Esta pesquisa foi parcialmente financiado pela National Science Foundation (NSF 1227359), WVU programa EPSCoR financiado pela National Science Foundation (EPS-1003907), WVU escritório ADVANCE patrocinado pela National Science Foundation (1.007.978), e WVU PSCoR, respectivamente. O trabalho foi feito em microfabricação WVU compartilhados Instalações de pesquisa (instalações de salas limpas) e Microfluidic Integrative Research celular no Laboratório Chip (microchip Lab) na West Virginia University. A imagem confocal foi feito em WVU Microscópio Facility Imaging.

1. Fotolitografia Fabricação de Moldes Mestre Photoresist O protocolo seguinte ilustra o processo para fabricar os microcanais com diâmetros entre 30-60 um. Para obter um microcanal com um diâmetro mais pequeno (inferior a 30 um), numa única rotação de revestimento de material fotosensitivo é necessário. <ol><li> Transferir o material fotosensitivo de refluxo do frigorífico a 4 ° C e a sala limpa 24 horas antes da utilização e permitir que aqueça até à temperatura ambiente. </li><li> Limpar uma bolacha de silício e cozê-la durante uma hora a 150 ° C, para permitir que a desidratar. A desidratação irá ajudar a adesão fotossensível à base de silício. </li><li> Spin-revestir a primeira camada de material fotosensitivo utilizando a seguinte receita: </li></ol><table border="1"><tbody><tr><td> Passo </td><td> Tempo (segundos) </td><td> Velocidade (rpm) </td><td> Aceleração </td></tr><tr><td> 1 </td><td> 2 &lt;/ Td&gt; <td> 300 </td><td> maior </td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td> maior </td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td> maior </td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td> maior </td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td> maior </td></tr></tbody></table><ol start="4"><li> Em seguida, coze a bolacha numa placa de aquecimento com uma temperatura de 110 ° C durante 90 seg. Após a cozer suave da espessura do material fotosensitivo será de 20-30 ^ m. </li><li> Girar revestimento de uma segunda camada de material fotosensitivo seguindo a mesma receita utilizada para a primeira camada. </li><li> Assar a bolacha Mole novamente, colocando-o numa placa de aquecimento com uma temperatura de 110 ° C durante 90 seg. Após este coza suave da espessura do material fotosensitivo será de 40-60 ^ m. </li><li> Gerar padrões de mestre positivos expondo o photoresist à luz UV com uma dose de exposição de 14.500 mJ / cm 2, por meio de uma máscara de filme. </li><li> Dilui-se com o desenvolvedor desionizada (DI), água (1:2 (v / v)). Lavar a bolacha repetidamente na solução até que o padrão está completamente desenvolvido. Em seguida, lave o wafer com água DI e seque-o com gás nitrogênio. </li><li> Refluxo: Coloque a bolacha numa placa de aquecimento com uma temperatura de 120 ° C durante 4 min e com uma tampa de uma placa de Petri de vidro para evitar a evaporação do solvente. Retire o wafer do fogão e deixe esfriar à temperatura ambiente. A espessura do material fotosensitivo após o refluxo será de 50-60 ^ m. </li></ol> 2. Fabricação de litografia suave de PDMS Microcanal Rede <ol><li> Preparar a solução de polidimetilsiloxano (PDMS) com a proporção em peso de 10:01 (base: agente de cura) e misture bem utilizando um misturador planetário centrífuga. </li><li> Lançai a solução PDMS para o molde mestre photoresist refluído. Colocar o PDMS fundido num exsicador durante 15 min para desgaseificar. Use azoto gasoso para remover quaisquer bolhas restantes, se necessário. </li><li> Asse o PDMS numa estufa a uma temperatura de 60 ° C durante 3 horas para permitir a cura. Em seguida, retire a camada de PDMS curada do molde mestre. </li><li> É um perfurador aguçado para criar os orifícios de entrada / saída por furos de perfuração na rede de canais. Limpar a superfície do PDMS usando gás azoto. </li><li> Trate duas camadas de PDMS com plasma de oxigênio por 30 segundos dentro de um aspirador de plasma a uma pressão de operação de 4,5 x 10 -2 Torr e uma taxa de fluxo de oxigênio de 3,5 pés 3 / min. Em seguida, alinhar as superfícies do PDMS manualmente sob um microscópio óptico. Use uma gota de água se necessário, para um melhor controlo do alinhamento. </li><li> Asse o dispositivo num forno a 60 ° C durante 30 min para conseguir a ligação permanente. </li></ol> 3. Cultura HUVEC e propagação no Chip <ol><li> Cultura das HUVECs com meio de cultura com L-glutamina, suplementado com 10% de soro fetal bovino (FBS). Por diaforam usadas passagens electrónicos experimentais entre 2 e 5. </li><li> Uma vez que as HUVECs são confluentes, a contagem das células e colhem-lhes, em primeiro lugar a lavagem das células com solução salina tamponada com HEPES (HEPES-BSS) e, em seguida, tratar as células com tripsina / EDTA e incubar durante 2-6 min a 37 ° C. Após a tripsinização é completa, neutralizar a tripsina / EDTA com uma solução de neutralização de tripsina. Centrifugar e suspender as células em meio de cultura com 8% de dextrano para recolhê-las. Dextrano foi usada para aumentar a viscosidade média para ajudar na melhor semeadura de células e do acessório. </li><li> Tratar o dispositivo com plasma de oxigénio durante 5 min com uma pressão de serviço de 4,5 x 10 -2 Torr e uma taxa de fluxo de oxigénio de 3,5 ft 3 / min. Em seguida, carregar o dispositivo com água DI e tratar com luz UV por 8 horas em um laminar capa de biossegurança para a esterilização. </li><li> Um dia antes das células estão prontas, o dispositivo de lavagem com 1 x tampão fosfato salino (PBS 1x) e depois o revestimento com fibronectina (100 ug / ml, dilbuído com PBS 1x) e incubar no frigorífico a 4 ° C durante a noite. </li><li> Depois do revestimento com a fibronectina, o dispositivo de lavagem com 1x PBS, em seguida, carregar com meio de cultura. Incubar o dispositivo a uma temperatura de 37 ° C durante 15 min. </li><li> Carregar as células HUVEC em meio de cultura de 8% de dextrano com uma concentração celular de 3-4 x 10 6 células / ml. Coloque uma gota de 20 ul de células a uma entrada do dispositivo e inclinar-se a introdução das células no interior do canal microfluídico. Depois de 15-20 minutos as células começarão a anexar as paredes laterais dos canais. Girar o dispositivo a cada 15 minutos para criar uma distribuição mais uniforme das células. Se necessário, uma carga adicional pode ser executada. </li></ol> 4. Configuração de perfusão de longo prazo Depois de 5-6 horas de cultura estática, os HUVECs anexados vai começar a espalhar totalmente. Configure perfusão usando um sistema de bomba de seringa controle remoto com um fluxo constante de 10 mL / h. A perfusão pode ser ajustada fou uma taxa de fluxo mais elevada, e pode durar por um período de tempo entre 4 dias a 2 semanas. 5. Coloração celular e Caracterização Microscópio <ol><li> Quando as células atingem confluência no interior do dispositivo, em primeiro lugar, o dispositivo de lavagem com 1x PBS para remover completamente o meio. Em seguida, carregar o aparelho com corante vermelho diluído com diluente (4 ul-1 ml). Carregar o corante semelhante ao procedimento de carregamento da célula. Incubar o dispositivo no escuro durante 5 min à temperatura ambiente, e, em seguida, o dispositivo de lavagem com meio de cultura para parar a coloração. Comprida incubação do corante pode causar toxicidade celular e disadhesion. </li><li> Carregar o dispositivo com o corante azul diluída com PBS 1x (2 gotas por ml). Incubar no escuro durante 5 min à temperatura ambiente e depois lavar com o dispositivo de 1x PBS. </li><li> Examine a coloração de células em microscópio óptico invertido. Se a coloração era bom, carregar os microcanais com meio de fixação (3,5% paraformaldeído diluído com PBS 1x), em seguida submergiro dispositivo de fixação no suporte e cobri-lo completamente com folha de alumínio. Armazenar o dispositivo no frigorífico a uma temperatura de 4 ° C para evitar que o dispositivo de secagem e fotobranqueamento. O dispositivo fixo está agora pronto para imagiologia confocal, o que pode ser feito por um microscópio de varrimento de laser confocal. </li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

<html> 1. Fabricação de moldes Mestre Um dos princípios orientadores para a concepção e morfometria vascular é conhecida como lei de Murray, 16, que afirma que a distribuição dos diâmetros dos vasos em toda a rede é regida por contrapartida mínima de energia. Afirma também que o cubo dos diâmetros de um navio-mãe a uma bifurcação é igual à soma dos cubos dos diâmetros dos vasos filha ( <img alt="Equação 1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/5...

Microvasos, como uma parte do sistema de circulação, medeiam as interacções entre o sangue e os tecidos, suporta as actividades metabólicas, definir microambiente do tecido, e desempenham um papel fundamental em muitos estados patológicos e de saúde. Recapitulação de microvasos funcionais in vitro pode fornecer uma plataforma para o estudo de fenômenos vasculares complexos. No entanto, em ensaios convencionais microvasos in vitro, tais como ensaios de migração de células endoteliais, ensaios de formação de tubos endoteliais e de rato e ensaios de anel da aorta do rato, são incapazes de recriar o microvasos in vivo no que diz respeito à geometria tridimensional (3D) e controlo de fluxo contínuo 1-8. Estudos de microvasos com modelos animais e em ensaios in vivo, tais como o ensaio de angiogénese da córnea, pinto corioalantóico ensaio de angiogénese da membrana e ensaio de tampão de Matrigel, são mais demorado, alto custo, um desafio no que diz respeito à observação e quantificação, elevantar questões éticas 1, 9-13. Avanços em Microfabricação e tecnologias de chips microfluídicos têm permitido uma variedade de insights sobre ciências biomédicas, enquanto reduzindo os altos custos experimentais e as complexidades associadas com animais e estudos in vivo de 14, como condições biológicas de forma fácil e rigidamente controlada e ambientes fluidos dinâmicos, que não teriam foi possível com as técnicas convencionais macroescala. Aqui, apresentamos uma abordagem para a construção de uma reendotelizados microcanais-on-a-chip que imita a geometria 3D in vivo microvasos e é executado sob fluxo de perfusão contínua controlada usando a combinação da técnica de fotolitografia reflowable photoresist, litografia macia e microfluídica.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

Os esforços têm sido focados no desenvolvimento de ensaios in vitro para o estudo de microvasos porque estudos em animais in vivo é mais demorado, caro e de observação e quantificação são muito desafiante. No entanto, em ensaios in vitro convencional microvasos têm limitações quando representar microvasos in vivo no que diz respeito a geometria tridimensional (3D) e fornecer o fluxo de fluido contínuo. Usando uma combinação de técnicas de fotolitografia reflowable photoresist, litografia macia e microfluídica, temos desenvolvido um multi-profundidade reendotelizados transversais circulares microcanais-on-a-chip, que imita a geometria 3D in vivo microvasos e é executado sob perfusão contínua controlada fluxo. Um fotosensitivo refluído positivo foi usada para fabricar um molde mestre, com uma rede de microcanais transversal semicircular. No alinhamento e união das duas (PDMS) microcanais polidimetilsiloxano replicated do molde mestre, uma rede de microcanais cilíndrico foi criado. Os diâmetros dos microcanais pode ser bem controlada. Além disso, as células primárias endoteliais da veia umbilical humana (HUVEC) semeadas dentro do chip mostraram que as células revestidas a superfície interior dos microcanais em perfusão controlada duradouro durante um período de tempo entre 4 dias a 2 semanas.

A nossa abordagem para fabricar a rede de microcanais múltiplos profundidade imita as geometrias complexas 3D de microvasos in vivo, em que os microcanais têm secções transversais arredondadas 15. Além disso, os diâmetros dos canais de ramificação-mãe e os canais filha aproximadamente obedecem à lei de Murray, para manter o fluxo do fluido em um nível requerido de modo que a resistência global do canal é baixa e velocidades de fluxo são mais uniforme em toda a rede de 16-18. ...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Uma plataforma de microcanais-on-a-chip foi desenvolvido pela combinação da técnica fotolitográfica refluído fotossensível, litografia macia, e microfluidos. A plataforma de microcanais reendotelizados imita a geometria tridimensional (3D) de microvasos in vivo, é executado sob fluxo de perfusão contínua controlada, permite a imagem de alta qualidade e em tempo real, e pode ser aplicado para a pesquisa microvascular.

Procedimento para o Desenvolvimento da Multi-profundidade Circular transversal reendotelizados microcanais-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

procedure-for-development-multi-depth-circular-cross-sectional

Research

JoVE Journal

Bioengineering

13.8K visualizações.  West Virginia University.  Uma plataforma de microcanais-on-a-chip foi desenvolvido pela combinação da técnica fotolitográfica refluído fotossensível, litografia macia, e microfluidos. A plataforma de microcanais reendotelizados imita a geometria tridimensional (3D) de microvasos in vivo, é executado sob fluxo de perfusão contínua controlada, permite a imagem de alta qualidade e em tempo real, e pode ser aplicado para a pesquisa microvascular. 

Vídeo: Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedimento para a Engenharia de pulmão

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Bioengenharia

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

A microfluídica Endothelialized para estudo das interacções microvasculares em Doenças Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedimento para decelularização do Coração Suínos por perfusão coronariana retrógrada

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Métodos de caracterização da Co-desenvolvimento de biofilme e Habitat Heterogeneidade

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanopartículas Lipídicas Sólidas (NLS) para intracelular Segmentação Aplicações

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caracterização qualitativa da fração aquosa de Hydrothermal Liquefação de algas por cromatografia gasosa 2D com Time-of-flight Espectrometria de Massa

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Desenvolvimento de um modelo organotípicas multicelular tridimensional da Mucosa Intestinal Humano cultivadas sob microgravidade

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Montagem e Rastreamento do Desenvolvimento de Comunidade Microbiana em uma Plataforma Microwell Array

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

Método de Teste Padrão ASTM D 7998-19 para o Desenvolvimento de Resistência Coesiva de Adesivos de Madeira

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Fracionamento de fluxo de fluxo assimétrico para dimensionamento de nanopartículas de ouro em suspensão

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...