@misc{
author = "Raskovic, Bozidar and Madureira, Tania Vieria and Lopes, Celia and Rocha, Eduardo",
year = "2022",
abstract = "In 2018, approximately 10.5 million experimental animals were used in the EU for various testing purposes, and 26% of that number accounted for fish. The scientific community, regulatory agencies, and national and EU policymakers endorse the refinement, reduction, and replacement (3Rs) of assays to reduce the number of sacrificed experimental animals. Despite the guidelines in fish exposure assays being well established (such as OECD and US EPA) and the number of test animals kept as low as feasible, each exposure study still requires at least 105 fish. For this reason, many in vitro methods were developed or improved for use in fish assays, which provided a good platform for testing the effects of chemicals, complementing, or replacing in vivo studies. Methods from mammalian in vitro testing were used to develop primary piscine cultures and cell lines, typically cultured in monolayers, viz. two-dimensional (2D). The advantages of 2D cultures are rapid exposure tests, being easy for handling and requiring low-cost maintenance. However, the uses of 2D cultures also have downsides: cells cannot last for a long time and cannot mimic the organization of tissues since they are cultured in the bottom of flasks and plates. To solve these problems, researchers created three-dimensional (3D) cultures. The microarchitecture and physiology of cells in 3D cultures are closer to in vivo systems. 3D primary cultures in fish have been seldom used, but they may provide an excellent research tool for the toxicological assessment of chemicals. To further use fish spheroids in toxicology, we are expanding a protocol for routine isolation and culture/co-culture of cells from brown trout (Salmo trutta) liver. The protocol is compatible with exploring differential centrifugation of isolated cells to change the composition of spheroids in terms of the ratio of hepatocytes and biliary epithelial cells. After plating in non-adhesive plates, cells were cultured for 12 days until maturation, using orbital shakers, in which they slowly aggregated and formed spheroids in the incubator (at 18 °C). DMEM enabled the formation of spheroids with sufficient size, with an absence of significant necrotic centres, which usually occur in spheroids due to hypoxia. From day 12 to 18, liver spheroids were exposed to single or mixtures of endocrine disruptors (17α-ethinylestradiol and the progestins levonorgestrel and megestrol acetate) to assess their advert effects on cells. Spheroids were evaluated using light and electron microscopy (routine staining and immunohistochemistry), gene expression (a set of genes related to lipid metabolism, yolk proteins and vitellogenin), and biochemical assays (lactate dehydrogenase and resazurin assays). The spheroids were able to respond to the stimuli.",
publisher = "Czechoslovak Microscopy Society",
journal = "16th Multinational Congress on Microscopy",
title = "Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research",
pages = "235-235",
url = "https://hdl.handle.net/21.15107/rcub_agrospace_6941"
}
