This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1 Criação de placas PNIPAAm revestidas <ol><li> Dissolve-se o 2,6 g de PNIPAAm em 2 ml de uma solução de hexano a 60% de tolueno / 40%. </li><li> Aquecer a mistura a 60 ° C durante 10 minutos agitou-se, até que a PNIPAAm é dissolvido. </li><li> Cortar papel de filtro para um círculo de diâmetro 60 mm e coloque o papel no funil de Buchner. </li><li> Filtrar a solução através de Buchner Funil para a pré-pesado copo de vidro (não use plástico, como o hexano vai derreter plásticos). </li><li> Colocar a proveta e o conteúdo para um vácuo de sino (24 psi) S / N (16 horas). Nota: Até o resíduo é reagido com isopropílico vai oxidar para ter certeza que não entra em contato com o oxigênio. </li><li> Pesar no copo para estabelecer o peso do PNIPAAm. </li><li> Adicionar álcool isopropílico para a PNIPAAm, criando uma 50/50 w / w solução. </li><li> Coloque 2 ml da solução sobre a superfície da placa de cultura de tecidos, e no revestimento, durante 5 min, sob luz UV. </li><li> Lava-se a placa com 2 ml de PBS quente duas vezesantes de usar para cultura de células. </li></ol> 2 Criação de Fichas de celulares Nota: as folhas da pilha de células primárias para o órgão-alvo podem ser criadas através de um número de métodos diferentes, ou por revestimento de superfícies de cultura de tecidos com o polímero termo-sensível, tal como descrito aqui. Placas termossensíveis Pré-revestidos também são oferecidos por um número de fornecedores. Nota: Este protocolo é para a cultura usando um prato de 35 mm. Resumidamente, as células são primeiro incubadas a 37 ° C durante um mínimo de 24 horas à confluência para estabelecer conexões laterais entre as células adjacentes. Para soltar as folhas de células, as placas são sujeitas a temperaturas inferiores a 32 ° C. A camada de células é então transferido para a matriz fibrosa base forte contendo um hidrogel permissiva neovascular com as células endoteliais vasculares. <ol><li> Isolar a população de células. Nota: Este método é dependente dos procedimentos de derivação individuais e o tipo de células. Rato mu liso aórticocélulas LECSA (RASMC) são utilizadas neste exemplo. Estas são células primárias de músculo liso isoladas a partir da aorta abdominal de um rato. </li><li> Lavam-se as células com 2 ml de PBS quente. </li><li> Adicionar 3 ml de tripsina (ou outro / solução dissociando clivagem) para as células durante 5 min. </li><li> Inibir a tripsina por adição de 3 ml do meio de cultura, ou uma solução tampão de fosfato (PBS) contendo 10% de soro fetal bovino (FBS). </li><li> Recolher as células num tubo cónico e contagem de uma alíquota. </li><li> Rodar as células a 1.000 rpm (228 xg) durante 5 min. </li><li> Aspirar o sobrenadante e ressuspender as células no seu meio de crescimento (mais SmGM2 kit bala meio de cultura é usada para RASMC). </li><li> Coloque a mídia que contém as células em um termo-sensível placa 35 mm - PNIPAAm placa revestida com uma concentração que vai atingir 100% de confluência. Nota: Para RASMCs que o número foi determinado como sendo de 100.000 células / cm 2. No entanto, devido à perda de células durante a passagem, 120% do que va<html> lue é usado. </li><li> Colocar numa incubadora a 37 ° CO / N. Nota: É importante manter as células a 37 ° C, para manter a adesão de células à placa. </li></ol> 3: Preparação da Matriz Fundamental Nota: Várias matrizes fibrosas em 3D podem ser utilizados para a camada de matriz fibrosa forte entre as camadas de células delicadas. Alguns exemplos incluem: gelfoam, biovidro, materiais naturais acellularized 26 ou nanospun materiais 27,28 A matriz de bexiga do porco (UBM) utilizada nesses estudos foi generosamente fornecidos a partir de nosso colaborador, o Dr. Badylak 29. <ol><li> Antes de usar, determinar as características da matriz, incluindo a falta de conteúdo celular se descelularizados matriz é usado, 27,28 viabilidade celular específica, e o espaço vazio 22. </li><li> Cortar a matriz pré-esterilizados em um tamanho e forma desejados. Nota: Aqui, um furador é utilizada para cortar um círculo diâmetro de 4 mm. </li></ol>ve_title &quot;&gt; 4. células endoteliais de semeadura em um Neovascular Permissivo Hidrogel Nota: As células endoteliais podem ser obtidos a partir de uma variedade de fontes, incluindo a diferenciação de células estaminais ou progenitoras. Aqui, são utilizadas células HUVEC. <ol><li> Use qualquer hidrogel permissiva (fibrina, géis de colagénio), enquanto o tempo de reticulação é suficientemente curto para permitir que as células permanecem viáveis. Nota: Aqui, um gel de hialuronano (HA), com base reticulados com uma ponte de dissulfureto é utilizado. </li><li> Prepare o hidrogel de HA, de acordo com o protocolo da empresa. </li><li> Recolher as células endoteliais e dispersam-se numa solução de uma única célula utilizando tripsina 1x. Nota: Accutase ou dissociação celular Tampão também poderia ser utilizada para a dispersão de células individuais. </li><li> Desactivar a enzima tripsina, utilizando uma quantidade igual de inibidor de tripsina de soja (que é importante que as células não entram em contacto com o soro) ou 10% de FBS em PBS, recolhendo a solução / células para um tubo de 15 ml. </li><li> CONT as células, e calcular o volume necessário para as dimensões de emenda (previamente quantificada). Nota: Para um patch 4 milímetros, aqui de 2 milhões de células endoteliais são usados. </li><li> Extrato de 2 milhões de células, e coloque em um novo tubo de 15 ml. </li><li> Giram em (228 xg) por 5 min. </li><li> Aspirar o sobrenadante, deixando as células como um sedimento num tubo cónico. </li><li> Misturar os materiais líquidos de HA e de gelatina numa proporção de 1: 1 ração. Em seguida, adicionar 80% do volume total dentro do tubo cónico contendo o sedimento. </li><li> Ressuspender as células endoteliais na mistura 1: 1 HA / Gelatina </li><li> Colocar as células em suspensão na mistura de HA / gelatina para a base de matriz fibrosa a partir do Passo 2. </li><li> Adicionar 1/5 (20 por cento) do volume total desejada do agente de reticulação </li><li> Incubar durante 1 hora a 37 ° C. </li></ol> 5. Isolamento de Folhas Celulares <ol><li> Retirar as placas tratadas com PNIPAAm 35 milímetros contendo as células a partir do incubador e colocar num capuz de cultura de células emRT. </li><li> Aspirar rapidamente o meio das células, e adicionar 2 ml de 6% de gelatina normal que foi aquecido a 37 ° C. </li><li> Enquanto que a gelatina está ainda quente, coloque a estrutura de metal em que a gelatina, submergindo-a abaixo da superfície da gelatina normal (Filme 1). </li><li> Coloque a totalidade da placa em gelo durante 5 a 7 minutos, permitindo que a gelatina endureça. </li><li> Após 7 min, uma espátula para separar cuidadosamente os bordos de gelatina do lado da placa, e em seguida utilizar uma pinça para levantar a estrutura de metal da placa Nota: A gelatina 6%, e a folha de célula deve levantar com a estrutura. </li><li> Mover a lâmina da célula para o prato e colocar na parte superior do conjunto de matriz de hidrogel de base fibrosa, ajustando cuidadosamente a estrutura no topo da construção. Nota: O lado apical da camada de células ainda estará na posição superior. </li><li> Adicionar 2 ml de meio morno (37 ° C). </li><li> Incubar S / N permitindo que a folha de células para aderirem à superfície do hidrogel. </li><li> Remover tele grade metálica após a solução aquece (aproximadamente 1 hora), ou no dia seguinte. </li></ol>

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We have nothing to disclose.

Os passos críticos no protocolo incluem: revestimento das superfícies das placas com o polímero thermoresponsive e manipular as folhas de células após o arrefecimento das placas. Como as células diferentes apresentam diferentes propriedades físicas, como adesividade, o tempo de elevação deve ser otimizado para cada tipo de célula diferente. O segundo, e mais importante desafio componente deste protocolo, centra-se na manipulação da camada de células, um aspecto crítico dos métodos para a montagem do tecid...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Calcein-AM</td><td>Invitrogen</td><td>C3099</td><td>Cell tracker / live dye</td></tr><tr><td>Lysotracker Red</td><td>Invitrogen</td><td>L7528</td><td>Cell tracker</td></tr><tr><td>Neutral Red</td><td>Sigma</td><td>N7005</td><td>Visible Cell dye</td></tr><tr><td>pNIPAAM</td><td>Sigma Aldrich</td><td>412780250</td><td>Poly(&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide)</td></tr><tr><td>Toluene</td><td>Sigma Aldrich</td><td>244511-1L</td><td></td></tr><tr><td>Hexane</td><td>Sigma Aldrich</td><td>296090-1L</td><td></td></tr><tr><td>RAOSMC</td><td>Lonza</td><td>R-ASM-580</td><td>Rat Aortic Smooth Muscle Cells</td></tr><tr><td>SmGM2</td><td>Lonza</td><td>CC-4149</td><td>Smooth Muscle Media</td></tr><tr><td>HUVEC</td><td>Invitrogen</td><td>C-003-5C</td><td>Human Venous Endothelial Cells</td></tr><tr><td>HyStem</td><td>Glycosan/Biotime</td><td></td><td></td></tr><tr><td>Isopropyl alcohol</td><td>VWR International</td><td>BDH1133-4LP</td><td></td></tr><tr><td>Trypsin</td><td>Corning Cellgro</td><td>25-053-C1</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14287-072</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16140-071</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Specific Equipment&lt;/strong&gt;</td></tr><tr><td>Filter paper</td><td>Ahlstrom&amp;nbsp;</td><td>6310-0900&amp;nbsp;</td><td></td></tr><tr><td>Buchner Funnel&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>Z247308&amp;nbsp;</td><td></td></tr><tr><td>UpCell Plates&amp;nbsp;</td><td>Nunc&amp;nbsp;</td><td>2014-11&amp;nbsp;</td><td></td></tr><tr><td>UV light</td><td>Jelight Company&amp;nbsp;</td><td>UVO Cleaner Model No.42</td><td></td></tr></tbody></table>

tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

Muitos tecidos, como os corações humanos adultos, são incapazes de se regenerar adequadamente após a lesão. 2,3 Estratégias em engenharia de tecidos propor inovações para ajudar o corpo a recuperação e reparação. Por exemplo, as abordagens TE pode ser capaz de atenuar a remodelação cardíaca após infarto do miocárdio (IM) e, possivelmente, aumentar a função cardíaca total a um próximo nível pré-MI normal. 4 Tal como acontece com qualquer tecido funcional, regeneração bem sucedida do tecido cardíaco envolve a entrega dos vários tipos de células com sinais ambientais que favoreçam a integração e sobrevivência do enxerto de células / tecido implantado. Tecidos artificiais devem abordar vários parâmetros, incluindo: sinais solúveis, interações célula-célula, e materiais de matriz avaliados como veículos de entrega, os seus efeitos sobre a sobrevivência da célula, resistência do material, e facilitação de organização célula-tecido. Estudos empregando a injeção direta de células do enxerto apenas ignorar esses elementos essenciais. 2,5,6Um projeto de tecidos combinando esses ingredientes ainda tem de ser desenvolvido. Aqui, apresentamos um exemplo de projetos integrados que utilizam camadas de folhas de células padronizadas com dois tipos distintos de materiais biológicos derivados contendo o tipo de célula para órgãos-alvo e células endoteliais para aumentar a formação de novos vasos no &quot;tecido&quot;. Embora esses estudos concentram-se na geração de tecido cardíaco semelhante, este projeto tecido pode ser aplicado a muitos outros do que o coração com design e material alterações mínimas órgãos, e é destinado a ser um produto off-the-shelf para terapias regenerativas. O protocolo contém cinco etapas detalhadas. Uma temperatura de poli sensível (N -isopropylacrylamide) (PNIPAAm) é usado para placas de cultura de tecidos revestimento. Em seguida, as células são cultivadas de tecido específico sobre a superfície das placas revestidas / superfícies micropattern para formar camadas de células com aderências laterais fortes. Em terceiro lugar, uma matriz de base, é criado para o tecido através da combinação de matriz porosa com Permissi neovascularve hidrogéis e células endoteliais. Finalmente, as folhas de células são levantados a partir dos pratos PNIPAAm revestidos e transferido para o elemento de base, fazendo com que a construção completa.

O diagrama de fluxo (Figura 1) mostra o método geral para fazer o penso multicamadas. Folhas de células são separadas da placa tratada PNIPAAm por baixar a temperatura abaixo de 32 ° C. Em seguida, a camada de células é colocada em cima do hidrogel reticulado contendo as células endoteliais semeadas na matriz fibrosa subjacente (Figura 1). As placas pré-tratados termossensíveis, também podem ser utilizados para criar as camadas de células. Superfícies topológicas especiais ...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

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

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

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

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

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.4K visualizações.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

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

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Manipulando células vivas para construir estável montagem celular 3D sem andaime Artificial

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Bioquímica

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Expansão de duas dimensões Electrospun nanofibras esteiras em três dimensões andaimes

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genética

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

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

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

Viabilidade de Bioprinted celulares Construções Usando uma impressora cartesiano Dispenser Três

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

Bioengenharia

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construção de Fichas de hidrogel modular para micropadronadas Macro-escalado 3D Cellular Arquitetura

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

A Facile e Eco-friendly rota para fabricar poli (ácido láctico) Scaffolds com Graded Pore Tamanho

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabricação de espumas de Matriz Extracelular derivados e Microcarregadores como cultura celular de tecidos específicos e plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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. ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Construção de uma folha de células-tronco mesenquimais em várias camadas com um sistema de cultura dinâmica 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Núcleo/Shell impressão scaffolds para engenharia de tecidos de estruturas tubulares

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...