Este trabalho é financiado pelo National Institutes of Health Grant R01 EB-008836 e R01 HL083895 (tanto para LEN). Poderíamos agradecer a Daryl Smith, o Glassblower University, para fazer o biorreatores para a nossa pesquisa.

Autoclave Montar e autoclave a tubulação para o sistema de fluxo e componentes biorreator (biorreator si ea tampa rolha de silicone) conforme as instruções na Figura 1 e Figura 2. Alimentação por sonda tem um conector macho em uma extremidade e uma extremidade aberta do outro lado. Três segmentos de tubos curtos são inseridos através de uma tampa de silicone para a troca de gás. 1. Costura PGA Malha <ol><li> Corte PGA malha para 1,1 centímetros x ~ folha de 8 cm (dependendo do tamanho biorreator). </li><li> Tubo de silicone Limpo (diâmetro interno de 3 mm) com água destilada (Dh20) e ar seco antes do uso. </li><li> Use sutura Dexon 6,0 a costurar PGA mesh ao redor do tubo de silicone limpa começando com três nós cirúrgico seguido de pontos simples. </li></ol> 2. PGA Andaimes Tratamento de Superfícies <ol><li> Dip andaimes PGA em NaOH 1M por 1-2min e registrar o tempo de tratamento e usar o mesmo tempo para todos os andaimes PGA. </li><li> Enxágüe PGA andaimes em banhos dH2O por 2 minutos 3 vezes. </li><li> Pat PGA andaimes seco com Kimwipes entre cada mergulho na dH 2 banheiros O. </li><li> Seca PGA andaimes sob a capa de cultura de tecidos para o ar seco por 15 minutos com ventilador ligado. </li></ol> 3. Costura braços Dacron <ol><li> Use sutura Prolene 4.0 para costurar pequenos pedaços de cuffs de Dacron (1cm) em cada extremidade da malha PGA com uma sobreposição de 2-3mm. Nota: tenha cuidado para não perfurar tubos de silicone (Figura 3). </li><li> Use a sutura mesmo a costurar três pontos ao redor da extremidade livre de algemas Dacron. Certifique-se de deixar suturas suficiente nas extremidades livres de sutura Prolene para o passo 5.1 (Figura 3). </li></ol> 4. Montagem de biorreator (dia antes do início da cultura biorreator) <ol><li> Mergulhe malha costurada e tubos de silicone, instrumentos cirúrgicos, e um fio fino em um banho de etanol 70% por 20-30 minutos. </li><li> Abrir a autoclave bolsas contendo o biorreator e submergir-lo em banho de etanol 70% por pelo menos 30 minutos antes da montagem. Certifique-se de lavar o biorreator exaustivamente com etanol. Exposição de todos os componentes biorreator para o etanol 70% é um passo além da esterilização. Esta etapa também remove endotoxinas, que não é removido por autoclavagem e é prejudicial para as células vasculares. </li><li> Puxe tubo de silicone pelo lado de armas usando fio fino para retirá-lo completamente. </li><li> Tome biorreator de banho de etanol (mesh manter submerso em etanol). Corrigir o andaime PGA dentro do biorreator pela fixação cuffs de Dacron sobre os lábios de vidro queimado apertando e amarrar as suturas Prolene (Figura 3). </li><li> Conecte um lado de braços laterais biorreator para conectores através de tubos de silicone. </li><li> Puxe outro lado da tubagem de silicone com tensão suficiente e inserir os restantes dois conectores para tubos de silicone. Segure-se em tubo de silicone firmemente ao inserir conectores! </li><li> Reinserir biorreator em banho de etanol e lave com etanol puxando conectores para fora dos braços laterais. </li><li> Virar biorreator mais e permitir que um molho por 10 minutos. </li><li> Biorreator virar o lado direito para cima e deixe de molho por mais 10 minutos. </li><li> Drene todo o etanol. </li><li> Criou três grandes pratos de Petri (10 cm) em série e biorreator lugar no prato centro para segurar o excesso de etanol pingava das extremidades dos tubos. </li><li> Lave biorreator e PGA malha com água de cultura de tecidos utilizando um 5ml ou 10ml pipeta. Também a água lave cultura de tecidos em tubos de silicone. </li><li> Completamente drenar toda a água em excesso em placas de Petri de cada lado do biorreator. </li><li> Biorreator secar durante a noite na capa com ventilador ligado e OFF UV. </li><li> Notas adicionais: certifique-se barra de agitação estéril é em biorreator. Certifique-se de não &quot;pairar&quot; sobre o biorreator deste ponto em diante, para evitar a contaminação. Corta-se a abundância de tiras de parafilme e mergulhe-os em um banho de etanol 70% pequenas (placa de Petri grandes funciona bem). </li></ol> 5. Dia 1: Configuração biorreator <ol><li> Coloque uma placa de Petri estéreis sobre abertura de cada biorreator para proteger a PGA andaime dentro de contaminantes. </li><li> Montar sistema de fluxo de biorreator, como indicado na figura 4 e parafilme todas as juntas de ligação. <ol><li> Limpe os conectores primeiro com algodão embebido em álcool. </li><li> Anexar porta de injeção no braço, o terceiro não utilizada do biorreator. </li><li> Remover tubos morsa e gravata azul off final da diluição salina definida como perto de Y-junção possível. Puxe fixação de tubo no local para garantir que não haja transferência de líquidos a esta parte do tubo </li><li> Remover saco IV, e anexar tubulação morsa (vermelha) para até final de saco IV. Certifique-se de limpar porta de inserção com álcool limpe primeiro. </li><li> Walrus anexar a um lado do sistema de fluxo (via tubo branco final). </li><li> Inserir torneira de 3 vias no sistema de fluxo. </li><li> Remover transdutor de pressão e conectar-se a torneira de três vias. </li><li> Ligue a outra extremidade do transdutor de pressão para abertura central no saco. </li></ol></li><li> Conectaro biorreator para o sistema de fluxo através do Y-junction. Use uma seringa de 60ml para adicionar 350ml de 1 Fungizone% (Fungizone mistura de 5 ml com 495ml de PBS) para bolsa de soro. </li><li> Aperte o saco IV para lavar o sistema de fluxo, ajustando os galos parar para permitir o fluxo de PBS. Nota: verificar dentro biorreator para garantir que não haja vazamento. </li><li> Re-suspender 8 x10 6 células musculares lisas (cerca de um confluentes T75) em 1.25ml de médio e de sementes em cada scafffold PGA. Certifique-se de suspensão de células tem sido uniformemente pingou sobre a junção PGA malha de Dacron, bem como para o lado inferior da PGA malha. </li><li> Limpe borda do biorreator com álcool limpe via rotação do biorreator para os lados evitar pairando. </li><li> Silicone conjunto da tampa rolha (Figura 2) <ol><li> Peel saco de autoclave para trás com cuidado, tomando cuidado para não expor a parte inferior da tampa. </li><li> Anexar porta de injeção ao tubo de alimentação no conector macho. </li><li> Anexar PTFE 0,20 mM filtros a cada uma das três portas ar. </li><li> Tenha cuidado para não expor / toque de fundo da tampa durante este processo. </li><li> Parafilm a porta de injeção. </li></ol></li><li> Insira a tampa rolha de silicone em biorreator de vidro e certifique-se que o tubo de alimentação no interior do biorreator não toca o andaimes semeado PGA. Parafilme em torno da tampa. </li><li> Lugar biorreator com o sistema de fluxo dentro da incubadora (de lado) e gire o biorreator a cada 5 minutos por 25-30 minutos. </li><li> Encher a câmara de biorreator com 400ml de nossos meios de cultura 10/04, conforme descrito no (Quadro 1). Este meio de cultura é &quot;otimizado&quot; para as artérias suína engenharia. </li></ol> 6. Dia 07/06: Ligar a bomba e primeira alimentação <ol><li> Crescer os andaimes semeado estaticamente, sem qualquer pulsátil bombeamento através do tubo de silicone para 07/06 dias. Não há necessidade de mudança médio ou suplementação de vitamina C durante este tempo. </li><li> Certifique-se que não há fugas de PBS ou dobras da tubulação do sistema de fluxo antes de ligar a bomba. </li><li> Ligue a bomba do sistema de fluxo e certifique-se de ajustar a configuração da bomba para que a pressão lê aproximadamente 270/-30mmHg. </li><li> Pressões registro diário em toda a cultura e manter a pressão na 270/-30mmHg. Transdutor de pressão pode ser conectado a um computador para ler e monitorar a pressão. </li></ol> Alimentação primeira <ol start="5"><li> Montar a injeção porta e PTFE filtro para alimentação de tampas tanto para a mudança de médio e médio fins resíduos destituição. </li><li> Coloque o tubo de alimentação firmemente na bomba e coloque uma extremidade à porta de alimentação do biorreator ea outra extremidade à tampa de alimentação. Certifique-se de limpar as portas de injeção com toalhetes de etanol. </li><li> Usar um dual-direcional bomba Masterflex a bomba de fora 200ml de meio. Em seguida, use um tubo de alimentação nova bomba de 200ml de meio fresco de volta para o biorreator. Sempre começar com uma velocidade muito lenta, especialmente quando o bombeamento de volta para o meio biorreator. </li><li> Mudança de médio e complementar 2x/semana ácido ascórbico. Para adicionar o ácido ascórbico, use uma seringa de 30ml para tirar 25ml de meio e colocá-la de lado na capa de tecido. Dissolver 25 mg de ácido ascórbico em 5ml de PBS e filtrá-la através de um filtro de 0,22 mM. Primeiro injetar o ácido ascórbico estéril no tubo de alimentação e adicionar novamente o meio de 25ml retirado anteriormente. A receita média é dada na Tabela 1. </li></ol> 7. Resultados representativos: <img src="/files/ftp_upload/2646/2646fig1.jpg" alt="Figura 1"/> Figura 1. A tubulação e conectores para a montagem do sistema de fluxo é mostrado acima. <img src="/files/ftp_upload/2646/2646fig2.jpg" alt="Figura 2"/> Figura 2. O conjunto da tampa rolha de silicone é mostrado acima. <img src="/files/ftp_upload/2646/2646fig3.jpg" alt="Figura 3"/> Figura 3. Esquemas de montagem biorreator são mostrados acima. Dentro do cuffs de Dacron biorreator são fixados para os braços de vidro com os nós de sutura azul. <img src="/files/ftp_upload/2646/2646fig4.jpg" alt="Figura 4"/> Figura 4. Sistema de fluxo conectado à tubulação e biorreator é mostrado acima. L/S18 tubulação será bombeada por uma bomba Masterflex e, portanto, dirigir o fluxo. O transdutor de pressão medirá a pressão antes de entrar no biorreator de fluxo superior. <img src="/files/ftp_upload/2646/2646fig5.jpg" alt="Figura 5"/> Figura 5. Imagem do navio colhido de engenharia. Vasos Engineered aparecerá para ser opaco e atingir uma espessura de parede de aproximadamente 250μm após 8 semanas de cultura em condições pulsátil. <img src="/files/ftp_upload/2646/2646fig6.jpg" alt="Figura 6"/> Figura 6. Hematoxilina e eosina secções transversais dos vasos de engenharia. A e B são de 8 semanas não-pulsado e pulsado vasos, respectivamente.C e D são quatro semanas de embarcações não-pulsado e pulsado, respectivamente. L indica o lado luminal dos vasos. A barra de escalas é 100μm. <img src="/files/ftp_upload/2646/2646fig7.jpg" alt="Figura 7"/> Figura 7. Manchas tricrômico de Masson para colágeno (azul) para seções transversais dos vasos de engenharia. A e B são de 8 semanas, os navios não-pulsado e pulsado, respectivamente. C e D são quatro semanas de embarcações não-pulsado e pulsado, respectivamente. Note-se que o navio de 4 semanas pulsado mostra mais colágeno do que suas contrapartes não-pulsado. Setas brancas apontam para fragmentos restantes PGA nos vasos. A barra de escalas é 100μm. <img src="/files/ftp_upload/2646/2646fig8.jpg" alt="Figura 8"/> Figura 8. Coloração imunoquímica de marcadores SMC em bovinos artérias de engenharia. Músculo liso α-actina, calponin-1, e de músculo liso cadeia de miosina pesada (SMMHC) são precoces, intermediárias e tardias SMC marcadores contrátil, respectivamente. Até o final de 12 semanas de cultura, as células da parede do vaso expressar SM α-actina e quantidades moderadas de Calponin-1 e SMMHC. A barra de escalas é 20Hm. <table border="1"><tbody><tr><td> Componente </td><td> Quantidade </td></tr><tr><td> DMEM (DME / baixa modificado) </td><td> 500 ml </td></tr><tr><td> FBS (soro fetal bovino) inativados pelo calor </td><td> 100 ml </td></tr><tr><td> HEPES 1,0 M </td><td> 5ml </td></tr><tr><td> Vitamina C (dissolvido em PBS ou DMEM) </td><td> 25 mg </td></tr><tr><td> Prolina / Glycine / Alanina 25 mg/25 mg/10 mg (dissolvido em 5ml de PBS) </td><td> 5ml </td></tr><tr><td> CuSO 4 1,5 g (dissolvido in1 ml de PBS) </td><td> 1ml </td></tr><tr><td> Penicilina G a 10.000 unidades / ml </td><td> 5ml </td></tr><tr><td> PDGF-BB (derivado de plaquetas-BB growth factor) em 10ng/ml </td><td> 5μg </td></tr><tr><td> bFGF (fator de crescimento básico de fibroblastos) em 10ng/ml </td><td> 5μg </td></tr></tbody></table> Tabela 1. Componentes de &quot;40-10&quot; médio são apresentados na tabela acima. Com a exceção de PDGF-BB e bFGF, todos os outros componentes estão a ser filtrado por um filtro de 0,2 Hm antes de usar.

<ol>
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A qualidade dos navios engenharia é em grande parte ditada pela qualidade do SMCs usado em cultura de tecidos. Os aspectos críticos do fenótipo contrátil SMC incluem morfologia, número de passagem baixa, ea capacidade de proliferar no interior do biorreator. Recomendamos que o número de passagem pode ser maior que P3, no momento da semeadura de células para o cadafalso polímero. Além disso, é crucial para confirmar que as fontes de SMC são livres de micoplasma antes do uso. Temos observado que as células con...

<table border="1"><tbody><tr><th> Nome do reagente / Material </th><th> Fornecedor </th><th> Número Catálogo </th></tr><tr><td> FBS (soro fetal bovino), inativado pelo calor </td><td> HyClone </td><td> SH30071 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11885 </td></tr><tr><td> rhFGF básica </td><td> R &amp; B </td><td> 234-FSE </td></tr><tr><td> rrPDGF-BB </td><td> R &amp; B </td><td> 520-BB </td></tr><tr><td> Penicilina G </td><td> Sigma </td><td> PENNA </td></tr><tr><td> Sulfato de cobre (II) </td><td> Sigma </td><td> C8027 </td></tr><tr><td> Gylcine </td><td> Sigma </td><td> C8790 </td></tr><tr><td> L-alanina </td><td> Sigma </td><td> A7469-25G </td></tr><tr><td> L-Proline </td><td> Sigma </td><td> P5607-25G </td></tr><tr><td> Ácido ascórbico </td><td> Sigma </td><td> A4544-25G </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375-100G </td></tr><tr><td> Stopper de silicone </td><td> Cole-Parmer </td><td> 06298-24 </td></tr><tr><td> Masterflex tubos L / S </td><td> Cole-Parmer </td><td> 06508-16, 06508-18 </td></tr><tr><td> Masterflex bomba </td><td> Cole-Parmer </td><td> 7553-80 </td></tr><tr><td> Cuff de Dacron </td><td> Maquet </td><td> 174406 </td></tr><tr><td> PGA sentiu </td><td> Concordia </td><td> MO000877-01 </td></tr><tr><td> 4-0 1,5 métrica Surgipro II sutura </td><td> Syneture </td><td> VP-557-X </td></tr><tr><td> 6-0 0,7 sutura Dexon métrica </td><td> Syneture </td><td> 7538-11 </td></tr><tr><td> 0.22μm PTFE filtros </td><td> Whatman </td><td> 6780-2502 </td></tr><tr><td> Três Way Parar cock- </td><td> Edwards Lifesciences </td><td> 593WSC </td></tr><tr><td> Transdutor de pressão </td><td> Edwards Lifesciences </td><td> PX212 </td></tr><tr><td> Sacos IV </td><td> Baxter </td><td> R4R2110 </td></tr><tr><td> Conjunto de diluição salina </td><td> Seta </td><td> W20030 </td></tr><tr><td> Tubo de silicone </td><td> Saint-Gobain </td><td> F05027 </td></tr></tbody></table>

engineering biological-based vascular grafts using a pulsatile bioreactor

Muito esforço tem sido dedicado a desenvolver e fazer avançar a metodologia para regenerar funcional de pequeno diâmetro arterial ignora. No ambiente fisiológico, tanto a estimulação mecânica e química são necessários para manter o bom desenvolvimento e funcionalidade de 1,2 vasos arteriais. Sistemas de cultura biorreator desenvolvido pelo nosso grupo são projetados para suportar a regeneração navio dentro de um ambiente controlado com precisão químico-mecânico que imita a de vasos nativos. Nossa assembléia biorreator e procedimentos de manutenção são bastante simples e altamente repetitivo 3,4. Células musculares lisas (PMEs) são semeados em um ácido poliglicólico tubular (PGA) de malha que é rosqueado ao longo tubo de silicone compatível e cultivadas em biorreator, com ou sem estimulação pulsátil por até 12 semanas. Há quatro principais atributos que distinguem o nosso biorreator de alguns antecessores. 1) Ao contrário dos sistemas outra cultura que simulam apenas o bioquímico em torno dos vasos sanguíneos nativo, nosso biorreator também cria um ambiente fisiológico pulsátil, aplicando tensão radial cíclica para os vasos em cultura. 2) Várias embarcações de engenharia podem ser cultivadas simultaneamente em diferentes condições mecânicas dentro de um ambiente químico controlado. 3) O biorreator permite uma camada mono de células endoteliais (CE) para ser facilmente revestidas no lado luminal dos vasos de engenharia para implantação de modelos animais. 4) Nossas biorreator também pode vasos cultura projetada com diâmetro diferentes variou de 1 mm a 3 mm, salvando o esforço para adaptar cada biorreator individuais para caber um diâmetro específico. Os vasos engenharia cultivadas em nosso biorreator assemelham vasos sanguíneos nativa histologicamente em algum grau. Células nas paredes dos vasos expressam marcadores madura SMC contrátil, como suave cadeia muscular miosina pesada (SMMHC) 3. Uma quantidade substancial de colágeno é depositado dentro da matriz extracelular, que é responsável pela resistência mecânica final das embarcações engenharia 5. Análise bioquímica também indica que o conteúdo de colágeno dos vasos engenharia é comparável à de artérias nativas 6. Importante, o biorreator pulsátil tem constantemente regenerado vasos que apresentam propriedades mecânicas que permitem experiências de implantação bem sucedida em modelos animais 3,7. Além disso, este biorreator pode ser modificado para permitir a avaliação em tempo real e controle de remodelação do colágeno ao longo do tempo, de forma não invasiva, utilizando um microscópio óptico não-linear (NLOM) 8. Para concluir, este biorreator deve servir como uma excelente plataforma para estudar os mecanismos fundamentais que regulam a regeneração funcional de pequeno diâmetro enxertos vasculares.

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Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Nosso grupo desenvolveu um sistema de cultura biorreator que imita as tensões pulsátil fisiológica do sistema cardiovascular para regenerar implantáveis ​​de pequeno diâmetro enxertos vasculares.

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 ...

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Bioengineering

16.9K visualizações.  Yale University. Nosso grupo desenvolveu um sistema de cultura biorreator que imita as tensões pulsátil fisiológica do sistema cardiovascular para regenerar implantáveis ​​de pequeno diâmetro enxertos vasculares.

Vídeo: Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

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  ...

Bioengenharia

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 ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Construção e Caracterização de um biorreator Fold Novel Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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.

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 ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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 ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

guiado por imagem, fabricação à base de laser de Redes microfluídicos Vascular derivadas

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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.