Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores

Detalhes bibliográficos
Autor(a) principal: Mendonça, Marlei Leandro de
Data de Publicação: 2013
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da UFSCAR
Texto Completo: https://repositorio.ufscar.br/handle/ufscar/7021
Resumo: The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing α-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics.
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spelling Mendonça, Marlei Leandro deSuazo, Cláudio Alberto Torreshttp://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4783900Z9http://lattes.cnpq.br/4040394501423588d4fce75e-7a0a-4952-be4a-21c58ad2ab1a2016-08-17T18:39:47Z2013-09-052016-08-17T18:39:47Z2013-04-29MENDONÇA, Marlei Leandro de. Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores. 2013. 197 f. Dissertação (Mestrado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2013.https://repositorio.ufscar.br/handle/ufscar/7021The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing α-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics.O crescente interesse na utilização de células mesenquimais estromais multipotentes (CMMs) para aplicações na engenharia de tecidos e na terapia celular é atribuído principalmente à sua plasticidade e capacidade regenerativa. As CMMs representam uma revolução no tratamento de inúmeras doenças e no entendimento dos mecanismos de reparo tecidual. Sua baixa disponibilidade nos tecidos (de 0,00001 à 0,0002%) associada à necessidade de elevadas doses para as aplicações terapêuticas (aproximadamente 2 x 106 células/kg paciente) e à ineficiência dos cultivos tradicionais (em monocamada) in vitro para atender à alta demanda existente para essas aplicações, tornaram indispensável o desenvolvimento de novas tecnologias de expansão ex vivo em larga escala. A utilização de biorreatores tipo frascos spinner com microcarregadores em suspensão é um sistema alternativo eficaz para esta expansão. O relato bibliográfico de cultivos com esse sistema, no entanto, tem indicado a formação de grandes aglomerados de microcarregadores com células como um possível obstáculo na obtenção de maior produtividade celular. Tais aglomerados dificultam a difusão de nutrientes, gases (principalmente o oxigênio) e reagentes de quantificação celular indireta em seu interior; e prejudicam a recuperação das células no final do cultivo. Microesferas de alginato tornaram-se interessantes para diminuir a frequência de colisões entre os microcarregadores, pois não se aderem às células nem aos microcarregadores podendo evitar a união dos mesmos. O sulfato de dextrana foi descrito na literatura tanto como inibidor do contato célula-célula, quanto do encontro entre microcarregadores em cultivo, por isso também passou a ser uma opção promissora para evitar a formação de aglomerados de microcarregadores. Assim, os objetivos deste trabalho foram desenvolver uma metodologia de cultivo de CMMs em frasco spinner com microcarregadores Cultispher-S para aplicação em terapia celular e avaliar a influência de microesferas de alginato e do sulfato de dextrana nesse cultivo. Para isso, a linhagem hMSC-TERT e 3 g/L Cultispher-S foram mantidos em spinner de 100 mL contendo meio de cultura α-MEM (suplementado com 15% de soro fetal bovino, glicose e glutamina), a 37ºC, com controle de pH. Os resultados mostraram que a adição de sulfato de dextrana (à 0,5% e 1%) e de 100% de microesferas de alginato (em relação ao número de partículas de Cultispher-S) ao cultivo provocaram morte celular considerável; e que o controle (sem aditivos), assim como a adição de 50% de microesferas de alginato promoveram crescimento celular. A formação de aglomerados foi observada nos cultivos, no entanto, ela foi atrasada com adição de 50% de microesferas de alginato. Esse fato permitiu a obtenção de um fator de expansão de 50,4 vezes (com a correção do fator de diluição) e de 13,1 vezes (sem a correção). As células recuperadas após a expansão mantiveram suas características imunofenotípicas.Financiadora de Estudos e Projetosapplication/pdfporUniversidade Federal de São CarlosPrograma de Pós-Graduação em Biotecnologia - PPGBiotecUFSCarBRBiotecnologiaCélulas-troncoBiorreatoresMicrocarregadores - aglomeradosMicroesferas de alginatoSulfato de dextranaStem cellBioreactorMicrocarrierClustersDextran sulfateAlginate microspheresOUTROSExpansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadoresinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-1-1a35fad8c-e6aa-4839-b89a-4a1eb6fcd885info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL5353.pdfapplication/pdf2805182https://repositorio.ufscar.br/bitstream/ufscar/7021/1/5353.pdfdbd89699431c4bbf40d6959bb375df30MD51TEXT5353.pdf.txt5353.pdf.txtExtracted texttext/plain0https://repositorio.ufscar.br/bitstream/ufscar/7021/4/5353.pdf.txtd41d8cd98f00b204e9800998ecf8427eMD54THUMBNAIL5353.pdf.jpg5353.pdf.jpgIM Thumbnailimage/jpeg6369https://repositorio.ufscar.br/bitstream/ufscar/7021/5/5353.pdf.jpg8d0bc12803e69bf618a73081754657e0MD55ufscar/70212023-09-18 18:30:34.225oai:repositorio.ufscar.br:ufscar/7021Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:30:34Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false
dc.title.por.fl_str_mv Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
title Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
spellingShingle Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
Mendonça, Marlei Leandro de
Biotecnologia
Células-tronco
Biorreatores
Microcarregadores - aglomerados
Microesferas de alginato
Sulfato de dextrana
Stem cell
Bioreactor
Microcarrier
Clusters
Dextran sulfate
Alginate microspheres
OUTROS
title_short Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
title_full Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
title_fullStr Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
title_full_unstemmed Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
title_sort Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores
author Mendonça, Marlei Leandro de
author_facet Mendonça, Marlei Leandro de
author_role author
dc.contributor.authorlattes.por.fl_str_mv http://lattes.cnpq.br/4040394501423588
dc.contributor.author.fl_str_mv Mendonça, Marlei Leandro de
dc.contributor.advisor1.fl_str_mv Suazo, Cláudio Alberto Torres
dc.contributor.advisor1Lattes.fl_str_mv http://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4783900Z9
dc.contributor.authorID.fl_str_mv d4fce75e-7a0a-4952-be4a-21c58ad2ab1a
contributor_str_mv Suazo, Cláudio Alberto Torres
dc.subject.por.fl_str_mv Biotecnologia
Células-tronco
Biorreatores
Microcarregadores - aglomerados
Microesferas de alginato
Sulfato de dextrana
topic Biotecnologia
Células-tronco
Biorreatores
Microcarregadores - aglomerados
Microesferas de alginato
Sulfato de dextrana
Stem cell
Bioreactor
Microcarrier
Clusters
Dextran sulfate
Alginate microspheres
OUTROS
dc.subject.eng.fl_str_mv Stem cell
Bioreactor
Microcarrier
Clusters
Dextran sulfate
Alginate microspheres
dc.subject.cnpq.fl_str_mv OUTROS
description The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing α-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics.
publishDate 2013
dc.date.available.fl_str_mv 2013-09-05
2016-08-17T18:39:47Z
dc.date.issued.fl_str_mv 2013-04-29
dc.date.accessioned.fl_str_mv 2016-08-17T18:39:47Z
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dc.identifier.citation.fl_str_mv MENDONÇA, Marlei Leandro de. Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores. 2013. 197 f. Dissertação (Mestrado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2013.
dc.identifier.uri.fl_str_mv https://repositorio.ufscar.br/handle/ufscar/7021
identifier_str_mv MENDONÇA, Marlei Leandro de. Expansão de células mesenquimais estromais em frasco spinner e avaliação de aditivos para diminuir a aglomeração de microcarregadores. 2013. 197 f. Dissertação (Mestrado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2013.
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