Este trabalho foi financiado pelos Institutos Nacionais de Saúde - National Heart Lung, and Blood Institute (Award # R00HL093358 a LDB).

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	<li>Stegemann, J. P., Hong, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+biochemical,+and+extracellular+matrix+effects+on+vascular+smooth+muscle+cell+phenotype.">Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.</a> Journal of applied physiology. 98, 2321-2327 (2005).</li><li>Guarnieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+RGD+gradient+on+PEG+hydrogel+scaffold+influences+cell+migration+parameters.">Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters.</a> Acta. 6, 2532-2539 (2010).</li><li>Krebs, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+poly(lactic-co-glycolic)+acid+scaffolds+with+in+situ+pore+formation+for+tissue+engineering.">Injectable poly(lactic-co-glycolic) acid scaffolds with in situ pore formation for tissue engineering.</a> Acta. 5, 2847-2859 (2009).</li><li>Zimmermann, W. -. 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Não há conflitos de interesse declarados.

O encapsulamento dos cardiomiócitos de rato neonatal no resultado de fibrina géis de forma consistente e viável tridimensional in vitro modelo do sistema do miocárdio. Fibrina é um biomaterial preferido porque quando as células são aprisionadas, elas são metabolicamente ativa e capaz de compactação, remodelação e recriando uma matriz extracelular que é consistente com o tecido do coração nativo 12. Porque nós permitimos que os cardiomiócitos a alinhar-se neste ambiente, sua funcionalidade é mais característico do músculo cardíaco, resultando em maior força de contração em comparação com tecidos isotrópico 6. Para potenciais aplicações terapêuticas, é necessário encapsular células dentro de um material que promove tanto a viabilidade e funcionalidade. Os protocolos aqui apresentados demonstram uma forma eficiente e precisa para a criação de uma rede de fibrina para controlar o comportamento de células cardíacas em um microambiente tridimensional. Alguns problemas potenciais podem surgir durante a criação ea cultura dessas construções. Um potencial problema é a manutenção da viabilidade celular antes de encapsulamento, o que irá afectar significativamente a funcionalidade da construção. Esforço deve ser feito para limitar a morte celular após o isolamento, reduzindo o tempo entre o isolamento eo encapsulamento das células dentro do gel de fibrina. Construções deve ser fornecida mídia todos os dias em um horário rigoroso. Além disso, é importante para assegurar a homogeneidade em todas as soluções utilizadas. Se a mistura de fibrinogênio, trombina e células produz um ambiente heterogêneo, a capacidade das células para remodelar o ECM, mecanicamente casal e contrato é potencialmente obstruído. Também é importante para prevenir a formação de bolhas de ar durante a injeção das construções, a fim de evitar perturbações na continuidade da engenharia de tecidos. Uma maneira de aliviar esta questão é chamar mais gel do que é necessário para fazer a construção com a seringa e injetar lentamente. Por último, uma vez que a matriz de fibrina definiu e foi em meio de cultura por 24 horas, é essencial destacar a construir a partir dos lados do molde do anel para promover a compactação baseada em células do gel de fibrina. Mantendo a construir no meio do molde facilita a troca de gases e nutrientes. Aderência para os lados do molde do anel podem também romper o alinhamento desejado celular. É importante notar que a decapitação consciente é usado como método de eutanásia neste protocolo, que é um método aceitável no âmbito das orientações de ambos os Institutos Nacionais de Saúde ea Associação Médica Americana Veterinária. No entanto, algumas instituições recomendam usar anestésico seguido por decapitação em ratos neonatal. Nós escolhemos a decapitação consciente porque garante a quantidade mínima de tempo sob condições de hipóxia para o tecido do coração extirpado / células. Quantidades relativamente pequenas de hipóxia pode levar à isquemia e morte dos miócitos potencialmente miócito, que poderiam afetar significativamente os resultados deste protocolo.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Cloreto de sódio </td><td> Sigma </td><td> S7653 </td><td> PBS </td></tr><tr><td> Cloreto de potássio </td><td> Sigma </td><td> P9333 </td><td> PBS </td></tr><tr><td> Fosfato de sódio dibásico </td><td> Sigma </td><td> S7907 </td><td> PBS </td></tr><tr><td> Fosfato de potássio monobásico </td><td> Sigma </td><td> P5655 </td><td> PBS </td></tr><tr><td> Glicose </td><td> Sigma </td><td> G5400 </td><td> Isolamento </td></tr><tr><td> hemostat </td><td> Multa Ferramentas Ciência </td><td> 91308-12 </td><td> Isolamento </td></tr><tr><td> pinça fina </td><td> Multa Ferramentas Ciência </td><td> 11251-20 </td><td> Isolamento </td></tr><tr><td> tesouras grandes </td><td> Multa Ferramentas Ciência </td><td> 91401-14 </td><td> Isolamento </td></tr><tr><td> micro-tesoura </td><td> Multa Ferramentas Ciência </td><td> 91501-09 </td><td> Isolamento </td></tr><tr><td> lidar com bisturi </td><td> Multa Ferramentas Ciência </td><td> 10008-13 </td><td> Isolamento </td></tr><tr><td> lâmina de bisturi </td><td> Fisher Scientific </td><td> 08-918-5A </td><td> Isolamento </td></tr><tr><td> Underpad banco absorvente </td><td> VWR </td><td> 56617-014 </td><td> Isolamento </td></tr><tr><td> drape estéril </td><td> Fisher Scientific </td><td> GM42526 </td><td> Isolamento </td></tr><tr><td> saco de autoclave </td><td> Pescador </td><td> 01-812-54 </td><td> Isolamento </td></tr><tr><td> compressa de gaze </td><td> Fisher Scientific </td><td> 13-761-52 </td><td> Isolamento </td></tr><tr><td> betadine </td><td> Purdue Produtos </td><td> 67618-150-01 </td><td> Isolamento </td></tr><tr><td> luvas estéreis </td><td> Fisher Scientific </td><td> 19-020 </td><td> Isolamento </td></tr><tr><td> pipeta de transferência estéril </td><td> Fisher Scientific </td><td> 9962 </td><td> Isolamento </td></tr><tr><td> colagenase </td><td> Worthington </td><td> CLS2 </td><td> Isolamento </td></tr><tr><td> Teflon haste 1 / 4 polegada de diâmetro </td><td> McMaster-Carr </td><td> 8546K11 </td><td> Parte do molde </td></tr><tr><td> Teflon ID tubo 1 / 4 de polegada, OD 1 / 2 polegada </td><td> McMaster-Carr </td><td> 8547K31 </td><td> Parte do molde </td></tr><tr><td> Silicone O-Ring ID 1 / 4 de polegada, OD 1 / 2 polegada </td><td> McMaster-Carr </td><td> 9396K204 </td><td> Parte do molde </td></tr><tr><td> Teflon tubo de 1 / 4 ID polegadas, 5 / 16 polegadas OD </td><td> McMaster-Carr </td><td> 52355K14 </td><td> Parte do molde </td></tr><tr><td> Seringas Kendall monoject 6cc </td><td> Fisher Scientific </td><td> 05-561-41 </td><td> Parte do molde </td></tr><tr><td> BD seringa 3cc </td><td> Fisher Scientific </td><td> 309585 </td><td> Parte do molde </td></tr><tr><td> Fibrinogênio bovino </td><td> Sigma </td><td> F8630 </td><td> Construir </td></tr><tr><td> A trombina bovina </td><td> Sigma </td><td> T7513 </td><td> Construir </td></tr><tr><td> 1 M HEPES </td><td> Sigma </td><td> H0887 </td><td> Construir </td></tr><tr><td> Cloreto de sódio </td><td> Sigma </td><td> S7653 </td><td> Construir </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 10569 </td><td> Construir </td></tr><tr><td> PLURONIC F-127 </td><td> Sigma </td><td> P2443 </td><td> Construir </td></tr><tr><td> Cloreto de cálcio </td><td> Sigma </td><td> 383147 </td><td> Construir </td></tr><tr><td> 0,2 micron filtro </td><td> Fisher Scientific </td><td> SCGVT05RE </td><td> Construir </td></tr><tr><td> 40 micron filtros celular </td><td> Fisher Scientific </td><td> 22-363-547 </td><td> Construir </td></tr><tr><td> 0,45 micron filtro de tampa de garrafa </td><td> Corning </td><td> 430627 </td><td> Construir </td></tr><tr><td> de vidro pré-filtro </td><td> Millipore </td><td> AP2007500 </td><td> Construir </td></tr><tr><td> 18G 1 1 / 2 polegada de comprimento agulha </td><td> Fisher Scientific </td><td> 14-826-5D </td><td> Construir </td></tr><tr><td> 21G agulha de 1 polegada </td><td> Fisher Scientific </td><td> 14-826C </td><td> Construir </td></tr><tr><td> construção de frascos </td><td> Fisher Scientific </td><td> 2116 </td><td> Construir </td></tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140 </td><td> Mídia </td></tr><tr><td> soro de cavalo </td><td> Sigma </td><td> H1138 </td><td> Mídia </td></tr><tr><td> Soro fetal bovino </td><td> Invitrogen </td><td> 16000 </td><td> Mídia </td></tr><tr><td> ácido aminocapróico </td><td> Acros Organics </td><td> 103305000 </td><td> Mídia </td></tr><tr><td> ácido ascórbico </td><td> Sigma </td><td> A5960 </td><td> Mídia </td></tr><tr><td> insulina </td><td> Sigma </td><td> I9278 </td><td> Mídia </td></tr><tr><td> Paraformaldeído, 16% </td><td> Microscopia Eletrônica de Ciências </td><td> 15710 </td><td> Histologia </td></tr><tr><td> Temperatura de corte ideal (OCT) </td><td> Ted Pella </td><td> 27050 </td><td> Histologia </td></tr><tr><td> 2-metilbutano </td><td> Pescador </td><td> 03551-4 </td><td> Histologia </td></tr><tr><td> Anticorpo primário do mouse MYH1/2/4/6 </td><td> Santa Cruz Biotechnology </td><td> SC-32732 </td><td> Histologia </td></tr><tr><td> Coelho Conexina 43 anticorpo primário </td><td> Tecnologia de sinalização celular </td><td> 3512 </td><td> Histologia </td></tr><tr><td> Dylight 549-burro conjugado anticorpo anti-rato-secundário </td><td> Jackson Laboratories ImmunoResearch </td><td> 715-505-151 </td><td> Histologia </td></tr><tr><td> Dylight 488-conjugados de anticorpos anti-coelho Donke secundário </td><td> Jackson Laboratories ImmunoResearch </td><td> 711-485-152 </td><td> Histologia </td></tr><tr><td> Vivo / morto ensaio </td><td> Invitrogen </td><td> L-3224 </td><td> Análise </td></tr></tbody></table>

encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering

A cultura de células em um ambiente de hidrogel tridimensional é uma técnica importante para o desenvolvimento de construções para a engenharia de tecidos, bem como estudar as respostas celulares em condições diversas da cultura in vitro. O ambiente tridimensional reproduz com mais exatidão o que as células observar in vivo devido à aplicação de estímulos mecânicos e químicos em todas as dimensões 1. Tridimensional hidrogéis podem ser feitas a partir de polímeros sintéticos como PEG-DA 2 e PLGA 3 ou um número de proteínas que ocorrem naturalmente, como o colágeno 4, 5 ou ácido hialurônico fibrina 6,7. Hidrogéis criado a partir de fibrina, uma proteína do sangue que ocorre naturalmente coagulação, pode polimerizar para formar uma malha que é parte da cura do corpo ferida processos naturais 8. Fibrina é célula-degradáveis ​​e potencialmente autólogo 9, tornando-se um andaime ideal temporário para engenharia de tecidos. Aqui nós descrevemos em detalhe o isolamento dos cardiomiócitos neonatal a partir de três dias os filhotes de rato velho e preparação das células para encapsulamento em construções de hidrogel de fibrina para engenharia de tecidos. Miócitos neonatal são uma fonte de células comuns utilizados para estudos in vitro na formação de tecido cardíaco e engenharia 4. Fibrina gel é criado pela mistura do fibrinogênio com a trombina enzima. A trombina cliva fibrinopeptídeos FPA e FPB de fibrinogênio, revelando os sítios de ligação que interagem com outros monómeros 10. Essas interações causam os monômeros para auto-montar em fibras que formam a malha de hidrogel. Porque o timing dessa reação enzimática pode ser ajustado alterando a relação de trombina ao fibrinogênio, ou a razão de cálcio para a trombina, pode-se molde de injeção constrói com um número de diferentes geometrias 11,12. Além disso podemos gerar o alinhamento do tecido resultante da forma como restringir o gel durante a cultura 13. Após o cultivo da engenharia construções de tecido cardíaco durante duas semanas sob condições estáticas, as células cardíacas começaram a remodelar a construir e pode gerar uma força de contração em condições de estimulação elétrica 6. Como parte deste protocolo, também descrevem métodos para analisar a engenharia de tecidos do miocárdio após o período de cultura, incluindo a análise funcional da força ativa gerada pelo músculo cardíaco construir sobre a estimulação elétrica, bem como métodos para determinar a viabilidade celular final (Live-Morto ensaio) e coloração imuno para examinar a expressão de proteínas e morfologia típica importante para a contração (Miosina Cadeia Pesada ou MHC) e acoplamento celular (Conexina 43 ou Cx43) entre os miócitos.

Watch this Scientific Journal Video about Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering at JoVE.com

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Descrevemos o isolamento de cardiomiócitos neonatal e na preparação das células para encapsulamento em construções de hidrogel de fibrina para engenharia de tecidos. Descrevemos métodos para analisar a engenharia de tecidos do miocárdio após o período de cultura, incluindo a força ativa gerada com a estimulação elétrica e viabilidade celular e coloração imuno.

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 ...

encapsulation-cardiomyocytes-fibrin-hydrogel-for-cardiac-tissue

Research

JoVE Journal

Bioengineering

24.1K visualizações.  Tufts University. Descrevemos o isolamento de cardiomiócitos neonatal e na preparação das células para encapsulamento em construções de hidrogel de fibrina para engenharia de tecidos. Descrevemos métodos para analisar a engenharia de tecidos do miocárdio após o período de cultura, incluindo a força ativa gerada com a estimulação elétrica e viabilidade celular e coloração imuno.

Vídeo: Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

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

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Engenharia de um hidrogel de camada dupla para controlar a diferenciação ASC

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Engenharia de Tecidos do Intestino num modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

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 ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Um protocolo para Decellularizing Cochleae Mouse para engenharia de tecidos do ouvido interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Produção de elastina, como proteína hidrogel para encapsulamento e imunocoloração de células em 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

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