Recent research studies on fish cell lines found many more application of cell lines pathological studies, toxicology, biomedical research, vaccine development etc

1. Fish cell lines and its applications
By
Bhukya Bhaskar
Acknowledgement:
Scientists efforts on fish
cell lines

2. Introduction
• Cell line: its arises from a primary culture at the time of first successful subculture. It consist of lineages
of cells originally present in primary culture.
• Cell culture: it refers to a culture derived from dispersed cells takenfrom original tissue, from a primary
culture, or from a cell line or cell strain by enzymatic,mechanical,or chemical disaggregation.
• Primary cell culture: The eukaryotic cells taken directly from an animal for culture purpose.
• Hybridoma: a clone of hybrid cells produced by fusion of a myeloma cell with an antibody producing
cell. Each hybridoma produces one type of monoclonal antibody only.
• Totipotent: its describe the cell that is not committed to a single developmental pathway, and thus it is
capable of formingall types of differntial cells.
• Transgenic: an organism that carries a foreign DNA (transgene).
• In situ hybridization: it’s a process of annealing a probe in order in order to screen a DNA library.
• Southern Hybridization: a technique used for the detection of specific DNA sequences(restriction
fragments).
• In vitro: living outside the organism or in an artificial environment, typically in glass vessels in which
cultured cells, tissues or whole plants may reside.
• In Vivo: natural condition in which organisms reside, refers to its biological processes that take place
within a living organism or cell under normal conditions.
• In Vivo Gene Therapy: Direct delivery of gene(s) to a tissue or an organ to alleviate genetic disorders.
• Somatic cell Gene Therapy: Delivery of gene(s) to somatic cells to correct genetic defects.
• First fish cell line reported in 1962(wolf &Quimby, 1962).
• Due to the good correlation reported between the in vivo fish data and in vitro data, the established fish
cell lines can represent alternative tool to acute fish bioassay for toxicity screening of chemicals.
• Rainbow Trout Gonad (RTG2) was the very first fish cell line to be developed and used for virus studies

3. Cont…
• The 19th-century English physiologist Sydney Ringer developed salt solutions
containing the chlorides of sodium, potassium, calcium and magnesium suitable for
maintaining the beating of an isolated animal heart outside of the body.
• Fish cell culture has developed rapidly since Wolf and Quimby established the first
fish cell line RTG-2 in 1960s for the first time. After then, fish cell culture has
become anessential research technology which has been used extensively, ranged
from virology, environmental toxicology, cytobiology, oncology, genomics, genetics
and environmental protection.
• Cultured fish cells have more advantages than live fish as the experimental material:
the materials are cheap and easy to obtain and the experimental condition could be
controlled accurately and the experiment could be repeated.
• Why cell culture is required?
• As the anatomy and physiology of fish is complex, there are many different cells,
Many different proteins which are interacting continuously for normal body
functioning. Being complex in nature, these events are difficult to watch
individually in vivo. Moreover, fish being delicate are usually harmed and get
stressed while observing biological events. Hence it is necessary to develop a
parallel cell observation system particularly in vitro in nature to which acell culture
system strongly supplements

4. Types of cell Cultures:
Cell cultures may contain the following three types of cells:
• 1. Stem cells,
• 2. Precursor cells and
• 3. Differentiated cells
• Stem cells are undifferentiated cells which can differentiate under correct inducing
conditions into one of several kinds of cells; different kinds of stem cells differ
markedly in terms of the kinds of cells they will differentiate into.
• Precursor cells are derived from stem cells, are committed to differentiation, but are
not yet differentiated; these cells retain the capacity for proliferation.
• Differentiated cells usually do not have the capacity to divide. Some cell cultures,
e.g., epidermal keratinocyte cultures, contain all the three types of cells. In such cell
cultures, stem cells constantly provide new cells which develop into precursors; the
precursor cells proliferate and mature into the differentiated cell types.
Cell cultures can be grown as:
• 1. Monolayers or as
• 2. Suspension Cultures
• Therefore, cells in culture need a surface or substrate to adhere to so that they are
able toproliferate. Cells that are unable to adhere to a substrate are unable to divide,
i.e., their growthis anchorage dependent. The surface, available for attachment of
cultured cells is calledsubstrate. The various kinds of substrates used in cell
cultures may be grouped into thefollowing 3 categories: (1) glass, (2) plastics,
(3) metals.

5. Types of suspension cultures :
• 1. Batch Cultures (fixed medium volume; as the cell grow, medium is gradually depleted;
eventually cells cease to divide),
• 2. Fed Batch Culture (gradual addition of fresh medium leading to an increase in culture
volume),
• 3. Semi-continuous Batch Culture (at regular intervals, a constant fraction of the
culture,including cells, is withdrawn and an equal volume of fresh medium is added to the
culture),
• 4. Perfmion Culture (at regular intervals, a constant volume of spent medium, without cells,
is withdrawn and an equal volume of fresh medium is added); and
• 5. Continuous-flow Culture (continuous withdrawl of culture along with cells and addition
ofequal volume of fresh medium so that the culture is maintained in a steady state).
• Freshly isolated cultures from fish tissues are known as primary cultures until sub-cultured.
• At this stage, cells are usually heterogeneous but still closely represent the parent cell types as
well as in the expression of tissue specific properties.
• After several sub-cultures onto freshmedia, the cell line will either die out or 'transform' to
become a continuous cell line.
• Such celllines show many alterations from the primary cultures including change in
morphology,chromosomal variation and increase in capacity to give rise to tumors in hosts
with weak immune systems.
• Fish cells can be grown either in an unattached suspension culture or attached to a solid
surface. Suspension cultures have been successfully developed to quite large bioreactor
volumes, with successful production of viruses and therapeutic proteins.

6. Materials
• Fish cell line of choice
• L-glutamine-200mM (100x)
• Minimum essential medium (Eagle) with 10% Fetal bovine serum
• Trypsin-EDTA
• Culture flask (75 cm2 or 25 cm2)
• Pipettes, sterile, cotton plugged, 1-ml, 5ml, 10 ml
 70% isopropanol,
• graduated cylinder, 100ml
• Glass bottle 100 ml
• Beaker, 500 ml
• Bleach.
• Cell counting using the Hemo-cytometer.
75 cm2 flask of cells, Trypan Blue (0.1% in PBS), Microscope,
Dilution tubes (12*75mm), Pasteur pipette, hanks balanced salt
solution, or MEM-0 (MEM w/o serum) Trypsin-EDTA, Pipettes-
1ml, sterile, cotton plugged 22*22 mm cover-slips.

7. Media and supplements used in fish cell culture:
• The nutrient media used for culture of animal cells and tissues must be
able to support theirsurvival as well as growth, i.e., must provide
nutritional, hormonal and stromal factors. Thevarious types of media
used for tissue culture may be grouped into two broad categories:
• 1. Natural Media and
• 2. Artificial Media.
• These media consist solely of naturally occurring biological fluids and
are of the following 3 types:
• (1) cagula or clots,
• (2) biological fluids and
• (3) tissue extracts.
• Biological Fluids. Of the various biological fluids used as culture
medium (e.g., amnioticfluid, ascitic and pleural fluid, aqueous humour
from eye, insect haemolymph, serumetc.),serum is the most widely used
Serum is the liquid exuded from coagulating blood.Different preparations
of serum differ in their properties; they have to be tested for sterility
andtoxicity before use. The natural biological fluids are generally used
for organ culture. For cellcultures, artificial media with or without serum
are used.

8. Artificial Media
• (1) immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate),
• (2) prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic
compounds),
• (3) indefinite growth, and
• (4) specialized functions.
• The various artificial media developed for cell cultures may be grouped as:
• (i) serum containing media
• (ii) serum-free media,
• Serum Containing Media:
• The various defined media, e.g., Eagle's minimum essential medium etc. (see, serum-freemedia) when supplemented with
5-20% serum are good nutrient media for culture of mosttypes of cells. It provides the basic nutrients for cells; the
nutrients are present both in thesolution as well as are bound to the proteins. A major role of serum is to supply proteins,
e.g.,
• fibrobnectin, which promote attachment of cells to the substrate. It also provides spreadingfactors that help thecells to
spread out before they can begin to divide.
• Serum-free media:
• 1. Improved reproducibility of results from different laboratories and over time since variationdue to batch change of
serum is avoided.
• 2. Easier downstream processing of products from cultured cells.
• 3. Toxic effects of serum are avoided.
• 4. Biassays are free from interference due to serum proteins.
• Specific media like Leibovitz L15 is used to eliminate the need of adding CO2 and NaHCO3.Leibovitz's-15
supplemented with 20% fetal bovine serum. After the initiation of primaryculture, fish serum is added at 1% final
concentration. To prepare fish serum, blood is drawnfrom the caudal vein of an adult fish and is kept for overnight at 4°
C. The donor fishes areobtained from a hatchery after observing for the absence of parasitic infections and
surfaceinjuries. The fish is disinfected and maintained as above and fed with boiled fish twice a day.The supernatant is
then centrifuged at 2290 g for 5-10 min to precipitate the blood cells. Serumis pre-filtered using a 0.45 μm membrane
and filter sterilized using a 0.22 mm membrane.After inactivation at 56° C for 30min, the serum is stored at - 20° C until
use.
• Basic Constituents of media: Inorganic salts;Carbohydrates;Amino Acids;Vitamins;Fatty acids and lipids;Proteins and
peptides;;Serum

9. Buffering Systems
• Most cells require pH conditions in the range 7.2 - 7.4 and close
control of pH is essential for optimum culture conditions. There are
major variations to this optimum. Fibroblasts prefer a higher pH
(7.4 - 7.7) whereas, continuous transformed cell lines require more
acid conditions pH (7.0 - 7.4). Regulation of pH is particularly
important immediately following cell seedingwhen a new culture is
establishing and is usually achieved by one of two buffering
systems;
• 1. a "natural" buffering system where gaseous CO2 balances with
the CO3 / HCO3 contentof the culture medium and chemical
buffering using a zwitterion called HEPES
• 2. Cultures using natural bicarbonate/CO2 buffering systems need
to be maintained in an atmosphere of 5-10% CO2 in air usually
supplied in a CO2 incubator. Bicarbonate/CO2 is low cost, non-
toxic and also provides other chemical benefits to the cells. HEPES
has superior buffering capacity in the pH range 7.2 - 7.4 but is
relatively expensive and can be toxic to some cell types at higher
concentrations. HEPES buffered cultures do not require a
controlled gaseous atmosphere.

10. Explant Preparation:
• Explant culture
• It is a technique used for the isolation of cells from a piece or pieces of tissue.
• Tissue harvested in this manner is called an explant. It can be a portion of the shoot,
leaves, orsome cells from a plant, and can be any part of the tissue from an animal.
In brief, thetissue is harvested in a sterile manner, often minced, and pieces placed in
a cell culturedish containing growth media.
• Primary cultures are derived directly from excised,normal animal tissue and cultured
either as an explant culture or following dissociationinto a single cell suspension by
enzyme digestion. Such cultures are initiallyheterogeneous but later become
dominated by fibroblasts.
• The preparation of primarycultures is labor intensive and they can be maintained in
vitro only for a limited period oftime. During their relatively limited life span
primary cells usually retain many of thedifferentiated characteristics of the cell in
vivo.
• A. Preparation of donor fish: As contamination is the major problem in
tissueculture experiments. Adequate care should be taken to minimize the
possibleroutes of contamination. The donor fish is usually starved for a day or two
toreduce the possibility of gross contamination from feces and regurgitated
feed.During this period, fish is allowed to swim in well aerated autoclaved water
forreducing the microbial load adhered on to the skin and gills, then sacrificed
byplunging in ice for 10-15 min.

11. Cont…
• B. Decontamination: The decontaminating solutions for this purpose includes
chlorinesolution (500 ppm available chlorine), 70% ethanol, iodophore solution (0.5
w/v iodine) etc.For external organs like gills, skin or fin intended for culture, strong
disinfectants are avoidedbecause they damage the tissue too. The commonly used
antibiotics in the presentexperiment were penicillin (400 IU/ml) and streptomycin
(400 μg / ml) with an anti-fungalamphotericin B (10 μg/ml). The tissue of interest is
aseptically picked up and washed three tofive times with the antibiotic solution.
• C. Dissection and / or Disagregation: Two major methods for initiating aprimary
culture followed are given below.
• (i) Tissues are disaggregated into its component cells. This is achieved by enzymatic
digestionusing trypsin or collagenase supplemented with EDTA. These enzymes
digest the extra celluarmatrix and EDTA chelates divalent cations like Ca2+ and
Mg2+ required for the integrity of thematrix. The tissue was also dispersed by
mechanical means like slicing, sieving, forcingthrough a needle and repeated
pipetting. The collected cells are then seeded in the vessel at 5x 105 cells per ml of
medium.
• (ii) The required tissue is picked up and cut in small pieces to prepare explants of 1
mm3 sizeand planted in culture dishes. 1 or 2 fragments of tissue are seeded in 1
cm2 area. The caudalfin, heart and gills are aseptically excised from fingerlings are
rinsed individually withphosphate-buffered saline (PBS), 70% ethanol and Iodine
antiseptic (0.5% w/v iodine). Thehead and gut of the fry should be carefully
discarded while the remaining tissue mass iswashed as above. Explants of 1mm3
sizes are prepared and washed thrice with PBS containingantibiotics for 5-10min.

12. • Culture of cells following seeding:
• The explants are seeded in 25 cm2 tissue culture flask
and kept semi dry for a few minutes.The adherence of
explants is accomplished by incubation with 0.5 mL of
FBS at 28° C. After 8-10 hrs, the growth medium, L-15
(Leibovitz) is added gently. Fifty percent of the media is
• recommended to be exchanged once in every 3 days.
Daily observations are made using aninverted
microscope. The dispersed cells adhere on the culture
substrate and start toproliferate. Dead cells can not
secrete substrate adhesion molecules and hence float.
They canbe removed by subsequent medium
exchange. The optimum pH and incubation
temperaturemaintained should be nearly 7.4 and 22-
28° C respectively for culture of fish cells.

13. Subculture and maintenance:
• When the primary culture attains confluency, it is subcultured using a
solution of 0.1% trypsinand 0.02% versene (EDTA) as detachment agents.
Cells intolerant to trypsin can be scrapedusing a cell scraper or dispersed by
rocking followed by gentle pipetting. Detached cells canthen be distributed
to 2 to 4 flasks containing fresh medium depending on the split
ratiorequired. Between every subculture the culture flask should be observed
for contamination,change in pH and healthy proliferation of cells.
• For the first subculture the cells are carefully detached from the flask surface
using TPVGsolution (0.1% Trypsin, 0.2% EDTA and 0.2% glucose in PBS
1X) without dislodging theexplants. The detached cells are harvested in 5mL
of growth medium and transferred to freshflasks. The explants are
maintained further to collect fresh migrating cells. When the
confluentmonolayer is formed in the primary culture, the old medium is
removed and cells are dislodgedby treatment with the above TPVG solution
twice for 30 seconds each. The detached cells areresuspended in 5mL of
fresh growth medium (L-15 plus 20% FBS) and seeded in 25 cm2plastic
culture flasks. From second passage onwards, a split ratio of1:2 is usually
maintainedfor subsequent passages.

14. • Maintenance: Cultures should be examined daily,
observing the morphology, the color ofthe medium and the
density of the cells.
• A. Growth pattern: Cells initially goes through a quiescent
or lag phase that depends onthe cell type, the seeding
density, the media components, and previous handling. The
cells willthen go into exponential growth where they have
the highest metabolic activity. The cells willthen enter into
stationary phase where the number of cells is constant, this
is characteristic ofa confluent population.
• B. Harvesting: Cells are harvested when they reach a
population density which suppressesgrowth. Ideally, cells
are harvested when they are in a semi-confluent state and
are still in logphase. Cells which are not passaged and were
allowed to grow to a confluent state cansometime lag for a
long period of time and some may never recover.

15. Cell culture lab and Inverted microscope A& CO2
Inccubator(b) used at cell culture
• Basic aseptic conditions to be maintained
in the cell culture labs:
• If working on the bench use a Bunsen
flame to heat the air surrounding the
Bunsen.
• Swab all bottle tops & necks with 70%
ethanol.
• Flame all bottle necks & pipette by passing
very quickly through the hottest part of
theflame.
• Avoiding placing caps & pipettes down on
the bench; practice holding bottle tops
withthe little finger.
• Work either leftto right or vice versa, so
that all material goes to one side,
oncefinished, Clean up spills immediately
& always leave the work place neat & tidy.

16. List of cell lines developed from warmwater and cold-water fish species and currently available from American Type
Culture Collection (ATCC)
and the European Collection of Authenticated Cell Cultures (ECACC) [157-159]. This is not an exhaustive list

17. Cell lines of marine fish origin. The cell lines (SAF, SaBE-1c) are
currently available from the cell culture repositories.
Cell lines originated from aquatic invertebrates. These are not currently available from the cell culture repositories.

18. Applications for Fish cell cultures
• Fish cell lines applied in Pathological studies, Toxicity
studies, Immunological applications, In vitro models,
Transgenesis,Biomedical research in addition recent
research findings states few more applications .
• To investigate the normal physiology or biochemistry of cells.
For instance, studies of cell metabolism.
• To test the effect of various chemical compounds or drugs on
specific cell types (normal or cancerous cells, for example).
• To study the sequential or parallel combination of various cell
types to generate artificial tissues.
• Therapeutic proteins can be synthesized in large quantities by
growing genetically engineered cells in large-scale cultures.
• Creation of viral vaccines from large scale cell cultures.
• Cytotoxicity and genotoxicity studies.
• Fish cell cultures prove a useful tool for the transfection (gene
delivery) studies.

19. Application in model systems
• Since in vitro cell cultures mimic the host animal in vivo, fish cell
cultures act as excellent research models. Also, these are not subjected to
interference from environmental disturbances to which animals are
sensitive. On the other hand, genetic manipulations of the cells can be easily
achieved to study differential expression of genes and or proteins.
Consistency and reproducibility of results are added advantages. Cell
cultures have been increasingly used as model systems to study basic cell
biology, physiology, cellular communications, signaling pathways,
expression profiling, apoptosis, interactions between cells and pathogenic
agents, effects of drugs, metabolic effects of nutritional elements, and
mutagenesis.
• They are also important model systems in embryology, neurobiology,
endocrinology, and environmental biology. Consequently, cultured cells are
vital for the identification of specific molecules and/or mechanisms used in
initial pathogen host cell interactions.
• For example, the macrophage cells from tilapia gill were used to investigate
the attachment of pathogens during infection [36]. Ease of manipulation and
homology with functional genes engaged in human diseases make zebra fish
cell lines, a potential in vitro model to study diseases as well as cellular
Processes Many fish-derived cell lines were used to explore the field of fish
endocrinology

20. Cell lines applications in Virology
• Being obligate intracellular parasites, viruses require host cell machinery for
replication and propagation.
• Cell cultures are considered ‘the gold standard’ due to their diverse roles in
virology such as detection, identification, propagation, isolation,confirmation,
and characterization of viruses.
• Due to the relevance of cells in virology, the OIE (Office International des
Epizooties) protocols require cell cultures, in viral disease diagnosis
and confirmation.
• Fish cell cultures can function as an effective replacement for animals,
especially in the field of virology.
• Cell cultures can be reliable sources of viruses when compared to the
uncertainties associated with obtaining viruses from infected animals for
research purposes.
• Susceptible cell lines are essential to determine the detailed etiology
of viruses as evidenced in the case of Infectious Pancreatic Necrosis (IPN) and
Infectious Hematopoietic Necrosis (IHN) viruses For the emerging fish
viruses, the infectious cycle, mode of infection, pathogenicity, potential host
range, and viral replication inhibition strategies need to be determined for
establishing

21. Cell lines applications in Research on antivirals
• Fish cell lines are routinely used for screening antiviral compounds
Hao K, et al. reported the efficacy of acyclovir, a
common antiviral to treat human herpesvirus infection, against
channel catfish virus infection in CCO cells.
• Acyclovir was also found to exert effective antiviral activity
against cyprinid herpesvirus-3 (CyHV-3) infection in Common
Carp Brain (CCB) and Koi Fin cells (KF-1).
• Exopolysaccharides isolated from the algae Arthrospira platensis
inhibited KHV replication in CCB cell lines.
• Similarly, polyinosinic polycytidylic acid (poly I:C) was
reported to induce an antiviral state in CHSE-214 cell line against
IPNV.
• Balmer BF, et al. studied the efficacy of a compound
against Infectious Hematopoietic Necrosis Virus (IHNV) using
EPC cell lines, which was found to hinder viral entry by inhibiting
virus-host cell membrane fusion.

22. Cell lines applications in Toxicology
• Being relevant representatives for the aquatic environment,fish cell
cultures function as apt alternative for animals and are extensively
used as in vitro models for environmental toxicology studies
especially cytotoxicity analysis.
• In addition to avoiding high costs and variability of results; the
genotoxicity of chemicals, metabolism, DNA binding, and mode of
action can be Evaluated .
• Fish hepatoma cell lines were found useful to test the xenobiotic
efflux activity of human drugs.
• Fish cell lines were used to evaluate the cytotoxicity of chromium,
Polycyclic Aromatic Hydrocarbons (PAH), and aflatoxins using
comet assays and or neutral red dye uptake method .
• Fish cell cultures were found sensitive to several bacterial or fungal
toxins/extracellular products .
• The EPC cell line was found to be a suitable substrate for the study
of intracellular antigens and virulence factors produced by
Renibacterium salmoninarum

23. Cell lines applications in Drug screening and development
• Cell-based assays have become an inevitable part
of the pharmaceutical industry for high throughput
screening of potential compounds and to test the
cytotoxicity of candidate drugs.
• Other related applications include dose
optimization, drug delivery, drug safety,
pharmacology, cellular targeting, pharmaceutical
analysis,and quality assurance.
• Fish cell cultures can potentially play an important
role in the research and development of drugs
aimed to benefit fish and also to identify
therapeutic targets such as receptors.

24. Cell lines applications in Production of biologicals
• Interferons, blood clotting factors, monoclonal antibodies
(mABs), interleukins, lymphokines, insulin, growth factors,
hormones, viruses, enzymes, and anticancer agents.
• Fish cell lines are less expensive and thus more economical
for the mass production of biologicals compared to
mammalian cell cultures.
• Fish cell cultures can act as miniature factories to express
substantial quantities of commercially important proteins after
being infected with genetically engineered baculoviruses.
• More than 90% of the mABs are produced using in vitro
methods due to the ease of culture and less economic
consideration compared with the use of animals.
• Human cell lines are used to produce numerous FDA-
approved therapeutic proteins. Similar efforts could be
ventured using fish cell cultures.

25. Cell lines applications in Genome editing
• Cell lines are amenable for genetic modifications. Hence, fish cell cultures
are used in knockout studies, where certain genes are inactivated and their
effects are traced.
• The first gene editing using CRISPR-cas9 system in fish somatic cell lines
was followed by several such studies.
• Chinook salmon embryo (CHSE-214) cell line capable of expressing
geneticin and hygromycin resistance was generated by knockout technology.
• Liu Q, et al. reported successful gene editing using gRNA-Cas9
Ribonucleoprotein (RNP) Complex in medaka embryonic cell lines.
• Gratacap RL, et al. developed protocols for successful CRISPR gene editing
in CHSE-214 cell line using lentivirus transduction which could be used to
manipulate disease resistance in salmonid species.
• Chang N, et al. and Hwang WY, et al. successfully carried out genome
editing with RNA-guided Cas9 nuclease in zebrafish embryos.
• Fish cells can be fused with one another and with mammalian cells.
• For example, microcells have been prepared from goldfish RBCF-1 and
fused with human cells

26. Embryonic stem cells
• Embryonic Stem (ES) cells are pluripotent (ability to differentiate
• into any cell type) and used in biodiversity conservation and biotechnology
studies.
• Extensive studies in fish ES have been done in small model fishes, such as
zebrafish (Danio rerio) and medaka (Oryzias latipes) due to the convenience
in combining
• embryological, genetic and molecular analysis of vertebrate development.
• Fish ES cell lines are used as a vector for the efficient transfer of foreign
DNA into the germ cells of an organism.
• Hong Y, et al. developed a spermatogonial cell line from the testis of adult
medaka fish which produced viable sperm via spermiogenesis.
• With the hybrid catfish (♀ channel catfish × ♂ blue catfish) production, the
blue catfish are sacrificed for sperm collection.
• Development of a blue catfish spermatogonial cell line could be of potential
benefit to the industry.
• Embryonic germ cell transplantation was successfully used for surrogate
production in salmonids.
• Embryonic cell lines have been established from catfish, Nile tilapia and
several marine fish species

27. Cell lines applications in Cancer research
• Normal cells can be transformed into cancer cells using
radiation,chemicals, and viruses to study the mechanism
and functions of various carcinogenic chemicals,
induction of cellular apoptosis, DNA methylation,
histone modifications, tumor suppressor gene
expressions, etc.
• Fish cell lines are used in cancer biology to study the
mechanism of activation of procarcinogens, molecular
damage, and DNA repair activity.
• Fathead Minnow Cells (FHM), goldfish
erythrophoromas, and goldfish fibroblast cell lines were
used to study the mechanism and activation of
procarcinogens and subsequently the damage and repair
of genetic materials

28. Cell lines applications in Parasitology
• Several fish cell cultures were used to study the development
• and pathogenesis of parasites.
• EPC cell line supported
• the attachment and transformation of various stages of a fish
• ectoparasite, Ichthyophthirius multifiliis.
• Buchmann K, et al.
• studied the non-specific response of EPC to encapsulate
• and degrade the fish parasite Gyrodactylus derjavini.
• Primary cell
• cultures derived from salmonid fish allowed investigation of the
• microsporidian parasite Loma salmonae.
• Primary cultures of rainbow trout kidney were used to study the
• comparative development of two microsporidians infecting AIDS
• patients and salmonid fish

29. Cell lines applications in Regenerative
therapy
• Cell culture systems can produce functional cells or
tissue analogues on a large-scale that can be used
as replacement tissue or organs.
• Reconstitution of skin following severe burns is
considered
• the most successful application of cell-based
regenerative therapy.
• In this regard, fish cell cultures are experimentally
utilized for producing artificial skin to treat patients
with burns and ulcers.

30. Cell lines applications in Three-dimensional cell cultures
• Since cells in 3D systems interact with their
surroundings in all three dimensions; these models
are physiologically similar to in vivo conditions
and provide more reliable data.
• The 3D spheroids of rainbow trout (Oncorhynchus
mykiss) cell lines, RTG-2 and RTS-11 were
successfully developed and tested for their
efficiency to propagate Saprolegnia parasitica
spores that resembled in vivo infection.
• The 3D cell cultures raise the possibility for the
study of complex physiological processes in vitro.

31. Cell lines applications in Cell-based fish
• Cell culture systems can function as an innovative way
of animal-free production.
• Considering the adaptation of fish cell culture to in
vitro growth conditions in terms of tolerance to
hypoxia, high buffering capacity, and low-temperature,
an advanced approach towards the sustainability of
global fishery resources is the production of cell and
tissue culture-based seafood through bioreactor
culture.
• Benjaminson MA, et al. used tissue engineering for the
in vitro culture of skeletal muscle of goldfish that
resembled the fillet from a fibroblast fish cell line to use
in space travel.

32. Other uses of fish cell lines
• A recent study by Morin G, et al. revealed the nutritionalresearch capabilities of fish cell lines.
• Another study by Lescat L, et al. used fibroblast cell line from medaka fish (Oryzias latipes) to demonstrate that
chaperone-mediated autophagy (CMA) pathway involving lysosomal proteolysis exists in fish, which was thought to be
present only in mammals and birds.
• This study was a breakthrough in fish metabolism and provided insight into the evolutionary relationship of vertebrates
including fish, mammals, and birds.
• The potential utility of fish cell lines for transgenic and genetic manipulation studies was identified from the fluorescent
signals produced, when transfected with pEGFP vector DNA.
• Toxins produced by fish species such as chimeras, sharks,sting-rays, silurid catfish, and surgeonfish, stone-fish, and
• rabbitfish exhibit enzymatic, antimicrobial, cytotoxic, hemolytic, cardiovascular, neuromuscular, and anti-cancerous
properties
• and have pharmacological and therapeutic applications.
• Maintenance of venom gland organoids via 3D technology can be used to produce venom for use in biomedical
research.
• While the applications of cell cultures are numerous, one must be mindful of the disadvantages as well. Cell lines are
prone
• to genotypic and phenotypic drift.
• Another concern is misidentification or cell line cross-contamination.
• Apart from these, several biological pathways cannot be represented by cell line, which limits their use in certain
research areas. Primary cells and cell lines could show variability in drug dose, thus the data acquired through cell lines
need to be adjusted or cannot easily be replicated in an in vivo model. Additionally, primary cell cultures have the
potential to harbor resident pathogens.
• In research involving fish cell cultures (in virology and toxicology), a common practice observed is to use non-specific
cells unlike in mammalian biology studies. Utilizing fish cell lines with specific functions (originated from specific
tissue type) will greatly advance fundamental knowledge in the respective fields

33. Advantages
• The major advantage of using cell culture for any of the
above applications is the consistency and
reproducibility of results that can be obtained from
using a batch of clonal cells.
• the materials are cheap and easy to obtain
• the experimental condition could be controlled
accurately
• Fewer animals are harmed
• Can control all external factors
• Can easily test what the cells are doing
• Cells are easy to manipulate and propagate
• All of the cells are the same hence results of
experiments will be consistent
• Cheaper to maintain.

34. Limitations
 After a period of continuous growth, cell characteristics can change and may
become quite different from those found in the starting population. Cells can also
adapt to different culture environments (e.g. different nutrients, temperatures, salt
concentrations etc.) by varying the activities of their enzymes.
 It necessitates expertise for handling and to check chemical contamination,
microbial contamination and cross contamination
 Require a control environment in the workplace, for incubation, pH control
containment and disposal of biohazards
 Quantity and cost involvement is more in capital equipment, consumables,
medium,serum, plastics which is ten times more costly than using animal itself.
 Genetic instability like heterogeneity and variability may appear. It is a major
problem with many continuous cell lines resulting from their unstable aneuploid
chromosomal constitution. Heterogeneity in growth rate and capacity to
differentiate within the population can produce variability from one passage to
another.
 Phenotypic instability: sometimes the phenotypic characteristics of the tissue may
getlost which is due to dedifferentiation (a process assumed to be the reversal of
differentiation) also due to overgrowth of undifferentiated cells. It also maybe due
to adaptation.
 Identification of the cell type: If the differentiated properties are lost, it is difficult
torelate the cultured cell with the functional cell in the tissue from where the tissue
were derived. For this stable markers are required.

35. ADVANCES IN CELL CULTURE :
• Three-dimensional (3D) cell cultures have been widely used in biomedical research since the
early decades of this century.
• Holtfreter and later Moscona pioneered the field by their research on morphogenesis using
spherical re-aggregated cultures of embryonic or malignant cells.
• One major advantage of 3D cell cultures is their well-defined geometry-whether planar or
spherical-which makes it possible to directly relate structure to function, and which enables
theoretical analyses, for example of diffusion fields.
• Combining such approaches with molecular analysis has demonstrated that, in comparison to
conventional cultures, cells in 3D culture more closely resemble the in vivo situation with
regard to cell shape and cellular environment, and that shape and environment can determine
gene expression and the bio-logical behaviour of the cells.
• One impressive example is the ectopic implant-ation of embryonic cells, which can result in
malignant transformation, whereas the same cells undergo normal embryogenesis in the
uterus.
• Conversely, terato-carcinoma cells may undergo normal development when implanted into an
embryo . One further example is the relative resistance of cancer cells to drugs in 3D culture
compared to the same cells grown as conventional mono-layer or in single cell suspension .
• In the last 4 decades cell culture has matured from being merely a research tool into being one
of the foundations of the biopharmaceutical industry, and its use is continuing to expand
rapidly.
• In vitro models are replacing animals in many tests and assays; Its enormous potential in the
fields of stem cell and regenerative medicine has hardly started tobe realized; and its utility in
research grows ever faster.

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