Criopreservação de tecido adiposo humano : caracterização de fração estromal vascular, células-tronco derivadas de tecido adiposo e enxerto de gordura
Arquivos
Data
2017-08-01
Autores
Fortoul, Fabiana Cristina Zanata 
Orientadores
Ferreira, Lydia Masako
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: Tecido adiposo é importante fonte de células-tronco mesenquimais, denotando um aspecto funcional, além do estrutural. A criopreservação é uma opção para a utilização de tecido armazenado, evitando efeitos adversos de coleta, facilitando a utilização imediata ou repetida. Objetivo: Analisar a criopreservação do tecido adiposo humano com caracterização de fração estromal vascular/células-tronco derivadas de tecido adiposo e enxerto de gordura. Métodos: Tecido adiposo de oito doadores foi processado por digestão enzimática, fresco ou após criopreservação com DMSO por quatro semanas. Fração estromal vascular de tecido fresco, células criopreservadas e células de tecido criopreservado foram caracterizadas por produção, viabilidade, proliferação e expansão em cultura, diferenciação e imunofenotipagem. Enxertos de tecido adiposo fresco e criopreservado em camundongos C57BL/6GFP foram analisados nove semanas após transferência no subcutâneo de camundongos por meio de histologia e imuno-histoquímica. Resultados: A criopreservação de tecido adiposo manteve rendimento e viabilidade celular, capacidade de diferenciação adipogênica e osteogênica, aumento da expressão dos marcadores celulares estromais e adipogênicos e redução dos marcadores hematopoiéticos. Enxertos de gordura mostraram aspectos morfológicos e estruturais semelhantes por quantificação de infiltrado celular (H&E) e de fibrose (tricrômio de Masson), presença de células positivas para PVF em áreas periféricas, adipócitos funcionais evidenciados por perilipina e positividade para CD31 evidenciando vascularização, sugerindo um comportamento de suporte estrutural do tecido enxertado no receptor imunocompetente, tanto para tecido fresco como criopreservado. Conclusões: O tecido adiposo humano criopreservado mantém rendimento, e viabilidade celular, aumenta marcadores celulares de superfície precursores estromais de células da FEV, mantém características das CTTAH in vitro e do enxerto de gordura in vivo.
Background: Adipose tissue is not only a structural and volume tissue but also brings a functional important role as a source of Adipose-Derived Stromal/Stem Cells. Cryopreservation can avoid the morbidity associated with repetitive liposuction, allowing the use of stored tissue after the initial harvest procedure avoiding the adverse effects that can be a limiting factor for immediate use. This paper focuses on the characterization of fresh and cryopreserved human adipose tissue. This paper focuses on the characterization of fresh and cryopreserved human adipose tissue with the stromal vascular fraction/adipose-derived stem cell characterization and fat graft. Methods: Lipoaspirates from 8 donors were processed (collagenase digested) as fresh or cryopreserved tissue in DMSO for 4 weeks. SVF from fresh and cryopreserved tissue and cryopreserved SVF were characterized (cell yield, viability, CFU-F, cell culture, differentiation potential and immunophenotype). Fresh and cryopreserved adipose fat grafting was performed in C57BL/6 GFP mice to analyze morphology using confocal microscopy, histology and immunohistochemistry nine weeks after transplantation. Results: Cryopreservation of adipose tissue maintained cell yield, viability and differentiation potential, and also showed reduction in the hematopoietic surface markers and increased expression of stromal and adipogenic cell surface markers. In vivo cryopreserved fat grafts have reduced weight, although both groups showed preservation of morphological aspects (cellular infiltration and fibrosis), positive GFP cells in the peripheral areas, perilipin positive viable adipocytes and vascularization (CD31 positive). Conclusion: Cryopreserved adipose tissue maintained cell yield, reduces viability, reduces expression of hematopoietic and increased stromal and adipogenic cell surface markers, manteined ASC function and morphology of fat grafts in vivo.
Background: Adipose tissue is not only a structural and volume tissue but also brings a functional important role as a source of Adipose-Derived Stromal/Stem Cells. Cryopreservation can avoid the morbidity associated with repetitive liposuction, allowing the use of stored tissue after the initial harvest procedure avoiding the adverse effects that can be a limiting factor for immediate use. This paper focuses on the characterization of fresh and cryopreserved human adipose tissue. This paper focuses on the characterization of fresh and cryopreserved human adipose tissue with the stromal vascular fraction/adipose-derived stem cell characterization and fat graft. Methods: Lipoaspirates from 8 donors were processed (collagenase digested) as fresh or cryopreserved tissue in DMSO for 4 weeks. SVF from fresh and cryopreserved tissue and cryopreserved SVF were characterized (cell yield, viability, CFU-F, cell culture, differentiation potential and immunophenotype). Fresh and cryopreserved adipose fat grafting was performed in C57BL/6 GFP mice to analyze morphology using confocal microscopy, histology and immunohistochemistry nine weeks after transplantation. Results: Cryopreservation of adipose tissue maintained cell yield, viability and differentiation potential, and also showed reduction in the hematopoietic surface markers and increased expression of stromal and adipogenic cell surface markers. In vivo cryopreserved fat grafts have reduced weight, although both groups showed preservation of morphological aspects (cellular infiltration and fibrosis), positive GFP cells in the peripheral areas, perilipin positive viable adipocytes and vascularization (CD31 positive). Conclusion: Cryopreserved adipose tissue maintained cell yield, reduces viability, reduces expression of hematopoietic and increased stromal and adipogenic cell surface markers, manteined ASC function and morphology of fat grafts in vivo.