O financiamento foi fornecido pelo National Institutes of Health (EB 001.013), Ciências Naturais e Pesquisa de Engenharia Council of Canada e Canadian Institutes of Health Research. Agradecemos ao Dr. AP McGuigan por sua experiência e ajudar no desenvolvimento de módulos.

1) Preparação de Tubos <ol><li> Corte 2-3 comprimentos m de tubos de polietileno (0,76 mm de diâmetro x 1,22 milímetros OD). </li><li> Um fio de agulha 20 G1 em uma das extremidades do tubo. </li><li> Tubos de bobina e coloque em uma bolsa de conversores auto-vedação (7 1 / 2 &quot;x 13&quot;). As duas extremidades do tubo devem ser colocados no final unsealed do saco e gravado no local com fita de esterilização a gás. </li><li> Saco de vedação e gás esterilizar. </li></ol> 2 Neutralização) de colágeno Nota: 1 ml de colágeno preenche cerca de 2 m de tubos de polietileno. <ol><li> Coloque a quantidade necessária de colágeno necessário para o comprimento desejado de tubo em um tubo cônico de 15 ml e manter em gelo. </li><li> Adicionar 128 uL de 10x α-MEM por mL de colágeno para o tubo e misturar por pipetagens repetidas da solução. Nota: (1) A solução deve girar o amarelo como o α-MEM é misturado na colágeno acidificado e (2) tentar minimizar bolhas, quando a mistura. </li><li> Neutralizar o colágeno, adicionando bicarbonato de sódio 0,8 M até que o colágeno tem um pH de 7,4. Para uma estimativa geral do bicarbonato de sódio pH adicionar até a solução de colágeno amarela vira uma cor rosa claro. Nota: Ele normalmente leva 30-60 mL por cada mL de colágeno. </li><li> Mantenha colágeno neutralizado em gelo até estar pronta para usar como o colágeno neutralizado vai gel se não forem mantidas a 4 ° C. </li></ol> 3) Incorporação de Cells (Opcional) Nota: Dependendo do tipo de célula que as células podem ser incorporados em uma concentração de entre 1 milhão de células por ml a 20 milhões de células por mL. Use o meio necessário para as células que você deseja incorporar. <ol><li> Remova a mídia a partir de células e lavar com 5 mL de PBS. </li><li> Remover PBS e adicionar 3 mL de tripsina-EDTA. </li><li> Incubar as células em tripsina-EDTA por 5 min a 37 ° C. </li><li> Ressuspender as células em 7 mL de mídia. </li><li> Contagem de células. </li><li> Transferir o número necessário de células para a incorporação de um tubo cônico de 15 mL. </li><li> Agregar as células a 300 xg por 5 min. </li><li> Aspirar mídia fora do pellet de células. </li><li> Ressuspender as células do colágeno neutralizada e manter em gelo. </li></ol> 4) gelificação do Colágeno <ol><li> Em um armário de segurança biológica corte a extremidade selada do saco que contém o tubo de polietileno. </li><li> Anexar uma seringa de 3 ml para a agulha 20G, no final, um dos tubos, usando uma pinça esterilizada. Manter o tubo no saco. </li><li> Insira a outra extremidade do tubo no colágeno neutralizado até que a ponta está no fundo do tubo cônico, usando uma pinça esterilizada. Manter o tubo cônico no gelo. </li><li> Puxe o colágeno na tubulação de polietileno retraindo o êmbolo da seringa devagar. Nota: Se o colágeno é puxado para dentro do tubo de forma rápida, em seguida, irá formar bolhas no colágeno. </li><li> Depois de puxar o colágeno através do tubo, puxe a agulha 20G de tubulação e colocar ambas as extremidades de volta para o saco. Saco de fita fechada. </li><li> Incubar a bolsa a 37 ° C por 60 min. Se o colágeno contém células, então, virar o saco sobre cada 15 minutos. para que as células não se contentar com um lado do módulo. </li></ol> 5) Corte de Tubos Contendo o colágeno coagulada <ol><li> Cabeçalhos autoclave e lâmina para o dispositivo de corte e bandeja para segurar a tubulação. </li><li> Montar o dispositivo de corte. </li><li> Tubulação lugar em uma bandeja estéril na frente do dispositivo de corte e carga do tubo no dispositivo de corte. </li><li> Configurar um tubo cônico de 50 mL contendo 25 mL dos meios de comunicação na saída do dispositivo de corte para recolher os módulos de corte. Nota: Use mídia contendo FBS mesmo para colágeno somente módulos como os módulos não se separam da tubulação sem FBS na mídia. Se os módulos contêm células usam os meios de comunicação necessários para as células incorporado. Se não houver células incorporados nos módulos usar a mídia para as células endoteliais. </li><li> Definir o dispositivo de corte para cortar 2 mm de comprimento tubos e cortar a tubulação. </li><li> Incubar módulos corte a 37 ° C por 60 min no tubo cônico de 50 mL. </li></ol> 6) A remoção do colágeno Módulos de Tubulação <ol><li> Vortex tubo cônico de 50 mL contendo módulos na tubulação por 10 seg. Nota: Seja gentil quando separar módulos integrados com alta densidade de células para evitar a ruptura. </li><li> Deixe módulos para liquidar o fundo do tubo. Leva cerca de 5 min. </li><li> Usando uma boca larga pipeta 10 mL sorológicos transferir os módulos resolvido a um tubo de 15 mL cônico. </li><li> Repita os passos 6,1-6,3 duas vezes. </li><li> Vá para a etapa 7 para módulos de revestimento com células endoteliais ou módulos de transferência para uma placa de cultura não-tecido tratado. </li></ol> 7) Revestimento os módulos com células endoteliais <ol><li> Settle módulos no fundo de um tubo cônico com 15 mL 5 mL dos meios de comunicação para poe incorporados células. </li><li> Remova a mídia a partir de células endoteliais e lavar com 5 mL de PBS. </li><li> Remover PBS e adicionar 3 mL de tripsina-EDTA. </li><li> Incubar as células em tripsina-EDTA por 5 min a 37 ° C. </li><li> Ressuspender as células em 7 mL de mídia. </li><li> Contagem de células. Precisa de 5x10 6 células endoteliais por mL de módulos embalado na parte inferior do tubo cônico. </li><li> Agregar as células a 300 xg por 5 min e ressuspender em 5 mL de mídia das células endoteliais. </li><li> Adicione o de 5 mL da mídia contendo células endoteliais para os módulos. Nota: Isto dá uma proporção de 50:50 de dois tipos de mídia que nós encontramos para trabalhar para a sobrevivência ea função de ambos os tipos de células. Testar o uso de um meio de co-cultura formulada para garantir a manutenção adequada dos fenótipos dos dois tipos de células antes de usá-lo. </li><li> Incubar os módulos com as células endoteliais tanto estática e dinamicamente. </li><li> Para os módulos de incubação estática misturar com células endoteliais por inversão e definir o tubo na posição vertical a 37 ° C por 15 min. Repetir. </li><li> Dinamicamente a semente células endoteliais-o suavemente o tubo a 37 ° C por 60 min. </li><li> Repita duas vezes de incubação estática. </li><li> Coloque os módulos em uma placa de cultura de tecidos não-tratados e incubar durante a 37 ° C. </li><li> O lugar no dia seguinte os módulos em uma placa nova cultura não-tecido tratado com meios frescos (mistura 50:50, se usar o sistema de co-cultura) para remover unattached células endoteliais. </li><li> Incubar módulos que são revestidos com células endoteliais até cobrir 100% da superfície dos módulos. Isso geralmente leva cerca de 7 dias. </li></ol> 8) Resultados Representante: Os módulos vão olhar cilíndrica quando eles são removidos do tubo. Se os módulos contêm células incorporados e / ou são revestidos com células endoteliais que podem contratar até 50% em volume e desenvolver uma forma oval (Figura 1). Os módulos também se tornará mais densa e mais opaco quando vistos por microscopia de luz (Figura 1). Além disso, quando as células endoteliais são confluentes na superfície dos módulos há uma formação de tight junctions que pode ser visto por imunofluorescência da VE-caderina (Figura 2). Análise de transferência de massa demonstrou que os módulos são capazes de suportar altas densidades celulares (8x10 7 células / cm 3), sem desenvolver um núcleo necrótico devido ao transporte de oxigênio inadequada, o que muitas vezes é problemático em tecidos maiores (por exemplo, módulos de grande diâmetro, D = 1,4 mm) (Figura 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Figura 1" /> Figura 1: Módulo de fabricação e contração. Contração módulo ocorreu durante os três dias seguintes HUVEC-C (linhagem de células endoteliais) semeadura resultados diâmetro módulo significativamente menor e comprimento (p &lt;0,001) (A). Incorporados células HepG2 foram distribuídos uniformemente dentro dos módulos no momento da fabricação e manteve alta viabilidade (B). [Live células verdes; células mortas red] [adaptado de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Figura 2" /> Figura 2: VE-caderina coloração de imunofluorescência de junções celulares CE apertado 10 dias após RAEC onde revestidas na superfície de um módulo. Barra de escala = 50 mm. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Figura 3" /> Figura 3: coloração tricrômico de Masson de módulos típicos (0,76 mm de diâmetro inicial) e grandes módulos (1,4 mm de diâmetro inicial) demonstrar o efeito das limitações de difusão de oxigênio. Sete dias após a fabricação, um grande número de células mortas tinham formado dentro do núcleo das grandes módulos (painel inferior direito), deixando apenas uma fina borda (~ 200μm de espessura) de células viáveis. Por outro lado, os módulos pequenos retida uma distribuição uniforme e elevado de células vivas. [Módulos incorporado com células HepG2 e revestidos com células endoteliais.] [Extraído de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Figura 4" /> Figura 4: (A) HMEC-1 semeado em poloxamine - módulos de colágeno. Após o dia 1 de semear os módulos poloxamine colágeno mantêm a sua forma cilíndrica e não há propriedades apego limitado de células do colágeno em comparação com os módulos apenas. Barra de escala = 200 mM. (B) imagem do microscópio de luz de um módulo de colágeno contendo PLGA baseado em microesferas biodegradáveis. Barra de escala = 500 mm. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Figura 5" /> Figura 5: Exemplos de ensaios in vitro. (A) A angiogênese ensaio: capilar, como formações no módulo semeadas com células endoteliais podem ser facilmente detectados e quantificados após 5 dias de incubação com células-tronco derivadas de tecido adiposo. (B) imagens de microscopia confocal de um ensaio ao vivo / morto em módulos, onde o sFORMAÇÃO PARA células vivas é verde e de células mortas é vermelho. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Figura 6" /> Figura 6: Módulos de colágeno contendo embutidos células-tronco mesenquimais e uma camada superficial de células endoteliais. Módulos foram expostos a tensão de cisalhamento (~ 0,64 dyn / cm 2) por 7 dias em uma câmara microfluídicos e começar a mostrar a fusão em pontos de contato. Células endoteliais estão manchadas com vWF (marrom) e núcleos de células mesenquimais aparecem em azul (H &amp; E). Barra de escala = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Figura 7" /> Figura 7: Centenas de módulos de colágeno contendo células-tronco derivadas de tecido adiposo (ASC) e revestidos com células endoteliais microvasculares Humanos (HMEC) são implantados sob a pele do rato para estudar ASC / HMEC interação in vivo para aplicações de regeneração de gordura.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Não há conflitos de interesse declarados.

Nós fabricamos diversas micro-tecidos usando módulos integrados com diferentes tipos de células 1-11. Conseguimos incorporados cardiomiócitos primária, ilhotas, células-tronco adiposas e células mesenquimais do estroma, bem como várias linhagens de células incluindo HepG2, NIH 3T3 e clone 9 células do fígado. Temos os módulos revestidos com vários tipos de células endoteliais de aorta de ratos, incluindo células endoteliais, células endoteliais da veia umbilical humana e humanos células endot...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
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fabrication of micro-tissues using modules of collagen gel containing cells

Este protocolo descreve a fabricação de um tipo de micro-tecidos chamados de módulos. A abordagem módulo gera uniforme, tecidos escalável e vascularizada. Os módulos podem ser feitas de colágeno, bem como outros materiais gelable ou reticulável. Eles são cerca de 2 mm de comprimento e 0,7 mm de diâmetro sobre a fabricação, mas encolher de tamanho com células incorporado ou quando os módulos são revestidos com células endoteliais. Os módulos individualmente são pequenos o suficiente que as células incorporados estão dentro do limite de difusão de oxigênio e outros nutrientes, mas módulos pode ser embalado para formar tecidos maiores que são perfusable. Estes tecidos são modulares na construção, porque tipos diferentes de células pode ser incorporado em ou revestidos nos módulos antes de serem embalados para formar tecidos complexos. Há três passos principais para fazer os módulos: (1) neutralizar o colágeno e incorporação de células-lo, (2) gelificação do colágeno no tubo e corte os módulos e (3) revestimento dos módulos com células endoteliais.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Criação de micro-tecidos utilizando gel de colágeno cilíndricos, chamados módulos, que contêm células embarcados e cuja superfície é revestida com as células endoteliais.

Fabricação de Micro-tecidos utilizando Módulos de Colágeno Gel células contendo

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.6K visualizações.  University of Toronto. Criação de micro-tecidos utilizando gel de colágeno cilíndricos, chamados módulos, que contêm células embarcados e cuja superfície é revestida com as células endoteliais.

Vídeo: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricação de hidrogéis micropatterned para Sistemas de Cultura Neural usando fotolitografia Projeção Dinâmica Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Bioengenharia

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imagiologia desnaturadas colágeno Vertentes<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via foto desencadeada Hibridização do Caged Colágeno mimético Peptídeos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparação e Fotoacústica Análise de Veículos celulares contendo ouro nanobastões

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formando polymersomes de tamanho gigante usando hidratação Gel-assistida

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricação e caracterização de espectros ópticos de tecido contendo Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparação, purificação e utilização de lipossomas contendo ácido graxo

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Captação de novas nanopartículas revestidos de lipídeos contendo Falcarindiol por células-tronco mesenquimais humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Produção microfluida de liposomos sensíveis à temperatura contendo lisolipóides

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...