New Protocol for Cell Culture to Obtain Mitotic Chromosomes in Fishes

Abstract

Cell culture is an excellent alternative for the maintenance of cell lines and to obtain quality chromosome preparations of fishes. However, this methodology is still little employed, mainly because of the difficulty of standardization of cell cultures. In this study, we describe a methodology for the rapid acquisition of cell lineages and mitotic chromosomes for cytogenetic studies of fish species from muscle tissue cells. Our methodology is based on the use of a gelatin film, which provides better adhesion of a large number of cells and appropriate conditions for multiplication. The cells of Astyanax altiparanae, used as an experimental model, with fibroblast-like morphology, showed rapid cellular proliferation, resulting in a great number of cells. Chromosomal preparations of cultured cells showed the diploid number of the species, 2n = 50 chromosomes, in 80% of the cells examined, with chromosomes intact and distended. Cell populations were cryopreserved and after being recovered, these cells maintained their proliferative capacity. The development of this methodology represents an innovation for the fish cytogenetics area and it may bring a significant contribution to the conservation and study of several groups due to the difficulty of obtaining good-quality chromosome preparations.

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes, Characidae)

“…This is the first time that cell cultures have been established for H. multifasciatus. Cell culture for Astyanax species reported results similar to our studies, with cells with fibroblast morphology and cell confluence in 10 days in primary culture and between two and four days after subcultivation (Paim et al, 2018).…”

“…The cells were isolated and cultivated according to Paim et al (2018). For this, tissue fragments were digested with a collagenase solution at 0.003 mg/mL (Millipore-Sigma, Burlington, MA, USA) for 60 min at 28 °C and then centrifuged at 1000 rpm.…”

“…Mitotic chromosomes were prepared using cells from the first to the fifth step of the cell cultures according to Paim et al (2018). When the cultures presented a high proportion of dividing cells, a colchicine solution (0.0016%) was added to the flasks for 100 min.…”

“…The technology of cell culture, very widespread in several areas of Biology, is still little explored in fish cytogenetics, despite being an excellent alternative to obtain quality chromosomal preparations. This little use is mainly related to the difficulty in standardizing the isolation and maintenance techniques of these cell cultures (Amemiya et al, 1984;Bejar et al, 1997;Zhang et al, 1998;Wang et al, 2012;Paim et al, 2018). However, Romanenko et al (2015) used the culture technique to study chromosomes of the species Acipenser ruthenus, a large species, widely used in aquaculture for the production of caviar, which is on the IUCN list of endangered fish.…”

New parameters for the in vitro development of cell lines from fish species

“…Despite that, some cells, as in Astyanax sp. (Paim et al, 2018), adapt only to DMEM medium with its complex chemical composition, and high concentrations of amino acids, vitamins, and glucose (Yao & Asayama, 2017).…”