Name: preparation of chitosan-based injectable hydrogels and its application in 3d cell culture
Uploaded: 2017-09-29T14:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Esta pesquisa foi apoiada pelo National Science Foundation da China (21474057 e 21604076).

atenção: favor consultar todas as fichas de dados de segurança (MSDS) antes do uso. Por favor, use as práticas de segurança adequadas ao realizar experimentos de química, incluindo o uso de uma coifa e equipamento de protecção pessoal (óculos de segurança, luvas de proteção, jaleco, etc.). O protocolo exige célula padrão manipulação técnicas (esterilização, recuperação da célula, célula passagem, congelamento de célula, coloracao celular, etc).
 1. preparação do hidrogel <ol><li>síntese do benzaldeído finalizado di-acrescida de polietileno glicol (PEG DF) <ol><li>pré-dessecação de PEG polímero <ol><li>pesar 4,00 g PEG (4.000 MW, 1.00 mmol), transferir para um balão de fundo redondo (250 mL) e adicionar tolueno (100 mL). 
			Nota: Outras PEGs com diferentes pesos moleculares pode trabalhar para a formação de hidrogel, mas levará a rigidez diferente.</li>
			<li>Aquecer a solução por uma pistola de calor (ou um prato quente em torno de 40 ° C) ligeiramente para ajudar a dissolver todos os polímeros. Depois de dissolvem todos os polímeros, use um evaporador para remover todos os solventes. Repita esse processo seco e dissolver duas vezes tornar o PEG secado polímeros. 
			Atenção: Esta deve ser executada em uma coifa com extremo cuidado. Tolueno é inflamável.</li>
		</ol></li> <li>Reação de terminação benzaldeído de PEG <ol><li>Adicionar um agitador magnético e tetrahidrofurano (THF, 100 mL) no balão e dissolver PEG, utilizando um agitador. Adicionar 4-carboxybenzaldehyde (0,90 g, 6,0 mmol) e 4-dimetilaminopiridina (0,07 g, 0,6 mmol) em sequência para as soluções para dissolver todos os sólidos completamente.</li>
			<li>Add N, N &#39;-dicyclohexylcarbodiimide (DCC, 1,25 g, 6,0 mmol) para a solução e adicionar um tubo de secagem que é preenchido com anidro CaCl 2 para o balão. Manter a reação sob agitação à temperatura ambiente (~ 20 ° C) por cerca de 12 h.</li>
		</ol></li> <li>Processo de pós-reação <ol><li>filtrar os sólidos brancos gerados na solução por vácuo, depois que a reação é terminada. Despeje a solução em éter dietílico frio (500 mL) sob agitação para precipitar os sólidos brancos. Reunir todos os sólidos brancos pelo filtro e seque os sólidos em uma coifa.</li>
			<li>Dissolver os sólidos brancos em THF novamente, filtrar qualquer sólidos insolúveis brancos, despeje a solução de éter dietílico frio fresco para precipitar os sólidos brancos e secá-lo. Repita este processo de duas a três vezes e em seguida, coloque os sólidos brancos em um forno de secagem a vácuo (20 ° C, 0.1 mbar) para secá-los completamente. Recolher o pó branco como o produto final: benzaldeído finalizado PEG DF.</li>
		</ol></li></ol></li> <li>Preparação do hidrogel <ol><li>pesar quantidades diferentes de PEG DF (0,11 g, 0,028 mmol g 0,22, 0.055 mmol; 0,44 g, 0.110 mmol) em um tubo (10 mL), adicionar água desionizada (5,0 mL) e use um vórtice ou agitador magnético para ajudar dissolver o polímero.</li>
		<li>Dissolver glicol quitosana (0,495 g, 6.18 x 10 -3 mmol) em água desionizada (15,0 mL) em um tubo (50 mL) e usar um vórtice por alguns minutos para homogeneizar a solução de quitosana (3% em peso).</li>
		<li>Adicionar a solução de quitosana glicol (0,2 mL, 3% em peso) e soluções DF PEG (0,2 mL) sequencialmente em um tubo (2,0 mL). Use um vórtice para misturar as soluções homogênea de hidrogel forma em vários minutos. Siga as proporções na tabela 1 tornar hidrogel de forças mecânicas diferentes. 
		Nota: Use o método do tubo-invertendo para determinar se o hidrogel já formou.</li>
	</ol></li> <li>Análises de reologia <ol><li>efectuar análises de reologia em um rheometer rotatória com uma placa de aço paralela (diâmetro: 20 mm). Para o teste de gelificação, espalhe o glicol solução de quitosana (0,2 mL, 3% em peso) em uma placa inferior. Em seguida, adicionar DF PEG soluções aquosas (0,2 mL, 2% em peso) gota uniformemente sobre a superfície da solução de quitosana e misture rapidamente com uma pipeta. Abaixe a placa superior e iniciar para teste</li>
		<li>De hidrogel &#39; s teste de rigidez, cortar um círculo um hidrogel (diâmetro: 20 mm) e medir o módulo de armazenamento (G &#39;) valores contra as análises de frequência na estirpe de 1%. G típico &#39; valores a 6,3 rad s &#8722; 1 estão listados na tabela 1. 
		Nota: Efectuar as análises de reologia do hidrogel 0,5 h após a formação de hidrogel para assegurar a estabilização de vínculo dinâmico do hidrogel.</li>
		<li>Para o hidrogel &#39; s auto reparáveis Propriedade testar, cortar um hidrogel em pedaços e juntar os pedaços de auto-recuperação de uma peça integrante. Em seguida, corte a peça de hidrogel para fazer um círculo (diâmetro: 20 mm) e colocá-lo sobre o rheometer para realizar a análise de reologia.</li>
	</ol></li></ol> 2. cultivo de célula 3D em hidrogel <ol><li>preparação de soluções de gelificação de hidrogel <ol><li>pesar DF PEG (0,44 g, 0.11 mmol) em um tubo estéril (4,0 mL), adicionar em meios de cultura celular (RPMI-1640, 2.0 mL) e usar um vórtice ou agitador para ajudar a dissolver o polímero para obter a solução de polímero (20% em peso). Carregar a solução em uma seringa (10,0 mL) e então ele esterilizar por passagem através de um filtro de bactérias-retentiva mícron (0,22 µm).</li>
		<li>Pesar a quitosana de glicol (0,165 g, 2,06 x 10 -3 mmol) em um tubo estéril (15,0 mL), adicionar em meios de cultura celular (RPMI-1640, 4,0 mL) e o vórtice para ajudar a dissolver o polímero para obter a solução de quitosana glicol (4,0% em peso). Carregar a solução em uma seringa (10,0 mL) e filtrá-la com um filtro de 0,22 µm bactérias-retentiva.</li>
	</ol></li> <li>Cultivo celular em hidrogel atenção: executar todos os procedimentos relacionados em uma capa de cultura de tecidos de células. Conhecimentos básicos de técnica estéril é esperado.
	<ol><li>Preparação da suspensão de células <ol><li>células L929 cultura RPMI-1640, suplementado com 10% FBS, 5% de penicilina (10 mL) em uma Petri prato (diâmetro 10 cm) e incubar a 37 ° C, 5% de CO 2. Mudar o meio todos os dias antes de usar.</li>
			<li>Colher as células L929 com PBS contendo tripsina (0,025% do w/v) e EDTA (0,01% w/v), centrifugar (70 x g, 5 min) e ressuspender as células em meio RPMI-1640 (1,0 mL). Realize a contagem de células usando a operação padrão da placa de contagem de sangue. Ressuspender as células para ajustar a concentração de células para ~ 3,75 × 10 6 células/mL.</li>
		</ol></li> <li>Encapsulamento de célula em hidrogel <ol><li>misturar as suspensões de células L929 (0,4 mL, 3,75 x 10 6 células mL -1) com solução de quitosana glicol (0,4 mL) em um tubo (4,0 mL) pelo vórtice. Pipetar a solução de quitosana L929/glicol (0,8 mL) para o centro de um prato de Petri confocal (diâmetro 2,0 cm). Pipetar as DF PEG soluções (0,2 mL) para o mesmo prato e pipetar delicadamente para misturar a solução e induzir a formação de hidrogel. 
			Nota: Avaliar a formação de hidrogel inclinando o prato de petri.</li>
			<li>Para cultura de células direta, adicionar quantidades adicionais de meios de cultura RPMI-1640 (1,0 mL) em cima do hidrogel. Colocar a célula incorporada hidrogel (1,0 mL, 1.5 wt % glicol quitosana, 4,0 wt % DF PEG, 1,5 × 10 6 células mL -1) em uma incubadora (37 ° C, 5% CO 2) e altere o meio todos os dias. Prepare-se para a imagem latente de célula no dia 1, 3, 5 e 7 após encapsulamento celular.</li>
		</ol></li> <li>Para a pós-cultura de células 3D em hidrogel após a injeção, preparar celular carregado hidrogel (1,0 mL, consulte a etapa 2.2.2) numa seringa (10,0 mL, agulha 48 G). Após as formas de hidrogel, injetar o hidrogel lentamente em uma placa de Petri para a imagem latente confocal. Adicionar um montante adicional de meios de cultura (1,0 mL) em cima do hidrogel e alterá-lo todos os dias. Coloque o prato de Petri em uma incubadora (37 ° C, 5% CO 2) e prepare-se para a imagem latente depois. 
		Atenção: Verifique e seguir o protocolo de operação de segurança de uma seringa.</li>
	</ol></li> <li>Análise de viabilidade de células <ol><li>Confocal observação <ol>
			<li>Enxaguar o hidrogel com PBS (1,0 mL) por duas vezes. Mancha o hidrogel com diacetato de fluoresceína (FDA, 0,5 mL, 0,05 mg/mL) e soluções de propidium iodeto (PI, 0,5 mL, 0,08 mg/mL) por 15 min. Depois da coloração, remover todos os solventes.</li>
			<li>Observar o hidrogel usando um microscópio confocal em comprimentos de onda de excitação de 488 nm e 543 nm a visualização ao vivo e mortos células, respectivamente. Leva pilhas através de cada profundidade de 2 µm do hidrogel para validar uma distribuição uniforme de células ao longo de z. 
			Nota: FDA manchas de células vivas enquanto PI manchas de células mortas.</li>
		</ol></li> <li>Degradar o hidrogel (1,0 mL) com ácido acético (HAc, 3% de v, 1,0 mL) por 5 min e pipetar para um tubo (4,0 mL). Coletar as células por centrifugação (70 x g, 5 min) e ressuspender as células em meio de cultura RPMI 1640 celular (1,0 mL). Executar célula contagem usando um sangue contagem placa.</li>
	</ol>
	</li>
</ol>

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O hidrogel apresentado neste protocolo (Figura 1) tem dois componentes principais: a polímero natural quitosana de glicol e um sintético benzaldeído encerrado gelator polímero PEG do DF, que são ambos os materiais biocompatíveis. Síntese do DF PEG é apresentado utilizando uma reação de modificação de uma etapa. PEG, de peso molecular 4.000 foi escolhida no presente protocolo em preocupações de solubilidade, eficiência de modificação, bem como rigidez de hidrogel. Uma série ...

Hidrogel é materiais de polímero reticulado com grandes quantidades de água e propriedades mecânicas macias, e eles têm sido usados em muitas aplicações bio-medical1,2. Hidrogel pode oferecer um ambiente suave e molhado, que é muito semelhante ao ambiente fisiológico para células em vivo. Portanto, hidrogel tornaram-se uma das plataformas mais populares para cultura de células 3D3,4. Em relação à cultura de pilha do prato de Petri 2D, cultura de células 3D avançou rapidamente para oferecer que uma matriz extracelular (ECM) imitou o microambiente para células de contato e montar para fins de proliferação e diferenciação5. Além disso, o hidrogel contendo polímeros naturais poderia oferecer biocompatível e promovendo ambientes para as células a proliferar e diferenciar3. Hidrogel derivado de polímeros sintéticos é preferidos para seus componentes simples e claras, que excluem influências complexas como as proteínas de origem animal ou vírus. Entre todos os candidatos de hidrogel para cultura de células 3D, hidrogel que facilmente é preparados e têm uma propriedade consistente é sempre preferidos. A facilidade para ajustar propriedades do hidrogel para atender necessidades diferentes pesquisas é importante como bem6.
Aqui apresentamos uma preparação superficial de um hidrogel de glicol baseado em quitosana usando química imina dinâmico, que se torna uma plataforma versátil de hidrogel para cultura de células 3D7. Neste método, conhecido biocompatível glicol quitosana são usados para estabelecer quadros de redes do hidrogel. Seus grupos aminoácidos são reagiu com um glicol de polietileno benzaldeído encerrado como o gelator de polímeros que formam ligações de imina dinâmico como crosslinkages de hidrogel8. Obrigações de imina dinâmico podem formar e decompor reversível e responsiva ao redor, dotando o hidrogel com quitosana mecanicamente ajustável redes9,10,11. Devido ao seu conteúdo de água de alto, materiais biocompatíveis e forças mecânicas ajustáveis, o hidrogel é aplicado com sucesso como um andaime para células L929 em cultura de células 3D12,13. O protocolo aqui detalha os procedimentos, incluindo a síntese de polímeros gelator, preparação de hidrogel, incorporação de célula e cultivo celular 3D.
O hidrogel também mostra várias outras características devido a sua crosslinkages de imina dinâmico, incluindo sua multi responsivo aos vários estímulos bio-(ácido e pH, piridoxal derivados de vitamina B6, papaína de proteína, etc), indicando que o hidrogel pode ser induzido a decompor-se sob condições fisiológicas8. O hidrogel é também auto reparáveis e injetável, o que significa que o hidrogel pode ser administrado através de um método de injeção invasiva mínima e ganhar uma vantagem em droga e célula entregas14,15. Pela adição de aditivos funcionais ou gelators de polímero preconcebidos específico, o hidrogel é compatível para obter propriedades específicas como magnética, temperatura, pH-responsivo, etc.16,,17, que pudesse cumprir um ampla gama de requisitos de pesquisa. Essas propriedades revelaram capacidade potencial do hydrogel ser um injetável várias transportadoras para drogas e células em ambos in vitro e em vivo pesquisa biomédica e aplicações.

<table><tbody><tr><td>Glycol chitosan</td><td>Wako Pure Chemical Industries</td><td>39280-86-9</td><td>90% degree of deacetylation</td></tr><tr><td>4-Carboxybenzaldehyde</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>619-66-9</td><td>99%</td></tr><tr><td>N, N&#039;-dicyclohexylcarbodiimide</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>538-75-0</td><td>99%</td></tr><tr><td>Calcium chloride anhydrous</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>10043-52-4</td><td>96%</td></tr><tr><td>4-dimethylamiopryidine</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>1122-58</td><td>99%</td></tr><tr><td>Polyethyleneglycol</td><td>Sino-pharm Chemical Reagent</td><td>5254-43-7</td><td>99%</td></tr><tr><td>Tetrahydrofuran</td><td>Sino-pharm Chemical Reagent</td><td>109-99-9</td><td>99%</td></tr><tr><td>Toluene</td><td>Sino-pharm Chemical Reagent</td><td>108-88-3</td><td>99%</td></tr><tr><td>Ethyl ether</td><td>Sino-pharm Chemical Reagent</td><td>60-29-7</td><td>99%</td></tr><tr><td>Acetic acid</td><td>Sino-pharm Chemical Reagent</td><td>64-19-7</td><td>99%</td></tr><tr><td>Anhydrous CaCl2</td><td>Sino-pharm Chemical Reagent</td><td>10043-52-4</td><td>99%</td></tr><tr><td>Fluorescein diacetate</td><td>Sigma</td><td>596-09-8</td><td>99%</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>25535-16-4</td><td>94%</td></tr><tr><td>RPMI-1640 culture media</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td></td><td>0.25%</td></tr><tr><td>PBS</td><td>Solarbio</td><td></td><td>0.01 M</td></tr><tr><td>Penicillin streptomycin solution</td><td>Hyclone</td><td></td><td>10,000 U/mL</td></tr><tr><td>Rheometer</td><td>TA Instrument</td><td></td><td>AR-G2</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>710-3channel</td></tr><tr><td>L929 Cells</td><td>ATCC</td><td></td><td>NCTC clone 929; L cell, L929, derivative of Strain L</td></tr><tr><td>Evaporator</td><td>EYELA</td><td></td><td>N-1100</td></tr><tr><td>48 guage needle</td><td>ShanghaiZhiyu Medical Material Co., LTD</td><td></td><td>48-guage</td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>DM3000 B</td></tr><tr><td>Microscope software</td><td>Imaris</td><td></td><td></td></tr><tr><td>Heat gun</td><td>Confu</td><td></td><td>KF-5843&amp;nbsp;</td></tr><tr><td>Petri dish</td><td>NEST</td><td></td><td></td></tr></tbody></table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

O protocolo apresenta um método fácil, eficiente e versátil para preparar hidrogel baseado em quitosana usando química imina dinâmico. O hidrogel é preparado misturando-se soluções de glicol quitosana com um gelator de polímero sintetizado benzaldeído finalizado e hidrogel com eficiência é obtidos em vários minutos à temperatura ambiente. Por razões diferentes entre quitosana glicol, gelator de polímero e conteúdo de água, obtêm-se versátil hidrogel com tempos diferentes de gelificação e rigidez. Quando danificadas, o hidrogel pode recuperar suas aparições e módulo de elasticidade, devido a reversibilidade das obrigações imina dinâmico como crosslinkages. Esta auto reparáveis propriedade permite que o hidrogel ser injetável, uma vez que pode ser auto curado de peças espremidas de um hidrogel em massa integral após o processo de injeção. O hidrogel é também multi sensível a muitos estímulos bio-ativos devido ao status de equilibração diferente das obrigações imina dinâmico. Este hidrogel foi confirmado como bio-compatível e células de fibroblasto de rato L929 foram incorporados procedimentos padrão e a proliferação das células foi facilmente avaliada por um processo de cultivo de células 3D. O hidrogel pode oferecer uma plataforma ajustável para diferentes pesquisas onde um mímico fisiológico de um ambiente 3D para as células é lucrou. Juntamente com suas propriedades multi responsivos, auto reparáveis e injetáveis, o hidrogel potencialmente pode ser aplicados como várias transportadoras para drogas e células em futuras aplicações biomédicas.

Uma apresentação esquemática do presente protocolo na preparação do hidrogel e seu uso como cultura de células 3D é oferecida na Figura 1. Informações do hidrogel conteúdo e rácios, preparados com diferentes forças mecânicas estão resumidas na tabela 1. O hidrogel é auto reparáveis e reologia Propriedade apresenta rigidez do hidrogel por módulo de armazenamento versus teste de frequência na Figura 2. As imagens confocal de cél...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Aqui nós descrevemos uma preparação superficial de hidrogel injetável à base de quitosana usando química imina dinâmico. São apresentados métodos para ajustar a força mecânica do hidrogel e sua aplicação em cultura de células 3D.

Preparação do hidrogel injetável à base de quitosana e sua aplicação em cultura de células 3D

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Universidad San Sebastian

Research

JoVE Journal

Chemistry

19.0K visualizações.  Tsinghua University. Aqui nós descrevemos uma preparação superficial de hidrogel injetável à base de quitosana usando química imina dinâmico. São apresentados métodos para ajustar a força mecânica do hidrogel e sua aplicação em cultura de células 3D.

Vídeo: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

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

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Construindo um hidrogel de colágeno para a entrega de células-tronco carregado microesferas

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bioengenharia

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hidrogel Andaimes para articular de condrócitos Cultura e Geração de cartilagem

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Formação de esferóides da célula do ligamento periodontal humano em filmes de quitosana

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Biologia do Desenvolvimento

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

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 ...

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 ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Hidrogel Bioprintable alginato/gelatina 3D em Vitro sistemas modelo induzem a formação de esferoide de célula

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocolos de bioimpressão 3D de bioinks baseados em hidrogel de gelatina

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Fabricação de microgéis chitosan-genipin controlados por tamanho e sem emulsão para aplicações de engenharia de tecidos

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...