Avaliação da integridade das células-tronco mesenquimais derivadas do tecido adiposo humano após o bioprocesso de criopreservação

Abstract

Resumo: As celulas-tronco de origem adulta surgem como promessa terapeutica na medicina regenerativa podendo ser encontradas em diversos tecidos como o adiposo. Com as perspectivas da criacao de um banco de celula-tronco para pesquisa e posterior uso terapeutico, o estudo da criopreservacao dessas celulas urge, a fim de garantir que essas celulas apos o descongelamento permanecam viaveis e funcionais. Objetivos: Comparar as celulas-tronco do tecido adiposo antes da criopreservacao e apos o seu descongelamento quanto a: a) indice de proliferacao e capacidade de formar colonias b) expressao das moleculas de superficies em especial a integrina ƒ¿4; c) capacidade de diferenciacao d) viabilidade e integridade celular. Metodos: Celulas-tronco adultas foram obtidas do tecido adiposo de 10 mulheres e 2 homens, saudaveis pela tecnica de lipoaspiracao, realizada por cirurgioes plasticos e apos o consentimento informado. O isolamento das celulas foi realizado atraves de digestao enzimatica com colagenase tipo I posterior cultivo em DMEM/F12 suplementado com 10% de SFB e 100U/mL de Penicilina/100ƒÊg/mL de Estreptomicina. Foi calculado o indice de proliferacao celular da primeira a terceira passagem para cada amostra. Realizadas analises de unidade formadora de colonia na segunda passagem, as celulas foram submetidas a diferenciacao adipogenica e osteogenica com auxilio de meios indutores. A Imunofenotipagem atraves de citometria de fluxo (FACS Calibur; Becton Dickinson, USA), utilizando-se marcadores: CD34, CD45, CD49d, CD73, CD90, CD105, anexina V e 7-AAD, seguidas do processo de criopreservacao, precedido de congelamento programavel (Nicool LM10). Vinte dias apos, as celulas foram descongeladas e os testes repetidos. Resultados: Rendimento celular: 9,09 } 4,55 X 106 celulas/mL isoladas. Indice de proliferacao antes da criopreservacao: Passagens 1= 0,24; 2= 21,33; 3= 13,03 (p=0,001), respectivamente; passagem 3=17,43 }4,11 apos descongelamento (p=0,07). Unidade formadora de colonia antes da criopreservacao: 28,08 % } 7,06%, apos o descongelamento 21,51% } 6,61% (p=0,001).Todas as amostras induzidas tiveram diferenciacao adipogenica e osteogenica, antes da criopreservacao e apos o descongelamento. A analise imunofenotipica demonstrou as seguintes expressoes antes da criopreservacao: CD34- (98,88% } 1,08%), CD45- (99,79% } 0,22%), CD49d+ (88,67% } 6,55), CD90+ (99,55 } 0,07%), CD73+ (99,57% } 0,43%) e CD105+ (99,4% } ,64%); Apos o descongelamento: CD34- (99,26% } 0,61%, p= 0,11), CD45- (99,8% } 0,14%, p= 0,79), CD49d+ (77,8% } 14,45%, p= 0,01), CD73+ (99,5% } 0,43%, p= 0,53), CD90+(99,45 } 0,7%, p= 0,62), CD105+ (98,26% } 2,07%, p= 0,05). Viabilidade celular (7-AAD-) e apoptose (Anexina V-) antes da criopreservacao: 91,39% } 5,85%, 91,34% } 4,54%; apos descongelamento das celulas: 76,31% } 13,33% (p= 0,001), 74,99% } 14,19% (p= 0,003), respectivamente. Conclusao: As celulas-tronco mesenquimais derivadas de tecido adiposo mantem suas caracteristicas imunofenotipicas, viabilidade, capacidades de proliferacao e de diferenciacao apos o descongelamento das celulas criopreservadas, a excecao da integrina ƒ¿4 (CD 49d), uma molecula de adesao que participa da integracao da celula no tecido hospedeiro.

Abstract: Adult stem-cells emerge as a therapeutic promise in regenerative medicine such as adipose. From the perspective of creation of a stem-cell bank for research and there after therapeutic use, the study of cryopreservation of these cells urge. However, to the safety of this technique, some criteria must be established in order to guarantee that cells after thawing remain viable and functional. Objective: To compare stem-cells of adipose tissue before cryopreservation and after thawing: a) proliferation index and capacity of colony forming; b) cell viability and integrity; c) differentiation capacity; d) expression of surface molecules, especially integrin á4; d) Methods: Adult stem-cells were obtained from adipose tissues of 12 adult healthy donors (10 female and two male) by the technique of liposuction, performed by plastic surgeons with informed consent. The isolation was performed by enzymatic digestion with collagenase type I, and cells were cultured on DMEM/F12 supplemented with 10% of Calf Fetal Serum, 100 units/mL of penicillin and 100 ìg/mL of Penicillin/Streptomycin. It was calculate the index of cellular proliferation from the first to the third passage to each sample and performed analyses of Colony Forming Units. At the second passage, cells were submitted to adipogenic and osteogenic differentiation using inducing media. Immunophenotyping using flow cytometer (FACS Calibur, Becton Dickinson, USA), utilizing the markers CD34, CD45,CD49d, CD73, CD90, CD105, annexin V and 7-AAD, followed of cryopreservation process, with programmed freezing (Nicool LM10). After 20 days, the cells were thawed and tests were repeated. Results: Cellular yield: 9.09 ± 4.55 X 106 cells/mL. Proliferation index, Passage 1 = 0.24 ± 0.12; 2 = 21.33 ± 8.48; 3 = 13.03 ± 5.38 (p=0.001); passage 3=17,43±4,11, after thaw (p=0.07). Colony forming units before cryopreservation: 28.08% ± 7.06%, after thaw 21.51% ± 6.61% (p= 0.001). All samples induced underwent adipogenic and osteogenic differentiation before cryopreservation and after thawing. The immunophenotypic analysis demonstrated the following expression pattern before cryopreservation: CD34- (98.88% ± 1.08%), CD45- (99.79% ± 0.22%), CD49d+ (88.67% ± 6.55), CD90+ (99.55 ± 0.07%), CD73+ (99.57% ± 0.43%) e CD105+ (99.4% ± 0.64%). After thawing: CD34- (99.26% ± 0.61%, p= 0.11), CD45- (99.8% ± 0.14%, p= 0.79), CD49d+ (77.8% ± 14.45%, p 0.01), CD73+ (99.5% ± 0.43%, p= 0.53), CD90+(99.45 ± 0.7%, p= 0.62), CD105+ (98.26% ± 2.07%, p= 0.05). Cell viability (7-AAD-) and apoptosis (Annexin V-) before cryopreservation: 91.39% ± 5.85%, 91.34% ± 4.54%; after cell thawing: 76.31% ± 13.33% (p= 0.001), 74.99% ± 14.19% (p= 0.003), respectively. Conclusion: Mesenchymal stem-cells derived from adipose tissue maintain their immunophenotypic characteristics as well viabilitiy, proliferation and differentiation capacities after thawing of cryopreserved cells. The exception is integrin á4 (CD 49d), an adhesion molecule involved in the integration of the cell in the host tissue.