The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOTA: Prematuro da Placenta devem ser coletadas de gravidezes saudáveis ​​Singleton, com uma preferência para cesarianas eletivas prazo. Escrito, o consentimento informado deve ser dada para a coleção de sua placenta. Seu comitê de ética em pesquisa em humanos relevantes devem aprovação totalidade da recolha e utilização de tecidos humanos. 1. Isolamento de Células Epiteliais Amnion <ol style="margin-left: 40px;"><li> Coloque a placenta em uma superfície estéril dentro de uma cabine de segurança biológica classe II. </li><li> Usando novelos estéreis solução salina equilibrada (HBSS), lavar o máximo de sangue possível da superfície da placenta e membranas. </li><li> Com a placenta colocada com o cordão umbilical voltada para cima, mecanicamente separar o bordo exterior das membranas de âmnio e cório. </li><li> Uma vez separados, retirar manualmente a membrana âmnio do cório membrana da placenta, trabalhando em torno da borda da membrana, para o cordão umbilical. Nota: Tenha careful para remover quaisquer peças de contaminar membrana cório do âmnio. </li><li> Com uma tesoura estéreis, cortar a membrana âmnio aproximadamente a 2 cm da base do cordão umbilical e introduzir num recipiente selado a 500 ml contendo 250 ml de HBSS. </li><li> Lavar cuidadosamente agitando o frasco lacrado contendo a membrana amnion. </li><li> Retire a membrana amnion do recipiente de coleta usando uma pinça estéril, e coloque em uma placa de Petri de 15 cm. </li><li> Corte a membrana amnion em pedaços de aproximadamente 5 cm de comprimento (quando mantido verticalmente com uma pinça), rejeitando pedaços sangrentos ou rasgadas. </li><li> Coloque em um recipiente de 500 ml selado e lavar com 250 ml HBSS aproximadamente 3-5 vezes ou até que a membrana amnion se torna translúcido com o sangue não contaminante. NOTA: Qualquer presença de sangue pode reduzir a eficiência da solução enzimática digerir. </li><li> Transferir todos os pedaços de membrana âmnio para um recipiente contendo 50 ml de solução enzimática a digerir, tendo o cuidado deminimizar transição de HBSS. </li><li> Incubar as peças em âmnio enzimática solução num banho de água quente ou quarto digerem a 37 ° C durante 15 min com agitação suave para remover todo o sangue restante. </li><li> Retire os pedaços de membrana amnion da solução enzimática e coloque em um novo recipiente estéril digerir. </li><li> Adicionar 100 ml de solução enzimática fresca digerir à membrana âmnio e incubar a 37 ° C durante 60 min num banho de água ou sala quente com agitação suave (primeiro a digestão). </li><li> Retire os pedaços de membrana âmnio e coloque em nova tomada recipiente cuidado para permitir a digestão enzimática solução para drenar a partir de cada pedaço de membrana. NOTA: Este pode ser conseguido arrastando pedaços âmnio ao longo da borda interior do recipiente. </li><li> Adicione 100 ml de enzimática fresco digerir solução para as peças de membrana âmnio e incubar 60 min em banho-maria ou sala quente a 37 ° C com agitação suave (segunda digestão). </li><li> Passe o suspensi celularem Passo de 1,14 por meio de um filtro de 70 ^ m e de centrifugação a 1000 xg durante 10 min. </li><li> Remover e descartar o sobrenadante e ressuspender o sedimento de células em 4 ml de HBSS contendo 1 mg / ml de inibidor de enzima à base de soja. Pipeta suavemente várias vezes com uma pipeta de 1 ml para se obter uma suspensão de célula única. NOTA: Nesta fase, as células podem ser deixadas em gelo até que as células da segunda digestão estão na mesma fase de processamento. </li><li> Retire os pedaços de membrana âmnio e coloque em nova tomada recipiente cuidado para permitir a digestão enzimática solução para drenar a partir de cada pedaço de membrana. NOTA: Este pode ser conseguido arrastando pedaços âmnio ao longo da borda interior do recipiente. </li><li> Passar a suspensão de células a partir do Passo 1,18 através de um filtro de 70 ^ m e centrifugar a 1000 xg durante 10 min. </li><li> Remover e descartar o sobrenadante e ressuspender o sedimento de células em 4 ml de HBSS contendo 1 mg / ml de inibidor de enzima à base de soja. Pipeta suavemente várias vezes com 1 mL de pipette para obter uma suspensão de célula única. </li><li> Piscina células das duas digestões, alternativamente, manter ou digestões de células separadas após a contagem de células. NOTA: Isso impede que a mistura de células potencialmente baixa viabilidade com células de alta viabilidade. </li><li> Adicionar HBSS para um volume final de 10-20 mL e realizar a contagem de células utilizando azul de tripano para medir a viabilidade. </li><li> Confirmar o fenótipo de células epiteliais por citometria de fluxo. Perfaz-se misturas de anticorpos apropriados utilizando anti-EpCAM-PE (diluição 1: 2) de anti-CD90-PeCy5 (1: 250) e anti-CD105-APC (1: 100) em HBSS. </li><li> Ressuspender um subconjunto de células a partir de uma ou ambas as digestões do coquetel de anticorpo numa concentração de 25 x 10 6 células / ml. </li><li> Por citometria de fluxo, manchas de 1 x 10 6 células com misturas de anticorpos contendo controlos do isotipo de controlo (por exemplo. EP-IgG de isotipo 1 + anti-CD90-PeCy5 + anti-CD105-APC). Mantenha ~ 1 x 10 6 células unstained para citômetro de fluxo set-up. </li><li> Incubar as células em anticorpococktails a 4 ° C durante 60 min. </li><li> Lavar as células 3 vezes em HBSS e ressuspender em 5 a 10 milhões de células por ml para citometria de fluxo. </li><li> Execute citometria de fluxo e os resultados de nota. A pureza do isolado de células (a percentagem de EpCAM positiva e CD90 / CD105 células negativas) deverá ser de 90-95% e &lt;1%, respectivamente. </li><li> Separe um número adequado de células para testes de critérios de controle de qualidade ou de libertação adequadas conforme determinado pela aplicação final. NOTA: Isso pode incluir doença / triagem viral, ou outro segurança ou critérios biológicos. </li><li> Para as restantes células, siga o protocolo de criopreservação, ou, alternativamente, coloque diretamente na cultura (direto à cultura passo 3.6). </li></ol> 2. Criopreservação de hAECs <ol style="margin-left: 40px;"><li> Determinar o número e viabilidade celular utilizando citometria de fluxo de dados ou contagem manual com exclusão do azul de tripan. </li><li> Centrifugar a 700 xg por 5 min. </li><li> Célula Resuspendas entre 1-10 milhões de células por ml em animais media criopreservação livre de produto. </li><li> Pipetar um volume adequado de células para frascos de crioconservação anéis-O e colocar em um aparelho de congelamento controlado arrefecimento onde ocorre a aproximadamente 1 ° C / min até -80 ° C é alcançada. </li><li> Transferir frascos em azoto líquido durante a armazenagem a longo prazo. </li></ol> 3. O descongelamento e Cultura da Cyropreserved hAECs <ol style="margin-left: 40px;"><li> Retirar os frascos de criopreservação de nitrogênio líquido e colocar no gelo. Nota: Somente Para transferência - proceder rapidamente à próxima etapa. </li><li> Descongelar as ampolas rapidamente criopreservação a 37 ° C até que apenas uma pequena parte (aproximadamente 5 x 5 mm) de gelo mantém-se. NOTA: É importante que a solução de células não aquecer para reduzir os efeitos tóxicos de DMSO no meio de criopreservação. </li><li> Diluir media criopreservação 10 vezes com (~ 4 ° C) meios livres de soro gelado, gota a gota, em um tamanho adequado adicionadod tubo ou recipiente. </li><li> Centrifuga-se a 700 xg durante 5 min à TA. </li><li> Ressuspender em meio isento de soro, a uma concentração aproximada de 1 milhão de células por ml. Determinar o número e viabilidade celular utilizando contagem automatizado ou manual, com exclusão do azul de tripan. Esperado viabilidade pós-descongelamento será 5-10% menos de viabilidade pré-criopreservação. </li><li> Células placa em células tratadas com cultura plásticas padrão, ou, alternativamente, usar colágeno tipo I em superfícies de cultura do casaco. </li><li> Para Tipo I revestimento Collagen seguir o protocolo abaixo <ol><li> Prepare ácido solúvel colágeno tipo I de placenta humana (colágeno 15 mg com 25 ml de água deionizada e 50 ácido acético glacial ul). </li><li> Cubra copo com Parafilm e uma agitação moderada a 37 ° C, até filamentos de colagénio são dissolvidos. </li><li> Solução de colagénio a filtração estéril utilizando um filtro de 0,22 um. </li><li> Diluir o colágeno estoque 01:10 filtrada com água deionizada. Este estoque diluída é o conce trabalhandontration solução para revestimento de superfícies de plástico e de membrana. </li><li> Revestimento de plástico colagénio, vidro e superfícies de membrana durante 2 horas à temperatura ambiente a 37 ° C, ou O / N a 2-8 ° C. </li><li> Retire o excesso de fluido da superfície revestida, e deixe-a secar O / N. </li><li> Enxágüe com água de grau de cultura de tecidos estéreis ou HBSS antes da introdução de células e meio. </li></ol></li><li> HAECs em placas a uma densidade de 25.000 células / cm2 em meio isento de soro, e para permitir a ligação ocorrer durante 72-96 hr. </li><li> Após a ligação, mudar mídia cuidadosamente aspiração e substituindo com um volume adequado de meio isento de soro a cada 2-3 dias. </li><li> Células de passagem utilizando o protocolo abaixo <ol><li> Aspirar os meios de cultura de células e lava-se com o volume de HBSS estéril 1x </li><li> Adicionar o volume 1x de solução enzimática digerir e incubar durante 5-15 minutos, ou até que as células sejam observadas separando-se da superfície da cultura de células. </li><li> Recolhe enzimática digerir solução contendo as células, garantindo cells são destacados da superfície da cultura de células pipetando repetidamente a solução enzimática contra a superfície de cultura de células digerir </li><li> Centrifugar a 700 xg por 5 min. </li><li> Ressuspender o sedimento de células em um volume apropriado de HBSS contendo 1 mg / ml de inibidor de enzima à base de soja. Pipeta suavemente várias vezes com uma pipeta de 1 ml para se obter uma suspensão de célula única. </li><li> Adicionar meio isento de soro para uma concentração aproximada de 1 milhão de células / ml. Determinar o número e viabilidade celular utilizando contagem automatizado ou manual, com exclusão do azul de tripan. </li><li> HAECs em placas a uma densidade de 25.000 células / cm2 em meio isento de soro, e continuar com métodos de cultura Standard. </li></ol></li></ol>

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The authors have nothing to disclose.

Existem vários parâmetros críticos que podem ter um impacto significativo no sucesso desta metodologia. Armazenamento da placenta ou âmnio por até 3 horas antes do isolamento do hAECs pode ser desejável para fins de logística ou programação, no entanto, recomenda-se que o tecido seja processado o mais rapidamente possível. Se o tecido é para ser armazenada, é recomendado que o armazenamento ser realizada seguindo esvaziamento e lavagem da membrana âmnio. Âmnio pode ser armazenado em HBSS estéril contendo ...

Células derivadas de fontes perinatais, como a placenta, membranas da placenta, do cordão umbilical e líquido amniótico têm atraído a atenção de pesquisadores e clínicos como uma fonte potencial de células para a medicina regenerativa 1,2. A razão para este interesse é que estes tipos de células todos possuem algum grau de plasticidade e imunomodulador capacidade 3, as propriedades que são fundamentais para as suas potenciais aplicações terapêuticas. hAECs são uma população heterogénea epitelial que podem ser derivados de termo ou pré-termo membrana âmnio 4, proporcionando uma fonte potencial abundante de material celular regenerativo. As propriedades que tornam hAECs atraentes como uma terapia celular incluem a sua multipotência, baixa imunogenicidade, e propriedades anti-inflamatórias. hAECs foram encontrados para ser altamente multipotentes in vitro e in vivo, capaz de se diferenciar em linhagens mesodérmica (cardiomyocytes, miócitos, osteócitos, adipócitos), linhagens endoderme (células pancreáticas, células hepáticas, células do pulmão) e linhagens ectodérmica (cabelo, pele, células neurais e astrócitos) 5-10. Reassuringly, apesar de suas hAECs multipotency parecem não quer formar tumores ou promover o desenvolvimento do tumor in vivo. Além disso, hAECs também são imunes privilegiado, expressando baixos níveis de classe II antígenos leucocitários humanos (HLA) 8. Esta propriedade provável subjaz a sua capacidade de fugir rejeição imunológica após o transplante alogênico e xenogênico, como demonstrado em estudos com macacos imunes competentes, coelhos, porquinhos da índia, ratos e porcos 11-13. hAECs exibir imunomodulador potente e propriedades imunossupressoras e, assim, oferecer vantagens práticas significativas para potenciais aplicações clínicas em terapia doença auto-imune. hAECs são acreditados para exercer funções imunomoduladoras em ambos os sistemas imune inata e adaptativa. Eme dos mecanismos sugeridos, é através da secreção de factores de 14 imunomodulador. As aplicações atuais de hAECs em modelos pré-clínicos de doenças animais incluem o tratamento de acidente vascular cerebral, esclerose múltipla, doença hepática, diabetes e doenças pulmonares crônicas e agudas. Pesquisadores demonstraram interesse em utilizar hAECs para tratar pós-AVC inflamação do cérebro, devido às suas propriedades únicas. Há evidências de que hAECs pode atravessar a barreira sanguínea do cérebro, onde eles podem enxertar, para sobreviver até 60 dias, diferenciar-se em neurónios, diminuir a inflamação e promover a regeneração de tecido danificado sistema nervoso central em modelos animais de doenças neurológicas 15. hAECs oferecer a capacidade de segmentar e reverter várias vias patológicas que contribuem para o desenvolvimento e progressão de esclerose múltipla. Por exemplo, os resultados de estudos animais pré-clínicos sugerem que hAECs são fortemente e imunossupressorapodem potencialmente induzir tolerância imunológica periférica e reverter respostas inflamatórias em curso. hAECs também têm mostrado ter a capacidade de se diferenciarem em células neuronais in vivo e aumentar neuroregeneração endógeno através da secreção de uma vasta gama de factores neurotróficos 16. Células epiteliais humanas e de roedores âmnio já demonstraram a sua eficácia terapêutica para o tratamento de doença do fígado em modelos animais. Em um modelo de indução de lesão tetracloreto de carbono de doença hepática, HAEC transplante liderança para enxerto de hAECs viáveis ​​no fígado, acompanhado de uma diminuição da apoptose dos hepatócitos, e diminuição da inflamação hepática e fibrose 17. hAECs podem ser estimulados com factores pancreáticos expressas incluindo insulina e transportadores de glicose. Diversos estudos têm investigado o potencial de hAECs para restaurar os níveis de glicose no sangue em ratos diabéticos 18. Em ratos que receberamhAECs, tanto a nível de peso e de glicose no sangue do corpo de animais diminuiu para níveis normais após a injeção das células. Estes estudos apresentam um caso forte para o uso de hAECs para o tratamento da diabetes mellitus. hAECs têm um papel comprovada na prevenção e reparação da lesão pulmonar aguda e crônica em adultos e modelos neonatais 19. Estes estudos revelaram que hAECs diferenciarem in vitro em células epiteliais do pulmão funcional que expressam múltiplas proteínas associadas à pulmão, incluindo fibrose cística da condutância transmembranar regulador (CFTR), o canal de iões, que é transformado em pacientes com fibrose cística 20. Além disso, quando hAECs são entregues para o adulto lesionado e pulmonar neonatal, que exercem os seus efeitos através da modulação de reparação de células imunitárias do hospedeiro, reduzindo o recrutamento de leucócitos pulmonar, incluindo neutrófilos, macrófagos e linfócitos 21-23. Dada a sua abundância,registro de segurança, e aplicações clínicas comprovadas para várias doenças, os ensaios clínicos utilizando hAECs é inevitável. Com o objetivo de acelerar a tradução de terapias em HAEC de ensaios clínicos, desenvolvemos métodos para isolar, criopreservar e hAECs cultura de uma maneira adequada para os ensaios clínicos, utilizando reagentes sem produtos de origem animal, de acordo com boas práticas de fabricação atuais (cGMP) orientações . Baseamos este protocolo um protocolo publicado anteriormente que estávamos usando com sucesso para isolar hAECs utilizando reagentes de origem animal 6. Nós alteramos o protocolo original para substituir os produtos derivados de animais com reagentes livre de produtos de origem animal, e posterior otimização foi realizada para otimizar o rendimento das células, viabilidade e pureza. Nosso objetivo foi desenvolver um protocolo que iria cumprir com as normas regulamentares de fabricação de células para testes clínicos em humanos.

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			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:220px;">Collection Kit</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protective Apparel</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bonnets</td>
			<td style="width:119px;">Medline</td>
			<td style="width:119px;">CRI1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;">Media and Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T3449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
			<td></td>
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			<td height="21" style="height:21px;">Consumables</td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Quando este procedimento é seguido corretamente, um rendimento médio de 120 milhões de hAECs deve ser esperado, com um alcance típico de 80-160000000 células. A partir destes rendimentos, uma viabilidade média de 83 ± 4% pode ser esperado. O aumento do rendimento e a viabilidade média ligeiramente inferior no método clínico pode ser devido a actividade da tripsina maior do que o produto derivado de animal, e, talvez, também devido à ausência de proteínas do soro. Isolado hAECs ter um perfil de superfície ...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K visualizações.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

Vídeo: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, cultura, e imagem de Fetal Humano pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptação do Semi-Automática de circulação Tumor de Células (CTC) Ensaios para aplicações clínicas e pré-clínica de Pesquisa

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e Cultura de Expansão de células endoteliais específicos de tumores

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional Cultura alginato-talão de células humanas adenoma pituitário

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e cultura de células endoteliais primárias de Artérias e Veias canina

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Um método de isolamento do romance grau clínico para rim humano Perivascular de células do estroma

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Cultura e Imagem de Organoides Epiteliais Nasais Humanos

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Isolamento e cultura de células-tronco derivadas de tecido adiposo humano com um método inovador livre de xenogênicos para terapia humana

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...