This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

Este protocolo segue as diretrizes LUMC do animal comitê de ética em pesquisa. 1. Preparação de Instrumentos, Meio de Cultura, e MTCS Nota: Execute todos os preparativos na capela de fluxo laminar. O MTCS câmara de perfusão, borbulhador e suporte são descritos na Lieber et al., 2010 11. <ol><li> Desinfectar os fórceps e micro tesoura com etanol 70%. Prepara-se uma seringa de 5 mL com uma agulha 21G com NaCl estéril de Tyrode Buffer (130 mM, KCl 5,4 mM, HEPES 6,0 mM, 1,2 MgSO 4 mM, 1,2 mM de KH 2 PO 4, glicose 20 mM, 1,5 mM de CaCl 2 .2H 2 O, pH = 7,2). Prepara-se uma seringa de 5 mL com uma agulha de 21G estéril com solução de KCl (100 mM). </li><li> Preparar 65 ml de meio por coração: DMEM suplementados com 10% de Soro Fetal de Vitela, antibiótico / antimicótico (A / A; 100 unidades de penicilina, 100 ug de estreptomicina, e 0,25 ug de anfotericina B / ml), insulina-Transferrin-O selénio (STI; 10 ug / ml de insulina, 5,5 ng / ml de transferrina, 6,7 ng / ml de selenito de sódio) e esterilizar filtro. </li><li> Esterilizar a câmara de perfusão e de retenção de bolhas por pulverização com 70% de etanol e seco com uma toalha de papel. Montar o MTCS (Figura 1D), no entanto, inicialmente sem a câmara de perfusão. Posicione a armadilha de bolhas no stand. Use uma bomba de roletes com cabeças de bomba peristáltica fácil de carga. <ol><li> Usando tubo flexível de silicone fazer ligações entre o reservatório (tubo de 50 ml) e a bomba, a bomba e a armadilha de bolhas, o borbulhador e reservatório (ver Figura 1D). Adicione a tubagem no interior da incubadora o tempo suficiente para permitir que forma a atingir o equilíbrio de gás com o gás na incubadora através da tubagem de gás permeável de silício (1 M) e o tubo exterior da incubadora tão curto quanto possível. </li></ol></li><li> Encha o tubo de 50 ml com etanol 70%, ligar a bomba (velocidade de fluxo de cerca de 1 ml / min para permitir a circulação adequada) e deixar o cir etanolcular por 30 min para esterilizar toda a tubulação. Remover o etanol por esvaziamento do reservatório e bombeando o etanol a partir do sistema. </li><li> Encha o tubo de 50 ml com água destilada estéril, ligar a bomba e deixe a água circule durante 30 minutos para se livrar do restante etanol. Remover a água por esvaziamento do reservatório e a bombear a água para fora do sistema. </li><li> Encha o tubo de 50 ml com 45 ml de meio, coloque o suporte na incubadora. Ligue o (velocidade de fluxo de cerca de 1 ml / min) e a bomba faz circular o meio para preencher toda a tubagem, remover todas as bolhas de ar e permitir que a forma para se adaptar à composição do gás na incubadora durante pelo menos 1 h. Manter a incubadora a condições-padrão de cultura de tecidos (5% CO 2 e 37 ° C). Assegure-se que não há bolhas de ar na tubagem. </li></ol> 2. Isolamento de rato coração <ol><li> Injectar o mouse com 500 unidades de heparina. Isto irá evitar a coagulação do sangue e permite a remoção de bLood a partir do coração. Após 10 min, anestesiar o rato numa câmara de indução com 4% de isoflurano utilizando um vaporizador de precisão. A anestesia é suficiente quando o mouse não responde aos pés pitada. </li><li> Transferir o rato para uma placa de dissecção e manter a anestesia por meio de uma máscara ligada a um circuito coaxial. Desinfectar a pele do rato com 70% de álcool. Com uma tesoura de abrir a cavidade abdominal para expor a veia cava e diafragma. </li><li> Para expor o coração, fazer incisões laterais a partir da última para a primeira costelas e refletem a caixa torácica sobre a cabeça de rato. Pare a anestesia como mouse não está respirando mais. Observe o coração batendo. </li><li> Inserir a agulha 21G ligada a uma seringa de 5 ml estéril contendo tampão de Tyrode para a veia cava inferior do abdominal para dentro da cavidade torácica (através do diafragma). Certifique-se de que o sangue pode sair da veia cava caudal a partir da inserção da agulha. Perfundir thTampão de Tyrode e suavemente e com uma pressão constante para a veia cava até que o coração perde parcialmente a sua cor vermelha. Nota: o coração permanece a bater, permitindo a remoção do sangue a partir do coração. </li><li> Substituir a agulha com a agulha 21G ligada a uma seringa de 5 ml contendo solução de KCl estéril. Suavemente perfundir a solução de KCl para a veia cava até o coração parar de bater e remover a agulha. Nota: A solução de KCl preserva o coração na fase diastólica relaxada, o que vai permitir uma inserção mais fácil das agulhas de perfusão e do sistema de perfusão coronária. </li><li> Levante ligeiramente o coração usando uma pinça curva e usando uma tesoura dissecar o coração livre do tecido circundante, mas deixar artérias e veias proximais ao coração intactos e anexado ao coração. Transferir o coração para um tubo de 15 ml contendo PBS arrefecido em gelo suplementado com antibiótico / antimicótico (A / D). Armazenar o coração em PBS gelado por até 3 horas. </li></ol> 3. Cannulation dos corações mouse na câmara de perfusão <ol><li> Efectuar todos os procedimentos em capela de fluxo laminar. Transferir o coração isolado a partir do tubo de 15 ml a um prato de 10 cm de Petri e adicionar PBS suplementado com A / A. </li><li> Sob um microscópio de dissecação, o uso de tesoura e micro fórceps para remover todo o tecido cardíaco, mas não preservar a aorta ascendente, pelo menos, da bifurcação com a artéria inominada e as veias pulmonares, 2 mm proximal ao coração. Cortar a extremidade da ponta do coração para criar um acesso para o lúmen do ventrículo esquerdo (tipicamente 2 mm). Coloque a câmara de perfusão em câmara de fluxo a sob o microscópio de dissecação. </li><li> Anexar uma seringa de 5 ml com meio à agulha de inserção usando o tubo de silicone. Encher a câmara de perfusão com 20 ml de meio e colocar o coração no estágio de rotação entre as agulhas embotadas 2 na câmara de perfusão (Figura 1). Ajustar a altura da fase de rotação de modo que o coração está posicionada em frente doagulhas. </li></ol> 4. A ligadura para a cultura das valva mitral (ver figura 1) <ol><li> Ligadura da aorta com sutura (seda 7.0). Ligadura do apêndice atrial esquerdo com sutura. Insira a agulha (nr. 1 na Figura 1) através da veia pulmonar para o átrio esquerdo e ligar com sutura proximal à sua entrada no átrio esquerdo. Certifique-se de que a agulha não é muito longe para o átrio esquerdo, pois isso iria danificar a válvula mitral. </li><li> Inserir a agulha (nr.2 na Figura 1) com um movimento linear para o ventrículo esquerdo. Transferir o meio a partir da câmara de perfusão para um tubo de 50 ml. Selar a agulha para o miocárdio com cola biocompatível. </li><li> Depois que a cola secar, injetar cuidadosamente em meio coração, ligando uma seringa cheia de médio a agulha nr.1 e verifique se há algum vazamento. Assegurar que as saídas médias de nr.2 agulha eo último sangue restante é perfundido para fora do coração. </li></ol> 5. Ligação para a cultura das Válvula Aórtica (veja a Figura 1) <ol><li> Inserir a agulha (nr.1 na Figura 1) na aorta e ligar com sutura. Certifique-se de que a agulha não é muito longe para a aorta como isso iria danificar a válvula aórtica. Inserir a agulha (nr.2 na Figura 1) com um movimento linear para o ventrículo esquerdo. Transferir o meio a partir da câmara de perfusão para um tubo de 50 ml. Selar a agulha para o miocárdio com cola biocompatível. </li><li> Depois que a cola secar, injetar cuidadosamente médio para o coração, ligando uma seringa cheia de médio a agulha nr.2 e verifique se há algum vazamento. Assegurar que as saídas médias de nr.1 agulha eo último sangue restante é perfundido para fora do coração. </li></ol> 6. Coloque Perfusão Secção de suporte <ol><li> Encha câmara de perfusão com os 20 ml de meio transferidos. Coloque a junta e tampa na câmara e aperte com lavadora e parafusos. </li><li> Remova os MTCS montados a partir do incubator. Fixe a câmara de perfusão no stand. Conecte o tubo (veja a Figura 1D). Colocar o suporte na incubadora (5% de CO 2 e 37 ° C). Conecte a tubulação à bomba. Ligue a bomba com velocidade de fluxo de cerca de 600 mL / min. </li><li> Certifique-se de que no reservatório e / ou o borbulhador o nível do meio permite que as visualizações da presença do fluxo pela queda das gotas médias de entrada. Após a cultura, o coração é removido do sistema, fixadas e processadas para exame histológico. </li></ol>

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	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, 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+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

As etapas críticas em cultura das válvulas cardíacas de rato incluem fazer o tempo entre a excisão do coração do rato e a ligação na câmara de perfusão tão curto quanto possível, para assegurar a viabilidade e ligação das agulhas perpendicular às válvulas para assegurar a direcção correcta de fluxo . Além disso, a verificação do fluxo após a ligadura na câmara de perfusão sem forma garante uma inserção correcta e ligadura de agulhas. É essencial para manter uma cultura estéril e evitar bolhas...

Doença valvar cardíaca é uma das principais causas de morbidade e mortalidade no mundo ocidental; sua prevalência aumenta com a idade e afeta mais de 10% da população de 75 anos ou mais 1. As válvulas da parte sistêmica do coração, as valvas aórtica e mitral, são os mais afetados. Válvula cardíaca doença é caracterizada pela perda da estrutura altamente organizada das válvulas, o que resulta na alteração das propriedades mecânicas 2. A integridade estrutural é, portanto, crucial para a função da válvula. Os folhetos da válvula são constituídos por células intersticiais valvulares (VIC), células endoteliais valvulares (VEC), e matriz extracelular, o que é altamente organizado num padrão em camadas 3,4. Os VICs são responsáveis ​​pela síntese de ECM, degradação e organização. Fatores que emana da corrente sanguínea, ECs ou residentes no ato ECM nas VICs orquestrando sua função. Além,forças mecânicas agir sobre o folheto durante o ciclo cardíaco, resultando em laminar ou tensão de cisalhamento oscilatório, tensões de compressão ou de tracção que influenciam o comportamento de VICs 5. A fim de compreender como a estrutura da válvula é regulado, ele deve primeiro ser compreendida como VICs responder ao conjunto diversificado de estímulos experimentadas durante o ciclo cardíaco. Estudos in vitro têm sido muito informativo sobre as características e capacidades das células valvulares. A resposta destas células, in vitro, no entanto, nem sempre podem mimetizar com exactidão a resposta in vivo 6; Por exemplo, a resposta a estímulos de VIC é dependente da presença de células endoteliais e a composição 5 ECM. Além disso, a resposta das células a estímulos valvulares depende da sua localização específica no folheto 7. Além disso a estímulos bioquímicos, o comportamento das células valvulares é determinada por forças mecânicas que actuam ón a válvula 8. Cada região da válvula é submetida a seu próprio conjunto específico de tensões hemodinâmicas. Embora atuais modelos ex vivo têm mostrado que as forças mecânicas são importantes determinantes da estrutura valvular 5, os mecanismos associados ainda não estão claros. Enquanto os modelos in vivo têm fornecido insights sobre os mecanismos moleculares subjacentes ao desenvolvimento valvular 9,10, insights sobre biologia valvular adulto ainda é indescritível. Por conseguinte, um modelo ex vivo de fluxo foi desenvolvido em que as válvulas cardíacas podem ser cultivadas na sua posição natural no coração por um período prolongado de tempo 11. Isto tem a vantagem de que as válvulas permanecem na sua configuração natural e os VICs experimentar o mesmo ambiente como in vivo, fazendo com que as respostas a estímulos VICs tão natural quanto possível. Além disso, a cultura das válvulas na sua posição natural no coração facilita a sujeição de cada umregião valvular às tensões hemodinâmicas relevantes. Neste modelo ex vivo, isto é, o sistema de cultura de tecidos em miniatura (MTCS), as válvulas podem ser submetidos a diferentes estímulos bioquímicos e hemodinâmicas, permitindo o estudo do seu papel na remodelação da válvula cardíaca.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

A válvula da aorta (Figura 2) da válvula mitral ou 11 podem ser cultivadas por pelo menos 3 dias. Através da cultura na posição aberta (que representa a posição sistólica para a válvula aórtica diastólica e a posição da válvula mitral para a), as células permanecem viáveis ​​valvulares. Não é observada a morte celular como determinado pela ausência de células de TUNEL positivo (Figura 2H, I) ou clivado expressão da caspase-3 (não mostrado e Lieber <e...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Cultura Rato Cardiac Valves no Sistema de Cultura de Tecidos Miniature

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

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Bioengineering

8.8K visualizações.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Vídeo: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Reconstruções anatômicas do sistema venoso cardíaco humano usando contraste Tomografia computadorizada de Amostras de perfusão-fixados

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo Relativo Avaliação hidrodinâmica de Tri-folheto polímero Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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

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

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

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

A<em&gt; In Vitro</em&gt; Modelo Cultura Órgão da Murino Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamíferos em andaimes tridimensionais do Peptide

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Um método baseado em molde líquido de criação livre de andaime tridimensional de tecido cardíaco

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...