FISH CELL LINES:

Till now, few fish cell lines are established and are available in different databases. Fish cell
lines are mainly used to test the toxicology of water, to examine the viral load or to produce
vaccines for other farmed fish. Some fish cell lines were used to generate edible fish (Rubio
et al., 2019). Fish cell lines are established from fish tissues to isolate the fish viruses or to
detect them. (Pandey, 2013).
The starter cultures of fish cell lines can be taken from different databases or culture
collection centers like American Type Culture Collection (ATCC) (Wolf, 1965). It can also
be taken from databases like FICELdb and Cellosaurus. FICELdb is fish cell database that
has many legacy lines (from 1962 to 1999). Next, Cellosaurus is also a fish cell database that
was established in 2012. It has different cell line repositories from all over the world. Total
100,000 cell lines are categorized in it. Out of which 558 are immortalized fish lines and out
of them 9 fish cell lines are taken from fish muscles.
From these 9 cell lines, two are obtained from salt water fish and are epithelial. Six cell lines
are obtained from fresh water fish. Rest three were obtained from white sturgeon, bluefin
trevally and barramundi. These cell lines are used for culturing fish pathogens. (Rubio et al.,
2019)
Table 1 Immortalized fish muscle-isolated cell lines available from Cellosaurus.
(Immortalized Fish Muscle-Isolated Cell Lines Available from Cellosaurus., 2019)

Fish Cell Lines from Different Tissues

Usually fish cell lines are obtained from tissues like liver, gill, kidney, skin, brain, bladder,
spleen or heart. The second most tissue used for culturing is Fin. Fin is used because its
regenerative ability is high. Moreover, ovary, skin and scales are also used to develop the cell
lines. A cell line XM was made from skin and fin of fish.
Larval and embryonic cells are very easy to culture because they are mitotically active.
Recently, many embryonic stem-like cells were also established from different fish species.
Studies on zebrafish and medaka were done to develop Embryonic stem cell lines (Pandey,
2013).
Methods of Cell Isolation
The methods to isolate fish cells are just same as mammalian cell isolation methods.
Generally, sterilization of fish is done and then it is anesthetized. After that a sample tissue is
excised with biopsy. The tissue is then digested enzymatically using trypsin or collagenase or
can be explanted. These isolating methods work well for adult fish.
Explanted tissues are attached to a culture plate and the cells will be transferred from tissue to
surface of culture. The culture plates are coated with proteins like collagen, gelatin or laminin
to increase the cell adhesion.
On the other hand, the tissues that are digested enzymatically are prepared in aqueous
solutions. By the action of trypsin and collagenase enzymes the cells are released into the
liquid medium. Then the cells are washed with buffer for contaminants removal. After that
filtration is done to separate cells from debris.
To isolate cells from embryo of fish different methods are used. The cells from the embryo of
wild fish species are difficult and challenging to isolate.
Many other different methods like magnetic beads that are coupled with muscle progenitor
specific antibodies are used that can rapidly isolate cells from tissues and the damage is also
very minimal. Another technique known as fluorescence-activated cell sorting (FACS) is also
a very efficient and effective method to isolate the cells. (Rubio et al., 2019)

Figure 1 Isolation of cells from fish (Isolation of Cells from Fish, 2018)

Cell culture conditions

Some of the conditions that are required to establish a fish cell culture are discussed below:
Extracellular environments and scaffolds
The fish cells need extracellular matrix (ECM) to survive in the cultures. The culture plates
can be coated with different proteins like laminin, fibronectin, collagen or elastin etc. Atlantic
salmon muscle cells were cultivated on a laminin coated culture plate. The fish cells need the
extracellular matrix to grow and proliferate. Moreover, some cells also require fish
glycoaminoglycans. (Rubio et al., 2019)
Growth media
In the growth media of fish culture, different variables like buffer, salt concentration, carbon
source, temperature and pH should be kept in mind. Eagle’s Medium, Medium 199 (M199),
Modified Eagle’s Medium (MEM), and Leibowitz’s 15 (L-15) were used but some additional
salts were added in them for the marine species. (Rubio et al., 2019)
Eagles’ minimal essential medium in which foetal bovine serum (FBS) acts as a media that is
suitable for the growth of all types of cells including mammalian, birds, amphibians, fish and
birds. Glasgow MEM and Hank’s MEM (HMEM) are routinely used media for fish cultures.
Leibovitz L-15 media is the media that do not need Carbon dioxide and cells grow efficiently
in this media. (Pandey, 2013). Depending on the consumption of the CO2, the media are used,
as some cells require 5% CO2, others do not need at all. (Rubio et al., 2019)
Then some growth factors like Fibroblast growth factor (FGF2) is also added in the media as
it helps in growth of muscle cells. (Rubio et al., 2019). other growth factors like mammalian
epidermal growth factor (mEGF) can also be used. (Pandey, 2013)
If the FBS is removed from the media, the proliferation slowdowns but if the vitamin E or
fatty acids are added then the growth becomes rapid. So FBS can be replaced as it costs more.
(Rubio et al., 2019)
Uses of Fish Cell Lines
As fish cell lines are available since 1960s, they are useful in the scientific research. But as
compared to mammalian cell lines, they are less commonly used. For the first time work on
RTG-2 fish cell line was done for virological studies. (Pandey, 2013)
Later, there use in fields like biomedical research, pathology, oncology and toxicology started
and they were also useful in studying diseases of fish. Genetic regulation, expression and
DNA repair and replication can also be studied using these cell lines. In almost 50 years,
several different fish cell lines of different varieties and species of fish were established.

Figure 2 Uses of fish cell lines (Uses of fish cell lines, 2016)
Fish cell lines have an important contribution in fish toxicology, virology, endocrinology,
disease control, ecotoxicology, aquaculture, biotechnology and radiation biology. In newly
developed cell lines, the transfection of DNA and gene expression can be studied efficiently.
Fish cell lines were transfected with pEGFP vector and they show fluorescent signals. So,
using this approach genetic manipulations can be studied. (Pandey, 2013)
To evaluate and study the presence of a virus in a healthy fish the cell line can be made and it
is isolated from others to study the virus. In an experiment, cell lines were treated with five
fish viruses to check the susceptibility, resultantly, nodavirus and MABV NC1 were infected
by the virus. CPE and RT PCR were run to confirm the infections. (Pandey, 2013)

REFERENCES:
1. Rubio, N., Datar, I., Stachura, D., Kaplan, D., & Krueger, K. (2019). Cell-Based Fish:
A Novel Approach to Seafood Production and an Opportunity for Cellular
Agriculture. Frontiers in Sustainable Food Systems, 3.
https://doi.org/10.3389/fsufs.2019.00043
2. Pandey, Govind. (2013). Overview of fish cell lines and their uses. 2. 580-590.
https://www.researchgate.net/publication/270174922_Overview_of_fish_cell_lines_a
nd_their_uses
3. Wolf, K. (1965). Some Recent Developments and Applications of Fish Cell and
Tissue Culture. The Progressive Fish-Culturist, 27(2), 67–74.
https://doi.org/10.1016/S1546-5098(08)60115-6

TABLE AND FIGURE REFERENCES:

1. Immortalized fish muscle-isolated cell lines available from Cellosaurus. (2019, June
11). [Table]. https://www.frontiersin.org/articles/10.3389/fsufs.2019.00043/full
FIGURE REFERENCES:
1. Isolation of cells from fish. (2018, July 28). [Photograph].
https://www.jove.com/t/58159/preparation-high-quality-primary-cell-culture-from-
fish

2. Uses of fish cell lines. (2016). [Photograph]. https://slideplayer.com/slide/9140398/