1. Estabelecimento de culturas de fibroblastos a partir de explantes de pele <ol><li> 4 milímetros biópsias do perfurador adquiridos de antebraço humano são colocados em MEM suplementado 100 unidades / ml de penicilina, 100 ug de estreptomicina / ml e 0,25 ng / ml Fungizone. </li><li> Apare qualquer gordura subcutânea e pique a biópsia em pequenos pedaços por um número de balanço 24 lâmina de bisturi de encontro ao fundo da placa de Petri. </li><li> Slurry lugar o tecido em um frasco de cultura de 25 centímetros de tecido com dois meios de fibroblastos primários de crescimento (MEM suplementado com FCS a 10%, 100 unidades / ml de penicilina, estreptomicina 100 ug / ml, e 25 ug / ml de Fungizone) adicionado até que a superfície do balão é cobertos, mas a profundidade de líquido é insuficiente para o tecido a flutuar. </li><li> Seguindo três dias de incubação a 37 ° C numa atmosfera humidificada de 5% de CO 2, adicionar 3 ml de meio de crescimento de fibroblastos primários. </li><li> Após mais 3 dias imprensa mudança de incubação com novos meios de crescimento de fibroblastos primários, ou se CELls são quase confluentes podem ser divididas 1:04 em meio fresco. (Fungizone pode ser omitida a partir deste ponto). </li></ol> 2. Fase I - Preparação de colágeno I de caudas de ratos Nota: Protocolo para aproximadamente 12-14 rabos de rato adolescente (frescas ou congeladas) <ol><li> Preparar da cauda de rato, por lavagem em etanol a 70% e remover os tendões da seguinte forma: </li><li> Remover a pele da cauda de rato por corte a meio da cauda a partir de cima para baixo com um. Bisturi e pealing ao longo do comprimento da cauda </li><li> Retire do tendão do núcleo da região proximal da cauda. </li><li> Remover tendão em direção à região distal da cauda evitando a bainha com pinças dentadas. </li><li> Extrair 1 g de tendão / 250 ml de 0,5 M de ácido acético com agitação a 4 ° C durante 48 h. </li><li> Centrifuga-se o extracto (7500 xg) durante 30 minutos e descartar o sedimento. </li><li> Adicionar um volume igual de 10% (w / v) de NaCl ao sobrenadante, e agita-se durante 30-60 min. </li><li&gt; Centrífuga (10.000 xg) durante 30 minutos, deitar fora o sobrenadante e re-dissolver o precipitado em 0,25 M de ácido acético em ~ relação de 1:1 por agitação durante 24 horas a 4 ° C. </li><li> Dialisar a solução de colagénio contra 6-8 mudanças de 6L ~ 17,5 mM de ácido acético (1 ml de ácido acético glacial por litro de água fria, a mudança 2 x ao dia). </li><li> Centrifugar colagénio dialisada a 30000 xg, durante 1,5 horas. </li><li> Remover o sobrenadante e colocar em frasco estéril. </li><li> Ajustar a concentração de colagénio I a ~ 2 mg / ml utilizando 0,5 mM de ácido acético a 4 ° C. </li></ol> 3. Fase II - Configurando a matriz 3D com fibroblastos incorporados (permitir a contratação de 8 dias). <ol><li> Montar a mistura, utilizando reagentes de frio a 4 ° C em um frasco pré-arrefecida. Mantenha colágeno I no gelo. </li><li> Para um frasco de T75 confluentes de fibroblastos primários (número de células de ~ 1 x 10 6) a utilização: <ul><li> 25 ml de colagénio da cauda de rato (cerca de conc 2mg/ml) </li><li> 3 ml de 10xMEM </li><li> 0,22 M NaOH: primeiro, adicionar 2 ml, em seguida, gota a gota, agitando até o colágeno fica laranja, </li><li> mas não de-rosa (geralmente até 3 ml). Aproximado p H = 7,2. </li><li> Nota: É importante assegurar que o meio / gel permanece neutro ou ligeiramente ácido, como os fibroblastos não irão contrair o gel, se forem expostos a condições, mesmo ligeiramente alcalinas. </li></ul></li><li> Trypsinise os fibroblastos, girar a 400 xg durante 5 minutos e remover o sobrenadante. </li><li> Durante 5 minutos girar acima: Prepare 12 x 35 milímetros pratos de plástico - normalmente atingir 12 pratos de um frasco de fibroblastos / Mix de colágeno. </li><li> Re-suspender fibroblastos em 3 ml FCS e imediatamente adicionar à mistura de colágeno e mexa. </li><li> Placa de aproximadamente 2,5 ml de colagénio / f ibroblastos, por prato tão rapidamente quanto possível. Tente evitar bolhas e definição de colágeno. </li><li> Deixe o colagénio-se a 37 ° C numa atmosfera humidificada de 5% CO 2 em ar durante 10 minutos. </li><li> Adicionar 1 ml de fibroblastos crescemth meios (DMEM + FCS 10%). </li><li> Separar a matriz de colagénio / f ibroblastos dos lados do prato, usando uma pipeta. </li><li> No dia seguinte: Adicione 1 ml de meio de crescimento de fibroblastos (DMEM + FCS 10%). </li><li> Alterar mídia todos os dias. Permitir colagénio / f ibroblastos matriz para contrair durante aproximadamente 8 dias, até que eles se encaixam em um prato de 24 cavidades (Contracção de ~ 3,5 cm a 1,5 cm de diâmetro, ver Figura 1). </li></ol> Nota: A concentração de colagénio deve ser ajustada de acordo com a aplicação. Quanto mais diluir a solução de colágeno, mais rápido ele será contratado pelos fibroblastos. Ligeiras diferenças na taxa de contracção será experimentada com lotes diferentes de colagénio na mesma concentração. De igual modo, as culturas de fibroblastos diferentes irão contrair os géis de colagénio em taxas diferentes, e os fibroblastos presentes mais, mais rapidamente a taxa de contracção. Concentração de colagénio e densidade de fibroblastos podem, pois, ajustado para modificar a taxa de contracção,ea densidade do final do gel de colagénio. 4. Fase II - chapeamento células de interesse no topo da matriz Nota: Esterilizar todos os fórceps e equipamentos com etanol antes do uso. <ol><li> Utilizando uma pinça sem corte, mova suavemente matriz contratada a 24 bem-prato. Certifique-se que não se dobra. </li><li> Preparar a suspensão de células de interesse em aproximadamente 4 x 10 1 ml 4 ml / placa e após centrifugação (trypsinising, as células de se livrar de tripsina) na parte superior da matriz. O número real de células necessárias irão variar, dependendo do tipo de células utilizado. Médio de células deve ser por meio de crescimento normal das células de interesse. </li><li> Permitir que as células a crescer até à confluência no topo da matriz, a cerca de 3-5 dias. </li></ol> 5. Fase III - A transferência da matriz para uma grade de invasão (aproximadamente 0-21 dias) <ol><li> Corte grades de aço inoxidável para criar um tripé e autoclave antes do uso (ver Figura 2). </li><li> Coloque grade estéril6 prato cm, adicionar o meio de crescimento a um nível acima da grelha (aprox 10.5ml). Matriz lugar na grelha, e aspirar cuidadosamente meios de modo que o fundo da matriz esteja em contacto com os meios, mas não submersa. Isto é referido como a interface ar / líquido, o que cria um gradiente que promove a invasão. Substituir médios a cada dois dias (ver figura 2). </li><li> Vivo de células, utilizando imagiologia contextual células fluorescentes pode ser trabalhada, nesta fase, ou mais cedo. O colágeno fibrilar contratado ou eu posso ser fotografada usando geração de segundo harmônico (SHG, veja a Figura 3). Usando multi-fotão excitação combinada com detecção de campo amplo descobrimos que SHG pode ser trabalhada pelo menos 100 um para a matriz, enquanto que as células que expressam GFP citoplasmático pode ser trabalhada, pelo menos, 200 um de profundidade. </li></ol> Nota: No que diz respeito à quantificação da invasão, o dia em que matricies são colocados sobre as grades define Dia 0. Colocação em grades gera um gradiente de meios de cultura celular que promove int invasão<html> oa matriz. As amostras podem ser visualizados ao longo do próximo 1 - 21 dias (ou mais) para avaliar processos biológicos, tais como invasão, proliferação de sobrevivência, ou diferenciação (Veja a lista de referência). 6. Estágio IV - Fixação <ol><li> Adicionar 5 ml de 4% de PFA em um tubo Falcon. </li><li> Transferir a matriz sobre uma superfície plana. Corte a matriz no meio com um bisturi limpa (como você vai estar manchando a seção transversal), levante a matriz com bisturi e transferência para a PFA 4%, corrigir durante a noite. </li><li> Matrizes organotípicas agora está pronto para manchar com anticorpo / mancha de escolha (veja a Figura 3). </li></ol> 7. Os resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> Figura 1 Exemplo de fibroblastos convencionalmente colagénio I. Mistura de fibroblastos e colagénio de cauda de rato separada da placa de petri e deixa-se contraem ao longo de 6-8 dias. Figura 2 &quot;src =&quot; / files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; Figura 2 progressão ensaio organotípicas. Um esquema, de um conjunto organotípica-se e progressão. B, Exemplo de colagénio / f ibroblastos no topo da matriz de aço inoxidável, rede estéril para criar interface ar / líquido. <img alt="Figura 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> Figura 3 Aplicações de ensaio organotípica. A, B não-invasivos e invasivos ductais pancreáticas adenocarcinoma (PDAC) invadindo células ao longo do tempo com a matriz organotípica. C, vivendo equivalente de pele mostrando uma epiderme estratificada em um fibroblasto-contratado componente colágeno dérmico. D, C8161 invadir células de melanoma em um fibroblasto -contratado equivalente dérmica de colágeno. E, a invasão de células PDAC (verde) que interagem e invadindo com fibroblastos (vermelho) dentro da matriz organotípica. F </strong&gt;, as células invasoras PDAC (verde) interagindo com um ambiente degradante matriz extracelular (roxo) visualizada usando multifotônica baseado em geração de segundo harmônico.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

Não temos nada a revelar.

Apresentamos aqui um método para a produção de uma matriz de 3-dimensional adequada para estudos de migração celular invasiva 6-8. A matriz é constituída por colagénio tipo 1 fibrilhas, que são contraídas ao longo de um período de vários dias, por fibroblastos humanos primários epidérmicos. O colagénio é obtido por extracção de ácido, não digestão enzimática, o que preserva a reactividade nas extremidades poli-peptídicas e promove a reticulação das fibras de colagénio em agregados maiores mediante neutralização das condições do tampão 9. Isso resulta em um gel reconstituído que mais de perto imita as características de colagénio in vivo e tem consequências importantes para as propriedades invasivas das células cultivadas nos géis. Não importa se fresco ou congelado material de partida é utilizado, mas a utilização de caudas de rato adolescentes é importante porque a ligação cruzada do colagénio é mais lábil em animais jovens. Colágeno I de animais mais jovens é, portanto, mais fácil de extrair e re-constitui wom maior fidelidade. A matriz de colagénio pode ser fixadas e coradas com anticorpos dirigidos contra as células tumorais, quer invasivos ou fibroblastos estromais 2,10 e são altamente úteis para testar a eficácia de fármacos que possam inibir a invasão 5,6. Embora este protocolo sugere a utilização de primários de fibroblastos de pele humana, é viável utilizar fibroblastos primários do outros tipos de tecidos, de acordo com o tipo de tecido sob investigação. Também é possível estabelecer culturas de fibroblastos imortalizados utilizando, incluindo as células que foram transfectadas estavelmente com conjugados de proteínas fluorescentes. Desta forma, ambas as células estromais e de tumor podem ser rotulados de visualizar as interacções célula-célula durante a invasão 11.

<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> MEM 10x </td><td> Gibco </td><td> 21430 </td><td> - </td></tr><tr><td> NaOH </td><td> Sigma </td><td> 367176-500G </td><td> Prepare estoque 0,22 M em água </td></tr><tr><td> FCS </td><td> PAA Laboratories </td><td> A15-101 </td><td> - </td></tr><tr><td> 35 pratos milímetros </td><td> Falcão </td><td> 353001 </td><td> Para a etapa 3,4 </td></tr><tr><td> 60 pratos milímetros </td><td> Falcão </td><td> 353004 </td><td> Para o passo 5,2 </td></tr><tr><td> Primavera fórceps sem corte </td><td> Samco </td><td> E003/02 </td><td> Dentada, não lisa </td></tr><tr><td> Paraformaldeído 16% </td><td> Electron Microscopy Serviços </td><td> 15710 </td><td> Diluir a 4% em PBS antes da utilização </td></tr><tr><td> Telas para cd-1, tamanho 40 mesh </td><td> Sigma </td><td> S07707-5EA </td><td> Grades de aço inoxidável, 5 por pacote </td></tr><tr><td> Tubagem de diálise </td><td> Medicell Internacional </td><td> 7607 2295 </td><td> 12-14 kD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> Ácido acético </td><td> Sigma </td><td> 242853 </td><td> - </td></tr><tr><td> 24 prato bem </td><td> Falcão </td><td> 353047 </td><td> Para passo 4.1 </td></tr><tr><td> Fungizone </td><td> In Vitrogen </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

A migração celular é essencial para muitos aspectos da biologia, incluindo o desenvolvimento de cicatrização de feridas, as respostas celulares do sistema imunitário, e as metástases de células tumorais. A migração tem sido estudada em lamelas de vidro, a fim de tornar a dinâmica celular passíveis de investigação por microscopia de luz. No entanto, tornou-se evidente que muitos aspectos da migração de células dependem de características do ambiente local, incluindo a sua elasticidade, a composição de proteína, e do tamanho dos poros, o que não é fielmente representado por dois substratos rígidos dimensionais, tais como vidro e de plástico 1. Além disso, a interacção com outros tipos celulares, incluindo fibroblastos do estroma 2 e 3, as células do sistema imunológico foi demonstrado desempenhar um papel importante na promoção da invasão das células cancerosas. Investigação ao nível molecular tem cada vez demonstrado que a dinâmica molecular, incluindo a resposta ao tratamento com droga, de células idênticas são significativamente diferentes quando comparados <em&gt; In vitro e in vivo 4. Idealmente, seria melhor para estudar a migração de células em seu contexto que ocorre naturalmente em organismos vivos, no entanto isso não é sempre possível. Sistemas de tecidos intermédios de cultura, tais como a matriz de células derivada, matrigel, cultura organotípica (descrito aqui) de explantes de tecidos, Organóides, e xenoenxertos, são, portanto, importantes intermediários experimentais. Estes sistemas aproximados determinados aspectos de um ambiente in vivo, mas são mais passíveis de manipulação experimental, tais como o uso de linhas de células transfectadas estavelmente, regimes de tratamento de drogas, a longo prazo e de alta resolução de imagem. Tais sistemas intermediários são especialmente úteis como provar motivos para validar sondas e estabelecer parâmetros necessários para a imagem da resposta dinâmica de células e repórteres fluorescentes antes de realizar imagens in vivo 5. Como tal, eles podem desempenhar um papel importante na redução da necessidade de experiências com o livinanimais g.

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Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

É descrito um método para a preparação de uma matriz de 3-dimensional constituído por colagénio do tipo I e fibroblastos humanos primários. Este gel organotípica serve como um substrato útil para avaliar a migração celular invasiva porque imita características básicas de estroma de tecido e é susceptível de muitas formas de microscopia.

Organotípica Colágeno Ensaio I: Uma Plataforma maleável para Avaliar Comportamento celular em um contexto de 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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22.3K visualizações.  University of Glasgow. É descrito um método para a preparação de uma matriz de 3-dimensional constituído por colagénio do tipo I e fibroblastos humanos primários. Este gel organotípica serve como um substrato útil para avaliar a migração celular invasiva porque imita características básicas de estroma de tecido e é susceptível de muitas formas de microscopia.

Vídeo: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Um método para Encapsulation ovário folículo e Cultura em um proteoliticamente Degradáveis ​​3 Sistema Dimensional

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Bioengenharia

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

Andaimes auto-relato para cultura de células 3-Dimensional

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

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Um método aperfeiçoado para a preparação de colágeno tipo I Da Pele

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Gradiente planar do sistema de difusão para investigar Quimiotaxia numa Matriz de Colagénio 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

O tecido triturado em Compressed Collagen: Um BioTransplant contendo celular para o reparo Reconstructive Single-encenado

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno peptídeo Microcarriers para expansão da célula e seu potencial uso como sistema de entrega de células em um bioreator modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.