This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative.

1. Entrar <ol><li> Ligue o biorreator. Clique em qualquer lugar para abrir a página de login. Selecione o nome de usuário e digite a senha e clique em &quot;Login&quot;. </li></ol> 2. Calibração <ol><li> Calibre o sensor de pH (2 pontos antes da autoclavagem). </li><li> Inspeccionar sensor de pH e confirmar ponta do sensor é preenchido com uma solução de electrólito. Prepare dois copos com soluções de calibração pH (eletrólito) pH 4 e pH 7 e tem disponível um frasco de lavagem com água destilada. </li><li> Conectar o cabo de pH para o sensor de pH. Navegue até a guia &quot;Ações&quot; na interface Olá e clique em &quot;calibrar&quot;. Digite temperatura tampão no campo Temp Solução de Calibração. </li><li> Colocar o sensor de pH em tampão 1 (pH 4) e inserir o valor no campo de &quot;zero&quot;. Aguarde até que o gráfico para estabilizar e clique no botão &quot;calibrar 1&quot;. Lavar o sensor de pH com água destilada. </li><li> Colocar o sensor em tampão 2 (pH 7). Digite o tampão2 valor no campo &quot;span&quot;. Aguarde até que o gráfico para estabilizar e clique calibrar 2 botão. </li><li> Clique em &quot;Salvar&quot; e depois clique em &quot;Fechar&quot;. </li><li> Calibre o sensor de oxigênio dissolvido (OD). </li><li> Verifique se o sensor de OD foi polarizada por ser ligado ao sistema por várias horas. Navegue até a guia &quot;Ações&quot; na interface Olá e clique em Calibrar. Clique no botão &quot;FAZER A&quot;. </li><li> Desligue o sensor de OD e digite 0 no campo &quot;zero&quot;. Aguarde até que o gráfico para estabilizar e clique no botão &quot;Calibrar 1&quot;. </li><li> Volte a ligar o sensor de OD e digite 100 no campo &quot;Span&quot;. Aguarde até que o gráfico para estabilizar e clique no botão &quot;Calibrar 2&quot;. </li><li> Clique em &quot;Salvar&quot; e clique em &quot;fechar&quot;. </li></ol> 3. autoclave e instalar sensores de e Vasos de reagente <ol><li> Após a calibração, sensores lugar e bem térmica em autocbolsas e lave autoclave durante 30 minutos a 121 ° C, 15 psi. </li><li> Higienizar bolsas de autoclave com 70% de álcool isopropílico (IPA) e bolsas de transferência para cabine de segurança biológica (BSC). Retire a embalagem exterior do navio. </li><li> Higienizar embalagem interior com 70% IPA e transferir navio para cabine de segurança biológica (BSC). Remova a embalagem interior e inspeccionar o navio e tubos por danos causados ​​durante o transporte. </li><li> Instalar o pH e DO sensores nas duas portas dianteiras. Instale o bem térmica para a porta traseira esquerda. Abra a tampa do sensor. </li><li> Guie o sensor através da porta sensor. Passe o sensor com força na porta. </li><li> Navio Transferência de BSC. </li><li> Pendure o DO e cabos dos sensores de pH fora da manga vaso e verifique se não há nada na manga. Deslize o navio na manga, primeiro os pés. </li><li> Ajustar cuidadosamente o sensor de temperatura para o poço térmico navio. Certifique-se de que o fundo do recipiente, assenta contra os aquecedores. </li><li> Removera tubulação fixa de suas malas. Jogo codificação de cores na tubulação para os conectores correspondentes e bombas na unidade de controle biorreator. </li><li> Instale a tubulação de gás principal pressionando o conector na sua saída de gás. Instale a linha de gás micro rodando no sentido horário conector na saída de gás. Instale o tubo de filtro de exaustão: </li><li> Abra o forno filtro. Fixe o filtro de exaustão no canal U para que sua tubulação atravessa os dois ganchos para o filtro e sair do forno. </li><li> Instalar o tubo de bolsa condensador no suporte de tubagem. Feche a porta. </li><li> Rota linhas de adição A e B, ambas as linhas de comunicação social, e da linha de colheita por trás do sensor de OD e para o banco ao lado da unidade de controle biorreator. </li><li> Conecte os cabos dos sensores de OD e pH. </li></ol> 4. adição de meio e Micro-portadores <ol><li> Navegue até a guia &quot;ações&quot; e clique em &quot;Controle de Bombas&quot; na interface do computador. </li><li> Forme um Sterile conexão entre uma linha não utilizado meio disso (1 faixa laranja) e da fonte de garrafa / saco médio por soldagem do tubo ou usar acessórios Luer. </li><li> Clique na barra deslizante para ligar a bomba de mídia on. Clique na barra para transformar a mídia bomba desligada após a adição desejado quantidade de meio. </li><li> Colocar em esferas microtransportadoras Ca 2 +, Mg 2 + livre de PBS durante 3 horas à temperatura ambiente. </li><li> Lavar grânulos várias vezes com Ca2 +, Mg2 + livre de PBS. </li><li> Autoclave durante 15 minutos a 115 ° C, 15 psi. NOTA: Adicionar 3 g / L (peso seco) nesta experiência. </li><li> Bomba em micro-transportadores que tenham sido hidratadas, lavado e autoclavado para o reactor da mesma forma que o meio foi adicionado no passo 4.1. </li></ol> 5. Equilíbrio e de um ponto FAZER Calibração <ol><li> Defina os controladores para Auto e digite os setpoints desejados. Aqui, use Agitação Set Point (SP) = 15 rpm, temperatura SP = 37,0 ° C, pH = 7,2, DO = 100%. Aguarde até que o pARÂMETROS equilibrar. </li><li> Confirmar sensor está totalmente polarizado. Confirme DO valor presente estabilizou. </li><li> Navegue até a guia &quot;Ações&quot; e clique em &quot;calibrar&quot;. Clique em &quot;fazer um&quot; clique &quot;de um ponto&quot;. </li><li> Digite &quot;100&quot; no campo &quot;Span&quot;. Clique no botão &quot;Calibrar 1&quot;, clique em &quot;Save&quot; e clique em &quot;Fechar&quot;: </li></ol> 6 Iniciar uma execução <ol><li> Navegue até a guia Ações. Clique em &quot;Batch&quot;. Use o teclado na tela ou um teclado externo para digitar um nome de lote 16 caracteres ou menos. </li><li> Clique no botão &quot;Hide&quot; do teclado no ecrã. Clique no botão &quot;Iniciar lote&quot; confirmar clicando em &quot;Iniciar&quot; na sobreposição. </li></ol> 7 Inocular com células <ol><li> Formar uma conexão estéril entre um (1 banda de laranja) não utilizado linha além médio ea / source garrafa saco célula por meio de soldadurao tubo ou usar os acessórios Luer. </li><li> Instale a seção de silicone da tubulação na bomba de mídia para que a seta aponta para a tubulação entre a bomba eo reservatório. </li><li> Verifique braçadeira tubo é aberto e sua braçadeira de tubulação ramificada está fechado. Clique no controle deslizante para ligar a bomba de mídia on e clique em &quot;Off&quot; após a adição de células. </li><li> Navegue até a guia &quot;Ações&quot; e clique em &quot;Bombas de controle&quot;. </li></ol> 8 Amostragem <ol><li> Após inoculação e tão frequentemente quanto desejado para monitorar a cultura, retirar uma amostra a partir da cultura da seguinte forma: </li><li> Navegue até a guia &quot;ações&quot; e clique em &quot;tomar de exemplo&quot;. Coloque o tubo de amostragem na bomba de amostragem e manipular a válvula de amostragem de acordo com as instruções na tela. </li><li> Realizar pelo menos uma observação microscópica diária e contagem de células nesses exemplos. </li><li> Quando as células atingiram a densidade desejada, neste case de 1,2 x 10 6 células / ml, infectar as células com a adição de um inoculo de vírus. </li><li> Adicionar assepticamente o inoculo para uma seringa de 20 ml, e ligar a seringa para uma das portas de adição de peças no reactor. Introduzir o inoculo para o reactor por meio de pressão sobre o êmbolo da seringa. </li><li> Continuar a amostragem e análise da cultura para a concentração intracelular de partículas de adenovírus. </li></ol>

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells A Manual of Basic Techniques and Specialized Applications. , (2010).</li><li>Healthcare, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+Principles+and+Methods.">Microcarrier Cell Culture Principles and Methods.</a> GE Healthcare Data File 18-1140-62. , (2005).</li><li>Simaria, A. S., 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=Allogeneic+cell+therapy+bioprocess+economics+and+optimization+Single-Use+cell+expansion+Technologies.">Allogeneic cell therapy bioprocess economics and optimization Single-Use cell expansion Technologies.</a> Biotechnol and Bioeng. 111 (1), 69-83 (2014).</li><li>Lee, B., Fang, D., Croughan, M., Carrondo, M., Paik, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+novel+pneumatic+mixing+for+single-use+bioreactor+application.">Characterization of novel pneumatic mixing for single-use bioreactor application.</a> BMC Proc. 5 (S8), O12 (2011).</li><li>Eibl, R., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+in+cell+culture-based+upstream+processing.">Disposable bioreactors in cell culture-based upstream processing.</a> BioProcess Int. 7 (S1), 18-23 (2009).</li><li>Chaubard, J. F., 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=Disposable+bioreactors+for+viral+vaccine+production+challenges+and+opportunities.">Disposable bioreactors for viral vaccine production challenges and opportunities.</a> Biopharm Int Supp. , (2010).</li><li>Croughan, M. S., Hamel, J. F., Wang, D. I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Effects+on+Animal+Cells+Grown+in+Microcarrier+Cultures.">Hydrodynamic Effects on Animal Cells Grown in Microcarrier Cultures.</a> Biotechnol and Bioeng. 95 (2), 295-305 (2006).</li><li>DePalma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-use+Equipment+on+Cusp+of+Industrialization.">Single-use Equipment on Cusp of Industrialization.</a> Genet Eng Biotechnol News. 32 (1), (2012).</li><li>Baltz, R. H., Demain, A. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Industrial Microbiology and Biotechnology. , (2010).</li><li>Sousa, M. F., Giroux, D., Clemente, J., Lee, B., Carrondo, M. J., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impact of Bioreactor Design on the Performance of Microcarrier Cultures. , (2012).</li></ol>

Autores D. Giroux e Y. Hashimura são funcionários da PBS Biotech que produz reagentes e instrumentos utilizados neste artigo.

Este sistema de biorreatores de uso único é relativamente simples de usar e oferece análises em tempo real para o acompanhamento e análise do reator. É extremamente bem adequado para a cultura de células de mamíferos e insetos com densidades celulares atingindo mais de 30 milhões de células / ml. Além de células A549 descrito neste relatório 11, que cresceram células SF-9 de insecto em bioreactor bem. A mistura suave fornecido pela roda pneumática reduz os danos celulares. Vários passos são fundamentais quando configurou este reator. Primeiro calibração, adequado do pH e DO sensores é importante para a monitorização óptima da cultura e para a adição de reagentes para ajustar o pH, ou o oxigénio no sistema. Em segundo lugar, as garrafas de reagente e de sementes devem ser preenchidos e os acessórios luer feito num ambiente estéril, tal como um BSC. Uma vez que os frascos de reagentes são movidos para fora do ambiente estéril, as conexões com as linhas de alimentação de biorreatores deve ser feita com cuidado para evitar a contaminação microbiana. <pclass = &quot;jove_content&quot;&gt; Embora este sistema biorreator funciona bem para linhas celulares de mamíferos e insetos, não é projetado para culturas bacterianas. O sistema não pode fornecer a mistura rápida e oxigenação, que é necessária para as células bacterianas. O crescimento bacteriano é melhor realizado em um biorreator de tanque agitado. Em comparação com outros bio-reactores de utilização única para a cultura de células de mamífero ou de insecto, o sistema é fácil de usar, que fornece dados suficientes para análise de pistas, e tem um crescimento celular semelhante ou melhor do que o outro sistema de uso único, nós avaliamos. O uso único sistema biorreator pneumático tem o potencial de atender a muitas das pesquisas e aplicações clínicas nas áreas de biotherapeutics, vacinas, células-tronco e medicina personalizada 4. Além disso, a flexibilidade do sistema permite Batch, Fed-batch, Perfusão, e transfecção aplicações baseadas biorreator 5. Finalmente, de uso único sistemas de biorreatores descartáveis ​​têm o potencial para atender às necessidades da produção industrial em grande escala e para aderir às diretrizes e recomendações dos órgãos reguladores nacionais e internacionais 6-10.

Linhagens de células de mamíferos podem ser classificados em uma das três categorias com base em suas características de crescimento: as células que crescem em suspensão, as células que crescem como agregados, e as células que crescem ancorados a um substrato. Embora o biorreactor ar rodas demonstrado neste vídeo é capaz de crescer todos os três tipos de células, este vídeo vai demonstrar a utilização do biorreactor de cultivo de células dependentes de ancoragem em micro-transportadores. Células de mamíferos dependentes de ancoragem podem ser cultivadas para fins de produção de mais células - onde as próprias células são o produto. Por exemplo, derivadas da medula óssea as células estaminais mesenquimais humanas estão actualmente a ser cultivadas com a finalidade de recolher as células e injectando-os em tecido doente. O bioreactor pneumático demonstrado neste vídeo provou adequado para a produção de tais células estaminais mesenquimais para esta aplicação (Serra et al., Comunicação pessoal, 2013). Anchorage depAs células de mamíferos são geralmente cultivadas endent pequena escala em recipientes de cultura em 2D, como placas de cultura de células, frascos de cultura de células, ou garrafas de rolo, onde eles aderem a uma superfície de crescimento especialmente tratado 1. Quando as células são mais desejado, as placas ou frascos pode ser expandida através da utilização de mais ou maiores navios. No entanto, para o cultivo mais eficaz em termos de custos de grandes quantidades de células dependentes de ancoragem, aumentando a área de superfície para a fixação das células pode ser conseguida pela utilização de pequenas esferas sólidas chamados micro-transportadores. Dependendo das características de ligação da célula, vários tipos diferentes de micro-transportadores estão disponíveis comercialmente, tais como dextrano, péptido, ou colagénio revestido. Os micro-transportadores têm uma grande área de superfície em relação ao volume proporcionando uma maior área de superfície para o crescimento celular; e os micro-transportadores podem ser mantidas em suspensão, com agitação, o que permite que as células a ser cultivadas a elevadas densidades em sistemas de biorreactor 2. Atualmente, os tipos de bioreactors onde as células aderentes são cultivadas em micro-veículos incluem frascos spinner e sistemas de tanque agitado, que utilizam rotores axiais para manter uma suspensão de células revestidas micro-portadores. Vários fatores são importantes para o cultivo bem sucedido de células, incluindo a tensão de oxigênio, tensão de cisalhamento, matriz de superfície, e de nutrientes e as concentrações do metabolito. O uso de biorreatores permite o monitoramento em tempo real das condições de crescimento e potencial para significativamente mais baixos custos de produção 1. Existem vários modelos de biorreatores comum para o cultivo in vitro de células, incluindo, suspensão agitada, girando navio parede, de fibra oca, saco de biorreator em uma plataforma central, e sistemas de leito fluidizado 3. Muitos destes sistemas apresentam problemas específicos para o cultivo de células e escalar -up, como alto custo, gradientes de concentração de nutrientes, cisalhamento hidrodinâmico, agregação celular, e dificuldade de amostragem, monitoramento e controle cell scale-up. Várias linhas de células aderentes são utilizados na produção de vírus, quer na produção de vacinas virais ou para a produção de vectores virais para as aplicações de terapia génica. Neste vídeo, utilizando o uso único pneumático (Ar-roda) do sistema biorreactor, que demonstram a cultura de células de carcinoma de pulmão humano (A549) em células de micro-transportadores para a produção de um adenovírus oncolítico. O desenho biorreator pneumático usa uma roda de agitação vertical que é alimentado pela flutuabilidade de gás aspergido na parte inferior do bioreactor. Este método suave agitação limita as forças de cisalhamento hidrodinâmicos, mas ainda garante ótima médio e célula de mistura 4. Em comparação com o reactor de tanque com agitação, o reactor de pneumático tem uma baixa tensão de corte na parede, mesmo com grandes volumes de sistemas de reactores biológicos de ar-roda (Figura 1). Em contraste com o bio-reactores de tanque agitado, o rotor vertical de uso único, este reactor é ligado por uma corrente de bolhas de gás dentroo recipiente, o que permite uma suave e uniforme de mistura média (Figura 2).

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 3&#160;</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Use Assembly</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Human Lung Carcinoma Cells (A549)</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:119px;">&#160;CCL-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose Medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA, 0.25%</td>
			<td style="width:124px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytodex 1 Microcarriers</td>
			<td style="width:124px;">GE</td>
			<td align="right">3781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifoam C</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">A8011</td>
			<td></td>
		</tr>
	</tbody>
</table>

cultivation of mammalian cells using a single-use pneumatic bioreactor system

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Na Figura 3, os parâmetros para o início de funcionamento do bioreactor são mostrados. Esta figura mostra a tela antes de definir os parâmetros de temperatura, oxigênio dissolvido, pH e agitação. Uma vez que os parâmetros são definidos, os parâmetros de execução são continuamente monitoradas e ajustes podem ser feitos para manter as condições exigidas. O software produz uma leitura contínua, que permite a fácil identificação de problemas. Neste experimento usando 2,5 L de DMEM contendo 10% de FBS, 250 ppm de RdC Anti-espuma C, 2 mm, L-glutamina e 3 g / L de micro-transportadores, o sistema é estabilizado assim a percentagem de oxigénio é de 50%, o temperatura é de 37 ° C, e o pH é 7,2. O reactor é inoculado com células A549 a uma concentração de 7 x 10 4 células / ml (ou 10 células / micro-transportador). Os parâmetros escolhidos para esta corrida resultaram na maioria das células que aderiram às micro-transportadores dentro de 2 horas (Figura 4). Após 12 horas, as células são demôniostrating sinais de achatamento e espalhando sobre a superfície da micro-transportador (Figura 5). Por 24 horas, micro-portadores têm uma distribuição relativamente uniforme de células, sem micro-operadoras sem células e sem grandes aglomerados de células no micro-transportadoras (Figura 6). A percentagem de colonização de micro-transportador de células A549 é de 75% em 24 horas e 90% em seguida (Figura 7). As células continuou a crescer exponencialmente a ~ 1 milhão de células / ml, após 48 horas (Figura 8). Após a infecção por uma dose de adenovírus oncolítico (2 x 10 8 viriões / ml) a 50 h, o aumento da densidade de 1,2 milhões de células / ml e depois começou a diminuir à medida que a infecção progrediu lítico. Houve ~ 10000 vezes amplificação do inoculo virai. Em experiências anteriores, verificou-se que a monitorização e ajuste de fluxo de gás é crítico para a maximização do crescimento de células (dados não publicados). Rapidamente células de crescimento pode esgotar o sistema de oxigênio com<html> o nível de OD de cair para zero. Enquanto as células continuaram a crescer, foi a uma taxa muito mais lenta. É fundamental para monitorar e ajustar o fluxo de oxigênio para atender as demandas de células durante a fase logarítmica de crescimento. Cada corrida pode ser analisado para o crescimento ideal comparando o pH, OD e temperatura com a contagem de células diárias. <img alt="Figura 1" src="/files/ftp_upload/52008/52008fig1highres.jpg" /> Figura 1: Dinâmica dos fluidos do biorreator sistema pneumático (PBS) em comparação com um biorreator tanque agitador medir forças de cisalhamento de parede em vários volumes de reatores. <img alt="Figura 2" src="/files/ftp_upload/52008/52008fig2highres.jpg" /> Figura 2 impulsor reator pneumático Air-Wheel. <html> 52008 / 52008fig3highres.jpg &quot;/&gt; Figura 3 Pneumático Roda screen shot de parâmetros de software para iniciar o biorreator prazo. <img alt="Figura 4" src="/files/ftp_upload/52008/52008fig4highres.jpg" /> Figura 4 colonização Micro-portadora com células A549 2 horas após a inoculação. (Média de diâmetro micro-carrier ~ 180 m.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig4highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/52008/52008fig5highres.jpg" /> Figura 5 12 h após o início da cultura, as células A549 são anexando, achatamento, e espalhando sobre os micro-transportadores.&quot;Target =&quot; 5highres.jpg _blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 6" src="/files/ftp_upload/52008/52008fig6highres.jpg" /> Figura 6. Micro-portadores revestidas com células após 24 horas de cultura. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig6highres.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 7" src="/files/ftp_upload/52008/52008fig7highres.jpg" /> Figura 7 Percentagem de colonização de micro-transportador de células A549 após a inoculação. <img alt="Figura 8" src="/files/ftp_upload/52008/52008fig8highres.jpg" /> Figura 8 O número de células A549 viáveis ​​em cultura de pré e pós-infecção com adenovirnos. Note-se que o passo de infecção virai ocorreu em 50 horas.

Watch this Scientific Journal Video about Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System at JoVE.com

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

O cultivo de células de mamíferos utilizando um uso único sistema pneumático Bioreactor

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

cultivation-mammalian-cells-using-single-use-pneumatic-bioreactor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

24.3K visualizações.  Johns Hopkins University. Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. 

Vídeo: Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Engenharia Biológica Baseado em enxertos vasculares Usando um biorreator pulsátil

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Bioengenharia

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacteriana Detecção e Identificação Utilizando sensores eletroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Funcionamento de um biorreator de bancada

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Um biorreator Novel de Cultivo de Alta Densidade de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Um Orbital agitando a cultura de células de mamíferos em vasos em forma do produzir agregados uniformes

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Construção de Plasmids CRISPR e Detecção de Eficiência de Nocaute em Células Mamíferas através de um Sistema de Repórteres Dual Luciferase

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...