The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

NOTA: Este protocolo descreve a utilização de uma contra-pressão assistida sistema de distribuição robótico (Figura 1A) como uma superfície de gravação (Figura 1B) e bioprinter à base de extrusão (Figura 1C) 10. 1. Alteração de uma superfície de poliestireno <ol><li> Use 1 folhas mm de poliestireno desde placas de cultura de tecidos de poliestireno tendem a se curvar para cima no centro, arruinando a consistência altura de ambos gravura e impressão. NOTA: Como as folhas de poliestireno não são modificados para a adesão celular, o tratamento de plasma é realizado. </li><li> Oxigénio tratamento com plasma. <ol><li> Primeiro, selecione uma pressão de 2 bar no regulador do cilindro de oxigênio acoplado à máquina de plasma. Em seguida, desligar a máquina de plasma e a entrada das seguintes condições para o painel de controlo do equipamento: 150 W, 30 sscm de oxigênio, 10 min (para o poder, o fluxo de gás e tempo respectivamente). </li><li> Coloque o substrat poliestirenode e para a câmara de plasma, selar a porta e pressione o botão Iniciar no painel de controle. Embeber o plasma de oxigénio substrato de poliestireno tratado em soro fetal bovino e incubar a 37 ° C durante 2 h antes de lavar três vezes com fosfato 1x solução salina tamponada (PBS). </li></ol></li></ol> 2. Padrões de programação de impressão <ol><li> Usar o Programa para a Primeira Etch a superfície com um estilete ou bisturi. <ol><li> Quando se utiliza um estilete, inserir uma agulha têxtil diâmetro de 1,5 mm (a partir do interior com grande cuidado) para dentro do bocal de uma seringa de distribuição (5 ou 10 ml) até que se torne em cunha e protegidas. Se usando um bisturi, escolher um bisturi rodada manipulados de modo que pode ser fixado no mecanismo de fixação do braço de impressão. NOTA: A caneta grava padrões curvos melhor do que a lâmina de bisturi. </li><li> Quando a primeira tentativa de criar um arranjo bioprinted, esboçar o padrão desejado no papel de gráfico com eixos numerados para gerar as coordenadas XY. Então eunput as coordenadas gravado padrão / bioprinted para o software de planilhas (Figura 2). NOTA: O &quot;padrão desejado&quot; pode ser em muitas formas, tais como linear, em forma de S, ou circular. A escolha depende do modelo experimental necessária, como linear linhas paralelas para a diferenciação das MSCs para cardiomiócitos, como demonstrado aqui. A função de gráfico de dispersão permite a visualização do padrão enredo proposto. </li><li> Abra o / software de distribuição de impressão. Selecione &quot;Programa&gt; Adicionar programa&quot; seguido de &quot;Editar&gt; Adicionar ponto&quot; para configurar o programa. Exportar o x e y valores de coordenadas obtidas a partir da folha de cálculo para a impressão / distribuição de software usando o &quot;Copiar e colar&quot; a função. </li><li> Calibrar a altura de &quot;z&quot; do robô antes de cada execução, a fim de colocar o bico stylus / impressão sobre a superfície. <ol><li> Na cópia do software / distribuição, selecione a opção &quot;Robot&quot;, clique em &quot;Alterar Modo &amp;# 34; e habilitar a opção &quot;Modo de Ensino&quot;. Uma vez selecionado, o software permite a função &quot;Jog&quot; do robô. </li><li> Para correr, primeiro inicializar o robô para sua posição padrão, selecionando os seguintes comandos a partir da barra de menu; &quot;Robot&gt; Meca Inicializar&quot; e selecione &quot;Robot&gt; Jog&quot;. Introduzir os valores numéricos (em mm) no &quot;slots de X e Y&quot; necessários para colocar a caneta exactamente sobre a origem do padrão. </li><li> Em seguida, a entrada de um valor numérico (em mm) no &quot;slot de Z&quot; para colocar o bico de caneta ou impressão em contato com a superfície, mas não flexível ou recuar a superfície. Este ponto é designado como &quot;Z&quot; valor de partida. NOTA: A profundidade de cada ranhura podem ser variadas facilmente utilizando o Z-altura do sistema. As ranhuras de 40, 80, e 170 ^ M são demonstrados para cortar a superfície de 1 mm de espessura folhas de poliestireno (Figura 4 e Tabela 1). </li></ol></li><li> Selecione o pinstruções rint para cada um dos pontos coordenados, ou seja, &quot;o início da linha Dispensar&quot; para definir o primeiro ponto eo início de impressão, &quot;Linha de Passe&quot; para designar os pontos intermédios e &quot;Fim da Linha Dispensar&quot; para sinalizar para o robô para terminar a Tiragem. </li><li> Comunique-se o programa para o robô, selecionando os comandos acompanhamento a partir da barra de menu superior: &quot;Robot&gt; Enviar C &amp; T de dados&quot;. </li><li> Iniciado o condicionamento run / impressão, alterando o robô para o modo &quot;Run&quot;. Faça isso selecionando &quot;Robot&gt; Modo Mudando&gt; Transferir o modo Run&quot; na barra de menu superior. </li><li> Iniciar o procedimento de impressão, premindo o botão &quot;start&quot; verde na consola dispensador robô. </li></ol></li></ol> 3. Preparação e Impressão de Gelatina Bioink contendo celular <ol><li> Dissolve-se 2% de gelatina em Meio Mínimo Essencial alfa Médio (aMEM) (suplementado com 10% de FBS e 2% de antibiótico / antimycotIC) a 60 ° C durante 2 horas para preparar as soluções bioink. </li><li> Pré-cultura da proteína vermelha fluorescente expressando células-tronco mesenquimais (RFP-MSCs) a 70% de confluência em 10 cm placas de cultura de tecido, utilizando aMEM / 10% FBS. Libertar as células ligadas em suspensão por remoção do meio de revestimento e com uma solução de tripsina-EDTA 1x durante 5 min a 37 ° C. </li><li> Agregar as células por centrifugação a 1000 xg durante 5 min e remover o sobrenadante. Ressuspender o sedimento de células em 0,5 mL de PBS 1x e contar a densidade de células utilizando um hemocitómetro. </li><li> Depois de deixar a bioink arrefecer até à temperatura ambiente, misturar suavemente num volume suficiente para a suspensão de RFP-MSCs no bioink para atingir uma concentração final de 5 x 10 6 mL -1 células. </li><li> Verter a bioink rolamento de células dentro de uma seringa de impressão com o bocal fechado. Arrefecer a seringa carregada a 4 ° C a fim de atingir uma viscosidade de impressão. </li><li> Colocar a seringa carregada no automatizadosistema de distribuição de robótica e anexar as linhas de pressão de ar. Retire a seringa Luer selo e anexar o bocal de impressão. </li><li> Faça a extrusão do bioinks celularizados em linhas finas usando 0,05 MPa pressão de retorno, a 5 mm / seg velocidade de escrita a partir de uma seringa de 10 ml através de um 27 G agulha / bico (afuniladas recomendado sobre cilíndrica) sobre uma superfície de película de poliestireno, após a deposição pré-programado padrão descrito no passo 2.1 a colocar o bioink celularizado para as ranhuras pré-gravadas. </li><li> Após incubação de 1 h à temperatura ambiente, adicionam-se 10 ml de meio de crescimento (suplementado com 10% FBS e antibióticos) e incuba-se as células durante 24 h (para permitir que as células para detectar e reagir às características gravadas) antes da visualização usando um microscópio de fluorescência invertido a 10X de ampliação. </li></ol>

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The authors declare they have no competing financial interest.

O passo crítico deste procedimento é a entrega bioprinting efectiva das células estaminais enquanto o processo deve permitir a sedimentação de células para as características, de impressão sem bioink espalhando / hemorragia, proporcionam células sem morte celular tensão de cisalhamento e não desencadeiam a diferenciação no sentido linhagem indesejado. Se o alinhamento de células esperado não ocorrer, então a viscosidade bioink deve ser avaliado quanto à sua aptidão para a i...

A padronização deliberada de posicionamento de célula permite a formação de culturas que imitam in vivo uma organização celular. Na verdade, a investigação sobre a interacção entre vários tipos de células pode ser assistido por organizar a sua 2,3 colocação espacial. A maioria dos sistemas de padrões dependem de procedimentos de modificação de superfície para promover ou prevenir a adesão celular com a deposição de células passiva subseqüente. Bioprinting oferece controle espacial e temporal sobre distribuições de células 1. Além destas funções, bioprinting foi descrito como sendo um método tecnicamente simples, rápido e de baixo custo para a geração de andaimes geometricamente complexas 4. Ele utiliza software de design de computador e permite a introdução de células para o processo de fabricação 4. Sistemas bioprinting foram classificados com base em seus princípios de trabalho como laser baseados, à base de jato de tinta ou extrusão baseados em 4. Extrusão bioprinting foi descrito como o mais promissor, uma vez que permite a fabricação de construções organizados de tamanhos clinicamente relevantes dentro de um período de tempo realista 4-6. É realizada por qualquer pressão mecânica ou para trás extrusão assistida de um hidrogel bioink rolamento celular. No método aqui apresentado, foi empregue a pressão de retorno. Como mencionado, as células são entregues em um bioink cytocompatible. Tal bioink deve suportar a entrega de células sem produzir esforço de corte deletério, e ser de uma viscosidade suficiente para manter a integridade do traço impressa, sem entrar em colapso ou espalhamento (referido como &quot;tinta de sangria&quot;) 7-10. A interacção das células com a superfície aderente é conhecido para influenciar o comportamento celular. A topografia da superfície pode controlar a forma das células, orientação 11, e ainda o fenótipo. Em particular, a fabricação de sulcos e canais têm demonstrado induzirum esticado, morfologia alongada em vários tipos de células. A adopção desta morfologia foi encontrado para influenciar o fenótipo de células multipotentes e pluripotentes. Por exemplo, quando alinhadas em ranhuras, as células estaminais mesenquimais (MSC) mostram evidências de diferenciação em direcção cardiomiócitos 12,13 e células musculares lisas vasculares adoptar o fenótipo contráctil sobre o sintética 10,14-17. A célula alinhando canais ou ranhuras pode ser gerado numa superfície polimérica por meio de uma série de métodos, por exemplo, gravação iónica reactivo profundo, litografia de feixe de electrões, impressão a laser directo, femtosecond laser, fotolitografia e ataque químico de plasma seco 18. Estas abordagens são frequentemente demorados, exigem aparelhos complexos e podem ser limitante na forma do padrão gerado. Além disso, eles não sincronizar padronização com bioprinting e não permitem celularização imediata. O movimento coordenado controlada de um automatizadoSistema de distribuição pode seguir padrões complexos para a deposição de soluções. Aqui demonstramos como o movimento controlado por microescala pode ser aproveitada para criar canais para orientação de células. Uma caneta ou bisturi afiada está ligado à cabeça de impressão em lugar da seringa e o equipamento de extrusão pode, em seguida, gravar a superfície de polímero sob a orientação do software trama. O método oferece uma versatilidade na concepção padrão e é aplicável a materiais poliméricos normalmente usados ​​em bioengenharia, tais como poliestireno, de PTFE, e policaprolactona. Como um passo subsequente para a decapagem, as células podem ser bioprinted directamente aos sulcos riscados. O bioink gelatina utilizada aqui foi capaz de manter tanto o traço e permitir que as células depositadas para detectar as características gravadas. As células estaminais mesenquimais bioprinted aos sulcos gravados foram demonstrados para alongar ao longo delas em linhas distintas.

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			<td style="width:104px;">Covance</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">USB Microscope</td>
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			<td style="width:104px;"></td>
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			<td height="21" style="height:21px;width:151px;">Optical Microscope</td>
			<td style="width:131px;">Olympus</td>
			<td style="width:104px;">IX71</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
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			<td height="21" style="height:21px;width:151px;">Software</td>
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			<td height="21" style="height:21px;width:151px;">Spreadsheet</td>
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			<td style="width:104px;">Excel</td>
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			<td height="41" style="height:41px;width:151px;">Printing Co-ordinate Software</td>
			<td style="width:131px;">Janome</td>
			<td style="width:104px;">JR C-Points</td>
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			<td height="41" style="height:41px;width:151px;">Imaging Software</td>
			<td style="width:131px;">National Institutes of Health (NIH)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Equipment</td>
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			<td height="21" style="height:21px;width:151px;">Stylus (Blade)</td>
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			<td style="width:104px;">AK-5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">5ml printing syringe</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH10LL-B</td>
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			<td height="21" style="height:21px;width:151px;">30G printing needle</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH30-0.25-B</td>
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			<td height="41" style="height:41px;width:151px;">1mm polystyrene sheets</td>
			<td style="width:131px;"></td>
			<td style="width:104px;">Purchased locally</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Fetal bovine serum</td>
			<td style="width:131px;">Invitrogen&#160;</td>
			<td style="width:104px;">10270-098</td>
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			<td height="41" style="height:41px;width:151px;">Phosphate buffered saline</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;"></td>
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			<td height="61" style="height:61px;width:151px;">Gelatin from porcine skin, Gel strength 300, Type A</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">9000-70-8</td>
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			<td height="21" style="height:21px;width:151px;">&#945;MEM</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;">41061-029</td>
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			<td height="21" style="height:21px;width:151px;">Antibiotc antimycotic</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">A5955-100ML</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:151px;">Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td>
			<td style="width:131px;">Cyagen Biosciences Incorporation</td>
			<td style="width:104px;">RASMX-01201</td>
			<td></td>
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automated robotic dispensing technique for surface guidance and bioprinting of cells

Este manuscrito descreve a introdução de características de orientação de células seguindo-se a entrega directa de células para estas funcionalidades de uma bioink hidrogel utilizando um sistema de distribuição robotizado e automatizado. O bioink particular foi selecionado como ele permite que as células para sedimentar direção e sentido os recursos. O sistema de distribuição bioprints células viáveis ​​em bioinks hidrogel usando uma cabeça de impressão de contrapressão assistida. No entanto, através da substituição da cabeça de impressão com um estilete ou bisturi afiado, o sistema de distribuição também pode ser empregada para criar pistas topográficas através de condicionamento de superfície. O movimento caneta pode ser programada em passos de 10 um nos sentidos de X, Y e Z. Os sulcos estampados foram capazes de orientar células-tronco mesenquimais, influenciando-os a adotar uma morfologia alongada em alinhamento com a direção dos sulcos. O padrão pode ser concebido usando o software de plotagem em linhas retas, círculos concêntricos, e ondas senoidais. Em um processo subsequente, fibroe blastos de células estaminais mesenquimais foram suspensos numa bioink gelatina a 2%, para bioprinting num cabeçote de extrusão accionado contrapressão. O bioink rolamento de células foi então impressa utilizando as mesmas coordenadas programadas utilizados para a decapagem. As células bioprinted foram capazes de detectar e reagir às características gravadas como demonstrado pela sua orientação alongada ao longo da direcção das ranhuras gravadas.

Os resultados representativos demonstram que o sistema de dispensa robótico contrapressão assistida pode ser usado como um bioprinter à base de extrusão para a realização de ambos gravura e impressão bioink superfície (Figura 1 A). Ele pode ser usado para a geração de ranhuras gravadas em superfícies de polímeros, e, subsequentemente, para imprimir uma bioink rolamento célula directamente para as características (Figura 1 B e C). <p cla...

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Técnica Automated Robotic dispensação de Orientação da superfície e bioprinting de Células

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

automated-robotic-dispensing-technique-for-surface-guidance

Research

JoVE Journal

Bioengineering

6.3K visualizações.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Vídeo: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Bioengenharia

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting celularizado Constrói Usando um específico do tecido Hidrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Robotic Espelho Sistema de Terapia de recuperação funcional de hemiplégicos Braços

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robótico líquido manipulação montagem de dispositivos modulares DNA automática

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de microfluidic Bioprinting para a engenharia de tecidos e Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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 ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Manipulação de células-tronco neurais únicas e neurônios em fatias cerebrais usando microinjeção robótica

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Bioimpressão 3D de astrócitos cortical de murina para engenharia de tecido neural

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.