centrifugar a 410 rcf durante 8 min. Aspirar o sobrenadante deixando sedimento celular. </li><li> Adicionar conteúdo da segunda 0% DMSO / media para pellet celular e ressuspender. Tome pequena amostra para contagem de células. Centrifugar a 410 rcf durante 8 min. Enquanto centrifugação células de contagem em um hemocitómetro utilizando Trypan Blue para rotular as células mortas. Quantidade de células Record, bem como a viabilidade. </li><li> Aspirar o sobrenadante deixando sedimento celular. Ressuspender em 10% mídia NRCM (opcional: adicionar 100 bromodeoxiuridina? M (BrdU)) e células da placa em pratos revestidos com fibronectina. </li></ol> Cultura 6. celular <ol><li> As células em placas a cerca de 100k células por cm2. Ajustar a densidade de revestimento com base na utilização prevista da cultura. Prepare a 2% de meio NRCM diluindo 20% em IMDM Meios numa proporção de 1:10. O que se segue é um esboço de uma cultura típica. &lt;/ Li&gt; <li> No dia 1, lavar as células mortas afastado com IMDM morna, adicione 10% NRCM (opcional 100 M BrdU). No dia 2, lavar as células mortas afastado com IMDM morna, adicione 10% NRCM. Dia 3-adicionar 2% NRCM. No dia 4, adicionar 2% NRCM. No dia 5, assegurar que as células estão confluentes e batimento. Manter as células em cultura durante cerca de uma semana. Nesse ponto fibroblastos podem começar a superar significativamente as NRCMs. </li></ol>

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The authors have nothing to disclose.

Este protocolo permite que os NRCMs a ser isolado, criopreservadas e descongeladas. A descongelação das células é uma parte essencial do processo. Uma série de diluições de DMSO é usado para remover lentamente DMSO a partir das células 14. É importante que a extracção de DMSO ser realizado mais rapidamente que as células são particularmente sensíveis a morrer imediatamente após a descongelação. Se as células mais ou menos são requeridos para a descongelação, os volumes das soluções em ...

Cultura de células de cardiomiócitos é uma ferramenta crítica na pesquisa cardíaca moderna. Cardiomiócitos de ratos neonatais (NRCMs) são comumente utilizados uma vez que o isolamento e a cultura é mais fácil do que a de cardiomiócitos de rato adulto 1. O método NRCM ainda tem várias limitações, incluindo um procedimento de isolamento e a proliferação celular a longo limitada no prato. Existem numerosos protocolos para o isolamento de NRCMs com mais geralmente requerem 4-48 horas de trabalho 2-6. Além disso, as células são frequentemente isoladas a partir de 1 a filhotes de 2 dias de idade de ratos 2,4-7; o momento do nascimento pode ser imprevisível e conflito com outro trabalho no laboratório. Os isolamentos pode ser ineficiente e dispendiosa se apenas uma pequena quantidade de células são necessários para as experiências. A maioria dos esforços na melhoria do fluxo de trabalho o foco na redução do tempo de isolamento, mas isso não resolve os problemas de cronometrar o nascimento dos filhotes. Como alternativas, muitos laboratórios utilizamcardiomiócitos derivados de células-tronco embrionárias (CES) ou células-tronco pluripotentes induzidas (iPSCs). No entanto, o processo de reprogramação e / ou a diferenciação pode ser muito demorado e dispendioso, bem. Podem existir outros problemas quando se utiliza estas células como modelos in vitro de miócitos. Ambos ESC- e iPSC- cardiomiócitos derivados têm sido mostrados para apresentar diferenças de eletrofisiologia de cardiomiócitos primários 8-10. NRCMs dissociadas são capazes de serem armazenados durante vários dias, usando refrigeração 11, no entanto, esta não permite a armazenagem a longo prazo. O nitrogénio líquido é tipicamente usado para preservar as células para um período de tempo maior, mas requer um crioprotector tal como sulfóxido de dimetilo (DMSO). Pesquisas anteriores mostraram que a concentração ideal entre 5-10% DMSO na mídia congelamento permite a criopreservação de NRCMs, mas mesmo assim a viabilidade permanece baixa 12. Embora DMSO ajuda a proteger as células durante a livreXing, que pode ser tóxico para células em concentrações acima de 1,5% 13. Estudos anteriores têm demonstrado que a remoção de DMSO lentamente a partir das células, pode melhorar a viabilidade celular 14. Procurou-se melhorar a eficiência de ensaios baseados em células NRCM por criopreservação das células após o isolamento. Isto permite que as células a ser descongelado e utilizado quando necessário, reduzindo a frequência de isolamento e o consumo dos animais. Usando este método, que mostram que é possível criopreservar NRCMs e descongelar-los para uso numa altura posterior. Depois de descongelar as células manter uma viabilidade aceitável e produzir culturas NRCM que são positivas para sarcomérica α-actinina (α-SA) e contraem espontaneamente.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
		</tr>
		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Seguindo o isolamento dos cardiomiócitos de ratos neonatais, as células são congeladas até azoto líquido, e pode ser armazenado durante pelo menos vários meses. Tipicamente, durante o descongelamento, a viabilidade determinada por análise de Azul de Tripano será entre 40-60%. Embora este seja mais baixa do que outros tipos de células, com a sementeira e a cultura adequado as células irão proliferar (Figura 1). A fim de verificar a contractilidade, as células podem ser visualizados utilizando...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

O isolamento e criopreservação de Neonatais Rato Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Research

JoVE Journal

Developmental Biology

12.4K visualizações.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Vídeo: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento de Neural Stem / células progenitoras da Região Periventricular do rato adulto e Medula Espinhal Humano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Biologia do Desenvolvimento

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caracterização de células-satélite de músculo de rato Cabeça branquiométrico

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Melhoria Geração de Induced Cardiomyocytes utilizando uma construção policistrónico Expressando proporção ideal de GATA4, MEF2C e Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolamento de células embrionárias de murino hegemônicos endoteliais

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Isolamento eficaz de ilhotas funcionais a partir de ratinho neonato Pâncreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualização de Variações do ciclo celular e Determinação de Nucleação no pós-natais Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Bottlenose Dolphin (Tursiops truncatus) espermatozoides: recolha, criopreservação e heterólogo fertilização In Vitro

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Maturação de cardiomiócitos estaminais humanas derivadas de células em Biowires Usando Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Isolamento e caracterização de rato primário válvula células intersticiais: um novo modelo para estudar a calcificação da válvula aórtica

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Manipulação e tratamento de macho Europeu enguias (Anguilla anguilla) para maturação Hormonal e criopreservação de esperma

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...