This work was funded by the National Science Foundation (CBET 0845754, AHH).

1. Preparação para a Formação de alginato de Discos <ol><li> Prepara-se uma 4% (w / v) em solução de alginato de 1x Solução Salina Tamponada com Fosfato de Dulbecco sem cloreto de cálcio adicionado ou cloreto de magnésio e colocar num banho de água a 37 ° C. As concentrações da solução de alginato pode variar, mas 1-4% (w / v) de soluções de alginato são recomendados. </li><li> Misturar a solução de alginato a uma razão de 1: 1 com a base de meios de cultura de célula quente (por exemplo, Modified Eagle Médium da Dulbecco.) Para o tipo de célula desejado. A concentração de alginato é agora metade da concentração original, isto é., 2% (w / v). Esterilizar solução de alginato / mídia usando filtros de seringa de nylon de 0,2 um estéreis. </li><li> Preparar um banho de cloreto de cálcio di-hidratado 102 mM estéril em água estéril com solução suficiente para submergir moldes (~ 200 - 300 ml). </li><li> Recolher o estéril &quot;molde de formação de gel&quot; (diâmetro x 3 mm poços cilíndricos de altura de 6 mm em um 3 in x 3 em chapa de alumínio, consulte <strong&gt; Figura 1) e placas terminais (inferior: um 3 em x 3 em chapa de alumínio, Top: um 1,5 em x 3 em chapa de alumínio) e preparar moldes da seguinte forma: <ol><li> Cortar papel grosso filtro (filtro de papel mata-borrão) ea membrana microsieve celular (10 de tamanho de poro) para os tamanhos de parte superior e placas terminais inferiores e colocá-los no banho de cloreto de cálcio até à saturação, a cerca de 1 min. Se desejado, esterilizar o papel de filtro grosso e a membrana microsieve através de autoclave ou luz ultravioleta durante 30 min antes da utilização. </li><li> Defina-se a metade (metade inferior) da construção de moldes. <ol><li> Colocar os seguintes itens cima da outra na seguinte ordem e suavizar usando uma espátula estéril: grande placa terminal (3 em x 3 pol), papel de filtro de espessura, e, em seguida, a membrana microsieve. </li><li> Invertido e pressionar o molde para uma pilha de toalhas de papel para assegurar a perda de excesso de solução de cloreto de cálcio. </li><li> Coloque a &quot;gel de molde de formação&quot;, com os poços cilíndricos no topo do cell membrana microsieve e suavemente aperte o molde juntos em dois lados usando grampos da pasta nos lados direito e esquerdo. Certifique-se de deixar espaço suficiente para a parte superior da placa terminal (1,5 in x 3 in) para cobrir os poços completamente mais tarde. </li></ol></li><li> Defina-se a segunda metade (metade superior) da construção de moldes. <ol><li> Coloque os seguintes itens em cima de um ao outro na seguinte ordem e suavizar: pequena placa terminal, papel de filtro grosso, membrana microsieve. </li><li> Invertido e pressiona esta metade do molde sobre uma pilha de toalhas de papel para assegurar a perda de excesso de solução de cloreto de cálcio. Não fixe nesta parte do molde utilizando grampos da pasta no momento. </li></ol></li></ol></li><li> células de cultura, tal como recomendado por instruções do fabricante. Colheita das células desejadas para incorporar nos hidrogéis de alginato em camadas. A densidade celular recomendada é de 1 - 2 x 10 6 células / ml, mas isto pode ser variado dependendo experiências desejadas. Nota: Ao usar este métodopara fazer camadas com diferentes tipos de células, certifique-se de cultura dois tipos de células em paralelo para mergulhar. As células estaminais mesenquimais (MSCs) semeadas a uma densidade celular de 1 x 10 6 células / ml será utilizado como um exemplo para o seguinte protocolo. O número de células e o número de disco pode ser dimensionado para as experiências conforme necessário. <ol><li> Uma a duas semanas antes da incorporação, as células estaminais mesenquimais semente a uma densidade celular de 3000 - 5000 células / cm2 em 10 mL de meio de crescimento basal (Dulbecco Modified Eagle Médium, 10% de Soro Fetal Bovino, 2% de L-Glutamina, 1 % Aminoácidos não essenciais, 1% de penicilina / estreptomicina) em frascos T-75. </li><li> Remova a mídia passou a cada 2 - 3 dias, e substituir com 10 ml de meio de crescimento basal até que as células são 80 - 90% confluentes. </li><li> Para colher as células, remova meio gasto e pipeta de 3 ml de 1x Dulbecco salina de tampão fosfato sem cloreto de cálcio adicionado ou cloreto de magnésio para lavar as células. Remover esta solução, pipeta 3 ml de 0,25% Trypsin-EDTA sobre as células que crescem em frascos T-75, e incubar durante 5 min a 37 ° C. Após as células têm levantado da superfície aderente, adicionar 6 - 9 ml de meio de crescimento basais. </li><li> MSCs centrifugue-as a 600 xg durante 5 min à temperatura ambiente, aspirar o sobrenadante e re-suspensão em 1-5 ml de meio de crescimento basais. Contar as células usando um hemocitômetro utilizando as instruções do dispositivo. </li><li> Retirar 1 x 10 6 células da resuspensão de células totais, de centrifugação utilizando as mesmas condições, e aspirar o sobrenadante. </li></ol></li></ol> 2. Formação de celulares Seeded alginato Discs <ol><li> Re-suspender o sedimento de células com 1 ml de solução de alginato / meios estéreis para atingir uma densidade celular de 1 x 10 6 células / ml. A mistura de células-alginato será homogeneamente um aspecto turvo, quando as células são misturadas de forma adequada. </li><li> Pipete 130 pi da mistura de células-alginato em poços seis altos 6 mm de diâmetro x 3 mm na metade inferior do moldeconstruir gota a gota, de modo a não criar quaisquer bolhas. Um ligeiro menisco convexo deve ser visível acima do bordo de cada poço. </li><li> Cuidadosamente suavizar a metade superior da construção do molde usando uma espátula estéril e entregá-lo, de modo que a membrana da célula é microsieve na parte superior dos poços. Coloque o molde na construção de topo dos poços, tendo o cuidado de cobrir os poços que contêm células e a mistura de alginato completamente. </li><li> Levante a construção do molde carregado e, enquanto pressiona firmemente no centro, Mola os restantes dois lados (superior e inferior) para prender as metades superior e inferior da construção do molde. As soluções de células-alginato deve ser firmemente situado nos poços neste momento. </li><li> Mergulha-se o construto de molde fixada no banho de cloreto de cálcio 102 mM, certificando-se que toda a construção é submerso. Incubar na capa de cultura de células à temperatura ambiente durante 90 min. </li><li> No final dos 90 minutos, remover a construção do molde a partir do cloreto de cálcio 102 mMbanho e coloque em uma pilha de toalhas de papel na capa de cultura de células. Remove todos os quatro grampos da pasta e separar as duas metades do molde de construção. Hidrogéis formados nos poços não deve ter quaisquer bolhas e deve encher os poços completamente. </li><li> Com uma espátula, rastrear cuidadosamente o bordo dos poços contendo os hidrogéis para soltar cuidadosamente e cunha os hidrogeles. Depois de retirar os hidrogeles a partir da construção de moldes, soltar os hidrogéis directamente para um banho de 1x Solução Salina Tamponada com Fosfato de Dulbecco, com o cloreto de cálcio e cloreto de magnésio. Cobrir os geles completamente para lavar o excesso de solução de cloreto de cálcio para 1-5 min. </li><li> Transferir os hidrogeles em solução basal meio de crescimento em prato desejado (por ex., Placas de 6 cavidades) que cobre completamente os hidrogeles. Incuba-se durante pelo menos 1 hora na incubadora de cultura de células a 37 ° C e 5% de CO 2 antes de continuar para o passo de estratificação. Nota: Os hidrogeles que contêm as células podem ser mantidas ema incubadora de cultura de células indefinidamente neste momento até camadas de géis com uma outra população celular é desejada, desde que mudanças de suporte são concluídos a cada 2 - 3 dias. </li></ol> 3. Camadas de alginato de Discos <ol><li> Durante a última meia hora da incubação de 1 hora, recolher e preparar moldes e solução estéril para o recozimento os géis. <ol><li> Prepara-se o &quot;molde de corte&quot; pela fixação de um molde que tem cavidades de metade da altura do &quot;molde de formação de gel&quot; (6 mm de diâmetro x 1,5 mm de poços altos em um 3 em x 3 na placa de alumínio) a uma placa terminal (3 em x 3 em chapa de alumínio) usando grampos da pasta nos lados direito e esquerdo. </li><li> Preparar o &quot;molde de recozimento&quot;, fixando um molde que é de 3 mm de diâmetro maior do que o &quot;molde de formação de gel&quot; (9 mm de diâmetro x 3 mm poços altos em um 3 in x 3 em chapa de alumínio) a uma placa terminal (3 in x 3 em chapa de alumínio) usando grampos da pasta nos lados direito e esquerdo. </li><li> Prepara-se uma solução decitrato de sódio 100 mM / EDTA 30 mM (di-hidrato de citrato de sódio, di-hidrato de sal tetrassódico etilenodiaminotetra-acético ácido) em água estéril. </li></ol></li><li> Remover géis de populações de células desejadas (neste exemplo, dois discos com hMSCs) dos meios de comunicação no prato e colocar nos poços &quot;corte do molde&quot;. Cada gel deve caber confortavelmente nas cavidades com metade do gel salientes acima do molde. </li><li> Utilizando um bisturi, cortar o gel ao longo da superfície do molde (isto reduzirá o hidrogel em meio). Virar a metade superior do gel e colocá-lo em um poço de molde aberto. A metade do gel também deve caber dentro do molde bem, mas agora ambos os géis metade deve ser a altura do molde, com a superfície interior cortado visível. Repita com o segundo gel. Nota: Atenção: Só as superfícies de corte interiores irá formar discos em camadas. Usando as superfícies exteriores resultará na separação metades. Suspeita-se que a textura da membrana microsieve transferida para a superfície do gel de evitarO sucesso do processo de recozimento. </li><li> Coloque um pedaço de membrana celular microsieve seco no topo das cavidades, certificando-se que a membrana microsieve está em contacto com todas as metades de gel a ser recozido e cobrindo-as totalmente. Coloque papel de filtro grossa no topo da membrana microsieve, certificando-se de cobrir completamente os géis. </li><li> Pipetar uma solução de citrato de sódio 100 mM / EDTA 30 mM (di-hidrato de citrato de sódio, di-hidrato de sal tetrassódico etilenodiaminotetraacético ácido) para o papel de filtro de espessura até que seja saturado. Cerca de 750 ul é suficiente para quatro poços. </li><li> Incubar os géis durante 1 min à temperatura ambiente. Em seguida, retire a membrana microsieve celular e papel de filtro grosso e descartá-las. Remova os grampos da pasta e abrir o molde. </li><li> Para cada gel recozido, transferir um citrato de sódio half / tratado com EDTA do hidrogel ao preparado &quot;molde de recozimento&quot;, com superfície de corte virada para cima. Isto será um meio de const recozidoruct. </li><li> Usando uma espátula, levantar e colocar a / half-gel tratado com EDTA citrato de segunda sódio (pode conter um tipo de célula diferente) sobre o gel já no &quot;molde de recozimento&quot;, lançando nesta segunda meia-gel para que a superfície de corte está em contacto com a superfície do corte do gel já no &quot;molde de recozimento&quot;. </li><li> Pressione com cuidado os dois géis usando uma espátula para remover quaisquer bolhas de entre os dois géis. Além disso, reposicionar os géis, conforme necessário para garantir que eles estão directamente em cima uns dos outros. </li><li> Levante cuidadosamente o &quot;molde de recozimento&quot; e submergi-lo no banho de cloreto de cálcio 102 mM durante 30 minutos. Não cubra os poços com uma placa terminal. </li><li> Após a incubação de 30 minutos, retire o &quot;molde de recozimento&quot; e colocá-lo para toalhas de papel. Remova os grampos da pasta e separar o molde da placa terminal. </li><li> Com uma espátula, recolher os géis recozidos e colocá-los em uma de 1x Solução Salina Tamponada com Fosfato de Dulbecco, com o cloreto de cálcio e mbanho de cloreto de agnesium para lavar os géis. Subsequentemente, transferir hidrogéis recozidos a meios de cultura de células em um prato desejado (por ex., Placa de 6 poços) durante a cultura. </li></ol>

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The authors have nothing to disclose.

Aqui, nós descrevemos um protocolo para a formação de discos de hidrogel de alginato em camadas para estudar a co-culturas de várias populações de células, tais como aqueles em tecidos fisiologicamente em camadas, por exemplo, cartilagem. estruturas em camadas, tal como a plataforma de cultura descrito, pode ser usado para examinar a interacção entre as duas populações de células distintas sujeitos ao mesmo ambiente de cultura ou sob carga. O alginato é um polissacaríd...

tecidos que suportam cargas de compressão, tais como a cartilagem articular ou discos intervertebrais consistem em regiões de tecidos heterogéneos que são críticas para a função tanto biomecânico e mecano-transdução adequada no tecido. Não só é a organização celular e a função distinta em diferentes regiões, mas as matrizes extracelulares (ECM) são também variou em composição e organização. Por exemplo, a cartilagem articular é constituído por três zonas primárias com morfologia, variando de células, função mecânica, e ECM. As diferenças na sua vantagem ECM para diferencial responsabilidades suporte de carga; a camada superficial está envolvida principalmente em resposta à tração para carregar, enquanto as zonas média e profunda são principalmente responsáveis ​​pela resposta à compressão 1. Da mesma forma, no disco intervertebral, um núcleo pulposo do tipo gel é rodeado por um anel fibrose lamelar e as células dentro dessas duas áreas distintas experimentar diferentes tipos de estímulos biofísicos2. Nestes tipos de tecidos, células e as matrizes extracelulares no interior das camadas de tecido interagir uns com os outros como o tecido sofre e responde a forças mecânicas. Recapitulação de tais estruturas de tecidos heterogêneos continua a ser um desafio em engenharia de tecidos e medicina regenerativa, e nossa compreensão de seu significado biológico é limitado. Existe uma necessidade para a cultura de plataformas para analisar tecidos estratificados, bem como co-culturas de diferentes populações de células dentro de uma construção. Na engenharia de tecidos de cartilagem articular, construções de andaime-menos camadas foram construídos através do aproveitamento da capacidade dos condrócitos zonal para depositar ECM variou para imitar as diferentes camadas desse tecido 3,4. No entanto, as construções de hidrogel em camadas proporcionam uma oportunidade para estudar a interacção de diversos tipos de populações de células que não possuem a capacidade para formar um tecido robusto independentemente. Por exemplo, pop diferenteulations de células estaminais mesenquimais podem ser co-cultivadas dentro de construções em camadas. Tais andaimes em camadas têm sido utilizados com ambas condrócitos e diferenciação de células-tronco mesenquimais para melhorar a engenharia de tecidos 5. Não só pode ser co-cultivadas em populações de células diferentes camadas de hidrogel semelhantes, mas um único tipo de células também podem ser cultivadas dentro de camadas que foram manipuladas para ter diferentes rigidez ou conteúdo bioquímico para desencadear respostas diferentes a partir de células de 6,7. Muitos hidrogeles biomaterial diferentes têm sido utilizados para a camada de populações de células para engenharia de tecidos de cartilagem, tais como aqueles utilizando polietileno-glicol ou álcool polivinílico bases 7-9. No entanto, os hidrogéis de alginato são um dos biomateriais mais simples do que para criar andaimes em camadas para o estudo de populações de células heterogéneas em co-cultura. Embora os hidrogéis de agarose são também facilmente formado, hidrogéis de alginato tem a vantagem adicional de permitir fácil éolation de células a partir da construção 3-D para a análise de células individuais como foi anteriormente descrito 10. Em estudos anteriores, os hidrogéis de alginato bi-camadas foram formadas em folhas finas e a partir destas folhas, secções foram cortados (por exemplo., Utilizando um punção de biópsia) para aplicações particulares, tais como por análise de conteúdo bioquímica ou propriedades de cisalhamento interfacial 11,12. Outro método para a formação de folhas finas de alginato tem sido descrita com o potencial para a estratificação em camadas múltiplas, mas mesmo assim iria requerer uma alteração ao hidrogel para utilização em testes mecânicos 13. Aqui, apresentamos um método para criar reproducibly discos de hidrogel de alginato bi-camadas para uso em co-cultura de diferentes populações de células. Esta plataforma disco de alginato possui várias vantagens. Em primeiro lugar, a forma reprodutível e tamanho pequeno é propício para a estimulação mecânica das células incorporados sem a necessidade de uma biópsia PUNCH ​​ou outra alteração física para o hidrogel para muitas aplicações. Além disso, a viabilidade das células permanece elevada durante o processo de separação e, após formação de gel uma clara separação das duas populações de células dentro do gel é visível sem região de sobreposição inicial.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Alginic Acid sodium salt</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">A1112</td>
			<td style="width:295px;">Solution made in wt% using DPBS (-/-)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (-/-)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (+/+)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11965</td>
			<td>Example. Use desired medium type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Syringe Filters (0.02um Nylon)</td>
			<td style="width:180px;">FisherBrand</td>
			<td style="width:119px;">0979C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Calcium Chloride Dihydrate</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP510</td>
			<td>Prepare solution in sterile water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Criterion Blotter Filter Paper</td>
			<td style="width:180px;">Biorad</td>
			<td style="width:119px;">1704085</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cell Microsieve Membrane (10um pore size)</td>
			<td style="width:180px;">Biodesign Inc of New York</td>
			<td style="width:119px;">N10R</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">0.25% Trypsin-EDTA (1x), phenol red</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25200</td>
			<td>Thaw in 37&#7506;C water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium citrate dihydrate</td>
			<td style="width:180px;">FisherScience</td>
			<td style="width:119px;">S93364</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td>
			<td style="width:180px;">FisherBioreagent</td>
			<td style="width:119px;">BP121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum&#160;</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">26140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicilin/Streptomycin (10000 U/ml)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">15140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-Glutamine (200mM)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25030081</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Non-essential Amino Acids (100x)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11140050</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
	</tbody>
</table>

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.

A Figura 1 descreve a formação e estratificação dos hidrogéis de alginato. Completado géis bi-camadas exibem uma separação inicial completa de populações de células, como mostrado na Figura 2. A viabilidade celular das células estaminais mesenquimais humanas) incorporado dentro desses hidrogeles e camadas continua a ser elevada e comparável com os hidrogeles a granel, como mostrado na Figura 3. A viabilidade foi avaliada ap?...

Watch this Scientific Journal Video about Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations at JoVE.com

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

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 ...

layered-alginate-constructs-platform-for-co-culture-heterogeneous

Research

JoVE Journal

Bioengineering

7.8K visualizações.  University of Maryland. 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. 

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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcápsulas de alginato como uma plataforma 3D para Propagação e Diferenciação de Células-Tronco Embrionárias (hESC) para diferentes linhagens

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Bioengenharia

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Padronização de células co-cultura Usando duas fases aquosas Sistemas

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

A plataforma automatizada versátil para experiências de estimulação Micro escala celulares

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Engenharia de Tecidos: Construção de um multicelular 3D andaime para a entrega de camadas celulares Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Uma plataforma Novel Alongamento para Aplicações em Celular e Tecidual mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Viabilidade de Bioprinted celulares Construções Usando uma impressora cartesiano Dispenser Três

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

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

Bioprinting celularizado Constrói Usando um específico do tecido Hidrogel Bioink

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

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Uma plataforma microfluídica para a isolação de alta taxa de transferência de célula única e Cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.