Name: constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres
Uploaded: 2012-06-01T12:00:00
Description: Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

DOZ é apoiado por uma subvenção concedida à Fundação de Genebra. SN foi apoiado por uma bolsa pós-doutorado da Iniciativa Engenharia de Tecidos Pittsburgh.

1. Isolando derivadas de tecido adiposo células-tronco (ASC) Nota: Todos os procedimentos foram realizados à temperatura ambiente, a menos que indicado de outra forma. <ol><li> Isolar adiposo epididimal e perirenal rato e lavar com solução de Hank estéril tamponada de sal (HBSS) contendo 1% de soro fetal bovino (FBS), como descrito anteriormente 6. </li><li> Picar o tecido e transferir 1-2 g em 25 mL de HBSS contendo FBS a 1% para um tubo de 50 mL e centrif...

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A., Aebischer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+as+a+matrix+for+mammalian+cell+encapsulation.">Chitosan as a matrix for mammalian cell encapsulation.</a> Biomaterials. 15, 1049-1056 (1994).</li><li>Girandon, L., Kregar-Velikonja, N., Bo&#382;ikov, K., Barli&#231;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Models+for+Adipose+Tissue+Engineering+with+Adipose-Derived+Stem+Cells+Using+Different+Scaffolds+of+Natural+Origin.">In vitro Models for Adipose Tissue Engineering with Adipose-Derived Stem Cells Using Different Scaffolds of Natural Origin.</a> Folia Biol. (Praha). 57, 47-56 (2011).</li><li>Baruch, L., Machluf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate-chitosan+complex+coacervation+for+cell+encapsulation:+effect+on+mechanical+properties+and+on+long-term+viability.">Alginate-chitosan complex coacervation for cell encapsulation: effect on mechanical properties and on long-term viability.</a> Biopolymers. 82, 570-579 (2006).</li><li>Wei, Y., Gong, K., Zheng, Z., Wang, A., Ao, Q., Gong, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/silk+fibroin-based+tissue-engineered+graft+seeded+with+adipose-derived+stem+cells+enhances+nerve+regeneration+in+a+rat+model.">Chitosan/silk fibroin-based tissue-engineered graft seeded with adipose-derived stem cells enhances nerve regeneration in a rat model.</a> J. Mater. Sci. Mater. Med. , (2011).</li><li>Wang, Q., Jamal, S., Detamore, M. S., Berkland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA-chitosan/PLGA-alginate+nanoparticle+blends+as+biodegradable+colloidal+gels+for+seeding+human+umbilical+cord+mesenchymal+stem+cells.">PLGA-chitosan/PLGA-alginate nanoparticle blends as biodegradable colloidal gels for seeding human umbilical cord mesenchymal stem cells.</a> J. Biomed. Mater. Res. A. 96, 520-527 (2011).</li><li>Alves da Silva, M. L., Martins, A., Costa-Pinto, A. R., Correlo, V. M., Sol, P., Bhattacharya, M., Faria, S., Reis, R. L., Neves, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+of+human+bone+marrow+mesenchymal+stem+cells+in+chitosan-based+scaffolds+using+a+flow-perfusion+bioreactor.">Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan-based scaffolds using a flow-perfusion bioreactor.</a> J. Tissue Eng. Regen. Med. , (2010).</li><li>Kang, Y. M., Lee, B. N., Ko, J. H., Kim, G. H., Kang, K. N., Kim da, Y., Kim, J. H., Park, Y. H., Chun, H. J., Kim, C. H., Kim, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+biocompatibility+study+of+electrospun+chitosan+microfiber+for+tissue+engineering.">In vivo biocompatibility study of electrospun chitosan microfiber for tissue engineering.</a> Int. J. Mol. Sci. 11, 4140-4148 (2010).</li><li>Bozkurt, G., Mothe, A. J., Zahir, T., Kim, H., Shoichet, M. S., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+channels+containing+spinal+cord-derived+stem/progenitor+cells+for+repair+of+subacute+spinal+cord+injury+in+the+rat.">Chitosan channels containing spinal cord-derived stem/progenitor cells for repair of subacute spinal cord injury in the rat.</a> Neurosurgery. 67, 1733-1744 (2010).</li><li>Leipzig, N. D., Wylie, R. G., Kim, H., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neural+stem+cells+in+three-dimensional+growth+factor-immobilized+chitosan+hydrogel+scaffolds.">Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds.</a> Biomaterials. 32, 57-64 (2011).</li><li>Altman, A. M., Gupta, V., RÃ­os, C. N., Alt, E. U., Mathur, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion,+migration+and+mechanics+of+human+adipose-tissue-derived+stem+cells+on+silk+fibroin-chitosan+matrix.">Adhesion, migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin-chitosan matrix.</a> Acta Biomater. 6, 1388-1397 (2010).</li><li>Altman, A. M., Yan, Y., Matthias, N., Bai, X., Rios, C., Mathur, A. B., Song, Y. H., Alt, E. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+Human+adipose-derived+stem+cells+seeded+on+a+silk+fibroin-chitosan+scaffold+enhance+wound+repair+in+a+murine+soft+tissue+injury+model.">IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model.</a> Stem Cells. 27, 250-258 (2009).</li><li>Machado, C. B., Ventura, J. M., Lemos, A. F., Ferreira, J. M., Leite, M. F., Goes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+chitosan-gelatin-chondroitin+porous+scaffold+improves+osteogenic+differentiation+of+mesenchymal+stem+cells.">3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.</a> Biomed. Mater. 2, 124-131 (2007).</li></ol>

Um grande obstáculo na terapia de células-tronco base é o desenvolvimento de métodos eficientes para a entrega de células para as regiões especificadas para a reparação. Devido ao paciente a variabilidade do paciente, o tipo de tecido, tamanho da lesão e da profundidade, a metodologia de células estaminais entrega deve ser determinada numa base de caso-a-caso. Embora a incorporação de células-tronco dentro de uma matriz e entregá-los para o local da ferida parece ser uma abordagem lógico seguinte para a e...

<table border="1" ><tbody><tr><td> Nome do reagente / equipamentos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Hanks BalancedSalt Solução (HBSS) </td><td> Gibco </td><td> 14175 </td><td> Consumível </td></tr><tr><td> Soro Fetal Bovino </td><td> Hyclone </td><td> SH30071.03 </td><td> Consumível </td></tr><tr><td> Colagenase do Tipo II </td><td> Sigma-Aldrich </td><td> C6685 </td><td> Consumível </td></tr><tr><td> 70-iM de nylon de malha de filtro </td><td> BD Biosciences </td><td> 352350 </td><td> Consumível </td></tr><tr><td> 100-iM de nylon de malha de filtro </td><td> BD Biosciences </td><td> 352360 </td><td> Consumível </td></tr><tr><td> MesenPRO Sistema Meio de Crescimento </td><td> Invitrogen </td><td> 12746-012 </td><td> Consumível </td></tr><tr><td> L-glutamina </td><td> Gibco </td><td> 25030 </td><td> Consumível </td></tr><tr><td> T75 frasco de cultura de tecidos </td><td> BD Biosciences </td><td> 137787 </td><td> Consumível </td></tr><tr><td> Chitosan </td><td> Sigma-Aldrich </td><td> 448869 </td><td> Consumível </td></tr><tr><td> Ácido Acético </td><td> Sigma-Aldrich </td><td> 320099 </td><td> Consumível </td></tr><tr><td> N-octanol </td><td> Acros Organics </td><td> 150630025 </td><td> Consumível </td></tr><tr><td> Sorbitano-mono-oleato </td><td> Sigma-Aldrich </td><td> S6760 </td><td> Consumível </td></tr><tr><td> Hidróxido de Potássio </td><td> Sigma-Aldrich </td><td> P1767 </td><td> Consumível </td></tr><tr><td> Acetona </td><td> Fisher Scientific </td><td> L-4859 </td><td> Consumível </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich </td><td> 270741 </td><td> Consumível </td></tr><tr><td> Trinitro benzenossulfónico </td><td> Sigma-Aldrich </td><td> P2297 </td><td> Consumível </td></tr><tr><td> Ácido clorídrico </td><td> Sigma-Aldrich </td><td> 320331 </td><td> Consumível </td></tr><tr><td> Éter etílico </td><td> Sigma-Aldrich </td><td> 472-484 </td><td> Consumível </td></tr><tr><td> 8 m de tecido Placa Insere Cultura </td><td> BD Biosciences </td><td> 353097 </td><td> Consumível </td></tr><tr><td> 1,5 ml tubos de microcentrífuga </td><td> Pescador </td><td> 05-408-129 </td><td> Consumível </td></tr><tr><td> Reagente MTT </td><td> Invitrogen </td><td> M6494 </td><td> Consumível </td></tr><tr><td> Sulfóxido de dimetilo </td><td> Sigma-Aldrich </td><td> D8779 </td><td> Consumível </td></tr><tr><td> Qtracker celular Labeling Kit (Q rastreador 655) </td><td> Sondas moleculares </td><td> Q2502PMP </td><td> Consumível </td></tr><tr><td> Colágeno tipo 1 </td><td> Travigen </td><td> 3447-020-01 </td><td> Consumível </td></tr><tr><td> Hidróxido de Sódio </td><td> Sigma-Aldrich </td><td> S8045 </td><td> Consumível </td></tr><tr><td> 12-Bem Cultura Pratos Tecido </td><td> BD Biosciences </td><td> 353043 </td><td> Consumível </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5417R </td><td> Equipamento </td></tr><tr><td> Orbital Shaker </td><td> New Brunswick Scienctific </td><td> C24 </td><td> Equipamento </td&gt; </tr><tr><td> Incubadora de CO umidificado com Air-5 2% </td><td> Thermo Scientific </td><td> Modelo 370 </td><td> Equipamento </td></tr><tr><td> Agitador </td><td> IKA </td><td> Visc6000 </td><td> Equipamento </td></tr><tr><td> Agitador Magnético </td><td> Corning </td><td> PC-210 </td><td> Equipamento </td></tr><tr><td> Exsicador de vácuo </td><td> - </td><td> - </td><td> Equipamento </td></tr><tr><td> Analisador de Tamanho de Partícula </td><td> Malvern </td><td> STP2000 Spraytec </td><td> Equipamento </td></tr><tr><td> Banho de água </td><td> Fisher Scientific </td><td> Isotemp210 </td><td> Equipamento </td&gt; </tr><tr><td> Espectrofotômetro </td><td> Beckman </td><td> Beckman Coulter Espectrofotômetro DU800UV/Visible </td><td> Equipamento </td></tr><tr><td> Vórtice </td><td> Diagger </td><td> 3030a </td><td> Equipamento </td></tr><tr><td> Leitora </td><td> Molecular Devices </td><td> Spectramax M2 </td><td> Equipamento </td></tr><tr><td> Luz / microscópio de fluorescência </td><td> Olimpo </td><td> IX71 </td><td> Equipamento </td></tr><tr><td> Microscópio confocal </td><td> Olimpo </td><td> FV-500 Laser Scanning microscópio confocal </td><td> Equipamento </td></tr><tr><td> Microscópio Eletrônico de Varredura </td><td> Carl Zeiss MicroImaging </td><td> Leo 435 VP </td><td> Equipamento </td></tr><tr><td> Microscópio eletrônico de transmissão </td><td> JEOL </td><td> JEOL 1230 </td><td> Equipamento </td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

As células estaminais multipotenciais têm sido mostrados para ser extremamente útil no campo da medicina regenerativa 1-3. No entanto, a fim de utilizar essas células eficazmente para a regeneração de tecidos, um número de variáveis ​​devem ser tidos em conta. Estas variáveis ​​incluem: o volume total e área de superfície do local de implantação, as propriedades mecânicas do tecido e do microambiente do tecido, que inclui a quantidade de vascularização e os componentes da matriz extracelular. Portanto, os materiais que estão sendo utilizados para entregar estas células deve ser biocompatível com uma composição química definida, mantendo uma força mecânica que imita o tecido hospedeiro. Estes materiais também deve ser permeável ao oxigénio e nutrientes para proporcionar um microambiente favorável para as células para anexar e proliferar. A quitosana, um polissacárido catiónico com excelente biocompatibilidade, pode ser facilmente modificada quimicamente e tem uma elevada afinidade para se ligarem a in vivo macromolecules 4-5. A quitosana imita a porção de glicosaminoglicano da matriz extracelular, permitindo-lhe funcionar como um substrato para a migração de adesão celular, e proliferação. Neste estudo, utilizam quitosano na forma de microesferas para entregar células-tronco adiposas (ASC) em uma base de colagénio tridimensional andaime 6. Uma razão de célula-a-microesfera ideal foi determinada com respeito ao tempo de incubação e densidade celular para atingir o número máximo de células que poderiam ser carregados. Uma vez ASC são semeados no microesferas de quitosano (CSM), eles são incorporados em um andaime de colagénio e pode ser mantido em cultura durante períodos prolongados. Em resumo, este estudo fornece um método para entregar precisamente células-tronco dentro de um andaime tridimensional biomaterial.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

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

Um grande obstáculo em terapias com células-tronco atuais é determinar o método mais eficaz para entregar essas células para tecidos do hospedeiro. Aqui, nós descrevemos um método de entrega de quitosana-base que é eficiente e simples na abordagem, ao mesmo tempo permitindo que as células-tronco adiposas para manter a sua 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 ...

Research

JoVE Journal

Bioengineering

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Organotípica Colágeno Ensaio I: Uma Plataforma maleável para Avaliar Comportamento celular em um contexto de 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Bioengenharia

Engineering a Bilayered Hydrogel to Control ASC Differentiation

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

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

An Improved Method for the Preparation of Type I Collagen From Skin

Um método aperfeiçoado para a preparação de colágeno tipo I Da Pele

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imagiologia desnaturadas colágeno Vertentes<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via foto desencadeada Hibridização do Caged Colágeno mimético Peptídeos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

Síntese de uma proteína artificial intein mediada Hidrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Desenvolvimento de Amelogenina-quitosana Hidrogel para<em&gt; In Vitro</em&gt; Esmalte Regrowth Com uma interface Densa

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Microescala Electroporator para entrega Sequential Molecular assistida por Vortex

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Um Supramolecular hidrogel injetável e carregado com a droga para a injeção local por cateter no coração de porco

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Recombinante colágeno peptídeo Microcarriers para expansão da célula e seu potencial uso como sistema de entrega de células em um bioreator modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...