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Applications and potential uses of fish gill cell lines:
examples with RTgill-W1
L. E. J. Lee &V. R. Dayeh &K. Schirmer &N. C. Bols
Received: 15 September 2008 / Accepted: 22 December 2008 / Editor: J. Denry Sato
#The Society for In Vitro Biology 2009
Abstract Gills are unique structures involved in respiration
and osmoregulation in piscinids as well as in many aquatic
invertebrates. The availability of the trout-derived gill cell
line, RTgill-W1, is beginning to make impacts in fish health
and toxicology. These cells are available from the American
Type Culture Collection as ATCC CRL 2523. The cells
have an epithelioid morphology and form tight monolayer
sheets that can be used for testing epithelial resistance. The
cells can be grown in regular tissue culture surfaces or in
transwell membranes in direct contact with water on their
apical surfaces. The ability of RTgill-W1 to withstand hypo-
and hyper-osmotic conditions and their optimal growth
capacity at room temperature, make these cells ideal sentinel
models for in vitro aquatic toxicology as well as model
systems to study fish gill function and gill diseases. RTgill-
W1 support growth of paramyxoviruses and orthomyxovi-
ruses like salmon anemia virus. RTgill-W1 also support
growth of Neoparamoeba pemaquidensis, the causative
agent of amoebic gill disease. The cells have been used to
understand mechanisms of toxicity, ranking the potencies of
toxicants, and evaluating the toxicity of environmental
samples. These cells are also valuable for high throughput
toxicogenomic and toxicoproteomic studies which are
easier to achieve with cell lines than with whole organisms.
RTgill-W1 cell line could become a valuable complement
to whole animal studies and in some cases as gill replace-
ments in aquatic toxicology.
Keywords Aquatic ecotoxicology .Cytotoxicity .
Fish cell culture .Gill cell line .Rainbow trout .RTgill-W1
Introduction
In vitro models have been invaluable in many areas of life
sciences. These experimental systems allow direct access
and evaluation of specific functions with higher control of
the conditions of assays, reducing variability of responses
due to unavoidable stress responses. Ready access to
functional cells without the constraints of non-target tissues,
provides the possibility for easy studies of cellular
mechanisms. However, special considerations must be
addressed to establish stable in vitro function. Primary
cultures are usually short-lived and require specific culture
conditions. Microbial contamination is more common and
difficulties in obtaining adequate tissue amounts, have
prompted interest in developing permanent cell lines which
can provide a much more convenient source of cells.
Nevertheless, depending on the question being asked, primary
cultures (cells freshly derived from organ of interest) or cell
lines (cells that have been passaged many times in vitro)
have been used in various aspects of scientific research.
The gills of aquatic organisms are unique organs
involved in gas exchange, osmoregulation, and other
critical functions (Evans et al. 2005) essential for survival
of piscinid and invertebrate species. Thus, damage to gills
may reflect in impaired functioning of the organism and
eventual death. Studies of gill function have traditionally
In Vitro Cell.Dev.Biol.—Animal
DOI 10.1007/s11626-008-9173-2
L. E. J. Lee (*)
Department of Biology, Wilfrid Laurier University,
Waterloo, Ontario, Canada N2L 3C5
e-mail: llee@wlu.ca
V. R. Dayeh :N. C. Bols
Department of Biology, University of Waterloo,
Waterloo, Ontario, Canada N2J 3G1
K. Schirmer
Eawag–Swiss Federal Institute of Aquatic
Science and Technology,
Dübendorf, Switzerland
been performed in vivo in experimental animals. However,
recent advancements in gill cell culture provide an alternate
model system to study the gill (Fernandes et al. 1995;
Wood and Part 1997; Sandbacka et al. 1999; Fletcher et al.
2000; Wood et al. 2002; Leguen et al. 2007). In these
systems, branchial cells are grown on solid or permeable
insert supports. Within permeable inserts, gill membranes
can be established where experimental manipulations of the
apical and basolateral sides can be made once confluent cell
layers are formed. This approach allows the activities of
ions to be experimentally set on both sides of an epithelium
and electrophysiological parameters such as transepithelial
resistance and transepithelial potential to be monitored.
Reconstructed branchial epithelia withstand prolonged
apical exposure to freshwater and show many physiological
and morphological characteristics similar to those of the
gill epithelium in vivo (Fletcher et al. 2000; Wood et al.
2002; Leguen et al. 2007). However, inherent drawbacks of
primary cultures: labor intensive, short life span, and not
always easy to obtain in a reproducible and quantitative
manner have limited their usage.
The availability of fish cell lines, since the 1960s, has
begun to make impacts in scientific research, but at a much
slower rate than with mammalian cell lines. Early work
with fish cell lines was initiated with RTG-2, a gonadal cell
line derived from rainbow trout (Wolf and Quimby 1962),
mainly for virological studies. In the almost 50 yr since
then, fish cell lines have grown in number covering a wide
variety of species and tissues of origin and an array of
applications. Fish immunology (Clem et al. 1996; Bols
et al. 2001), toxicology (Babich and Borenfreund 1991;
Segner 1998), ecotoxicology (Fent 2001; Castano et al.
2003; Schirmer 2006), endocrinology (Bols and Lee 1991),
virology (Wolf 1988), biomedical research (Hightower and
Renfrow 1988), disease control (Villena 2003), biotechnol-
ogy and aquaculture (Bols 1991), and radiation biology
(Ryan et al. 2008) are some of the areas in which fish cell
lines have made significant contributions.
Many fish cell lines have been derived from dissociated
adult or embryonic tissues (Fryer and Lannan 1994) but
few have been characterized for tissue of origin with
appropriate markers (Bols and Lee 1994). Inasmuch as fish
comprise more than half of all vertebrate species together, it
is surprising that so few cell lines have been established
from piscinid species in comparison to mammals. To date,
of the more than 3,400 cell lines deposited at the American
Type Culture Collection (ATCC), only 31 cell lines could
be found that are of fish origin (see Table 1). Astounding
as well, is the fact that although greater than 70% of the
earth mass is covered with water and that aquatic
organisms prevail with a distinct organ such as the gills,
very few cell lines have been initiated from such a unique
organ, although a lot of research has been done at the
organismal level. Research with cells derived from the
gills of fish, a conspicuous organ involved in gas exchange
and ionoregulation, have been steady, yet, most of the work
involves use of primary cultures despite the reported
difficulties for their isolation, maintenance, and reproduc-
ibility (Wood et al. 2002; Leguen et al. 2007). The
reluctance to use cell lines stems from researcher’s
misconception that cell lines are mostly derived from
transformed cells and that differentiated characteristics of
the tissues of origin are not maintained (Sato 2008). This
may be the case for many mammalian cell lines, but most
cell lines derived from fish tissues have been from normal
tissues with a few exceptions, most notably EPC and
RTH-149 cells which were derived respectively from an
epithelioma and a hepatoma. Fryer and Lannan (1994)
noted that 14 out of 159 fish cell lines reported up to 1994
were initiated from tumorigenic tissues, which is less than
10%. Furthermore, from the 31 fish cell lines listed at
ATCC, only three were derived from tumorigenic tissues.
This contrasts with mammalian cell lines where over
50% of listed cell lines at the ATCC were derived from
cancerous tissues or transformed cells.
Of the 12 gill cell lines reported to date (Table 2), none
have been described that were initiated from neoplastic or
cancerous tissues and only the FG-9307 gill cell line has
been reported to undergo spontaneous neoplastic transfor-
mation in vitro (Guo et al. 2003). Nevertheless, literature on
the use of fish gill cell lines have been scarce and most
work performed to date involved two gill cell lines: RTgill-
W1 derived from gill explants of adult rainbow trout
(Oncorhynchus mykiss) (Bols et al. 1994), is representative
of gills from freshwater species; whereas, FG-9307, derived
from flounder (Paralichthys olivaceus) (Tong et al. 1997),
is representative of gills from marine fish. In this review,
we provide examples of applications and potential uses
mainly for the rainbow trout gill cell line RTgill-W1, since
RTgill-W1 is readily available from the American Type
Culture Collection as ATCC number CRL 2523, unlike FG-
9307, which is only available from Dr. Zhang, QingDao,
China.
RTgill-W1: Origins, Cell Culture Conditions, and Growth
Characteristics
RTgill-W1 is an epithelial cell line derived from the gill
explants of normal adult rainbow trout (Oncorhynchus
mykiss) (Bols et al. 1994). These cells have been authen-
ticated as derived from rainbow trout by microsatellite
analysis (Perry et al. 2001). The cells are routinely cultured
at room temperature in 75 cm
2
tissue culture flasks with
10 ml of Leibovitz-15 media (L15) with added fetal bovine
serum at 10% (v/v), 100 U/ml penicillin and 100 µg/ml
LEE ET AL.
streptomycin. Conditions for their routine growth, mainte-
nance, and use in toxicity assays was reported by Dayeh et
al. (2005a). The cells exhibit epithelial morphology (Fig. 1)
and are believed to have derived from undifferentiated
precursor gill stem cells. Given appropriate conditions,
mucus-secreting goblet-like cells and cells with abundant
mitochondria could be selected (Fig. 2). Additionally, these
cells form tight epithelia and withstand exposure to fresh-
Table 2. Fish gill cell lines reported in the literature up to September 2008
Cell line ATCC no. Morphology Species of origin Reference
G1B CRL-2536 Pleomorphic Walking catfish Clarias batrachus Noga and Hartmann 1981
G1 NA Epithelial Common carp Ciprinus carpio Chen and Kou 1988
BG/G NA Fibroblastic Bluegill sunfish Lepomis macrochirus Borenfreund et al. 1989
CCG NA Epithelioid Color carp Ciprinus carpio Ku and Chen 1992
ZG NA ND Zebrafish Danio rerio Collodi et al. 1992
RTgill-W1 CRL-2523 Epithelial Rainbow trout Oncorhynchus mikiss Bols et al. 1994
MG-3 NA Fibroblastic Mrigal Cirrhinus mrigala Sathe et al. 1995
RG-1 NA Fibroblastic Rohu Labeo rohita Sathe et al. 1997
FG-9307 NA Epithelioid Flounder Paralichthys olivaceus Tong et al. 1997
RGE-2 NA Epithelial Atlantic salmon Salmo salar Butler and Nowak 2004
RGF NA Fibroblastic Atlantic salmon Salmo salar Butler and Nowak 2004
CF-4 NA ND Zebrafish Danio rerio Hogstrand et al. 2007
NA not available, ND not described.
Table 1. Fish cell lines deposited at the American type culture collection as of September 2008
Fish species Designation ATCC no. Cell type/morphology Tissue source
Carassius auratus goldfish CAR CCL-71 Fibroblast Normal fin
Clarias batrachus walking catfish G1B CRL-2536 Pleomorphic Gill
Clupea pallasi Pacific herring PHL CRL-2750 Epithelial Larvae
Cyprinus carpio carp EPC CRL-2872 Epithelial Epithelioma papullosum
Danio rerio zebrafish ZF4 CRL-2050 Fibroblast Embryo fibroblasts
Danio rerio ZEM2S CRL-2147 Fibroblast Embryo fibroblasts
Danio rerio SJD.1 CRL-2296 Fibroblast Caudal fin
Danio rerio AB.9 CRL-2298 Fibroblast Caudal fin
Danio rerio ZFL CRL-2643 Parenchymal Normal liver
Fugu rubripes Tora fugu Fugu eye CRL-2641 Epithelial Eye
Fugu niphobles Kusa fugu Fugu fry CRL-2642 Fibroblast Fry
Haemulon sciurus blue striped grunt GF CCL-58
a
Fibroblast Fin
Ictalurus nebulosus brown bullhead BB CCL-59 Fibroblast CT and muscle
Ictalurus punctatus channel catfish 1G8 CRL-2756 Lymphoblast Blood cells
Ictalurus punctatus 3B11 CRL-2757 Lymphoblast Blood cells
Ictalurus punctatus 28S.3 CRL-2758 T lymphoblast Blood cells
Ictalurus punctatus 42TA CRL-2759 Macrophages Blood cells
Ictalurus punctatus G14D CRL-2760 T lymphocytes Blood cells
Ictalurus punctatus CCO CRL-2772 Fibroblast Ovary
Lepomis macrochirus bluegill BF-2 CCL-91 Fibroblast Caudal trunk
Morone chrysops white bass WBE CRL-2773 Epithelial Embryo
Oncorhynchus keta chum salmon CHH-1 CRL-1680 Fibroblast Heart
Oncorhynchus mykiss rainbow trout RTH-149 CRL-1710 Epithelial Hepatoma
Oncorhynchus mykiss RTG-2 CCL-55 Fibroblast Fry testis and ovary
Oncorhynchus mykiss RTG-P1 CRL-2829 Fibroblast Gonad
Oncorhynchus mykiss SOB-15 CRL-2301 Epithelial Liver
Oncorhynchus mykiss RTgill-W1 CRL-2523 Epithelial Gill
Oncorhynchus tshawytscha chinook salmon CHSE-214 CRL-1681 Mixed Embryo
Pimephales promelas fathead minnow FHM CCL-42 Epithelial CT and muscle
Poeciliopsis lucida topminnow PLHC-1 CRL-2406 Hepatocyte Hepatocellular cacinoma
Salmo salar Atlantic salmon ASK CRL-2747 Epithelial Kidney
a
This cell line is no longer available from ATCC.
FISH GILL CELL LINES
or saltwater when grown in transwell membrane chambers
(Fig. 3). Sandbichler and Pelster (2005) noted that RTgill-
W1, like primary trout gill epithelial cells, expressed proteins
involved in osmotic stress response when these cells were
grown in transwells and were exposed to freshwater on the
apical chambers. RTgill-W1 cells are quite tolerant of hypo-
and hyper-osmotic conditions and could be grown for
prolonged periods in direct exposure with media of varying
salinities (Fig. 4). RTgill-W1 are also quite sensitive to cor-
tisol exposure which inhibits cell proliferation and changes
cellular morphology (Fig. 5) (Lee, unpublished data).
Gill cell lines in basic research. Most physiological work
performed to date involved the use of either perfused gills,
primary gill cultures, or whole organisms. Little use of the gill
cell lines for basic research have been performed to date.
However, Ebner et al. (2007) recently reported using RTgill-
W1 to study activation pattern and subcellular distribution of
ERK, a mitogen-activated protein kinase; and Krumschnabel
et al. (2007) evaluated RTgill-W1 cells for their apoptotic
mechanisms, evaluating activation of effector caspases,
nuclear condensation, mitochondrial membrane potential,
and overall apoptotic volume decrease (cell shrinkage). These
authors reported that the RTgill-W1 cells behaved similarly to
mammalian cells albeit some differences were noted.
Gill cell lines in fish health research. Fish gill membranes
provide a thin entry route to pathogens. Mechanisms of
pathogen entry into gill epithelia is poorly understood and
although cell lines have been commonly used with mamma-
lian pathogens for mechanisms of host–pathogen relation-
ships, comparatively little usage of gill cell lines have been
made for the study of pathogens, except for viruses.
Figure 1. Morphology of RTgill-W1. Phase contrast micrograph of
RTgill-W1 monolayer at passage 86. Arrow indicates mitotic figure.
Bar= 100 µm.
Figure 2. Gill cell types.
(A) TEM of rainbow trout gill
lamella with common cells
found at base of lamella:
cchloride cell, pv pavement
cells, ggoblet cell, uundifferen-
tiated cell, ppillar cell, rbc red
blood cell. (B) TEM of RTgill-
W1 monolayer with proliferating
cell shown in telophase. (C)
TEM of RTgill-W1 monolayer
with goblet-like cell with coa-
lescing vesicles. (D) Fluores-
cence micrograph of RTgill-W1
monolayer stained with Rhoda-
mine 123 depicting mitochondria
rich cells. (E) Light micrograph
of RTgill-W1 monolayer stained
with periodic acid Schiff (PAS)
to demonstrate mucopolisacchar-
ide accumulation (arrows) in
goblet-like cells.
LEE ET AL.
Several diseases have been reported to affect the gills of
fish, among these, viruses have been quite problematic.
Viruses, as the ultimate intracellular pathogens, require host
cells, and cell lines have been essential in mammalian
virology. RTgill-W1 has been shown to support the growth
of a novel paramyxovirus isolated from the gills of disease
seawater-reared Atlantic salmon (Kvellestad et al. 2003).
The complete genome sequence of the virus, dubbed
Atlantic salmon paramyxovirus or ASPV, was recently
made possible by RTgill-W1’s ability to support their
growth (Nylund et al. 2008). Characterization of infectious
salmon anemia virus or ISAV, an orthomyxo-like virus
Figure 4. Morphology of RTgill-W1 under varying osmotic conditions. Phase contrast micrographs at ×40. Bar =100 µm.
Figure 3. Morphology of
RTgill-W1 grown in cell culture
inserts for 4 wk with weekly
changes of upper and lower
chambers. A,CFreshwater in
upper chamber. B,DSeawater in
apical chamber. Lower chambers
contained normal L-15 culture
media. Mag=×40 for A,Band
×100 for C,D.
FISH GILL CELL LINES
(Falk et al. 1997), was also made possible by their ability to
grow inside RTgill-W1 cells.
While viruses are the ultimate parasites using host cell’s
mechanisms for reproduction, the study of obligate intra-
cellular bacterial and/or protozoan parasites could also be
facilitated with the aid of cell lines. For instance, RTgill-
W1 cell line could be useful for studies of gill infecting
microsporidia such as Loma salmonae (Kent and Speare
2005), the causative agent for microsporidial gill disease of
salmonids which is among the most significant infectious
diseases affecting aquaculture-raised chinook salmon in
Canada (Speare et al. 2007). The usefulness of fish cell
lines to study microsporidia is addressed in a separate
manuscript by Monaghan et al. (this issue).
The study of ectopic parasites infecting gills could also be
investigated using gill cell lines. Noga (1987) used the G1B
cell line to study Amyloodinium ocellatum, a dinoflagellate
commonly found in fish gills and evaluated the effectiveness
of an antiprotozoal drug in vitro. The RGE-2 cell line from
Atlantic salmon gills (Butler and Nowak 2004), was devel-
oped to facilitate study of amoebic gill disease caused by
Neoparamoeba species. Lee et al. (2006) demonstrated rapid
growth and high yield of a lab strain of Neoparamoeba
pemaquidensis using RTgill-W1 and also demonstrated the
specificity of the amoeba for a gill cell line over nine other
fish cell lines derived from other tissues. Thus, gill cell lines
could be very useful for studying organ-specific pathogens.
Gill cell lines in toxicology research. The gills of aquatic
organisms are the primary target and uptake sites of water
contaminants (Evans 1987). As such, gills are exquisite
organs for the study of aquatic toxicant effects. However,
gills in vivo are difficult to evaluate or manipulate, thus, gill
cells in vitro could represent ideal systems for the study of
aquatic contaminants. While primary gill cultures have
been used for the evaluation of aquatic toxicants (Lilius et
al. 1995; Pärt 1995; Sandbacka et al. 1999; Wood et al.
2002), the difficulty of their isolation, maintenance, and
reproducibility makes them cumbersome to use. Permanent
cell lines, on the other hand, are easy to maintain and
manipulate and produce highly reproducible results. Thus,
the use of fish cell lines in toxicology and ecotoxicology
has been relatively broad and several reviews have been
published on the topic: Segner (1998); Fent (2001); Castano
et al. (2003); Bols et al. (2005); and Schirmer (2006).
Gill cell lines provide an opportunity to study both
cytotoxicity and biotransformation of chemicals at the
branchial level in much more detail than is possible in
vivo. Both, FG-9307 and RTgill-W1 have been used in
toxicity testing of various chemicals. For instance, the
toxicity of organophosphorous pesticides were evaluated
with FG-9307 cells (Li and Zhang 2001;2002), while
RTgill-W1 have been used to evaluate the toxicity of
industrial effluents (Dayeh et al. 2002), including petroleum
refinery effluents (Schirmer et al. 2001), and have also been
used to evaluate the toxicity of polycyclic aromatic hydro-
carbons (Schirmer et al. 1998a,b,1999) and metals (Dayeh
et al. 2005b) including Cu, Cd, Zn, Fe, and Ni. Most
recently, Bopp et al. (2008) used RTgill-W1 cells to further
evaluate toxicity of copper and hypothesized that copper-
induced loss in viability and genotoxicity in trout gills may
partially be triggered by the generation of reactive oxygen
species. Similarly, the toxicity of polibrominated diphenyl
ethers (PBDEs) when tested with RTgill-W1 (Shao et al.
2008) as well as with the trout liver cell line RTL-W1,
suggest that these compounds mediate cell injury through a
mechanism that may involve oxidative stress.
The biggest advantage of using RTgill-W1 though, is
that environmental samples can be directly evaluated on
these cells. Whole water samples can be evaluated on
RTgill-W1 by direct exposure without extraction or concen-
tration steps. The environmental water samples could be
added to gill cells for whole effluent testing (Lee et al. 2008).
For instance, industrial effluents (Dayeh et al. 2002) or oil-
sands process affected waters (Lee et al. 2008), were mixed
with L-15 media components and the toxicity of these
samples were evaluated using RTgill-W1 cells. The
evaluations were done in blind studies without knowledge
of sample content, yet the toxicity of the samples correlated
with their in vivo toxicity to whole fish (Dayeh et al. 2002)
Figure 5. Phase contrast micro-
graphs of RTgill-W1 grown for
21 d in the absence (A) or
presence of cortisol at 100 ng/ml
(B). Mag= ×40. Bar = 100 µm.
LEE ET AL.
or to potentially toxic chemical content such as naphthenic
acid and salinity content (Lee et al. 2008).
Studies with cultured cells permit the determination of
molecular and cellular mechanisms through which patho-
gens cause disease or pollutants lead to toxic effects in
organisms at sublethal and chronic levels. Gill cell lines are
proving useful for evaluating the effects of aquatic samples,
pathogens, drugs, and toxicants on cellular functions. The
availability of the permanent cell line RTgill-W1 is
therefore beginning to make major impacts in fish research.
Acknowledgements This study was funded by a Long Range
Initiative grant from CEFIC (European Chemical Industry Council)
and DEFRA-UK (project: CEllSens-Eco8). Continuing funding from
the Natural Sciences and Engineering Research Council of Canada
(NSERC) and from the Canadian Water Network to LEJL and NCB
are gratefully acknowledged. LEJL also thanks CEMA, the Cumulative
Environmental Management Association (Canada) for recent funding
on the use of fish cell lines to evaluate oil-sands process affected waters
and Mount Desert Island Biological Laboratory (MDIBL) for a New
Investigator Award allowing further work with RTgill-W1 and amoeba
interactions.
References
Babich, H.; Borenfreund, E. Cytotoxicity and genotoxicity assays with
cultured fish cells: a review. Toxicol. In Vitro. 5: 91–100; 1991.
doi:10.1016/0887-2333(91)90052-F.
Bols, N. C. Biotechnology and aquaculture: the role of cell cultures.
Biotechnol. Adv. 9: 31–49; 1991. doi:10.1016/0734-9750(91)
90403-I.
Bols, N. C.; Barlian, A.; Chirino-Trejo, M.; Caldwell, S. J.; Goegan, P.;
Lee, L. E. J. Development of a cell line from primary cultures of
rainbow trout, Oncorhynchus mykiss Walbaum, gills. J. Fish Dis.
17: 601–611; 1994. doi:10.1111/j.1365-2761.1994.tb00258.x.
Bols, N. C.; Brubacher, J. L.; Ganassin, R. C.; Lee, L. E. J.
Ecotoxicology and innate immunity in fish. Dev. Comp. Immunol.
25: 853–873; 2001. doi:10.1016/S0145-305X(01)00040-4.
Bols, N.C.; Dayeh, V. R.; Lee, L. E. J.; Schirmer, K. Use of fish cell
lines in the toxicology and ecotoxicology of fish. In: Moon, T.W.;
Mommsen, T. P. (eds) Biochem. Molec. Biol. Fishes. Vol. 6:
Environmnetal Toxicology. Amsterdam. Elsevier Science. 43–84;
2005.
Bols, N. C.; Lee, L. E. J. Technology and uses of cell cultures from
the tissues and organs of bony fish. Cytotechnol. 6: 163–187;
1991. doi:10.1007/BF00624756.
Bols, N. C.; Lee, L. E. J. Cell lines: availability, propagation and
isolation. In: Hochachka, P. W.; Mommsen, T. P. (eds) Biochem.
Molec. Biol. Fishes. Vol. 3: Analytical Techniques. Amsterdam.
Elsevier Science. 145–159; 1994.
Bopp, S. K.; Abicht, H. K.; Knauer, K. Copper-induced oxidative
stress in rainbow trout gill cells. Aquat. Toxicol. 86(2): 197–204;
2008. doi:10.1016/j.aquatox.2007.10.014.
Borenfreund, E.; Babich, H.; Martin-Alguacil, N. Effect of methyl-
azoxymethanol acetate on bluegill sunfish cell cultures in vitro.
Ecotoxicol. Environ. Saf. 17: 297–307; 1989. doi:10.1016/0147-
6513(89)90050-X.
Butler, R.; Nowak, B. F. A dual enzyme method for the establishment
of long- and medium-term primary cultures of epithelial and
fibroblastic cells from Atlantic salmon gills. J. Fish Biol. 65:
1108–1125; 2004. doi:10.1111/j.0022-1112.2004.00521.x.
Castano, A.; Bols, N. C.; Braunbeck, T.; Dierickx, P.; Halder, M.; Isomaa,
B.; Kawahara, K.; Lee, L. E. J.; Mothersill, C.; Part, P.; Repetto, G.;
Riego Sintes, J.; Rufli, H.; Smith, R.; Wood, C.; Segner, H. The use
of fish cells in ecotoxicology. ATLA. 31: 317–351; 2003.
Chen, S. N.; Kou, G. H. Establishment, characterization and
application of 14 cell lines from warm-water fish. In: Kuroda, Y.;
Kurstak, E.; Maramorosch, K. (eds) Invertebrate and fish tissue
culture. Tokyo: Japan Sci. Soc. 1988: 218–227.
Clem, L. W.; Bly, J. E.; Wilson, M.; Chinchar, V. G.; Stuge, T.;
Barker, K.; Luft, C.; Rycyzyn, M.; Hogan, R. J.; van Lopik, T.;
Miller, N. W. Fish immunology: the utility of immortalized
lymphoid cells—a mini review. Vet. Immunol. Immunopathol. 54:
137–144; 1996. doi:10.1016/S0165-2427(96)05682-6.
Collodi, P.; Kamei, Y.; Ernst, T.; Miranda, C.; Buhler, D. R.; Barnes,
D. W. Culture of cells from zebrafish (Brachydanio rerio)
embryo and adult tissues. Cell Biol. Toxicol. 8: 43–61; 1992.
doi:10.1007/BF00119294.
Dayeh, V. R.; Lynn, D. H.; Bols, N. C. Cytotoxicity of metals
common in mining effluent to rainbow trout cell lines and to the
ciliated protozoan, Tetrahymena thermophila.Toxicol. In Vitro.
19: 399–410; 2005b. doi:10.1016/j.tiv.2004.12.001.
Dayeh, V. R.; Schirmer, K.; Bols, N. C. Applying whole-water
samples directly to fish cell cultures in order to evaluate the
toxicity of industrial effluents. Water Res. 36: 3727–3738; 2002.
doi:10.1016/S0043-1354(02)00078-7.
Dayeh, V. R.; Schirmer, K.; Lee, L. E. J.; Bols, N. C. Evaluating the
Toxicity of Water Samples with the Rainbow Trout Gill Cell Line
Microplate Cytotoxicity Test, In: Small-Scale Freshwater Toxic-
ity Investigations. Vol I. Ed. C. Blaise and JF Ferard. Springer.
473–504; 2005a.
Ebner, H. L.; Blatzer, M.; Nawaz, M.; Krumschnabel, G. Activation and
nuclear translocation of ERK in response to ligand-dependent and -
independent stimuli in liver and gill cells from rainbow trout. J. Exp.
Biol. 210: 1036–1045; 2007. doi:10.1242/jeb.02719.
Evans, D. H. The fish gill: site of action and model for toxic effect of
environmental pollutants. Environ. Health Perspect. 71: 54–58;
1987. doi:10.2307/3430412.
Evans, D. H.; Piermarini, P. M.; Choe, K. P. The multifunctional fish
gill: dominant site of gas exchange, osmoregulation, acid-base
regulation, and excretion of nitrogenous waste. Physiol. Rev. 85:
97–177; 2005. doi:10.1152/physrev.00050.2003.
Falk, K.; Namork, E.; Rimstad, E.; Mjaaland, S.; Dannevig, B. H.
Characterization of infectious salmon anemia virus, an orthomyxo-
like virus isolated from Atlantic salmon (Salmo salar L.). J. Virol.
71(12): 9016–9023; 1997.
Fernandes, M. N.; Eddy, F. B.; Penrice, W. S. Primary cell culture
from gill explants of rainbow trout. J. Fish Biol. 47: 641–651;
1995. doi:10.1111/j.1095-8649.1995.tb01931.x.
Fent, K. Fish cell lines as versatile tools in ecotoxicology: assessment
of cytotoxicity, cytochrome P4501A induction potential and
estrogenic activity of chemicals and environmental samples. Toxicol.
In Vitro. 15: 477–488; 2001. doi:10.1016/S0887-2333(01)00053-4.
Fletcher, M.; Kelly, S. P.; Pärt, P.; O’Donnell, M. J.; Wood, C. M.
Transport properties of cultured branchial epithelia from fresh-
water rainbow trout: a novel preparation with mitochondria-rich
cells. J. Exp. Biol. 203: 1523–1537; 2000.
Fryer, J. L.; Lannan, C. N. Three decades of fish cell culture: a current
listing of cell lines derived from fishes. J. Tiss. Cult. Meth. 16:
87–94; 1994. doi:10.1007/BF01404816.
Guo, H. R.; Zhang, S. C.; Li, H. Y. Spontaneous neoplastic transformation
of the gill cell line FG-9307 from the olive flounder Paralichthys
olivaceus.North Am. J. Aquacult. 65: 44–48; 2003. doi:10.1577/
1548-8454(2003)065<0044:SNTOTG>2.0.CO;2.
Hightower, L. E.; Renfro, J. L. Recent applications of fish cell culture
to biomedical research. J. Exp. Zool. 248(3): 290–302; 1988.
doi:10.1002/jez.1402480307.
FISH GILL CELL LINES
Hogstrand, C.; Feeney, G.; Walker, P.; Zheng, D.; Kille, P. Tran-
scriptomic analysis of metal responses in cultured fish gill cells.
Comp. Biochem. Physiol. A: Molec. Integ. Physiol. 146(4),
Suppl. 1: S7; 2007. doi:10.1016/j.cbpa.2007.01.072.
Kent, M. L.; Speare, D. J. Review ofthe sequential development of Loma
salmonae (Microsporidia) based on experimental infections of
rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O.
tshawytscha). Folia Parasitol. (Praha). 52(1–2): 63–68; 2005.
Krumschnabel, G.; Maehr, T.; Nawaz, M.; Schwarzbaum, P. J.;
Manzl, C. Staurosporine-induced cell death in salmonid cells: the
role of apoptotic volume decrease, ion fluxes and MAP kinase
signaling. Apoptosis 12(10): 1755–1768; 2007. doi:10.1007/
s10495-007-0103-7.
Ku, C. C.; Chen, S. N. Characterization of three cell lines derived
from color carp, Cyprinus carpio.J. Tiss. Cult. Meth. 14: 63–72;
1992. doi:10.1007/BF01404746.
Kvellestad, A.; Dannevig, B. H.; Falk, K. Isolation and partial
characterization of a novel paramyxovirus from the gills of
diseased seawater-reared Atlantic salmon (Salmo salar L).
J. Gen. Virol. 84(8): 2179–2189; 2003. doi:10.1099/vir.0.18962-0.
Lee, L. E.; Van Es, S. J.; Walsh, S. K.; Rainnie, D. J.; Donay, N.;
Summerfield, R.; Cawthorn, R. J. High yield and rapid growth of
Neoparamoeba pemaquidensis in co-culture with a rainbow trout
gill-derived cell line RTgill-W1. J. Fish Dis. 29: 467–480; 2006.
doi:10.1111/j.1365-2761.2006.00740.x.
Lee, L. E. J.; Dayeh, V.; Schirmer, K.; Bols, N. C. Fish cell lines as
rapid and inexpensive screening and supplemental tools for whole
effluent testing. Integr. Environ. Assess. Manag. 4: 372–374;
2008. doi:10.1897/1551-3793(2008)4[372:FCLARA]2.0.CO;2.
Leguen, I.; Cauty, C.; Odjo, N.; Corlu, A.; Prunet, P. Trout gill cells in
primary culture on solid and permeable supports. Comp. Biochem.
Physiol. A. 148: 903–912; 2007. doi:10.1016/j.cbpa.2007.09.007.
Li, H.; Zhang, S. In vitro cytotoxicity of the organophosphorus
pesticide parathion to FG-9307 cells. Toxicol. In Vitro. 15(6):
643–647; 2001. doi:10.1016/S0887-2333(01)00090-X.
Li, H.; Zhang, S. In vitro cytotoxicity of the organophosphorus
insecticide methylparathion to FG-9307, the gill cell line of
flounder (Paralichthys olivaceus). Cell Biol. Toxicol. 18(4): 235–
241; 2002. doi:10.1023/A:1016050911012.
Lilius, H.; Sandbacka, M.; Isomaa, B. The use of freshly isolated gill
epithelial-cells in toxicity testing. Toxicol. In Vitro. 9: 299–305;
1995. doi:10.1016/0887-2333(95)00010-6.
Noga, E. J. Propagation in cell culture of the dinoflagellate
Amyloodinium, an ectoparasite of marine fishes. Science. 236
(4806): 1302–1304; 1987. doi:10.1126/science.236.4806.1302.
Noga, E. J.; Hartmann, J. X. Establishment of walking catfish (Clarias
batrachus) cell lines and development of a channel catfish
(IctaIurus punctatus) virus vaccine. Can. J. Fish. Aquat. Sci. 38:
925–930; 1981. doi:10.1139/f81-125.
Nylund, S.; Karlsen, M.; Nylund, A. The complete genome sequence
of the Atlantic salmon paramyxovirus (ASPV). Virology 373:
137–148; 2008. doi:10.1016/j.virol.2007.11.017.
Pärt, P. Primary cultures of epithelial cells from rainbow trout gills as
a tool in toxicological research. Mar. Environ. Res. 39: 369–370;
1995. doi:10.1016/0141-1136(95)98426-D.
Perry, G. M. L.; McDonald, G. J.; Ferguson, M. M.; Ganassin, R. C.;
Bols, N. C. Characterization of rainbow trout cell lines using
microsatellite DNA profiling. Cytotechnol. 37: 143–151; 2001.
doi:10.1023/A:1020516804173.
Ryan, L. A.; Seymour, C. B.; O’Neill-Mehlenbacher, A.; Mothersill,
C. E. Radiation-induced adaptive response in fish cell lines.
J. Environ. Radioact. 99(4): 739–747; 2008. doi:10.1016/j.
jenvrad.2007.10.001.
Sandbacka, M.; Pärt, P.; Isomaa, B. Gill epithelial cells as tools for
toxicity screening—comparison between primary cultures, cells
in suspension and epithelia on filters. Aquat. Tox. 46: 23–32;
1999. doi:10.1016/S0166-445X(98)00109-X.
Sandbichler, A. M.; Pelster B. Osmolarity-induced changes in trout
gill epithelial cells on permeable supports. Comp. Biochem.
Physiol. A: Molec. Integ. Physiol. 141(3): S197–S198; 2005.
Sathe, P. S.; Basu, A.; Mourya, D. T.; Marathe, B. A.; Gogate, S. S.;
Banerjee, K. A cell line from the gill tissues of Indian cyprinoid
Labeo rohita.In Vitro Cell. Dev. Biol. Anim. 33(6): 425–427;
1997. doi:10.1007/s11626-997-0059-5.
Sathe, P. S.; Mourya, D. T.; Basu, A.; Gogate, S. S.; Banerjee, K.
Establishment and characterization of a new fish cell line, MG-3,
from gills of mrigal, Cirrhinus mrigala.Indian J. Exp. Biol. 33:
589–594; 1995.
Sato, G. Tissue cultures: the unrealized potential. Cytotechnol. 57:
111–114; 2008. doi:10.1007/s10616-007-9109-9.
Schirmer, K. Proposal to improve vertebrate cell cultures to establish
them as substitutes for the regulatory testing of chemicals and
effluents using fish. Toxicology 224: 163–183; 2006. doi:10.1016/
j.tox.2006.04.042.
Schirmer, K.; Chan, A. G. J.; Greenberg, B. M.; Dixon, D. G.; Bols,
N. C. Ability of 16 priority PAHs to be photocytotoxic to a cell
line from the rainbow trout gill. Toxicology 127: 143–155;
1998b. doi:10.1016/S0300-483X(98)00031-6.
Schirmer, K.; Dixon, D. G.; Greenberg, B. M.; Bols, N. C. Ability of
16 priority PAHs to be directly cytotoxic to a cell line from the
rainbow trout gill. Toxicology 127: 129–141; 1998a. doi:10.1016/
S0300-483X(98)00030-4.
Schirmer, K.; Herbrick, J. A. S.; Greenberg, B. M.; Dixon, D. G.;
Bols, N. C. Use of fish gill cells in culture to evaluate the
cytotoxicity and photocytotoxicity of intact and photomodified
creosote. Envi ron. Toxicol. Chem. 18: 1277–1288; 1999.
doi:10.1897/1551-5028(1999)018<1277:UOFGCI>2.3.CO;2.
Schirmer, K.; Tom, D. J.; Bols, N. C.; Sherry, J. P. Ability of
fractionated petroleum refinery effluent to elicit cyto- and
photocytotoxic responses and to induce 7-ethoxyresorufin-O-
deethylase activity in fish cell lines. Sci. Total Environ. 271(1–3):
61–78; 2001. doi:10.1016/S0048-9697(00)00831-7.
Segner, H. Fish cell lines as a tool in aquatic toxicology. EXS. 86: 1–
38; 1998.
Shao, J.; Eckert, M. L.; Lee, L. E. J.; Gallagher, E. P. Comparative
oxygen radical formation and toxicity of BDE 47 in rainbow
trout cell lines. Mar. Environ. Res. 66: 7–8; 2008. doi:10.1016/j.
marenvres.2008.02.007.
Speare, D. J.; Markham, R. J.; Guselle, N. J. Development of an
effective whole-spore vaccine to protect against microsporidial
gill disease in rainbow trout (Oncorhynchus mykiss) by using a
low-virulence strain of Loma salmonae. Clin. Vaccine Immunol.
14(12): 1652–1654; 2007. doi:10.1128/CVI.00365-07.
Tong, S. L.; Li, H.; Miao, H. Z. The establishment and partial
characterization of a continuous fish cell line FG-9307 from the
gill of flounder Paralichthys olivaceus.Aquacult. 156: 327–333;
1997. doi:10.1016/S0044-8486(97)00070-7.
Villena, A. J. Applications and needs of fish and shellfish cell culture
for disease control in aquaculture. Rev. Fish Biol. Fish. 13: 111–
140; 2003. doi:10.1023/A:1026304212673.
Wolf, K. Fish viruses and fish viral diseases. Cornell University Press,
New York1988.
Wolf, K.; Quimby, M. C. Established eurythermic line of fish cells in vitro.
Science 135: 1065–1066; 1962. doi:10.1126/science.135.3508.1065.
Wood, C. M.; Kelly, S. P.; Zhou, B.; Fletcher, M.; O’Donnell, M.;
Eletti, B.; Pärt, P. Cultured gill epithelia as models for the
freshwater fish gill. Biochim. Biophys. Acta. 1566: 72–83; 2002.
doi:10.1016/S0005-2736(02)00595-3.
Wood, C. M.; Pärt, P. Cultured branchial epithelia from freshwater fish
gills. J. Exp. Biol. 200: 1047–1059; 1997.
LEE ET AL.
