Name: isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications
Uploaded: 2014-12-21T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

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 ...

<ol>
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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 style="width:184px;">Comments/Description</td>
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			<td style="width:119px;"></td>
			<td></td>
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		<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>
		</tr>
		<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>
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		<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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		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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 ...

Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

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

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

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

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

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 ...