Este trabalho foi financiado pelo Fundo de Pesquisa do Câncer de ovário [conceder LT/UIC/01.2011], o Centro para a Ciência UIC clínica e translacional, do Centro de Câncer UIC, e os Institutos Nacionais de Saúde conceder C06RR15482. Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo AAALAC ao abrigo de protocolos de cuidados animais aprovados pelo Animal Care e do Comitê Use a UIC. Animais para este projeto estão alojados em salas de barreira no Laboratório de Recursos Biológicos (BRL) da Universidade de Illinois em Chicago. O BRL tem uma equipe totalmente veterinária que monitora os animais pelo menos duas vezes por dia e fornece conselhos sobre cuidados com animais.

1. Dissecção e isolamento do tecido ovariano <ol><li> Preparar uma solução 0,5% (w / v) de alginato de sódio, prepare de uma versão modificada do protocolo descrito anteriormente 5, usando PBS estéril e calor a 37 ° C. Misture por inversão ou de balanço, mas não vortex. Desenhe o alginato em uma seringa de 1 ml com agulha 25 G e manter a 37 ° C. </li><li> Prepare estéril 10mM CaCl 2 solução para o alginato de reticulação em encapsulamento dos tecidos e quente a 37 ° C. </li><li> Prepare 1mL de meio de cultura (αMEM + 5 unidades de penicilina e estreptomicina 5 mg / ml) por bem em uma placa de 24 poços para cada experimento. Incluem vários fármacos, peptídeos ou vírus no meio de cultura. Para transfecção antes de encapsulamento de órgãos, tecidos incube com Lipofectamine 2000 e DNA plasmidial por quatro horas a 37 ° C. </li><li> Dissecar os órgãos do animal, remover qualquer tecido adiposo ou residual, puxe fora da membrana bursa redor do ovário, e cortar o ovário ou oviduto em quatro pedaços por que atravessa o órgão. Animais foram sacrificados por asfixia CO 2 seguido por deslocamento cervical. Para o oviduto, gentilmente desenrolar o tecido puxando para além das membranas de ligação e estendendo as extremidades do tubo em direções opostas com a pinça antes de cortar. </li><li> Coloque uma gota de alginato única (~ mL 1-3) em um de malha de fibra esterilizados. </li><li> Utilizando uma pinça estéril, mover uma peça de órgão no gota de alginato e do centro da gotícula usando uma pinça ou uma agulha esterilizada. </li><li> Inverter a gota contendo o pedaço de órgãos (organóides) segurando os de malha de fibra com uma pinça estéril e transformá-lo de cabeça para baixo para permitir que a gravidade para forçar o alginato em uma gota de suspensão. </li><li> Toque a pinça na borda de um prato de petri contendo 10 centímetros 10mM CaCl 2 solução de modo que a gota cai em solução. </li><li> Incubar por 2 minutos, retire com uma colher estéreis, contendo poros para liberar extras 10mM CaCl 2, e transferir para o meio de cultura. Até quatro géis de alginato podem ser transferidos para um único poço de uma placa de 24 poços contendo meio de cultura. Cultura por um período de tempo definido. </li></ol> 2. Degradação de alginato e coleta de células <ol><li> Para a solubilização do gel de alginato, incubar as culturas em liase alginato (3,4 mg / ml) dissolvido em L-15 Leibovitz media a 37 ° C por 30 minutos ou até gel é completamente dissolvido. </li><li> Remover o tecido da liase alginato. </li><li> Para a coleta da OSE, incubar os organóides com 500 unidades de colagenase em L-15 Leibovitz da mídia por 1 hora 8. Vortex em 2 pulsos segundo (2 segundos vortex e 2 segundos de descanso) em potência média por 1 minuto, seguido por um segundo pulsos (1 vortex segundo e um resto segundo) por 1 minuto. Deixe pedaços de órgãos caem ao fundo do tubo de microcentrífuga e sobrenadante pipeta no tubo de microcentrífuga estéril fresco. Centrífuga 3 minutos a 3000 rpm para sedimentar epitélio de superfície do ovário. </li><li> Para a coleta do epitélio tubário, cortar o tubo comprido e coloque em plástico de cultura de tecidos em DMEM com 5 unidades de penicilina e 5 mg / ml de estreptomicina e 4% (v / v) FBS. Mais de 3-5 dias, as células vão &quot;arrastar&quot; para fora do estroma tubária e crescer em cultura de tecidos plastic9. </li></ol> 3. Fixação de culturas alginato encapsulado órgão para parafina <ol><li> Recolher o alginato encapsulado pérola do meio de cultura no ponto de tempo selecionado. </li><li> Recrosslink o alginato para endurecer o gel, largando a cultura estéril em 10mM CaCl 2 solução por dois minutos. </li><li> Corrigir o órgão alginato encapsulado em paraformaldeído 2% contendo 50 mM de sacarose, 10 mM CaCl 2 e 50 mM cacodilato de sódio para garantir que o gel permanece sólida e totalmente encapsula tecido durante a fixação. </li><li> Após processamento de tecidos, incorporar todo o alginato encapsulado pedaço da cultura de órgãos em parafina e corte com um micrótomo para gerar 5 seções mM. </li><li> O alginato será dissolvido e removido do slide se realizar imunohistoquímica devido ao calor recuperação antigênica baseada. O alginato permanecerá e mancha de eosina compreensão hematoxilina e eosina. </li></ol> 4. Resultados representativos: Um exemplo de uma cultura de órgãos em três dimensões do ovário e oviduto é mostrado na Figura 1. Ovários podem ser cortados em pedaços de tamanhos diferentes, e OSE proliferar e migrar para reparar a superfície que foi ferido a partir da incisão com o bisturi. Durante este processo, a orientação correta da OSE em relação ao estroma subjacente é mantida, com a OSE encapsular o ovário. Conforme mostrado na Figura 2, a OSE dos ovários cortado ao meio encapsular o ovário de forma mais completa em comparação com ovários cortado em quartos ou oitavos. O reparo de feridas do ováriosuperfície podem ser estudadas no contexto do ovário inteiro e pode lançar luz sobre como reparar a ovulação ea superfície induz a transformação da superfície do ovário. O ponto final do experimento depende da informação que é necessário. Arquitetura do tecido, a morfologia celular, e taxas específicas de crescimento celular são facilmente monitoradas utilizando-se secções de parafina. Por exemplo, uma única camada de epitélio de superfície do ovário é observada após o cultivo em soro livre de αMEM por 7 dias (Figura 3A) enquanto que a adição de 5 mg / ml de insulina por 7 dias induz hiperproliferação do epitélio de superfície ovariano e formação de camadas múltiplas de células (Figura 3B). Adicionando bromodeoxiuridina para as culturas em uma concentração final de 10 mM 24 hr antes da fixação, a proliferação de células pode ser medido (3A e 3B inset). Para culturas mantidas em soro livre de αMEM, cerca de 3-5% da OSE sofrem proliferação durante o período de rotulagem 24 horas (3A inset), enquanto uma porcentagem maior de células da granulosa proliferam. Além de insulina para a cultura de mídia aumenta a porcentagem de proliferação OSE a aproximadamente 30% da OSE total (3B inset). Imunofluorescência da proteína verde fluorescente (GFP) podem ser visualizados através do gel de alginato com microscopia seguinte norma transfecção cDNA (Figura 3C). Enquanto a cultura de órgãos alginato prevê crescimento e análise de superfície epitélio ovariano e folículos primários, deve-se notar que a madura, folículos secundários não sobreviver neste sistema de cultivo (Figura 3D). Cultura de folículos individuais de maturidade dentro de uma matriz de hidrogel de alginato tem sido descrito em outros lugares 10. Alterações na proteína ou RNA pode ser analisado a partir de todo o órgão ou a partir de tipos específicos de células usando degradação enzimática (Figura 4). Tratamento de ovário Organóides cultivados com colagenase deixa o estroma subjacente intacto (Figura 4C), permitindo para o enriquecimento da OSE na célula pellet obtido após o tratamento, apesar de algumas células do estroma também estão isolados (Figura 4D). <img alt="Figura 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> Figura 1. Rato ovário sistema de cultura tridimensional usado para propagar OSE após ferindo. A) Sob o microscópio, a bursa ea almofada de gordura ao redor foram removidos. B) ovário intacto (O) com tubo oviductal (T) e gordura pad (F). C) Ovário com bursa e gordura abdominal removido (à esquerda) e desenrolado oviduto (direita). D) Organóides ovário ou oviductal foram colocados em uma gota de alginato. E) Organóides alginato envolto foram lançadas em uma solução de CaCl 2 crosslinking do alginato para formar um gel. F) alginato encapsulado organóides de ovário. G) alginato encapsulado organóides oviductal. H) Organóides alginato encapsulado incubadas em meio de cultura. <img alt="Figura 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> Figura 2. Superfície regenerado pela superfície do ovário medido pelo citoqueratina 8. Encapsulamento de Organóides ovário pela OSE depois de ser cortado em oitavos, quartos e metades, cultivadas em meio BSA, e medido pela análise imuno-histoquímica usando citoqueratina 8 (CK8) de anticorpos. <img alt="Figura 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> Figura 3. Alginato sistema de cultura permite a passagem de fatores de crescimento e transfecção de cDNA para a OSE, mas não suporta o rápido crescimento e maturação dos folículos. A) organóides ovário cultivadas por 7 dias em meio basal analisadas para CK8 expressão, que marca o OSE. Seção, Inset série coradas para incorporação de BrdU. B) Adição de insulina ao meio de cultura induz hiperplasia da OSE. Seção, Inset série coradas para incorporação de BrdU. C) organóides ovário transfectadas com cDNA para expressar GFP. D) sistema de cultura alginato suporta o crescimento de folículos primordiais (vermelho ponta de seta), mas não maduro folículos secundários (seta preta). <img alt="Figura 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> Figura 4. OSE pode ser isolado para a cultura seguinte análise. A) organóides cultivadas em meio por até duas semanas. B) organóides Encapsulated é tratada com liase alginato de deixar organóides intacta. Ovário é manchada com CK8 para marcar OSE. C) organóides é tratada com colagenase a OSE separado do estroma subjacente. Pedaço de tecido remanescente foi corado com CK8 para destacar a remoção da superfície do ovário. D) as células coletadas após o tratamento são enriquecidos por OSE como coradas para CK8 (vermelho). Células são contracorados com DAPI para marcar núcleos.

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Não há nada a revelar.

O desenvolvimento de um sistema de cultura tridimensional de órgãos utilizando hidrogéis de alginato representa um método versátil para a análise de muitos tipos de tecidos diferentes de uma grande variedade de organismos. O uso de culturas 3D pode ser estendido para os campos da engenharia de tecidos e medicina regenerativa, pois proporciona um andaime sobre o qual as células podem crescer 11. Atualmente, nosso laboratório está passando por estudos iniciais usando de ovário e de tecidos humanos tro...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Leibovitz L-15 de médio </td><td> Gibco </td><td> 11415 </td><td></td></tr><tr><td> αMEM médio </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> Alginato de sódio </td><td> Novamatrix </td><td></td><td></td></tr><tr><td> Cacodilato de sódio </td><td> Sigma Aldrich </td><td> C0250 </td><td></td></tr><tr><td> Malha de fibra de polipropileno </td><td> McMaster Carr </td><td></td><td> 45 &quot;roll de largura; 0,0041&quot; abertura </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Liase alginato </td><td> Sigma Aldrich </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> Sigma Aldrich </td><td> C9697 </td><td> De Clostridium histolyticum </td></tr><tr><td> Albumina de soro bovino </td><td> Biomedicals MP </td><td> 103700 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> Sua solução </td><td> Roche </td><td> 11074547001 </td><td> Insulina / transferrina / selenito de sódio </td></tr><tr><td> Citoqueratina de anticorpos 8 </td><td> Estudos do Banco de desenvolvimento Hibridoma </td><td> Troma-1 </td><td> Use a 1:200 </td></tr><tr><td> DAPI </td><td> Vector Labs </td><td> H1200 </td><td></td></tr><tr><td> Bromodeoxiuridina </td><td> Sigma </td><td> B50002-1G </td><td> Concentração final 10 mM </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Câncer de ovário é a quinta causa de morte por câncer em mulheres e tem uma taxa de mortalidade de 63% nos Estados Unidos 1. O tipo de célula de origem para o câncer de ovário ainda está em questão e pode ser tanto o epitélio de superfície do ovário (OSE) ou do epitélio distal do fímbrias tuba uterina 2,3. Cultura as células normais como uma cultura primária in vitro permitirá aos cientistas modelo de mudanças específicas que podem levar ao câncer de ovário no epitélio distintas, assim, definitivamente determinar o tipo de célula de origem. Isto irá permitir o desenvolvimento de biomarcadores mais precisos, modelos animais com alterações de tecidos específicos de genes, e das melhores estratégias de prevenção específicas para esta doença. Manter as células normais em hidrogéis de alginato promove a curto prazo na cultura in vitro de células no seu contexto tridimensional e permite introdução de DNA plasmídeo, siRNA, e pequenas moléculas. Pelos órgãos de cultura em pedaços que são derivados de cortes estratégicos usando um bisturi, várias culturas a partir de um único órgão podem ser gerados, aumentando o número de experimentos a partir de um único animal. Estes cortes aspectos do modelo da ovulação levando a proliferação da OSE, que está associado com a formação de câncer de ovário. Tipos de células, como a OSE, que não crescem bem em superfícies de plástico podem ser cultivadas usando este método e facilitar a investigação sobre os processos celulares normais ou os primeiros eventos na formação do câncer 4. Hidrogéis de alginato pode ser usado para suportar o crescimento de muitos tipos de tecidos 5. Alginato é um polissacarídeo linear composto por unidades repetitivas de ácido D-mannuronic β e α-L-ácido gulurônico que podem ser reticulados com íons de cálcio, resultando em uma ação gelificante suave que não danificar os tecidos 6,7. Como outras matrizes tridimensionais de cultura de células, tais como Matrigel, alginato fornece suporte mecânico para os tecidos, no entanto, as proteínas não são reativos com a matriz de alginato e, portanto, funções de alginato como uma matriz extracelular sintética que não inicia sinalização celular 5. O hidrogel de alginato flutua no meio padrão de cultura celular e suporta a arquitetura do crescimento de tecidos in vitro. Um método é apresentado para a preparação, separação e incorporação de ovário e pedaços de órgãos oviductal em hidrogéis de alginato, que pode ser mantidas em cultura por até duas semanas. A liberação enzimática de células para análise de proteínas e amostras de RNA a partir da cultura de órgãos é também descrito. Finalmente, o crescimento de tipos de células primárias é possível sem imortalização genética de ratos e permite que os pesquisadores de usar nocaute e camundongos transgênicos.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Cultura de células normais em seu contexto tridimensional representa um método alternativo para estudar os eventos iniciais necessárias para a transformação celular e tumorigênese. Este método é usado para crescer ovariano normal e células oviductal para estudar os eventos iniciais na formação de câncer de ovário.

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K visualizações.  University of Illinois at Chicago. Cultura de células normais em seu contexto tridimensional representa um método alternativo para estudar os eventos iniciais necessárias para a transformação celular e tumorigênese. Este método é usado para crescer ovariano normal e células oviductal para estudar os eventos iniciais na formação de câncer de ovário.

Vídeo: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impressão de planar e tridimensional de tintas condutoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Bioengenharia

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparação de Intact Discos cauda bovina Intervertebral de Cultura de Órgãos

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Um modelo tridimensional de Cultura Celular para avaliar os efeitos de fluxo de fluido intersticial na invasão da célula tumoral

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tridimensional Tecnologia Biomimético: Novel Biorubber Cria Definido micro e macro-escala Arquitecturas em colágeno hidrogéis

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabricação de Inverted coloidal de cristal poli (etileno-glicol) Andaime: Uma Plataforma Cultura celular tridimensional para Liver Engenharia de Tecidos

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Em camadas de alginato Constrói: Uma Plataforma para Co-cultura de populações de células heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...