RESUMO
Zidovudine (3'-azido-3'-deoxythymidine; AZT) is a nucleoside analogue widely used for the treatment of acquired immune deficiency syndrome (AIDS). Medical guidelines recommend the use of AZT by pregnant women in order to reduce risk of HIV vertical transmission. Although it is efficacious, little is known about the side effects of AZT on embryonic development. In this sense, we used murine embryonic stem (mES) cells as a model to investigate the consequences of AZT exposure for embryogenesis. Firstly, mES colonies were incubated with AZT (50 or 100 µM) and cell cycle profile was evaluated. While 27.7 ± 5.43% of untreated mES cells were in G2/M phase, this percentage raised to 45.96 ± 4.18% after AZT exposure (100 µM). To identify whether accumulation of cells in G2/M phase could be related to chromosome missegregation with consequent cell cycle arrest, aneuploidy rate was evaluated after AZT treatment. Untreated colonies presented 39.6 ± 8.4% of cells aneuploid, while after AZT 100 µM treatment, the proportion of aneuploid cells raised to 67.8 ± 3.4% with prevalence of chromosome loss. This event was accompanied by micronuclei formation as AZT 100 µM treated mES cells presented a 2-fold increase compared to untreated ones. These data suggest that AZT exerts genotoxic effects and increases chromosome instability at early stages of embryonic development.
Assuntos
Aneuploidia , Fármacos Anti-HIV/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Feminino , Camundongos , GravidezRESUMO
Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.
Assuntos
Animais , Humanos , Camundongos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Dextranos/farmacologia , Células-Tronco Embrionárias/citologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Imuno-HistoquímicaRESUMO
Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.