Name: recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model
Uploaded: 2018-02-07T15:00:00
Description: Watch this Scientific Journal Video about Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model at JoVE.com

A pesquisa que conduz a estes resultados recebeu financiamento da União Europeia sétimo quadro programa FP7/2007-2013 sob contrato de concessão n ° 607051 (BIO-INSPIRE). Agradecemos Carolien van Spreuwel-Goossens da Fujifilm fabricação Europe B.V., a assistência técnica durante a fabricação de RCP e Werner Stracke do Instituto Fraunhofer para silicato pesquisa ISC, para obter assistência com a análise de SEM.

hBMSCs foram isolados da cabeça do fêmur de pacientes de osteoartrite submetidos à cirurgia de substituição de cabeça do fêmur. O procedimento foi realizado sob a aprovação do Comitê de ética Local da Universidade de Wuerzburg e consentimento informado dos pacientes. Células endoteliais microvasculares primárias foram isoladas a partir de biópsias do prepúcio de doadores juvenis. Seu representante legal fornecido completo consentimento informado por escrito. O estudo foi aprovado pelo Conselho de ética lo...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+serum-free+microcarrier+expansion+of+mesenchymal+stromal+cells:+Parameters+to+be+optimized.">Recent advances in serum-free microcarrier expansion of mesenchymal stromal cells: Parameters to be optimized.</a> Biochemical and Biophysical Research Communications. 473, 769-773 (2016).</li><li>de Soure, A. M., Fernandes-Platzgummer, A., da Silva, C. L., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+microcarrier-based+manufacturing+of+mesenchymal+stem/stromal+cells.">Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.</a> J Biotechnol. 236, 88-109 (2016).</li><li>Schop, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+human+mesenchymal+stromal+cells+on+microcarriers:+growth+and+metabolism.">Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism.</a> J Tissue Eng Regen. Med. 4, 131-140 (2010).</li><li>Carmelo, J. G., Fernandes-Platzgummer, A., Diogo, M. M., da Silva, C. L., Cabral, J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+human+mesenchymal+and+pluripotent+stem+cell+microcarrier+cultures+in+cellular+therapy:+achievements+and+future+direction.">Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction.</a> Biotechnol Adv. 31, 1032-1046 (2013).</li><li>Confalonieri, D., La Marca, M., van Dongen, E., Walles, H., Ehlicke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Injectable+Recombinant+Collagen+I+Peptide-Based+Macroporous+Microcarrier+Allows+Superior+Expansion+of+C2C12+and+Human+Bone+Marrow-Derived+Mesenchymal+Stromal+Cells+and+Supports+Deposition+of+Mineralized+Matrix.">An Injectable Recombinant Collagen I Peptide-Based Macroporous Microcarrier Allows Superior Expansion of C2C12 and Human Bone Marrow-Derived Mesenchymal Stromal Cells and Supports Deposition of Mineralized Matrix.</a> Tissue Eng Part A. , (2017).</li><li>Davidenko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+crosslinking+for+tailoring+collagen-based+scaffolds+stability+and+mechanics.">Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics.</a> Acta biomaterialia. 25, 131-142 (2015).</li><li>Jin, G. Z., Park, J. H., Seo, S. J., Kim, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+cell+culture+on+porous+biopolymer+microcarriers+in+a+spinner+flask+for+bone+tissue+engineering:+a+feasibility+study.">Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study.</a> Biotechnol Lett. 36, 1539-1548 (2014).</li><li>Bae, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+Vascular+Networks.">Building Vascular Networks.</a> Science Translational Medicine. 4 (160), 160ps23 (2012).</li><li>Cao, L., Wang, J., Hou, J., Xing, W., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+and+bone+regeneration+in+a+critical+sized+defect+using+2-N,+6-O-sulfated+chitosan+nanoparticles+incorporating+BMP-2.">Vascularization and bone regeneration in a critical sized defect using 2-N, 6-O-sulfated chitosan nanoparticles incorporating BMP-2.</a> Biomaterials. 35 (2), 684-698 (2014).</li><li>Novosel, E., Kleinhans, C., Kluger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+is+the+key+challenge+in+tissue+engineering.">Vascularization is the key challenge in tissue engineering.</a> Advanced Drug Delivery Reviews. 63 (4-5), 300-311 (2011).</li><li>Cartmell, S. H., Porter, B. D., Garc&#237;a, A. J., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medium+perfusion+on+cell-seeded+three-dimensional+bone+constructs+in+vitro.">Effects of medium perfusion on cell-seeded three-dimensional bone constructs in vitro.</a> Tissue Engineering. 9 (6), 1197-1203 (2004).</li><li>Schanz, J., Pusch, J., Hansmann, J., Walles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularised+human+tissue+models:+a+new+approach+for+the+refinement+of+biomedical+research.">Vascularised human tissue models: a new approach for the refinement of biomedical research.</a> Journal of biotechnology. 148 (1), 56-63 (2010).</li><li>Steinke, M., Gross, R., Walles, H., Sch&#252;tze, K., Walles, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+3D+human+airway+mucosa+model+based+on+a+SIS+scaffold.">An engineered 3D human airway mucosa model based on a SIS scaffold.</a> Biomaterials. 35 (26), 7355-7362 (2014).</li><li>Moll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+of+a+Human+3D+in+vitro+Tumor+Test+System.">Tissue Engineering of a Human 3D in vitro Tumor Test System.</a> J. Vis. Exp. (78), e50460 (2013).</li><li>Groeber, F., Kahlig, A., Loff, S., Walles, H., Hansmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioreactor+system+for+interfacial+culture+and+physiological+perfusion+of+vascularized+tissue+equivalents.">A bioreactor system for interfacial culture and physiological perfusion of vascularized tissue equivalents.</a> Biotechnology Journal. 8, 308-316 (2013).</li><li>Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+and+endothelial+cells.">Blood vessels and endothelial cells.</a> Molecular Biology of the Cells. , (2002).</li><li>Logsdon, E. A., Finley, S. D., Popel, A. S., Gabhann, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systems+biology+view+of+blood+vessel+growth+and+remodeling.">A systems biology view of blood vessel growth and remodeling.</a> Journal of Cellular and Molecular Medicine. 18 (8), 1491-1508 (2014).</li><li>Scheller, K., Dally, I., Hartmann, N., M&#252;nst, B., Braspenning, J., Walles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upcyte&#174;+Microvascular+endothelial+cells+repopulate+decellularized+scaffold.">Upcyte&#174; Microvascular endothelial cells repopulate decellularized scaffold.</a> Tissue Engineering Part C: Methods. 19 (1), 57-67 (2012).</li><li>Nietzer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+Metastases+Including+Tumor+Stroma:+A+New+Technique+to+Generate+a+Three-Dimensional+Colorectal+Cancer+Model+Based+on+a+Biological+Decellularized+Intestinal+Scaffold.">Mimicking Metastases Including Tumor Stroma: A New Technique to Generate a Three-Dimensional Colorectal Cancer Model Based on a Biological Decellularized Intestinal Scaffold.</a> Tissue Engineering Part C: Methods. 22 (7), 621-635 (2016).</li><li>G&#246;ttlich, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Combined+3D+Tissue+Engineered+In+Vitro/In+Silico+Lung+Tumor+Model+for+Predicting+Drug+Effectiveness+in+Specific+Mutational+Backgrounds.">A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds.</a> J. Vis. Exp. (110), e53885 (2016).</li><li>Stratmann, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+human+3D+lung+cancer+model+based+on+a+biological+tissue+matrix+combined+with+a+Boolean+in+silico+model.">Establishment of a human 3D lung cancer model based on a biological tissue matrix combined with a Boolean in silico model.</a> Molecular oncology. 8 (2), 351-365 (2014).</li><li>Groeber, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+first+vascularized+skin+equivalent+for+as+an+alternative+to+animal+experimentation.">A first vascularized skin equivalent for as an alternative to animal experimentation.</a> Altex. 33 (4), 415-422 (2016).</li><li>Plunkett, N., O'Brien, F. 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Um dos principal objetivos da microcarrier é a expansão das células, mantendo a sua diferenciação para entregar as células para o local de necessidade. O método representado introduzir RCP microcarriers onde as células foram capazes de anexar, proliferar e colonizar o microcarriers com densidade de pilha alta. Isto foi observado pelo live/dead coloração, em que mais de 90% de células viáveis foram detectados enquanto apenas poucas células mortas foram obtidas após 7 dias de culturas dinâmicas. Da mesma for...

Um problema geral em aplicações de engenharia de tecido é produzir uma massa de células alta com o fenótipo de diferenciação correta na localidade de necessidade. A aplicação de microcarriers para abordar esta questão começou em 1967 com o aumento da importância à data nos campos tais como a engenharia de tecidos ortopédicos para geração em grande escala de pele, ossos, cartilagens e tendões1. Eles permitem a manipulação de culturas aderentes em maneiras semelhante da suspensão de culturas2 expandindo células em substratos (3D) tridimensionais de microescala. Assim, as células experimentam um fornecimento de nutrientes homogêneo e interações célula-matriz, que levam a melhor manutenção na vivo3,4 diferenciação que muitas vezes é perdida ao longo do tempo em 2D se aproxima de5. Uma maior taxa de superfície e o volume - levando-o à célula maior rendimentos6,7, superior de gás e nutrientes taxas de câmbio, comparando a sistemas estáticos8, a possibilidade de regular e sujeita a cultura a física estímulos de9e o potencial para intensificação do processo de expansão7 são ainda mais vantagens. Vários recursos tais como o diâmetro, densidade, porosidade, carga de superfície e propriedades de adesão10,11 distinguem as diferentes disponíveis comercialmente micro e macro-operadoras. No entanto, dentre a principal vantagem é sua entrega potencial como microtissues com defeito de local ou demanda.
Para aplicações da tecnologia de microcarrier em engenharia de tecido ósseo, ilustramos em um anterior relatório12 a produção de um novo microcarrier tipo constituído de um colágeno recombinante peptídeo (RCP, comercialmente disponível como Cellnest). Esta nova microcarrier permite o GMP-compatível com a escala de produção de andaime e célula, conforme necessário para a entrega da célula em um cenário clínico. Neste contexto, ajuste de estabilidade do andaime, taxa de degradação e propriedades de superfície através da escolha adequada de uma estratégia adequada de reticulação permite adaptar a técnica para o aplicativo selecionado, tipo de interesse de célula ou tecido13-alvo. Em particular, o emprego potencial desta microcarrier como um sistema de entrega de celular injetável para aplicação terapêutica14 torna particularmente interessantes em um ambiente clínico.
Neste trabalho, ilustramos, por conseguinte, o processo de cultivo para o isolamento e a expansão de medula óssea-derivado mesenquimais estromais células humanas (hBMSCs) e dérmicas microvasculares endoteliais células humanas (HDMECs) na recombinação de colágeno-I-baseada microcarriers peptídeo-baseado e sua preparação para a entrega em um ambiente clínico. Além disso, descrevemos a protocolos adicionais úteis para a manutenção da viabilidade celular após a implantação.
Viabilidade celular após o implante na verdade é fortemente dependente da vascularização15,16,,17, que garante a troca de oxigênio e nutrientes e facilita a remoção do lixo. Biorreatores constituem uma abordagem para superar os desafios de vascularização em engenharia de tecidos e manter a viabilidade celular, através de perfusão de meio de cultura, fornecendo assim, oxigénio e nutrientes18. Aqui, podemos ilustrar um in vitro método para avaliar a capacidade de migração das células endoteliais microvasculares do microcarriers a RCP para um biomatriz e sua capacidade de contribuir para o novo de vascularização e angiogênese. Este biomatriz é um segmento decellularized de suínos jejuno denominado BioVaSc (andaime biológico vascularizado), rica em colágeno e elastina e com conservas de estruturas vasculares, que inclui uma artéria alimentação uma drenagem veia de19 que tem sido aplicado para questões de implantação20.

<table>
	<tbody>
		<tr>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>20395.01</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-phenylindoldihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid 100%</td>
			<td>Sigma-Aldrich</td>
			<td>533,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance Kern EG 2200-2NM</td>
			<td>Kern &#38; Sohn GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope Axiovert 40C</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellnest</td>
			<td>Fujifilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL, 50 mL)</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen R solution 0,4%</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>47254.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Modified, without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth GmbH</td>
			<td>T171.4</td>
			<td>Denatured</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Bio&#38;SELL</td>
			<td>FCS.ADD.0500</td>
			<td>not heat-inactivated</td>
		</tr>
		<tr>
			<td>Fluorescence microscope BZ-9000</td>
			<td>Keyence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Morphisto</td>
			<td>10231.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma-Aldrich</td>
			<td>440191</td>
			<td>Reagent grade, &#8805;99%</td>
		</tr>
		<tr>
			<td>Incubator for bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Double Staining Kit</td>
			<td>Fluka</td>
			<td>04511KT-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer plate</td>
			<td>2Mag</td>
			<td>80002</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M0650</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>Microplate reader 
			Tecan Infinite M200</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 21G 16mm</td>
			<td>VWR</td>
			<td>613-5389</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma-Aldrich</td>
			<td>P4762</td>
			<td>lyophilized powder, &#8805; 10 units/mg protein</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Carl Roth GmbH</td>
			<td>6642.6</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quanti-iT PicoGreen dsDNA assay kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>Histofix 4%</td>
			<td>Carl Roth GmbH</td>
			<td>P087</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope Supra 25</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flasks (25 mL)</td>
			<td>Wheaton</td>
			<td>356879</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>VWR</td>
			<td>720-2561</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks (25 cm2, 75 cm2, 150 cm2)</td>
			<td>TPP Techno Plastik Products AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF-Mv</td>
			<td>Lifeline cell technology</td>
			<td>LL-0005</td>
			<td></td>
		</tr>
	</tbody>
</table>

recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model

Engenharia de tecidos é um campo promissor, focado no desenvolvimento de soluções para a crescente demanda em tecidos e órgãos em relação a fins de transplante. O processo para gerar tais tecidos é complexo e inclui uma combinação apropriada de tipos de células específicas, andaimes e estímulos físicos ou bioquímicos para orientar a diferenciação e crescimento celular. Microcarriers representam uma ferramenta atraente para expandir as células em um microambiente de tridimensional (3D), uma vez que fornecem maior superfície-para rácios de volume e imitar mais de perto a situação na vivo em comparação com métodos tradicionais bidimensionais. O sistema vascular, fornecendo oxigênio e nutrientes para as células e garantindo a remoção de lixo, constitui um importante bloco de construção quando gerando engenharia de tecidos. Na verdade, a maioria das construções falharem após ser implantado devido a falta de suporte vascular. Neste estudo, apresentamos um protocolo para a expansão de células endoteliais na recombinante microcarriers baseados em colágeno sob condições dinâmicas no balão do girador e biorreatores, e explicamos como determinar nesta viabilidade celular de configuração e funcionalidade. Além disso, propomos um método de entrega de celular para fins de vascularização sem etapas de desprendimento adicionais necessárias. Além disso, nós fornecemos uma estratégia para avaliar a vascularização celular potencial em um bioreator de perfusão em uma matriz biológica decellularized. Acreditamos que o uso dos métodos apresentados pode levar ao desenvolvimento de novas terapias baseadas em células para uma grande variedade de aplicações na prática clínica de engenharia de tecidos.

Como mostrado na figura 1A, obtivemos o elevado número de células viáveis na microcarriers a RCP após 7 dias da cultura, determinado pela coloração ao vivo/morto. Esses resultados foram confirmados por análise de SEM, em que microcarriers completamente colonizados foram observados ao redor dos poros, parcialmente sobrecrescimento-los (figura 1B). Por outro lado, experiências em que as células não foram semeadas uniforme...

Watch this Scientific Journal Video about Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model at JoVE.com

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

Propomos um protocolo de expansão celular na se microcarriers e seu uso como sistema de entrega em um bioreator de perfusão para propagar uma matriz de tecido decellularized. Nós também incluímos diferentes técnicas para determinar a proliferação celular e a viabilidade das células cultivadas no microcarriers. Além disso, demonstramos a funcionalidade das células após culturas de biorreator.

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

The overall goal of this procedure is to expand the cells on a high available surface area and to further deliver these cells to sites of defect or demand for clinical applications. This method can help answer key questions in the tissue engineering field, such as cell expansion, cell delivery, and vascularization. The main advantage of this technique is that we can expand the cells and deliver them, without any harvesting step, into defect sites, in a biocompatible way.Visual demonstration of this method is critical, as working microcarriers and bioreactors can be complicated, at first, due to the many steps involved. Helping with the procedure will be Jihyoung Choi, a PhD student from our laboratory. One day before the experiments, set up the spinner flasks and prepare them for sterilization by autoclave.Wrap the spinner flasks with aluminum foil around the side caps separately to avoid contamination during unwrapping. Wrap the flasks with one to two layers of aluminum foil for autoclaving to minimize contamination risks after sterilization. Leave the caps of the spinner flasks loose and when possible, sterilize them in an upright position.Next, weigh the required amount of RCP macroporous microcarriers in 20 milliliters of Dulbecco's phosphate buffered saline. Let them hydrate for at least one hour before heat sterilization by autoclave. Use the bioreactor system referenced in text protocol to produce vascularized tissue equivalents.This bioreactor consists of a vessel in which the biomatrix is placed, a medium reservoir, and a pressure bottle. Connect the vessel with the medium reservoir and the pressure bottle through silicone tubes. Leave the caps loose and place the system in an autoclave plastic bag.Close the bag well and place this in a second autoclave plastic bag before sterilizing by autoclave. Let the sterilized RCP macroporous microcarriers settle to the bottom and wash them with 10 milliliters of culture medium. Repeat this process three times.Also wash the spinner flasks with 10 milliliters of culture medium. Next, add 30 milligrams of RCP macroporous microcarriers per spinner flask in eight milliliters of culture medium. Equilibrate the spinner flasks RCP macroporous microcarriers at 37 degrees Celsius and 5%carbon dioxide for 30 minutes before seeding the cells.Then, seed half a million human bone marrow derived mesenchymal stromal cells, or human dermal microvascular endothelial cells per spinner flask in two milliliters of culture medium. Place the spinner flasks on a stirrer plate at 37 degrees Celsius and 5%carbon dioxide and start stirring at 90rpm for five minutes. Then, rest for 55 minutes for a total of one hour static dynamic incubation.Repeat this incubation cycle four times. Now, add 10 milliliters of cell culture medium per spinner flask. Then start continuous stirring at 95rpm while incubating at 37 degrees Celsius and 5%carbon dioxide.One day before starting the bioreactor cultures, cut open the biomatrix longitudinally on the antimesenteric side and fix it in a previously disinfected polycarbonate frame. After detaching and counting the cells, use a two milliliter syringe to inject 10 million HDMECs in one thousand microliters of culture medium through the preserved vasculature of the biomatrix. Inject approximately 700 microliters through the arterial inlet, and approximately 300 microliters through the vein outlet.Incubate the biomatrix with injected cells at 37 degrees Celsius and 5%carbon dioxide for three hours to allow cell attachment. Following incubation, the connection of the biomatrix to the bioreactor has to be done by two individuals, taking care that the arterial and the venous pedicles are connected to the medium flow correctly. Fill the bioreactor system with 350 milliliters of the vascular endothelial growth factor, 1%penicillin streptomycin, and incubate at 37 degrees Celsius and 5%carbon dioxide.Place the frame inside the vessel of the bioreactor. Then, carefully connect the arterial and venous pedicles to the vessel. Start perfusion by connecting the bioreactor system to a peristaltic pump.Set the pressure at 10 millimeters of mercury amplitude plus minus one. Increase the pressure stepwise every hour until reaching a pressure of 100 millimeters of mercury amplitude plus minus 20. Mix 330 microliters of 0.4%Collagen R solution, 170 microliters of 0.1%acetic acid, and 50 microliters of medium M199 in a 15 milliliter Falcon tube.Keep the mixture on ice. Then, take the RCP microcarriers from the spinner flasks. Remove the culture medium completely.Next, add the volume of sodium hydroxide required to neutralize the collagen mixture. Once neutralized, immediately mix the mixture with the RCP microcarriers. It is important to allow gelation of the collagen gel before starting medium flow.Otherwise, the gel will detach and the microcarriers will be washed away from the lumen of the biomatrix. Add the collagen gel RCP microcarrier mixture on the lumen of the biomatrix. Allow gelation for 30 minutes.Then, connect the bioreactor to the peristaltic pump and set up the pressure at 100 millimeters of mercury amplitude plus minus 20. Finally, perform analysis and read outs of bioreactor cultures as described in the text protocol. Representative results of cell proliferation on microcarriers is shown by live dead staining in which many living cells can be observed on the microcarriers after seven days of dynamic cultures.These results were confirmed by scanning electron microscopy analysis showing completely colonized microcarriers after seven days of dynamic cultures. HDMECs kept their functionality after seven days of dynamic cultures on RCP microcarriers. Here, bright field microscopy reveals sprouts growing from the colonized microcarriers when cultured on a collagen gel.RFP labeled HDMECs were observed in the vascular structures of the biomatrix after 21 days of bioreactor cultures, indicating their migration from the RCP microcarriers to the biomatrix. Furthermore, after 21 days of bioreactor cultures, the scanning electron microscopy image shows that the microcarriers were in close proximity to the biomatrix. Interestingly, they are not colonized by cells anymore.After watching this video, you should have a good understanding on how to expand the cells on microcarriers to prepare them in an injectable cell delivery system for therapeutic application. Once mastered, this technique can be done in four hours. This procedure represents a promising strategy in regenerative medicine to obtain and maximize cell expansion and to deliver cells to implantation sites where they could improve vascularization for tissue engineered grafts.The implications of this technique extend toward vascularization because it can be used on any cell type for a tissue specific application in a clinical scenario. This method can also provide understanding into the potential employment of microcarriers as an injectible cell delivery system for therapeutic purposes. Don't forget that working with primary cells can be extremely hazardous, and precautions such as wearing gloves and lab coat, and working in a biosafety cabinet should always be taken when performing this procedure.After its development, this technique paved the way for researchers in the field of tissue engineering to explore cell migration in vitro which could help to understand or predict in vivo situations.

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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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

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 ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Pele a tatuagem como uma nova abordagem para a Campanha de Vacinação de DNA

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

O cultivo de células de mamíferos utilizando um uso único sistema pneumático Bioreactor

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

O tecido triturado em Compressed Collagen: Um BioTransplant contendo celular para o reparo Reconstructive Single-encenado

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

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

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

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