Nós gostaríamos de agradecer fazendas Smithfield pela doação do tecido pulmonar porcina intacta. Também gostaríamos de agradecer ao Dr. Hu Yang, Dr. Christina Tang e do Departamento de Cirurgia Plástica VCU por nos permitir usar o seu equipamento. amostras de hidrogel e de tecido foram preparados para MEV no Departamento de Anatomia e Neurobiologia Microscopia Facility VCU apoiado, em parte, pelo financiamento do NIH-NINDS Centro Núcleo Grant 5 P30 NS047463 e, em parte, pela forma de financiamento NIH-NCI Cancer Center Support Grant CA016059 P30. SEM imagem de amostras no VCU Nanotechnology Núcleo Caracterização Facility (NCC). Este trabalho foi financiado pela National Science Foundation, CMMI 1.351.162.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Solução </td><td> filtro esterilizado </td><td> Instruções </td></tr><tr><td> 2 O DIH </td><td> sim </td><td> DIH 2 O; esterilizada por filtração </td></tr><tr><td> 0,1% de solução de Triton X-100 </td><td> sim </td><td> Sob hotte adicionar 100 uL de Triton X-100 a 100 ml solução DIH 2 O e agitar até à dissolução; filtro esterilizado. </td></tr><tr><td> 2% de desoxicolato Solução </td><td> sim </td><td> Sob exaustor adicionar 2 g solução de desoxicolato de sódio por 100 ml DIH 2 O e agitar até à dissolução; Filtrar em condições estéreis </td></tr><tr><td> 1 M NaCl </td><td> sim </td><td> Adicionar 58,44 g de NaCl para 1 L de DIH 2 O; agitar até se dissolver; filtro esterilizado </td></tr><tr><td> solução de DNase </td><td> sim </td><td> Adicione 12.000unidades de ADNase a 1 L DIH 2 O; Adicionar 0,156 g MgSO4 (Anidro), e 0,222 g de CaCl 2; agitar até se dissolver; filtro esterilizado </td></tr><tr><td> PBS </td><td> sim </td><td> Combine 27,2 g de Na 2 HPO 4 · 7H 2 O (dibico hepta-hidratado), 80 g de NaCl e 2 g de KCl com 10 L DIH 2 O; agitar até se dissolver; ajustar o pH para 7,4; filtro esterilizado </td></tr></tbody></table> Tabela 1: soluções necessárias para Decelularização Tissue. Faça as soluções acima para o processo de descelularização. Armazenar a 4 ° C. Cerca de 2,5 L será necessário para um pulmão de porco. 1. Formação Hidrogel <ol><li> Porcina Lung Decelularização (adaptado a partir de referências 9,10) <ol><li> Obter, um en bloc pulmão de porco normal, de um matadouro, com coração e vasculature intacta. </li><li> Use tesouras de tecidos ou um bisturi para dissecar afastado do coração, vasculatura tão próximo quanto possível do coração de corte, mantendo-se a vasculatura irá ser usado em passos posteriores para perfusão. </li><li> Dissecar cuidadosamente afastado o tecido conjuntivo em torno da traqueia, brônquios e vasculatura utilizando um bisturi ou tesoura. NOTA: Determinar o melhor pulmão para usar para o resto do procedimento, escolhendo um pulmão sem grandes cortes ou perfurações através da pleura e grandes quantidades de atelectasia ou oclusão vascular que podem impedir a eficácia do processo de descelularização. </li><li> Cortar o pulmão subóptima deixando tanto brônquio ligados à traqueia quanto possível (coração, o tecido conjuntivo e os lóbulos dos pulmões removidos podem ser eliminados). Deve haver um pulmão que continuem ligadas ao traqueia e vasculatura. NOTA: Manter seções para histologia, se desejar. 9 </li><li> Feche brônquios desconectados com grampos ou suturas para prevent excesso de refluxo. </li></ol><ol start="6"><li> Preparar as soluções de decelularização e refrigerar a 4 ° C até serem necessárias (Tabela 1). </li><li> Usando uma bomba manual canulada para se ajustar a artéria pulmonar, a perfusão do tecido pulmonar 3 vezes com água desionizada, tanto através da artéria pulmonar e traqueia. Perfundir vasculatura primeiro de cada vez. Comece com cerca de 1 L para a vasculatura e 1,5 L em cada traqueia para perfusão, mas como os detritos celulares são removidos mais líquido pode ser perfundido através do sistema. </li><li> Perfundir tanto vasculatura e traquéia com Triton solução X-100. </li><li> Submerge tecido pulmonar em solução de Triton X-100 durante 24 h a 4 ° C. </li><li> Perfundir vasculatura e traquéia 3 vezes com água DI para enxaguar. </li><li> Perfundir tanto vasculatura e traquéia, com solução de desoxicolato. </li><li> Submergir as secções de tecido em desoxicolato durante 24 h a 4 ° C. </li><li> Perfundir vasculatura e traquéia 3 vezes com água DI para enxaguar. </li><li>Perfundir tanto vasculatura e traquéia, com solução de NaCl. </li><li> Submergir o tecido em solução de NaCl filtrada durante 1 hora a 4 ° C. </li><li> Perfundir vasculatura e traquéia 3 vezes com água DI para enxaguar. </li><li> Perfundir tanto vasculatura e traquéia, com solução de DNase. </li><li> Submergir o tecido em solução de ADNase filtrada durante 1 hora a 4 ° C. </li><li> Perfundir tanto vasculatura e traqueia 5 vezes com PBS. </li><li> Dissecar o tecido cartilaginoso perceptível, traqueia e todos os túbulos de 2 mm ou maior em diâmetro (encontrado principalmente em torno do hilo e porções mediais dos pulmões) de condução das vias aéreas, deixando apenas a zona respiratória (principalmente nas áreas periféricas). </li><li> Dissecar tecido em 1 seções polegadas ou menores. Orientação do tecido não interessa para este passo. </li><li> Deitar fora o excesso de tecido líquido e coloque em tubos de 50 ml cônico. Congelar o tecido a -80 ° C. NOTA: Manter seções para histologia para assegurar a remoção de células e debri celulars, se desejado. Usamos H &amp; E, α-galactosidase, PicoGreen, hidroxiprolina, ninidrina, azul de alcian, SDS-PAGE e espectrometria de massa para caracterizar. 9 </li></ol></li><li> Lung Processing <ol><li> Retirar as tampas de tubos congelados contendo tecido pulmonar decelularizado. </li><li> Coloque papel de filtro sobre a abertura do tubo e prenda com elástico. NOTA: Tubo e conteúdo ainda deve ser congelado qualquer outro modo, em -80 ° C até congelados. </li><li> Liofilizar o tecido até que todo o excesso de líquido está desaparecido, usando um secador de gelo de acordo com as instruções do fabricante. Armazenar a -80 ° C até estar pronto para moinho. </li><li> Antes de iniciar adicionar nitrogênio líquido para fábrica de freezer para esfriar isolamento e componentes internos às condições de trabalho. </li><li> Retirar bar mill magnético do tubo moinho e adicionar o tecido para cobrir parte inferior. </li><li> Substitua bar moinho e adicionar o tecido ligeiramente comprimido. O bar moinho ainda deve mover-se livremente. </li><li> Fechar o tubo moinho e place no moinho congelador. </li><li> Preencha nitrogênio líquido para linha de preenchimento máximo. </li><li> Moinho congelador todo o tecido em pó fino (aproximadamente 5 min, taxa de compactação de ~ 600 min-1), em um cilindro de policarbonato com um pêndulo de aço inoxidável, bem como tampões de extremidade de aço inoxidável usando o moinho de congelador de acordo com as instruções do fabricante. Armazenar a -80 ° C até que esteja pronto para uso. </li></ol></li><li> A formação do gel micro-porosa (8 mg / ml) (adaptado a partir de referências 9,11) <ol><li> Adicionar 1% (w / v) do pó descelularizado e 0,1% (w / v) de pepsina a HCl 0,01 M, sob agitação constante (deve ser capaz de ver o fluxo no nível superior do líquido), à temperatura ambiente, durante 48 hr. NOTA: O pó é estaticamente carregada, então a adição do HCl, depois do pó proporciona a oportunidade para lavar o excesso de fora das paredes do tubo. </li><li> Após a digestão durante 48 h, e colocar a solução de reagentes em gelo durante 5 min. </li><li> Usando refrigerado 10% (v / v) de NaOH 0,1 M, umad 11,11% (v / v) de PBS 10X (para trazer toda a solução de concentração 1x), levar a solução de proteína digerida a pH fisiológico de 7,4. NOTA: A solução pode agora ser armazenada a 4 ° C durante até uma semana. Execute cinética de gelificação utilizando reometria 9 se desejar. </li></ol></li></ol> 2. Cross-ligando hidrogéis para melhorar a força mecânica <ol><li> Soluções de 1%, 0,1%, e 0,01% w / v solução de genipina de reticulação foram preparadas por dissolução da quantidade necessária de pó de genipina em 10% de DMSO. </li><li> Vortex a solução cada 15 minutos durante 1 hr. </li><li> Adicionar solução genipina para cobrir hidrogéis ECM (100 uL para cada poço de uma placa de 96 poços) que foram previamente montados na parte 1.3.4. </li><li> Deixar cada hidrogel ECM para reticular durante 24 h a 37 ° C. </li><li> Lavar o hidrogel ECM 3 vezes com PBS até a solução de lavagem não é mais azul. Os hidrogéis reticulados então estará pronta para posterior caracterização e ceEstudos de cultura II. </li></ol> 3. Cultura de Células com microporosa Gel <ol><li> Revestimento Bidimensional <ol><li> Usando a solução de 1.3.3, adicionar 20 ul de solução Pregel a cada poço de uma placa de cultura de tecidos de 96 poços não tratados. </li><li> refrigere durante a noite a 4 ° C para permitir a adsorção de proteína à placa. </li><li> solução Aspirar Pregel e enxaguar com PBS. </li><li> Células de passagem 9. </li><li> Adicionar células 10.000 / cm 2 a poços. </li><li> Aumentar a mídia para 100 jil por poço e incubar a 37 ° C. </li></ol></li><li> Três de cultura de células tridimensional 9 <ol><li> Utilizando uma solução a partir de 1.3.3, ressuspender as células peletizadas a uma concentração de 1.000.000 células / ml de solução Pregel. </li><li> dispensar rapidamente 16 ul de suspensão de células Pregel em cada poço de uma placa de 96 poços, de modo a formar um hidrogel de espessura ~ 500 um para a cultura de células em 3D. </li><li> Incubar a 37 ° C durante 30min para o gel para formar. </li><li> Adicionar 100 ul de meios de comunicação para o topo de hidrogeles formados e incubar a 37 ° C. </li></ol></li><li> Três de cultura de células tridimensional em geles de rigidez variável <ol><li> Utilizando uma solução a partir de 1.3.3, pipeta 100 uL de solução Pregel em cada poço de uma placa de 96 poços. </li><li> Incubar a 37 ° C durante 30 minutos para formar gel. Nota: Cross-ligando passos da parte 2 podem ser usados ​​aqui. </li><li> Células de passagem 9. </li><li> Adicionar células 10.000 / cm 2 a poços. </li><li> Aumentar a mídia para 100 jil por poço e incubar a 37 ° C. </li></ol></li></ol>

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Os autores não têm nada para revelar.

Um dos aspectos integrais da biologia é a auto-organização das moléculas em estruturas hierárquicas que executam uma tarefa específica. 13 No laboratório, a auto-montagem depende de numerosos factores tais como a concentração de sal, pH, e a duração de digestão. Como mostrado, uma auto-organização de formas de hidrogel quando as proteínas solubilizadas retornar a uma temperatura fisiológica. O hidrogel formado é capaz de promover a adesão celular e a proliferação in vitro. <p ...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>Rheometer</td><td>Discovery</td><td>HR-2</td><td>Use gloves and eye protection.</td></tr></tbody></table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Aqui é apresentado um método para o estabelecimento de múltiplos componentes hidrogéis de cultura de células para a cultura de células em pulmão in vitro. Começando com saudável en bloc tecido pulmonar de porcino, rato ou ratinho, o tecido é submergido perfundidos e em detergentes químicas subsequentes para remover os detritos celulares. Comparação histológica do tecido antes e depois do processamento confirma a remoção de mais de 95% de ADN de cadeia dupla coloração e alfa-galactosidase sugere a maioria dos detritos celulares são removidos. Após a descelularização, o tecido é, em seguida, liofilizado e cryomilled em um pó. O pó de matriz é digerido durante 48 horas numa solução de pepsina a digestão ácida e, em seguida, neutralizou-se para formar a solução Pregel. A gelificação da solução Pregel pode ser induzida através da incubação a 37 ° C e pode ser utilizada imediatamente após a neutralização ou armazenado a 4 ° C durante até duas semanas. Os revestimentos podem ser formados utilizando a solução Pregel sobre uma chapa não tratada para Capego ell. As células podem ser suspensas no Pregel antes de auto-montagem para alcançar uma cultura 3D, plaqueadas sobre a superfície de um gel formado a partir do qual as células podem migrar através do andaime, ou revestida sobre os revestimentos. Alterações à estratégia apresentada pode ter impacto na temperatura de gelificação, força ou tamanhos dos fragmentos de proteína. Além formação de hidrogel, o hidrogel de rigidez pode ser aumentada usando genipina.

Usando este método, temos produzido a partir de hidrogéis de porco normal, rato, e pulmões de ratinho (Figura 1). pulmões transformados fornecer um valor estimado de 5 mg, 40 mg, e 10 g de ECM em pó respectivamente. Uma visão geral do processo é mostrado na Figura 2. visualizações chave durante o processo incluem: aparência branca dos pulmões após a lavagem desoxicolato; após a formação Pregel, a solução deve ser opaco e a solução deve...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Este é um método para criar uma estrutura de suporte de cultura de células 3-dimensional a partir da matriz extracelular pulmonar. pulmão intacto é transformado em hidrogéis que podem suportar o crescimento das células em três dimensões.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.6K visualizações.  Virginia Commonwealth University.  Este é um método para criar uma estrutura de suporte de cultura de células 3-dimensional a partir da matriz extracelular pulmonar. pulmão intacto é transformado em hidrogéis que podem suportar o crescimento das células em três dimensões. 

Vídeo: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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

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

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

Bioengenharia

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Fácil manipulação das arquiteturas em hidrogel à base de proteínas para aplicações de cultura de células

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Bioquímica

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 ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

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

Bioprinting celularizado Constrói Usando um específico do tecido Hidrogel Bioink

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

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D para terapia regenerativa injetável e triagem de drogas de alto rendimento

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Cultura de células de hidrogel ultrafinos Porated elástico como uma membrana do porão biomimético para Dual

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

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 ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Tensão controlada de hidrogéis 3D sob imagem de microscopia ao vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Matrizes de hidrogel permitem aumento de rendimento para efeitos de triagem de componentes matriciais e terapêuticos em modelos de tumor 3D

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Hidrogéis bioimpressos fotosintonizáveis para sondar as respostas celulares ao enrijecimento da MEC

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Autor em destaque: Compreendendo doenças pulmonares crônicas usando hidrogéis fototáveis impressos em 3D 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Bioimpressão 3D de hidrogéis fototáveis para estudo da ativação de fibroblastos

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...