Desenvolvimento de cultura de células aderentes em peixes neotropicais e sua aplicação em estudos citogenéticos
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Data
2018-03-28
Autores
Paim, Fabilene Gomes
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Embora a ictiofauna Neotropical seja considerada a mais diversa do mundo, apenas 12,2% das espécies de peixes tiveram cariótipos descritos. A dificuldade de se obter preparações cromossômicas de boa qualidade para algumas espécies de peixes Neotropicais ainda é um dos grandes desafios na área de Citogenética, principalmente para peixes de pequeno e grande porte. Uma alternativa é uso de metodologias indiretas, tais como cultura de células, amplamente utilizada e estabelecida em mamíferos, entretanto para peixes ainda é pouco empregada pela dificuldade de padronização da técnica de isolamento e manutenção das culturas celulares. Dessa forma, o presente estudo teve como objetivo o desenvolvimento de uma metodologia de cultura de células aderentes para obtenção de cromossomos mitóticos em peixes neotropicais, estabelecendo linhagens celulares de diferentes espécies e a determinação de suas características citogenéticas. Seis espécies de peixes Neotropicais da ordem Characiformes foram utilizadas para obtenção das culturas primárias, desenvolvimento e criopreservação das linhagens celulares e analise citogeneticamente. Para obtenção das linhagens, células de tecido muscular foram isoladas usando enzimas proteolíticas. As células isoladas foram cultivadas em frascos ou placas tratadas com um filme à base de gelatina e mantidas à 27,5ºC, 5% CO2 em meio completo (DMEM+soro fetal bovino+antibióticos+antimicóticos). Quando as células cobriram toda a superfície do substrato, elas foram transferidas para novos frascos para obtenção das preparações cromossômicas e criopreservação. As preparações cromossômicas das linhagens celulares foram realizadas em diferentes passagens nas espécies e os cromossomos metafásicos obtidos apresentaram morfologia nítida e definida com grande número de metáfases por lâminas e, o número modal (2n) nas espécies foi mantido mesmo após o processo de criopreservação e sucessivos subcultivos. Além disso, o uso dessa tecnologia possibilitou a localização de sequencias repetitiva de DNA em Colossoma macropomum e Piaractus mesopotamicus. No total, 28 linhagens celulares foram congeladas em diferentes passagens para as seis espécies com o número de células viáveis superiores 90% e depois de descongeladas a viabilidade celular foi superior a 50%. Esses resultados representam uma grande inovação para área de citogenética de peixes neotropicais e a aplicação desta metodologia poderá sanar algumas lacunas existentes de grupos de peixes ainda poucos explorados citogeneticamente pela dificuldade de obter preparações cromossômicas de boa qualidade que são cruciais para o sucesso de várias experiências. Além disso, essa técnica tornou possível a criação do primeiro banco de células de peixes neotropicais para América Latina.
Although Neotropical ichthyofauna is considered the most diverse in the world, only 12.2% had karyotypes described. The difficulty of obtaining good quality chromosome preparations for some Neotropical fish species is still one of the major challenges in the area of cytogenetics, especially for small and large fish. An alternative is the use of indirect methodologies, such as cell culture, widely used and established in mammals, however for fish is still little used for the difficulty of standardization of the technique of isolation and maintenance of cell culture. Thus, the present study aimed to develop a methodology for adherent cell culture to obtain mitotic chromosomes in Neotropical fish, establishmenting cell lines of different species and the cytogenetic characteristics of these lines. Six species of Neotropical fish of the order Characiformes were used to obtain primary cell, development and cryopreservation of the cell lines and cytogenetic analysis. To obtain the cell lines, muscle tissue were isolated using proteolytic enzymes. The isolated cells were cultured in flasks or plates treated with a gelatin-based film and maintained at 27.5 °C, 5% CO2 in complete medium (DMEM + fetal bovine serum + antibiotics + antimycotics). When the cells covered almost all surface of the substrate, they were transferred to new flasks to obtain chromosome preparations and cryopreservation. The chromosome preparations of the cell lines were performed in different passages in the species and the metaphase chromosomes obtained showed clear and defined morphology with a large number of metaphases per slide and the modal number (2n) in the species was maintained even after the cryopreservation process and successive subcultures. In addition, the use of this technology enabled the localization of repetitive DNA sequences in Colossoma macropomum and Piaractus mesopotamicus. In total, 28 cell lines were cryopreserved at different passage levels for the six species with viable cell numbers higher than 90% and after thawing the cell viability was greater than 50%. These results show a great innovation for the cytogenetic area of Neotropical fishes and the application of this methodology might correct some existing gaps in fish groups which have been a little explored cytogenetically due to the difficulty of obtaining good quality chromosome preparations that are crucial for the success of several experiments. In addition, this technique made possible the creation of the first Neotropical fish cell bank in Latin America.
Although Neotropical ichthyofauna is considered the most diverse in the world, only 12.2% had karyotypes described. The difficulty of obtaining good quality chromosome preparations for some Neotropical fish species is still one of the major challenges in the area of cytogenetics, especially for small and large fish. An alternative is the use of indirect methodologies, such as cell culture, widely used and established in mammals, however for fish is still little used for the difficulty of standardization of the technique of isolation and maintenance of cell culture. Thus, the present study aimed to develop a methodology for adherent cell culture to obtain mitotic chromosomes in Neotropical fish, establishmenting cell lines of different species and the cytogenetic characteristics of these lines. Six species of Neotropical fish of the order Characiformes were used to obtain primary cell, development and cryopreservation of the cell lines and cytogenetic analysis. To obtain the cell lines, muscle tissue were isolated using proteolytic enzymes. The isolated cells were cultured in flasks or plates treated with a gelatin-based film and maintained at 27.5 °C, 5% CO2 in complete medium (DMEM + fetal bovine serum + antibiotics + antimycotics). When the cells covered almost all surface of the substrate, they were transferred to new flasks to obtain chromosome preparations and cryopreservation. The chromosome preparations of the cell lines were performed in different passages in the species and the metaphase chromosomes obtained showed clear and defined morphology with a large number of metaphases per slide and the modal number (2n) in the species was maintained even after the cryopreservation process and successive subcultures. In addition, the use of this technology enabled the localization of repetitive DNA sequences in Colossoma macropomum and Piaractus mesopotamicus. In total, 28 cell lines were cryopreserved at different passage levels for the six species with viable cell numbers higher than 90% and after thawing the cell viability was greater than 50%. These results show a great innovation for the cytogenetic area of Neotropical fishes and the application of this methodology might correct some existing gaps in fish groups which have been a little explored cytogenetically due to the difficulty of obtaining good quality chromosome preparations that are crucial for the success of several experiments. In addition, this technique made possible the creation of the first Neotropical fish cell bank in Latin America.
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Palavras-chave
linhagem celular, cromossomos, FISH, criopreservação, Peixes dulcícolas, Freshwater fish, cell lines, chromosomes, cryopreservation