Doing away with animal models using fish primary cultures

Doing away with animal models using fish primary cultures

The use of animals in scientific experiments involves ethical issues as well as regulations specific to each country. An example is the Animal (Scientific) Procedures Act, 1986 that needs to be followed in the UK when lab work is conducted on vertebrates (https://www.legislation.gov.uk/ukpga/1986/14/contents). There is increased awareness in using the 3Rs (Reduce, Refine and Replace) in terms of using animal models. This allows for lowering or refining the use of animals in labs.

The development of in vitro systems to lower the use of fish in labs is the need of the hour. For example, the early 1990s saw the development of cultures of fish liver and gills developed from Oncorhynchus my kiss or the rainbow trout. These cultures served to represent the physiology at a closer level than that of isolated cells. They have been used to assess the metabolism and transport of chemicals and also environmental monitoring systems. The fields of toxicology and bioconcentration assays also benefit from the use of such primary cultures to study the uptake, metabolism, and excretion of chemicals. These observations can be extrapolated from in vitro to in vivo hence, replace the use of live animals to assess toxicology. Such models especially, primary intestine cultures become important given that environmental contaminants can be taken up by humans who eat fish in their diet!

There are tests available for assessing chemicals such as the OECD Test No. 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) that use animals. However, such tests take up lots of time, are expensive and there are of course ethical issues when using animals. Other aspects include the challenge of testing low soluble and hydrophobic compounds. The development of intestinal cultures can address all these challenges.

As the response of the intestines are specific and also have a brief life span apart from complex associations with other cells and microbes. A team led by Laura published the culturing of primary intestinal epithelial cells cultured from the rainbow trout, Oncorhynchus mykiss in Biology Open. This organism was chosen as it is a model species to assess toxicology thus opening up the use of this culture to test the ADME (absorption, distribution, metabolism, and excretion) of pharmacological molecules.

An interesting point is variations in proliferation, differentiation, and responses to toxins across segments of the intestine. This is seen especially with fish systems. The authors suggested increased survival of intestinal epithelial cells when the top layer of enterocyte cells like a sheet that allows the maintenance of cell-cell contacts.  As far as media are concerned, anterior, mid and posterior intestinal cells grew well on L-15 medium while minimum essential medium (MEM) supported the pyloric caeca. There are also reports of the use of Dulbecco’s modified Eagle medium (DMEM) for good results.

Metabolic studies of 7-ethoxycoumarin-O-deethylation (ECOD) were at par with that of zebrafish larvae. Additionally, the expression of CYP3A was more in the posterior intestine. Overall, the primary culture takes a step ahead in the 3Rs- reducing the number of fish used to study dietary uptake, replace the use of live animals and refine the fact that live fish are not subjected to the exposure to toxic chemicals.

Reference:

Laura M. Langan, Stewart F. Owen, Awadhesh N. Jha. Establishment and long-term maintenance of primary intestinal epithelial cells cultured from the rainbow trout, Oncorhynchus mykiss. Biology Open 2018 7: bio032870doi: 10.1242/bio.032870.

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