Apoiado pelo NIH concessão HD052670.

1. Revestimento de placas com ECM proteínas <ol><li> Sob condições estéreis, misturar 120 ug de laminina com 12 ml de frio estéril 1X PBS por placa. </li><li> Tome Bioflex placas não tratadas e adicionar 2 ml da solução a cada poço (concentração final de laminina é 2 ug / cm 2). Alternativamente, outras proteínas de ECM podem ser utilizados, tais como colagénio-1 [10 ug / cm 2], fibronectina [5 ug / cm 2], vitronectina [0,5 ug / cm 2] ou elastina [10 ug / cm 2]. A técnica de revestimento é semelhante ao que é descrito para a laminina-revestimento. Assegure-se os poços são completamente cobertas pela solução. </li><li> Enrole as placas de plástico e colocado a 4 ° C sobre uma superfície plana durante a noite para permitir que o laminina para adsorver para o fundo dos poços. Nestas condições, as placas podem ser armazenadas durante pelo menos uma semana, mantendo a função de ECM boa. </li><li> No dia seguinte, sob condições estéreis, lavar os poços 3 vezes com PBS 1X. </li><li&gt; Adicionar 1 ml de BSA a 1% em PBS 1X a cada poço e incubar a 37 ° C durante 1 h para bloquear não-locais de ligação específicos nas membranas. </li><li> Lavam-se as placas 3 vezes com PBS 1X para remover as proteínas unadsorbed. </li><li> Manter as placas a 37 ° C em DMEM até que as células são isoladas a partir do passo 2,14. </li></ol> 2. Isolamento de células fetais de roedores pulmão do tipo II epiteliais Um dia antes de isolamento têm os copos de tela com malhas de diferentes tamanhos de nylon autoclavado e pronto. <ol><li> Obter pulmões fetais de timed-grávida mouse / rato em E17-19 da gestação. Transferir o tecido para um tubo de 50 ml cónico contendo meio DMEM e colocá-lo em gelo. </li><li> Fazer tampão de digestão num tubo cónico de 50 ml: (10 ml). Para este volume, o tecido de pulmão a partir de cerca de 20 fetos de rato / rato é usado. 8,5 ml de DMEM 0,5 ml 500mM pH 7.4 HEPES 5 mg de colagenase 1 5 mg de colagenase 1A 1 ml de galinha soro, inactivado pelo calor </li><li>Misturar bem até que todos os componentes são dissolvidos e filtrar através de um filtro de seringa de 0,2 micron dentro do capuz estéril. Colocar o tubo cónico contendo o tampão de digestão num banho de água a 37 ° C. </li><li> Remover o meio DMEM a partir do tubo cónico a partir do passo 2.1 e transferir o tecido em uma placa de Petri estéril. </li><li> Picar os pulmões bem com uma tesoura estéreis ou lâmina de barbear para fazer peças de tecido inferior a 1 mm e transferir o tecido para dentro do tubo cónico contendo o tampão de digestão de pré-aquecida a partir do passo 2.3. </li><li> Digerir o tecido pipetando cima e para baixo: 100 vezes com 10 ml de pipeta 100 vezes com 5 ml de pipeta 100 vezes com uma pipeta de Pasteur </li><li> Centrifugar o homogeneizado a 1300 rpm durante 5 minutos à temperatura ambiente. </li><li> Cuidadosamente remover o sobrenadante por aspiração e re-suspender o sedimento contendo as células em 15 ml de DMEM mais 20% de FBS. </li><li> Retire os copos de tela com 100 -, 30 - e malhas de 15 mícron de náilon e po lugarem em estéreis de 150 ml copos. </li><li> Adicionar homogenato celular a partir do passo 2,8 e sequencialmente filtrar através de 100 -, 30 -, e 15 micras copos tela de malha. </li><li> Collect agregada não filtrados células a partir da 30 - e 15 micron-malhas após várias lavagens com DMEM mais 10% FBS para facilitar a filtração de células não-epiteliais. </li><li> Descartar o filtrado a partir da malha 15 micron, que contém principalmente fibroblastos. </li><li> Purifica-se células do tipo II adicionais por incubação de células a partir do passo 2,11 em 75-cm 2 frascos para 30 min (approximatelly10 ml de suspensão de células por frasco). </li><li> Recolher o sobrenadante e centrifugar a 1300 rpm durante 5 minutos à temperatura ambiente para sedimentar as células. </li><li> Cuidadosamente remover o sobrenadante por aspiração e re-suspender o sedimento contendo as células em DMEM isento de soro. O volume de ressuspensão depende da quantidade de tecido processado. Aproximadamente, nós chapa do equivalente de células obtido a partir de um feto em cada poço (2 ml), a fim de achieve entre 60-70% de confluência no dia seguinte. </li><li> Suspensão placa da célula em Bioflex seis poços pré-revestidos com laminina ou fibronectina. </li></ol> 3. Isolamento de fibroblastos de pulmão de feto de roedores <ol><li> Siga os mesmos passos descritos acima para o tipo de isolamento das células II até 2,11. </li><li> Tome-se o filtrado a partir da malha 15-micron (sem lavagem anterior; volume aproximado é de 10-15 ml) e da placa em 75 cm de 2 a 37 ° C durante 30-60 min para permitir que os fibroblastos de aderir. </li><li> Aspirar o supernantant e substituir com DMEM isento de soro e incubar durante a noite. </li><li> No dia seguinte, a colheita das células com 0,25% (peso / vol) de tripsina em 0,4 mM de EDTA e da placa-as em placas Bioflex pré-revestidas com fibronectina. </li></ol> 4. Sistema experimental para proporcionar a estimulação mecânica para as células do pulmão <ol><li> De sementes as células (cerca de 50% confluentes) em placas de cultura de Bioflex em DMEM isento de soro (2 ml / poço) e deixá-los anexar e SPREanúncio por pelo menos 6h antes dos experimentos. Em geral nosso laboratório mantém células em cultura por cerca de 24 horas antes de aplicar tensão mecânica. Se um número específico de células são necessárias para as experiências, após isolamento, as células do tipo II são mantidas em DMEM isento de soro de 75-cm 2 frascos e no dia seguinte, as células são tripsinizadas, contadas e então semeadas. </li><li> No dia seguinte, os meios são substituídos com fresco DMEM isento de soro (2 ml / poço) e as placas Bioflex são montados numa Unidade Strain Flexcell FX-5000. Monocamadas não deve ser maior do que 80% confluente antes do início das experiências. </li><li> Estirpe Equibiaxial é aplicada às membranas. O regime de tensão varia dependendo de simulação de forças mecânicas in vivo (ver discussão). </li><li> Células cultivadas em não-esticado membranas cultivadas em paralelo são utilizados como controlos. </li><li> No final dos ensaios, as monocamadas pode ser processada para analisar as mudanças na expressão de genes (por exemplo C de proteína surfactante)por abundância de proteínas em tempo real-PCR, por Western blot, etc Além disso, as monocamadas pode ser fixado para experiências imunocitoquímica. Para esta técnica, após a fixação, as membranas de silastic são cortados a partir das placas e montadas em lâminas de vidro usando 10-20 ul de água como agente de montagem antes permeabilização e incubação com anticorpos. Sobrenadante pode também ser usado para investigar a presença de factores de crescimento, citocinas, etc </li></ol> 5. Os resultados representativos A Figura 1 ea Figura 2 mostram representativos contraste de fase fotografias de E18 fetais de rato células de tipo II isolado utilizando a técnica descrita no presente manuscrito. A Figura 3 demonstra que a esforços mecânicos induz a diferenciação de células de tipo II fetais epiteliais utilizando surfactante proteína C, como um marcador. <img src="/files/ftp_upload/3543/3543fig1.jpg" /> Figura 1. Repfotografia de contraste de fase representativa tirada logo após o isolamento mostrando a aparência agregado de células fetais do tipo II. <img src="/files/ftp_upload/3543/3543fig2.jpg" /> Figura 2. E18 células de tipo II fetais epiteliais foram isolados como descrito aqui e plaqueadas em placas de Bioflex revestidas com laminina. Fotografia foi tirada no dia seguinte após não esticados as células foram fixadas em paraformaldeído. A pureza das células foi determinada ser de 90 ± 5% por análise microscópica da morfologia das células epiteliais e imunocoloração para SP-C. <img src="/files/ftp_upload/3543/3543fig3.jpg" /> Figura 3. Células de tipo II Fetal epiteliais foram expostas a estirpe cíclico de 5% a 40 ciclos / min durante 16 h. Um blot) Northern de surfactante de proteína C (SP-C) a expressão do mRNA que mostra que a estirpe tipo induz a diferenciação das células II utilizando diferentes substratos ECM . Sinais + / - representam a exposição ou não streinar, respectivamente. Os dados são apresentados como média + / - SEM, n = 3; * P &lt;0,05 B) imagens de fluorescência imunocitoquímica demonstrando níveis de proteína SP-C (verde) em células de tipo II fetais expostos ou não (controle) a uma tensão mecânica.. Os núcleos foram contrastadas com DAPI (azul). Bar, 10 uM. C) Os resultados de Western blot de três experiências que mostram que estiramento mecânico aumenta SP-C da proteína. N = 3; * P &lt;0,05.

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Neste artigo, nós descrevemos um método para isolar as células fetais do tipo II epiteliais e fibroblastos e expô-los a estimulação mecânica usando o aparelho Strain Flexcell. Usámos esta técnica para avaliar a diferenciação de células epiteliais 1,2 e para estudar receptor e vias de sinalização activados por estiramento 3-9. Além disso, este método também pode ser usado para investigar as respostas das células induzidas por lesão mecânica 10,11. O procedimento baseia...

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase 1</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>Collagenase 1A</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Chicken serum</td><td>Sigma-Aldrich</td><td>C5405</td><td></td></tr><tr><td>Screen cups</td><td>Sigma-Aldrich</td><td>CD1-1KT</td><td></td></tr><tr><td>Syringe filters</td><td>Fisher Scientific</td><td>09-754-25</td><td></td></tr><tr><td>100 micron nylon mesh</td><td>Small Parts, Inc.</td><td>CMN-100-D</td><td></td></tr><tr><td>30 micron mesh</td><td>Small Parts, Inc.</td><td>CMN-30-D</td><td></td></tr><tr><td>15 micron mesh</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX15</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Collagen-1</td><td>Collagen Biomed</td><td>PC0701</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Vitronectin</td><td>Sigma-Aldrich</td><td>V-0132</td><td></td></tr><tr><td>Elastin</td><td>Sigma-Aldrich</td><td>E-6402</td><td></td></tr><tr><td>Bioflex plate</td><td>Flexcell International</td><td>BF-3001U</td><td>Uncoated</td></tr><tr><td>Flexcell Strain Unit</td><td>Flexcell International</td><td>FX-5000</td><td></td></tr></tbody></table>

an experimental system to study mechanotransduction in fetal lung cells

Forças mecânicas geradas in utero pelos repetitivos movimentos como de respiração e pela distensão líquido são essenciais para o desenvolvimento do pulmão normal. Um componente-chave do desenvolvimento do pulmão é a diferenciação de células alveolares do tipo II epiteliais, a principal fonte de surfactante pulmonar. Estas células também participar na homeostase de fluido no lúmen alveolar, de defesa do hospedeiro, e reparação de lesão. Além disso, as células do pulmão distais do parênquima pode ser directamente exposta ao estiramento exagerada durante a ventilação mecânica após o nascimento. No entanto, os mecanismos precisos moleculares e celulares, através da qual as células do pulmão detectam estímulos mecânicos para influenciar o desenvolvimento do pulmão e para promover a lesão pulmonar não são completamente compreendidos. Aqui, nós proporcionar um método simples e de alta pureza para isolar células de tipo II e de fibroblastos a partir de pulmões de roedores fetais. Em seguida, nós descrevemos um sistema in vitro, A Unidade de Strain Flexcell, para proporcionar a estimulação mecânica para células fetais, simulando forças mecânicas emdesenvolvimento pulmonar fetal ou lesão pulmonar. Este sistema experimental fornece uma excelente ferramenta para investigar os mecanismos moleculares e celulares em células de pulmão de feto expostos a esticar. Usando essa abordagem, nosso laboratório identificou vários receptores e proteínas sinalizadoras que participam mecanotransdução no desenvolvimento do pulmão fetal e lesão pulmonar.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Forças mecânicas desempenham um papel chave no desenvolvimento de pulmão e lesão pulmonar. Aqui, nós descrevemos um método para isolar roedores fetais de pulmão de células do tipo II epiteliais e fibroblastos e expô-los a estimulação mecânica utilizando um<em&gt; In vitro</em&gt; Sistema.

Um sistema experimental para estudar Mecanotransdução em células do pulmão fetal

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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Research

JoVE Journal

Bioengineering

12.9K visualizações.  Alpert Medical School of Brown University. Forças mecânicas desempenham um papel chave no desenvolvimento de pulmão e lesão pulmonar. Aqui, nós descrevemos um método para isolar roedores fetais de pulmão de células do tipo II epiteliais e fibroblastos e expô-los a estimulação mecânica utilizando um In vitro Sistema.

Vídeo: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Usando<em&gt; Ex Vivo</em&gt; Culturas Vertical Gota de toda fetais Órgãos para estudar processos de desenvolvimento durante Rato Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Biologia do Desenvolvimento

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medicina

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Imagens de células vivas durante Mecânica estiramento

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Bioengenharia

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura do mouse pulmão embrionário, um sistema para avaliar os mecanismos moleculares de Ramificação

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Biologia

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Geração de organoides pulmonares 3D inteiros de células-tronco pluripotentes induzidas para modelagem da biologia e doença do desenvolvimento pulmonar

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Diferenciação e estimulação mecânica de células endoteliais derivadas de hiPSC

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Autor em destaque: Estudando células endoteliais derivadas de hiPSC cultivadas sob estimulação mecânica mediada por fluidos 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

In vitro Modelo de Embarcação Humana Fetal On-chip para Estudo da Mecanobiologia do Desenvolvimento

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

Mecanoestimulação de organismos multicelulares através de um sistema de compressão microfluídica de alto rendimento

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

Um sistema experimental para estudar Mecanotransdução em células do pulmão fetal

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