Os autores gostariam de agradecer Gabriel Arom e Michael Yeranosian por suas contribuições técnicas para iterações anteriores deste projeto. Este projecto foi financiado pelo OREF Clínico Scientist Training Grant (NL), HH Lee Surgical Research Grant (NL), Veterans Administration BLR &amp; D Mérito comentário 1 I01 BX00012601 (DM) e Musculoskeletal Transplantation Foundation Young Investigator Award (FP).

NOTA: Todas as cirurgias animais foram aprovados pela equipe e animal veterinário comitê de uso local, antes do início dos experimentos. 1. Preparação de electrospun Policaprolactona Andaimes <ol><li> Pesar e dissolver de grau médico de poli éster terminado (ε-caprolactona) em forma de grânulo em 1,1,1,3,3,3-hexafluoro-2-propanol, para criar uma 10% w / w solução do polímero PCL. Deixe a solução agitar utilizando uma placa de agitação durante pelo menos 3 h para assegurar uma solução homogénea. </li><li> Electrospin a solução PCL para criar um manguito de fibras PCL altamente alinhadas para a fabricação de andaime. <ol><li> Prepare a configuração electrospinning em um exaustor química com o ventilador ligado em todos os momentos. Este consiste em uma grande caixa de acrílico, que servirá como um meio isolado a vácuo para o processo de electrospinning e tem pontos de entrada para a solução de fonte PCL impulsionado por uma fonte de tensão, o mandril coletando a motor, e uma porta de vácuo. Limpe o acrylicaixa c cuidadosamente com etanol e cobrir todas as superfícies com folhas de Parafilm para remover quaisquer impurezas que podem comprometer a qualidade do produto electrospun. </li><li> Carga de aproximadamente 3 ml da solução anterior em uma seringa de 10 ml com um rombo 18 L, 1 ½ polegada agulha. Remova as bolhas de ar, empurrando a seringa para cima. Solução de bloqueio em uma bomba de seringa programável. Insira a agulha através de um pequeno buraco na caixa de acrílico, deixando cerca de ½ polegada da agulha fora da caixa de acrílico para fixação do fio de fonte de tensão. </li><li> Use a 30 milímetros girando mandril Torno como o coletor para as fibras do LCP altamente alinhados; cobrir o mandril firmemente com uma fina tira de papel alumínio. Bloquear o mandril no interior do motor, no lado oposto da caixa de cerca de 15 cm de distância a partir da agulha de uma seringa. </li><li> Insira a mangueira de plástico na porta de vácuo e se conectar à fonte de vácuo coifa. Ligar a fonte de vácuo e cobrir a caixa de acrílico com uma tampa. </li><li> Set a velocidade da bomba de seringa programável de 2,5 ml / h de infusão. Ligue o motor para operar o mandril a 3.450 rpm e anexar o fio positivo da fonte de tensão para a ponta da agulha fora da caixa usando um jacaré. </li><li> Uma vez que a infusão da solução PCL começou, ligue a fonte de tensão e definir a tensão de operação de 20 kV. </li><li> Infundir a solução durante 12 minutos para criar uma braçadeira homogénea a partir de 0,5 ml da solução de PCL. NOTA: Em média, cada canhão tem material de electrospun suficiente para criar duas folhas de seis lâminas, que podem ser utilizados para criar um total de três suportes de quatro camadas. </li></ol></li><li> Laser cortar o manguito PCL para formar várias pequenas folhas em um VersaLaser Cortador 2.3 operado a configuração de baixa vácuo, tensão pouso 10,0 keV, 6,4 mm de distância de trabalho, e diâmetro da sonda de 3,0. NOTA: Neste exemplo, um desenho assistido por computador foi utilizado para instruir o cortador para produzir várias folhas de 1,5 mm x 35 mm x 150 mm com andaimesuniformemente distribuídos 150 mm de diâmetro nos 15% de área de poros. </li><li> Plasma gravar os scaffolds PCL usando um aspirador de plasma para induzir a hidrofilicidade da superfície PCL com aceleração de íons. Defina a vácuo para 450 mTorr e o deleite dos andaimes para 30 segundos a alta potência (29,6 W). </li><li> Banhe os andaimes em etanol a 70% em um ambiente estéril. </li><li> Brasão os andaimes individuais com colágeno para facilitar a adesão e proliferação celular in vivo. <ol><li> Criar uma solução de revestimento de colagénio por diluição de um 8: solução estéril de 2,5 Purecol colagénio de 3 mg / ml de solução-padrão, 10x PBS e NaOH 0,1 N 1:: 1 9 em 1x PBS a 4 ° C. Misturar bem com a solução de assegurar homogeneidade. </li><li> Casaco indivíduo 1,5 milímetros x 35 mm x 150 mm andaimes com uma película de camada fina da solução de colagénio acima. Deixa-se secar durante 24 horas em um ambiente estéril. </li></ol></li><li> Usando 5-0 Vicryl, empilhar e apor quatro indivíduo 1,5 milímetros x 35 mm x 150 mm scaffoldsusando um Krackow pontos para criar uma final 0,6 milímetros multi-camadas, andaime de espessura, revestido de colágeno, que está pronto para a implantação. </li></ol> 2. Rat Cirurgia Protocol <ol><li> Induzir a anestesia, colocando rato na câmara de inalação e entrega de 2% de isoflurano com 2 L / min de oxigênio. Confirme o rato está devidamente anestesiados pela aplicação de pressão a pata traseira e avaliação para qualquer resposta. </li><li> Na mesa de volta não estéril, subcutaneamente injectar 25 mg / kg de ampicilina e 0,03 mg / kg de buprenorfina. </li><li> Aplicar pomada oftálmica para os olhos. Clipe da pele do membro posterior operatório e preparar o sítio cirúrgico com três esfrega alternadas de clorexidina e etanol 70%. </li><li> Transfira o rato para a mesa de operação, em uma almofada de aquecimento para evitar a hipotermia. Nariz cone seguro e manter a anestesia através do procedimento com 2% de isoflurano em 2 L / min de oxigênio, entregue cone do nariz. Drapejá de uma forma estéril, deixando o membro operativo exposta. </li><li&gt; Antes de iniciar a cirurgia, primeiro, confirmar anesthetization adequada por falta de uma resposta à pressão do pé traseiras. </li><li> Adicione uma 2 cm de comprimento incisão média vertical para o joelho, centrado no nível da rótula. Retirar a pele lateralmente até a incisão é centrado sobre o joelho. </li><li> Usar um bisturi para fazer uma artrotomia parapatelar medial cortando apenas medial para a patela e estendendo-se proximalmente para o nível da junção músculo do quadríceps e distal ao nível da inserção do tendão patelar sobre o tubérculo tibial. Tome cuidado para não cortar os patelares ou quadríceps tendões. </li><li> Patela libertação lateralmente através de uma incisão vertical 1 cm para dentro da cápsula do joelho imediatamente lateral do tendão patelar. </li><li> Verifique se o joelho é estendido. Pegue um par de tesouras finas e passar sob a patela de lateral para medial. Espalhe a tesoura algumas vezes para que o mecanismo extensor pode ser traduzido para ambos os lados. </li><li> Enquanto flexionando tele joelho, traduzir a patela lateralmente para expor o interior da articulação do joelho. Certifique-se de clara visualização da fossa intercondilar e côndilos do fêmur. Usando um bisturi, transecto a ACL e PCL no entalhe. </li><li> Carga furadeira com uma k-wire 1,6 milímetros. Coloque ponta k-fio no ACL origem na fossa intercondilar. Broca superolaterally e visualizar o ponto de saída na face lateral do fêmur, a remoção de todo o tecido macio, se necessário com um bisturi. Passe o fio k dentro e fora algumas vezes para garantir a passagem livre para o enxerto. </li><li> Coloque o fio k sobre a pegada ACL no platô tibial. Broca anterolateralmente e visualizar ponto de saída na tíbia proximal ântero-lateral. Use bisturi para limpar tecidos moles como necessário de modo que o ponto onde o fio de k-sai da tíbia é completamente visualizada. </li><li> Passe uma agulha encurtado Keith (o ideal é não mais do que 2 centímetros de comprimento) através do túnel do osso femoral. Passe as duas pontas de sutura de uma extremidade do enxerto através do olho da Kagulha eith. Usar a agulha para puxar uma extremidade do enxerto através do túnel femoral. </li><li> Repetir o passo anterior para passar a outra extremidade do enxerto através do túnel tibial. </li><li> Use 4-0 Vicryl sutura para fixar o fim femoral do enxerto ao redor periósteo ou outros tecidos moles com um ponto a figura de oito. Tensão manualmente o enxerto com o joelho em extensão. Apor o fim tibial do enxerto de periósteo circundante ou outro tecido mole com um ponto figura em forma de oito. </li><li> Utilize uma tesoura para cortar o excesso de enxerto em ambas as extremidades, deixando 1-2 mm em cada extremidade passado o ponto figura em forma de oito. </li><li> Estenda o joelho e reduzir a patela. Usando 4-0 vicryl, coloque um ponto único figura de oito para fechar a cápsula articular medial, impedindo subluxação lateral da patela. </li><li> Feche a pele com um subcuticular 5-0 Monocryl ou Vicryl sutura contínua, com cuidado para não suturar o músculo subjacente ou ter qualquer sutura visível uma vez que a pele é fechada. </li><li> Inject ratos por via subcutânea com buprenorfina cada 12 horas para um total de três dias de pós-operatório. Verificar local cirúrgico para a drenagem ou deiscência da ferida no momento da injecção. Mancando e algum inchaço é normal nos primeiros dias após a cirurgia, mas prontamente quaisquer preocupações pós-operatórias em conjunto com o pessoal veterinário. O animal pode ser retornar à habitação social em 2 semanas de pós-operatório, quando incisões cirúrgicas são completamente curado. </li></ol> 3. Protocolo de Coleta de Dados <ol><li> No momento do sacrifício, asfixiar ratos individualmente numa câmara fechada de CO 2, seguida por toracotomia. </li><li> Colha os dois membros cirurgicamente reconstruídos e contralateral, separando na articulação do quadril. <ol><li> Para os membros reconstruídos, remover todo o tecido mole, incluindo o ligamento cruzado posterior e os remanescentes da ACL nativa interrompido cirurgicamente, por dissecção fina para isolar apenas o fêmur, tíbia, e enxerto. </li> &lt;li&gt; Para os membros contralaterais, remover todo o tecido mole, exceto o ACL nativa assim como o fêmur ea tíbia por dissecção bem. </li><li> Utilize uma ferramenta rotativa, tais como Dremel, para remover todos, mas ¾ de 1 cm de osso a partir de cada extremidade do fémur complexo tíbia-enxerto. </li><li> Durante este processo, e ao longo do teste biomecânico, regular e frequentemente pulverizar as regiões ligamentares com salino normal para evitar a dessecação do joelho colhidas que podem alterar os resultados falsamente. </li></ol></li><li> Para a análise histológica, fixar cada joelho individualmente em solução de paraformaldeído a 4% a 25 ° C durante 48 h. Em seguida, submergir o joelho em uma solução de reagente para Immunocal descalcificação completa; este processo é dependente do conteúdo de calcificação da amostra e pode levar até cinco dias. Verifique amostras individuais diariamente para avaliar o progresso como descalcificação incompleta pode reduzir a qualidade da amostra. Uma vez completo, executar o corte, montagem de slides, e coloração se o desejar. </li><li> Perform testes biomecânicos para avaliar a capacidade funcional do ligamento da engenharia de tecidos. <ol><li> Prenda o fêmur ea tíbia enrolando 28 G arame de aço galvanizado em torno da epífise de cada osso em separado. Isso é para evitar que os dados de testes biomecânicos imprecisas de falha de tração prematura da amostra com o osso em vez de no ligamento de interesse. </li><li> Pot o fêmur em uma mistura de cimento de polimetilmetacrilato (PMMA) osso. Para fazer isto, misturar os dois componentes de cimento e utilizar imediatamente a mistura viscosa para fixar o fémur no interior do metal, que encerra completamente a diáfise do osso no pote cimentada com a epífise e ligado ligamento saliente livremente. Permitir espontânea polimerização por radicais livres de transformar gradualmente os componentes viscosos misturadas a um material pastoso e, eventualmente, em uma matriz sólida endurecido. NOTA: Este processo leva vários minutos e pode ser monitorado por avaliação manual da temperatura de um bolo feito a partir deo restante cimento; a temperatura deve aumentar transitoriamente durante a reacção de polimerização exotérmica e diminuem para a TA, depois os material solidifica. </li><li> Repita o mesmo processo acima para cimentar a tíbia, com exceção, mantendo o ligamento do joelho em 20 ° de flexão para o teste mecânico ideal. </li><li> Monte o complexo fêmur-enxerto-tibial cimentada em um aparelho de ensaio de tração, e se preparar para carga recorde e deslocamento em função do tempo a partir do início de tensão ao fracasso. Neste exemplo, foi utilizado um modelo Instron 5564 com uma célula de carga de 1 kN. </li><li> Pré-tensão do enxerto a 2 N a uma velocidade de rampa de 0,5 N / min e, em seguida, para testar o enxerto falha a uma velocidade de deformação de 0,5 mm / seg. Durante o processo, certifique-se que o ligamento está falhando em meados da década de substância e que o fêmur e da tíbia óssea são seguras e não prematuramente falhando, o que pode inaccurately avaliar as propriedades biomecânicas do ligamento testado. </li><li> Use a carga-deslocamento geradocurvas para calcular a carga de ruptura e de rigidez do ligamento testado. </li></ol></li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-platelet+composite+enhances+biomechanical+and+histologic+healing+of+the+porcine+anterior+cruciate+ligament.">Collagen-platelet composite enhances biomechanical and histologic healing of the porcine anterior cruciate ligament.</a> Am J Sports Med. 37 (12), 2401-2410 (2009).</li><li>Leong, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+evaluation+of+heparin+mediated+growth+factor+release+from+tissue-engineered+constructs+for+anterior+cruciate+ligament+reconstruction.">In vitro and in vivo evaluation of heparin mediated growth factor release from tissue-engineered constructs for anterior cruciate ligament reconstruction.</a> J Orthop Res. 10, (2014).</li><li>Seo, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+cell+migration+and+angiogenesis+in+a+composite+silk+scaffold+for+tissue-engineered+ligaments.">Increase in cell migration and angiogenesis in a composite silk scaffold for tissue-engineered ligaments.</a> J Orthop Res. 27 (4), 495-503 (2009).</li><li>Freeman, J. W., Woods, M. D., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+the+anterior+cruciate+ligament+using+a+braid-twist+scaffold+design.">Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.</a> J Biomech. 40 (9), 2029-2036 (2007).</li><li>Bashur, C. A., Shaffer, R. D., Dahlgren, L. A., Guelcher, S. A., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter+and+alignment+of+electrospun+polyurethane+meshes+on+mesenchymal+progenitor+cells.">Effect of fiber diameter and alignment of electrospun polyurethane meshes on mesenchymal progenitor cells.</a> Tissue Eng Part A. 15 (9), 2435-2445 (2009).</li><li>Dash, T. K., Konkimalla, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly-&#1108;-caprolactone+based+formulations+for+drug+delivery+and+tissue+engineering:+A+review.">Poly-&#1108;-caprolactone based formulations for drug delivery and tissue engineering: A review.</a> J Control Release. 158 (1), 15-33 (2012).</li><li>Yoshimoto, H., Shin, Y. M., Terai, H., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable+nanofiber+scaffold+by+electrospinning+and+its+potential+for+bone+tissue+engineering.">A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering.</a> Biomaterials. 24 (12), 2077-2082 (2003).</li><li>Xu, Y., Ao, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+biomechanical+studies+of+inter-strand+healing+in+four-strand+autograft+anterior+cruciate+ligament+reconstruction+in+a+rabbit+model.">Histological and biomechanical studies of inter-strand healing in four-strand autograft anterior cruciate ligament reconstruction in a rabbit model.</a> Knee Surg Sports Traumatol Arthrosc. 17 (7), 770-777 (2009).</li><li>Shino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+the+anterior+cruciate+ligament+by+an+allogeneic+tendon+graft.+An+experimental+study+in+the+dog.">Replacement of the anterior cruciate ligament by an allogeneic tendon graft. An experimental study in the dog.</a> J Bone Joint Surg Br. 66 (5), 672-681 (1984).</li><li>Stasiak, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+device+to+apply+controlled+flexion+and+extension+to+the+rat+knee+following+anterior+cruciate+ligament+reconstruction.">A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction.</a> J Biomech Eng. 134 (4), 041008 (2012).</li><li>Brophy, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+short-duration+low-magnitude+cyclic+loading+versus+immobilization+on+tendon-bone+healing+after+ACL+reconstruction+in+a+rat+model.">Effect of short-duration low-magnitude cyclic loading versus immobilization on tendon-bone healing after ACL reconstruction in a rat model.</a> J Bone Joint Surg Am. 93 (4), 381-393 (2011).</li></ol>

The authors declare that they have no competing financial interests.

Lesões do LCA é uma condição comum em cirurgia ortopédica de esportes, com opções limitadas de Reconstrução, com o tempo presente. A fim de desenvolver um substituto apropriado da engenharia de tecidos para a ACL que irá permitir a regeneração in vivo, num modelo animal adequado é necessária. Neste estudo, a fabricação de um enxerto de engenharia biodegradável é descrita, como é o seu implante in vivo utilizando um modelo reprodutível de reconstrução ACL em um rato atímico. Este ...

A ruptura do ligamento cruzado anterior (LCA) é uma das mais comuns de lesões ligamentares do joelho 1. Porque a ruptura do LCA é incapaz de curar sem intervenção cirúrgica, as limitações nas atividades da vida diária, bem como a participação em esportes de carro mais de 175.000 pacientes submetidos à cirurgia a cada ano 2, com um custo estimado de um bilhão de dólares por ano 3. Atualmente, qualquer auto-enxerto ou enxerto de tendão é usado para a reconstrução do ligamento. Embora altas taxas de sucesso pode ser alcançado com tanto auto-enxerto e enxerto de substituição, complicações graves estão associados com estas opções de reconstrução 4. Tecido auto-enxerto é associado com área doadora morbidade e é limitado em oferta, especialmente em casos de re-ruptura ou lesão multi-ligamentar. Por outro lado, o tecido do enxerto está ligada com a integração do enxerto retardada, a resposta inflamatória adverso, o risco infeccioso teórica, e supp limitadoly 5. Enxertos não degradáveis ​​sintéticos foram desenvolvidos na década de 1970 e 1980, mas foram prejudicados pela ruptura prematura do enxerto, reações de corpo estranho, osteólise, e sinovite 6. Como resultado dessas preocupações sérias, não existem actualmente enxertos sintéticos disponíveis para uso clínico nos Estados Unidos. Devido a essas limitações com opções de enxerto existentes e aos desenvolvimentos recentes em biologia, engenharia e medicina regenerativa, tem havido grande interesse em uma solução de engenharia de tecidos para ACL enxertia. Estratégias de engenharia de tecidos actuais empregam materiais biológicos e sintéticos biodegradáveis ​​para permitir o crescimento interno de tecido do hospedeiro, evitando as limitações associadas com o implante de material sintético permanente 7. Policaprolactona (PCL) é um polímero biodegradável que é aprovado pela FDA para um número de aplicações médicas, incluindo barreira de adesão e curativos 8, que tem sido used em uma ampla variedade de aplicações, incluindo vasculares, osso, cartilagem, nervo, pele, tecido esofágico e engenharia 5,9-16. Biocompatibilidade favorável, relativamente longa in vivo meia-vida, a força mecânica adequada, e uma elevada elasticidade contribuir para a popularidade deste polímero em engenharia de tecidos. Em um modelo de roedor de cicatrização de feridas, implantado PCL electrospun foi demonstrado ser não imunogénico e para integrar no tecido local, sem reacções adversas 13. Uma imagem SEM de electrospun PCL é mostrado na Figura 1. Com atual FDA regulamentar normas, eficácia e segurança em ambos os pequenos e grandes modelos animais seria necessário para um PCL ou qualquer outra ACL enxerto projetado para mover-se em ensaios clínicos nos Estados Unidos. Além disso, condições in vivo pode muitas vezes aumentar as propriedades de um ACL enxerto de tecido in vitro engenharia. Um modelo de rato de reconstrução do LCA autólogo com flexor digitorhum longus tendão foi descrito anteriormente, em que o ACL nativa foi cortada, femorais e tibiais túneis foram perfurados, e o enxerto foi aprovada e fixada no local com sutura 17-22. Neste artigo, iremos descrever uma modificação deste modelo para a avaliação de substituições ACL engenharia em vez de para a reconstrução à base de auto-enxerto (Figura 2). Apesar de existirem muitos modelos animais para a engenharia de tecidos ligamento, o rato é vantajosa em relação aos modelos maiores para um número de razões. Essas vantagens incluem mais fácil criação e manipulação, menos considerações éticas, e reduziu 17,23 custo. Além disso, o modelo do rato tem sido utilizada extensivamente como um modelo para a regeneração do tecido ortopédico, incluindo cartilagem, tendão, e engenharia de tecidos ósseos 24. Em particular, os ratos nus atímicos foram escolhidos devido à sua falta de resposta mediada por células imunes de 25, permitindo a eventual implantação océlulas dadoras xenogénicas f neste modelo para aumentar ainda mais o enxerto engenharia no futuro. Neste trabalho métodos, descrevemos a fabricação e implantação cirúrgica de um acelular, enxerto polímero biodegradável em um modelo de rato atímica de reconstrução do LCA.

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
		</tr>
	</tbody>
</table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Em nossa experiência de 92 cirurgias de ratos por um único cirurgião, o tempo médio operatório da incisão para a conclusão da ferida foi de 16,9 min, com um desvio padrão de 4,7 min. No momento do sacrifício, os ratos pesavam 356 ± 23 g. Todos os ratos toleraram bem a cirurgia, e experiente sem complicações. Imediatamente após a cirurgia, os ratos foram anotados para suportar o peso sobre a extremidade operatório, mas exibiu um ligeiro coxear. Por uma semana de pós-operatório, todos os ratos foram deambu...

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Atímica Rat Modelo de Avaliação da Engineered Ligamento Cruzado Anterior Enxertos

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

athymic-rat-model-for-evaluation-engineered-anterior-cruciate

Research

JoVE Journal

Bioengineering

10.3K visualizações.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Vídeo: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodologia Elastografia por Ressonância Magnética para a Avaliação de engenharia de tecidos Crescimento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Bioengenharia

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Recuperação cirúrgica, isolamento e<em&gt; In vitro</em&gt; Expansão de humanos anteriores Cruciate células do ligamento derivados para aplicações em engenharia de tecidos

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Técnica Cirúrgica para a Implantação de engenharia de tecidos enxertos vasculares e Subsequente<em&gt; In Vivo</em&gt; Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

A escala do Engineered enxertos vasculares Usando 3D Impresso Guides and the Ring método de empilhamento

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamento de Estruturas Ligamentares com Soro Condicionado por Exercício: Um Modelo Translacional de Engenharia de Tecidos

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Uso do elevado-Throughput Microbioreactor sistema automatizado para produção de IgG1 modelo em células CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...