Tissue and Cell 45 (2013) 159–174

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Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

Cytological, immunocytochemical, ultrastructural and growth characterization of

the rainbow trout liver cell line RTL-W1
F. Malhão a,b , R. Urbatzka b , J.M. Navas c , C. Cruzeiro a,b , R.A.F. Monteiro a,b , E. Rocha a,b,∗
a
Laboratory of Histology and Embryology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto,
Portugal
b
Laboratory of Cellular, Molecular and Analytical Studies, Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associated Laboratory (CIMAR LA),
University of Porto (U. Porto), Rua dos Bragas, 289, 4050-123 Porto, Portugal
c
Endocrine Disrupters and Toxicity of Contaminants (DETC), Department of Environment, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), A6 Km7,
E-28040 Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Despite its wide use in toxicology, a detailed characterization of RTL-W1 cell line lagged behind leaving
Received 24 May 2012 ambiguities about its cell origin. We aimed to better characterize the line regarding cell phenotype and
Received in revised form 25 October 2012 tumorigenic state. We studied RTL-W1 cells in monolayers and in (4–22-week-old) aggregates consid-
Accepted 27 October 2012
ering: (a) morphology (light and electron microscopy); (b) immunophenotype using AE1/AE3, vimentin,
Available online 7 January 2013
Cam5.2, CK7 and CK19 and e-cadherin antibodies and (c) growth behavior. RTL-W1 organelle content is
constituted basically by mitochondria and abundant free ribosomes, with no (cytochemically) detectable
Keywords:
peroxisomes and lysosomes. Immunocytochemistry showed a strong marking for AE1/AE3 and vimentin
RTL-W1
Cell line
(in a cell subset). Since AE1/AE3 stained biliary epithelial ducts in trout liver, and considering the mor-
Microscopy phological characteristics and long term culture, RTL-W1 cells seem more similar to bile preductular
Immunocytochemistry epithelial cells (considered as stem cells in teleost liver). Also, we observed abnormal nuclear features
Growth described for both malignant cell lines and stem cells, so we could not conclude about tumorigenicity. Cell
aggregates had signs of hepatocytic differentiation, such as the development of RER and microvillus-like
projections into intercellular spaces. The morphological resemblance to the original tissue suggests that
aggregates could have an added value in metabolic as well as in cell-to-cell interaction studies.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction cell lines in ecotoxicology we recommend the workshop report by

The importance of studying the effects of pollutants in aquatic
organisms is a main issue for the environment but also regarding
the potential impacts on human health. In this vein, the use of fish
cell culture have gained relevance in the disclosure of the mech-
anisms involved in the toxicity processes, as well as in ranking
toxicants and developing biomarkers of exposure (Bols, 2005). The
advantages of working with cell cultures are related with the reduc-
tion in costs, animal use, space and toxic waste, together with a
higher reproducibility and a fast results (Fent, 2001; Schnell et al.,
2009b). Indeed, cell lines are easy to be manipulated and stan-
dardized in experiments (Thibaut and Porte, 2008). Moreover, their
use is demanded by European legislation to enhance the “Princi-
ple of the Three Rs”. For detailed information about the use of fish
∗ Corresponding author at: Laboratory of Histology and Embryology, Institute of
Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U. Porto), Rua de Jorge
Viterbo Ferreira no. 228, 4050-313 Porto, Portugal.
E-mail address: erocha@icbas.up.pt (E. Rocha).
Castano et al. (2003).
Besides having a broad range of applications and the significant
number of scientific works using fish cell lines, data are still scarce
and frequently not very precise or detailed when compared to those
of mammals. For instance in the cell repository ATCC (American
Type Culture Collection) of almost 3400 cell lines available only
31 are fish cell lines (Lee et al., 2009). The same is true for ECACC
(European Collection of Cell Cultures) in which only 21 of 40,000
correspond to fish cell lines. However, the number of reported
fish cell lines is much higher, and it was estimated that there are
nearly 283 cell lines (Lakra et al., 2011). Most fish cell lines lack a
detailed characterization, the cell origin and the passage numbers
are seldom referred in the literature. All put together, can lead to
misinterpretations and even to erroneous conclusions.
Although fish cell lines have some similarities in physiology and
culture protocols with mammalian cell lines (Lakra et al., 2011),
they possess some particular characteristics that differ significantly
to mammalian lines (Segner, 1998); nevertheless, the criteria used
for both cell lines classification are the same.
The vast majority of fish cell lines are epithelial or fibroblast-
like and derived from freshwater fish, namely from salmonids and

0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tice.2012.10.006
160 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174

cyprinids (Castano, 2005). Most fish cell lines are continuous, and
there are only a few examples of finite cell lines (Bols and Lee,
1991). Interestingly, most fish cell lines derived from normal tis-
sues, only about 10% of all fish cell lines came from neoplastic
tissues, contrary to what happens in mammals where about 50%
came from neoplastic or transformed cells (Lakra et al., 2011). In
fish, the process of becoming continuous or immortalized seems
to happen spontaneously (Bols, 2005), and this has been described
in cultures of a variety of organs (Clem et al., 1996; Marques et al.,
2007). Fish seem to be the vertebrate group in which cell lines arise
with higher frequency (Nicholson et al., 1987). In the case of rain-
bow trout, spontaneous immortalization can be partially explained
by the high levels of telomerase expression in their tissues (Klapper
et al., 1998). Another interesting point about fish cell lines is that
they do not seem to be prone to senesce; e.g., the cell line CHSE-214
was reported to have been passaged 385 times, in 20 years, without
signs of senescence (Lannan et al., 1984).
RTL-W1 (rainbow trout liver-Waterloo 1) is an epithelial cell line
reported in 1993 (Lee et al., 1993). It was originated from a primary
culture of a normal liver taken from a 4-year-old male rainbow
trout. According to the original descriptions, from the initial pri-
mary culture some cells continued to grow slowly until confluence
was reached (5 months later). At that time, cells were firstly sub-
cultured using a common trypsinization procedure. However, only
after 14 months in culture, the number of cells were enough to test
the presence of mycoplasm and to cryopreserve (Lee et al., 1993).
For 8 years, cells were passaged around one hundred times and
the time between passages started to decrease, reaching a period
of 7–10 days. RTL-W1 probably acquired spontaneous immortality
but it was not considered neoplastic as it did not form colonies in
agar (Lee et al., 1993). This cell line has always been considered
(to this date) as a normal liver cell line no matter which passage
number was used for experiments.
Since RTL-W1 was reported, more than 50 scientific works have
been published using this line for different aims (See Table 1).
RTL-W1 has been mostly used for evaluating the induction of
cytochrome P4501A (CYP1A) and the dependent enzyme activity
7-ethoxy resorufin-O-deethylase (EROD) caused by toxicant expo-
sure (Babín and Tarazona, 2005; Brack et al., 2002; Fent, 2001;
Navas and Segner, 2001; Schirmer et al., 2000). This cell line was
also used to evaluate the toxicity of natural compounds such as afla-
toxin (Bechtel and Lee, 1994). After the exposure to this mycotoxin,
the authors reported to have obtained sublines of RTL-W1 with
neoplastic behaviors including: morphologic changes, increased
proliferation, lost of growth inhibition and growth in soft agar.
The genotoxicity of some compounds was tested with RTL-W1
cells, using viability tests and evaluating DNA damage by comet
assay (Boettcher et al., 2010; Klee et al., 2004; Kosmehl et al., 2008;
Nehls and Segner, 2001). The RTL-W1 cell line was also tested for
the presence of estrogen receptors and inducible production of
vitellogenin, but none of them were found under the tested con-
ditions (Fent, 2001).
To the above contextualization it is important to add that liver
epithelial cell lines have been numerous times developed from nor-
mal rat liver using proteolytic enzymes in minced pieces of tissue,
but the cellular origin of those cell lines is commonly unknown (Lee
et al., 1993). In fish, the development of liver cell lines follows the
same tendency. Apart from the lower degree of responses observed
in liver cell lines when compared to primary cultures, they can still
exhibit different patterns of metabolism according to the cell type
in culture. Indeed, it is easy to accept that depending on the cell
origin, the results obtained can differ.
This study aims to offer more detailed information about
RTL-W1 cell line, in terms of morphology, at light and elec-
tron microscopy, the origin of the cell type and the state of
tumorigenicity. Additionally, to help disclosing the cellular nature
and behavior of RTL-W1 we described some aspects never stud-
ied before, namely: (i) the immunophenotypic profile by using
a panel of antibodies specific for different liver epithelial cell
types; and (ii) the spontaneous formation of three dimensional
(3D) arrangements with morphological features of hepatocytic
differentiation.

Table 1
Main applications of RTL-W1 cell line.

Reference Information Reference Information

(Lee et al., 1993)
(Bechtel and Lee, 1994)
(Bols et al., 1994)
(Clemons, 1996)
(Clemons et al., 1998)
(Whyte et al., 1998)
(Bols et al., 1999)
(Schirmer et al., 2000)
(Whyte et al., 2000)
(Behrens et al., 2001)
(Fent, 2001)
(Nehls and Segner, 2001)
(Perry et al., 2001)
(Billiard et al., 2002)
(Brack et al., 2002)
(Teneva et al., 2003)
(Dayeh et al., 2004)
(Gustavsson et al., 2004)
(Rastall et al., 2004)
(Schirmer et al., 2004)
(Babín et al., 2005)
Characterization and EROD activity (Bols, 2005) Ranking dioxin-like compouds
Effects of aflotoxin (Brack et al., 2005) EROD activity
Study glutamine requirement for in vitro (Dayeh et al., 2005) Toxicity of metals
growth
EROD activity (Wagg and Lee, 2005) Identification of fish cell lines using a
proteomic approach
EROD activity (Bopp et al., 2006) EROD activity
EROD activity (Embry et al., 2006) Study p53 protein and apoptosis induction
EROD activity (Olsman et al., 2007) EROD activity
EROD activity (Kosmehl et al., 2008) DNA damage using comet assay
EROD activity (Alonso et al., 2008) EROD activity
EROD activity (Schnell et al., 2009b) Test effect of ibuprofen in viability and
proliferation
Presence of Estrogen receptor Beta, induction (Seitz et al., 2008) DNA damage using comet assay
of expression of vtg with estradiol exposure
analyzed by RT PCR
Detection DNA damage using comet assay (Schnell et al., 2009a) Test toxicity of pharmaceuticals and personal
care products
DNA profiling (Thibaut et al., 2009) EROD activity
EROD activity (Boettcher et al., 2010) DNA damage using comet and micronucleus
assay
EROD activity (Kawano et al., 2010) Expression of major histocompatibility genes
complex genes
Study toxicity of a cyanobacterium (Kramer et al., 2010) EROD activity
Study toxicity of Triton X-100 with fluorescent (Fischer et al., 2011) Effect of mRNA expression and protein activity
dyes indicators of cell viability in ABC efflux transporters
EROD activity (Boettcher et al., 2011) Toxicity of trenbolone
EROD activity (Cofalla et al., 2012) EROD activity
EROD activity (Kienzler et al., 2012) EROD activity
EROD activity (Wetterauer et al., 2012) Toxicity of dioxins
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174
2. Materials and methods
2.1. RTL-W1 cultivation
Unless otherwise stated, all reagents and chemicals were pur-
chased from Sigma–Aldrich. All experiments reported in this
work were performed with cells between passages 100 and 120.
Cells were cultivated in 25 cm3 flasks (Orange Scientific, Belgium)
with gas exchange and in Leibovitz’s (L15) culture medium, sup-
plemented with 5% of FBS, 1% penicillin (10,000 U/ml) and 1%
streptomycin (10 mg ml−1 ), at 19 ◦ C, without CO2 atmosphere.
Cells were subcultured using 0.05% trypsin with 0.02% ethyl-
ene diaminetetraacetic acid (EDTA) for detachment. Medium was
replaced twice a week, until the flasks reached confluence to be
passaged again (7–10 days interval). In every passage, cells were
counted and the viability was assessed using 0.1% of trypan blue
exclusion dye in PBS. Viability was always higher than 95%.
2.2. Contamination detection
In relation to detection of bacteria and fungi, cells were allowed
to grow without antibiotics for one week. Mycoplasma detection
was performed by polymerase chain reaction using the primers
MSGO (5 TGC ACC ATC TGC CAC TCT GTT AAC CTC 3 ) and GPO1
(5 ACT CCT ACG GGA GGC AGC AGT 3 ) that allow the amplifica-
tion of a fragment of 717 bp, corresponding to a common region of
mycoplasma DNA. The amplification products were analyzed in an
electrophoresis 1% agarose gel.
2.3. RTL-W1 morphology
2.3.1. Light microscopy
The information about cell morphology was obtained first
by phase contrast microscopy with an Olympus CKX41 inverted
microscope. Then, for preparation of cytospins, cells were
trypsinized and centrifuged in a Sartorius 2–16 K centrifuge (160 g
for 5 min at 4 ◦ C). After removing the supernatant, the cell pellet
was resuspended in culture media. About 100 ␮l of the cell suspen-
sion were centrifuged in a StatSpin CytoFuge 2 (188 g for 6 min at
RT). Just for morphological comparative purpose we also prepared
cytospins of a human ductal breast epithelial tumor cell line (T47D)
purchased from Sigma–Aldrich, USA the passage number used was
7.
The obtained slides were allowed to dry at room temperature
for 1 h. Afterwards, it was performed a Romanovsky-type stain,
using methanol as fixative (1 min), followed by Hemacolour solu-
tions 2 and 3 (Merck, Germany) (2 and 3 min respectively). After
drying, slides were mounted with DPX mounting media, observed
with an Olympus BX50 light microscope and photographed with
an Olympus Camedia C-5050 digital camera.
2.3.2. Transmission electron microscopy
Cell pellets were obtained as described for cytospin procedure
and were immediately fixed in 2.5% glutaraldehyde in sodium
cacodylate-chloridric acid buffer 0.1 M, pH 7.2, for 1 h at 4 ◦ C; then
twice washed in the same buffer, 10 min each. Post-fixation was
performed in 1% osmium tetroxide in sodium cacodylate-chloridric
acid buffer 0.1 M, pH 7.2, for 2 h at 4 ◦ C. Following the routine
cell processing for electron microscopy, cells were dehydrated in
increasing series of ethanol (from 50% to absolute), two changes of
propylene oxide (15 min each) and mixtures of propylene oxide and
epoxy resin (3:1; 1:1; 1:3, in this order, for 1 h each). Final embed-
ding was made in the same resin. After 2 days of polymerization in
the oven at 60 ◦ C, the blocks were trimmed and cut with a Diatome
diamond knife, in an ultramicrotome Leica Reichert Supernova. One
␮m thick semithin sections were stained with a mixture 1:1 of 1%
161
methylene blue and 1% azur II. Ninety nm thick ultrathin sections
were placed into 200 mesh hexagonal copper grids and contrasted
with uranyl acetate and lead citrate. Sections were observed and
photographed with an electron microscope JEOL 100CXII.
2.3.3. Cytochemical detection of enzyme activities
In order to identify lysosomes and peroxisomes, and to adapt
procedures used in our laboratories for fish liver and other orga-
nisms and organs (Lobo-da-Cunha, 2002; Rocha et al., 1999), we
made cytochemical detections of acid phosphatase and catalase,
respectively. Cell pellets were fixed in 1.5% or in 2.5% glutaralde-
hyde, in 0.1 M sodium cacodylate-chloridric acid buffer pH 7.2, for
1 h at 4 ◦ C, for acid phosphatase and catalase detection, respec-
tively. Afterwards, two washings were performed with the same
buffer (30 min each at 4 ◦ C). From this point on, each pellet fol-
lowed the specific processing/incubation protocol for the desired
enzyme. Trout liver tissue was included as positive control for both
techniques.
2.3.3.1. Acid phosphatase. Before incubation, cells were rinsed with
sodium acetate-chloridric acid buffer 0.1 M pH 5.0. The material
was incubated for 30 min at 37 ◦ C in the same buffer with 13.9 mM
sodium ␤-glycerophosphate and 3.6 mM lead nitrate, under dark
condition with constant and gentle mixing. In order to stop the
reaction, the medium was replaced by other with 10 mM sodium
fluoride, at room temperature for 10 min. For washing, two rinses
were made in acetate buffer and one in sodium cadodylate buffer
(15 min each).
2.3.3.2. Catalase. Catalase – (as optimized by (Rocha et al., 1999)).
Prior to incubation cells were washed with 0.1 M tris-chloridric
acid buffer pH 8.5. The cell pellet was incubated for 2 h at 37 ◦ C
in a medium containing 0.12% hydrogen peroxide and 2 mg ml−1
of diaminobenzidine (DAB) in 0.1 M tris-chloridric acid buffer pH
8.5, under dark condition and with constant and gentle mixing.
Then pieces were rinsed twice, 15 min each, firstly in tris-chloridric
buffer and secondly in sodium cacodylate buffer.
Post-fixation was made in 1% osmium tetroxide buffered in
0.1 M sodium cacodylate buffer, pH 7.2, with 1.5% potassium ferro-
cyanide, for 2 h at 4 ◦ C. The sequent routine processing was done
routinely, as above detailed for transmission electron microscopy.
2.4. Immunocytochemistry
Cell pellets were obtained by centrifugation as described pre-
viously. Then they were fixed in buffered 10% formaldehyde for
24 h, and routinely processed for paraffin embedding. Sections with
5 ␮m were cut with a Leica 2155 microtome, then mounted on
3-amino propyltriethoxysilane treated slides, and left overnight
in the oven at 37 ◦ C. Slides were deparaffinized and rehydrated.
Prior to incubation with the primary antibodies, antigen retrieval
was performed as well as blocking of endogenous peroxidase by
immersion in 3% hydrogen peroxide in methanol, for 10 min. The
panel of antibodies was chosen according to their current use in
liver cell identification and cohesion (see details in Table 2). The
kits used as visualization systems were used according to man-
ufacture’s instructions. The chromogenic reaction was developed
with DAB and cell counterstaining with hematoxylin. Slides were
mounted with DPX synthetic mounting media, observed with an
Olympus BX50 light microscope and photographed with an Olym-
pus Camedia C-5050 digital camera.
All antibodies were tested simultaneously in the cell line pellet,
and in three controls: (a) a negative control, where the primary
antibody was omitted; (b) a set of human or canine tissues indicated
162 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174

Table 2
Antibodies tested for RTL-W1 characterization.

Antibody Supplier Host (anti-human) Dilution Incubation time Antigen retrieval Visualization system Marker

AE1/AE3 Zymed Mouse 1:600 ovn (4 ◦ C) citrate buffer pH 6.0 Novolink Max Polymer, Epithelial cells but not
Novocastra hepatocytes
CK7 Cell Markers Mouse 1:200 1 h (rt) PT link Trilogy®(Dako) Novolink Max Polymer, Biliary epithelial cells
Novocastra
CK19 Neomarkers Mouse 1:100 1 h (rt) PT link Trilogy®(Dako) Novolink Max Polymer, Biliary epithelial cells
Novocastra
Cam5.2 Becton Dickinson Mouse 1:5 1 h (rt) citrate buffer pH 6.0 LSAB Labvision Hepatocytes
Vimentin Dako Mouse 1:500 ovn (4 ◦ C) citrate buffer pH 6.0 Novolink Max Polymer, Mesenchymal cells
Novocastra
e-cadherin Dako Mouse 1:1000 ovn (4 ◦ C) citrate buffer pH 6.0 Novolink Max Polymer, Cell adhesion molecule
Novocastra

ovn: overnight; rt: room temperature.

as positive controls for the antibodies; and (c) a normal liver of with an Olympus SZX10 stereo microscope, fitted with a DP21 dig-
rainbow trout, to check the species specificity. ital camera.

2.5. Growth curve of the cell line RTL-W1

3. Results

In order to check the growth kinetics of RTL-W1, cells were

3.1. Contamination detection
plated at a density of 60,000 cells/well, 500 ␮l/well, in 24-well
plates (PAA). Medium was changed every two days. Cells were
In the culture flasks that were allowed to grow without antibi-
trypsinized and counted in a Neubauer chamber every 24 h, until 20
otics, no contamination was observed. Also, the PCR test to
days in culture. For each day of sampling, three wells were counted.
investigate mycoplasm presence was negative.

2.6. Growth in agar

RTL-W1 cells were cloned in 0.3% (w/v) agar (Merck) in the same
culture medium used for cultivation, forming at room temperature
a semi-solid medium. Different cell concentrations were tested:
100, 1000, 6000 and 20,000 cells/well. This assay was performed in
a 24-well plate (PAA) with 500 ␮l of medium per well, with trip-
licates for each cell concentration. Medium was replaced once a
week. Cells were allowed to grow for eight weeks. Cultures were
regularly observed and pictures were taken with an Olympus IX-
71 inverted microscopy equipped with an Olympus DP71 digital
camera.
2.7. Growth without serum
For studying cell growth without serum, the cells were seeded as
for the growth curve, but the objective was only to check whether
cells were able to survive without serum.
2.8. Long term culture
Cells were plated at the same density used for the growth curve
(60,000 cells/well), 500 ␮l of medium per well, in 24-well plates
(PAA), and were allowed to grow in culture without any passage
(even after reaching confluence). Medium was replaced twice a
week and the cells were observed regularly by phase contrast
microscopy up to the formation of three dimensional aggregates.
The aggregates were harvested in different times in culture and
their morphology studied by both light and electron microscopy.
Immunocytochemistry was also performed with the same panel of
antibodies and protocols used for the monolayer cultures.
Disaggregation of cells for cytospins was made by a common
trypsinization procedure, with a pipette with gentle up and down
suction. However, for histology, immunocytochemistry and elec-
tron microscopy, cell aggregates were maintained intact and the
protocols were similar to those used for cell pellets obtained from
monolayers.
Pictures were taken with an Olympus IX-71 inverted micro-
scope, equipped with an Olympus DP71 digital camera, and also
3.2. RTLW-1 morphology
3.2.1. Phase contrast microscopy
The morphological study of the RTL-W1 cells by phase con-
trast microscopy revealed cells that were predominantly polygonal
(Fig. 1a–c). Mitotic figures were frequently observed. Based on cell
size, two clearly different cell types were observed: one small (S
type) and other much bigger, made of cells with a larger and paler
cytoplasm (B type). In the latter type, it was common the presence
of multinucleated cells with prominent nucleoli. In cells seeded at
low density we also noted fibroblast-like cells (F type) (Fig. 1d).
This cell type number was in descending tendency as confluence
was reached. The morphology and behavior of RTL-W1 was sys-
tematically monitored and revealed very stable within this study.
3.2.2. Cytospins
The above mentioned findings were confirmed when the cells
were observed in cytospin slides (Figs. 2 and 3). One cell type was
relatively small (likely S type), with a high nucleus/cytoplasm ratio,
hyperbasophilic scanty cytoplasm, and round to oval nucleus. In
this cell type, anisocytosis and anisokaryosis were rarely seen. The
other cell type was comparatively larger (probable B type), but
in much lower number, having a lower nucleus/cytoplasm ratio
and, in general, a slightly less basophilic cytoplasm. Some cells
presented a transitional morphology (Tr type) between the two
already mentioned cell types. This intermediate morphology was
not clearly observed in phase contrast microscopy.
In the B cell subtype, it was frequently observed high anisocy-
tosis and anisokaryosis with nuclei varying from round and oval, to
reniform and irregular shapes. Multinucleated cells were regularly
present, and even within the same cell, anisokaryosis was evident.
Nuclei revealed prominent and sometimes multiple nucleoli. Chro-
matin pattern varied between euchromatic, reticular and coarse
(Fig. 3).
Some atypical features were encountered in the RTL-W1 cell
line. These were basically related to nuclear alterations: atypical
mitotic figures with an uneven distribution of chromosomes (tripo-
lar and tetrapolar) (Fig. 4a and b); cells with multiple fragments
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174 163

Fig. 1. Phase contrast morphology of RTL-W1 in culture – pictures a, b and c correspond to 4–6 days after passaging while d corresponds to the day after passaging. (a) and
(b) Mitotic figures (arrows). (b) and (c) Presence of cells with different sizes: the smaller (S) and the bigger ones (B). B cell type appears as polygonal multinucleated cells (c)
and (d) showing that the two different sizes of cells were present in different cell densities. In lower density (d) it was also observed a type of fibroblast-like cells (F) with
spindle shape.

Fig. 2. General morphology of RTL-W1 in cytospins – pictures correspond to cell pellet 4–6 days after passaging. (a) General aspect of the RTL-W1 cell line: two different
types of cells (S) and (B) and also cells with transitional phenotypes (Tr). (b) Type B multinucleated cell. Compare the difference in size and staining affinity between B and S
type.

Fig. 3. Nuclear detail of B cell type – pictures corresponds to cell pellet 4–6 days after passaging. (a) B cell type with four nuclei. It is noticeable the anisokaryosis and nuclear
molding. (b) B cell with irregular flower shape nucleus and coarse chromatin.
164 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174

Fig. 4. RTL-W1 nuclear features – pictures correspond to cell suspension of 4–6 days after passaging. (a) Tripolar and (b) tetrapolar mitotic figures. (c), (d) and (e) Display
cells with multiple nuclear fragments (circles), quite diverse in size and aspect. (f) Arrows points micronuclei.

of nucleus (Fig. 4c–e) and micronuclei were present in many cells
(Fig. 4f).
After an overall comparison between the RTL-W1 cell line
and a human breast cancer epithelial cell line (T47D) and taking
into account the nuclear morphology, it was possible to realize
that atypical morphological features such as those present in a
malignant epithelial cell line (T47D) (abnormal mitotic features,
cells with multiple fragmented nuclei, multinucleated cells with
anisokaryosis, and micronuclei) were found in the presumed “nor-
mal” liver cell line (RTL-W1). However, it is worth to mention that
in the T47D cell line, the nucleoli were much more numerous and
also remarkably more prominent (compare Fig. 4 and Fig. 5).
3.2.3. Transmission electron microscopy
The ultrastructure of RTL-W1 revealed two main types of cells,
probably corresponding to the S and B types described before
(Fig. 6). Like in cytospin observation, some cells exhibited an inter-
mediate morphology.
Cell type B had a large amount of cytoplasm but it was scarce
in organelles basically constituted by: free ribosomes, rare pro-
files of rough endoplasmic reticulum (RER), a few mitochondria
and, finally, filaments were distributed along the cytoplasm and
sometimes were arranged in bundles (Fig. 7).
The S cell type was smaller than the B variety, but richer in
organelles: abundant free ribosomes and mitochondria and some
RER profiles. A very characteristic feature was the presence of an
abundant number of structures constituted by coiled filaments
forming agglomerates, named herein as filamentous bodies (Fig. 8).
In S type cells the filaments also appeared distributed along the
cytoplasm, but they were not so easily seen as in B cells.
3.2.4. Cytochemical detection of enzyme activities
In relation to cytochemistry for detection of catalase and
acid phosphatase activities, both techniques showed negative
results, this resulted in a lack of identification of peroxisomes and
lysosomes, respectively. In trout liver tissue positive controls per-
oxisomes and lysosomes were identified.
3.3. Immunocytochemistry
In immunocytochemistry, none of the negative controls
revealed unspecific reaction. On the opposite, positive controls
showed the expected tagging. Positive reactions in both the cell line
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174 165

Fig. 5. Cytospins of human ductal breast epithelial tumor cell line (T47D) – pictures correspond to cell suspension of 4–6 days after passaging. (a) Note that most cells display
several prominent nucleoli. Additionally, the biggest cell (center–right) is multinucleated, with anisokaryosis, and shows also micronuclei (arrows). The cell at center–left
shows a fragmented nucleus. (b) Cell with a very fragmented nuclei.

3.4. Growth curve

The cell line growth curve pattern did not reveal a distinct lag
phase. Cell proliferation started immediately after subculturing.
The growth followed an exponential trend (R2 = 0.99). Within the
analyzed time period of 20 days, the cell line did not show signs of
a stationary phase (Fig. 10).

3.5. Growth in agar

After 1 week in culture, it was possible to notice a small group

of cells (around ten cells) in one of the wells of the seeding den-
sity 20,000 cells/well. With time, this group started to grow and
other groups appeared in cells cultured at other densities, except
Fig. 6. Two main cell types in RTL-W1 seen as seen in transmission electron
microscopy – picture corresponds to cell pellet 4–6 days after passaging. Image
illustrating the difference in the amount of organelles between the S and B cell
types (rich versus poor content). Nucleus (Nu).

and the rainbow trout reference liver tissue were detected only for
AE1/AE3. Strikingly, the strong positivity for AE1/AE3 was observed
for all cells in the line, but only cells from biliary passages in the nor-
mal trout liver resulted in positive staining for this antibody (Fig. 9).
Immunodetection with anti-vimentin antibody resulted in a strong
positive reaction only in a subset of the RTL-W1 cells, but not in
the normal trout liver tissue. Antibodies anti-e-cadherin resulted in
positive marking in sections of trout liver (hepatocytes and biliary
cells) but not in the cell line. All the other antibodies were negative
in both the cell line and the normal rainbow trout reference liver
tissue.

Fig. 7. B type cell – picture corresponds to cell pellet 4–6 days after passaging. The
poor organelle content embedded in a pale hyaloplasm is well patent in this image.
Filaments (Fil), mitochondria (Mi), nucleus (Nu), nucleolus (Ncl), Rough endoplasmic
reticulum (RER).
Fig. 8. S type cell with rich organelle content – pictures correspond to cell pellet
4–6 days after passaging. (a) General aspect of S cell type, in which the organelle
content is basically constituted by abundant mitochondria, filamentous bodies and
free ribossomes. Arrows point to filamentous bodies. (b) High magnification of fila-
mentous bodies. Note the convoluted arrangement of the filaments, much like in a
wool ball. Filamentous bodies (Fb), mitochondria (Mi), nucleus (Nu).
166 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174

Fig. 9. Immunocytochemistry data from RTL-W1 line and normal trout liver – pictures (a) and (e) correspond to cell pellet of cells 4–6 days after passaging. (a) Positive
staining for AE1/AE3 in the cell line pellet. (b) In the normal trout liver all biliary cells had positivity for this antibody. Smaller branches of the biliary tree (pre-ductules)
among hepatocytes. (c) biliary duct in normal trout liver. (d) e-cadherin positive marking on trout liver, i.e., intercellularly. Images (e) and (f) show vimentin positivity in the
cell line and no signal in trout liver, respectively.

Fig. 10. Growth curve of the RTL-W1 line. The values plotted in the final growth curve correspond to the average of the three replicas.
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174
Fig. 11. RTL-W1 growth in agar. (a) After 5 weeks it was possible to observe some small focuses of cell growth, which within 8 weeks progressed to cell aggregates (b).
for 100 cells/well density. At 5 weeks-time, in the well in which the
first group appeared, it was possible to observe monolayer areas in
which the first signs of cell aggregates arose (Fig. 11a). By the time
of 8 weeks, some cell aggregates had emerged from the mono-
layer and formed 3D aggregates (Fig. 11b), which reached up to
400–500 ␮m in diameter.
3.6. Growth without serum
Until 8 days in culture, cells seemed to grow similarly to those
cultivated with 5% of FBS in culture medium. After that initial
period, cells started to detach from the culture flask and within
twenty days in culture all cells had died.
3.7. Long term culture
In the wells where cells were left in culture without being pas-
saged, some multilayer growth foci were observed. This occurred
even before confluence was reached (Fig. 12a). These foci devel-
oped, and within 4 weeks in culture 3D cell aggregates attached
to the bottom of the flask were detected (Fig. 12b and c). In the
periphery of these aggregates a large number of spindle-like cells
appeared spread in all directions, as they were trying to make con-
tact with cells from neighboring aggregates (Fig. 12c). These started
to establish contact with each other by bridges of cells (Fig. 12d).
After 8 weeks in culture, it was noticed an increase in the number
of aggregates, as well as in the size of the first appearing aggregates.
Afterwards, it was observed a kind of nest formed by the cell aggre-
gates linked by bridges of cells. A monolayer of cells continued to
exist underneath the aggregates (Fig. 12e and f).
As the weeks went by, the aggregates merged (Fig. 13a) and
concomitantly the monolayer underneath gradually disappeared.
The aggregates remained attached to the culture flask for at least
22 weeks. From this time on we stopped registering the events in
the context of this work.
3.7.1. Aggregates morphology
The dimension of the aggregates assumed particular impor-
tance. In bigger aggregates, especially in those having more than
500 ␮m in diameter, a necrotic/apoptotic core started to spread
to the periphery of the aggregates. The suggested working time-
frame is 9–14 weeks. After that time, the cells from the center of
the aggregates started to show too many degenerative aspects.
The inspection of cytospin slides revealed a pool of cells with
very similar morphology, the presence of the two types of cells was
not that evident as previously described in monolayer cultures. The
vast majority of cells were morphologically identical to what was
described early as S type cells. The aggregates were still prolifer-
ating, as mitotic figures were often seen. Nevertheless, apoptotic
167
and/or necrotic cells (not frequent in monolayer cytospins), were
seen very frequently (probably corresponding to the necrotic core
of aggregates) (Fig. 14).
3.7.2. Aggregates immunophenotype
The immunocytochemistry pattern obtained was very similar to
that observed in monolayers: cells were positive only for AE1/AE3
and a subset of cells were positive for vimentin (Fig. 15).
3.7.3. Electron microscopy
Cells in long term culture aggregates were more homogeneous
in their ultrastructure when compared to cells found in short-term
monolayer cultures. Most cells from aggregates had a euchro-
matic round nucleus, with one or two nucleoli. The cytoplasmic
organelles were more developed in relation to the cells seen in
monolayers. One striking aspect was the number and arrangement
of RER cisterns, which appeared arranged in parallel rows. At the
same time, we continued to observe free ribosomes and polyribo-
somes, but in lower number (Fig. 16). This organelle configuration
was in contrast to what we found in the monolayer cells, where only
rare profiles of RER existed. Other relevant aspect was the absence
of the filamentous bodies found in abundance when cells were held
in monolayer. It was noticeable the accumulation of lamellar bodies
and the appearance of dense bodies of diverse sizes.
When observing the cell borders in the aggregates, there were
features revealing cell cohesion-adherence, such as desmosomes
(in large numbers), tight junctions and membrane interdigitations
(Fig. 17a). Another vital finding was the formation of canalicular-
like structures in intercellular spaces within the aggregates, with
apical cell blebs and even slender projections (often closely resem-
bling typical microvilli) projecting toward the core/lumen (Fig. 17b,
Fig. 18).
4. Discussion
When the RTL-W1 cell line was reported by Lee et al. (1993),
the type of cell from which the line emerged was not clarified. The
authors tried to figure out the exact cell origin presenting argu-
ments for and against possible liver cell type sources (epithelial
versus stromal), but no final conclusion was reached. Since then,
scientific papers have been published using this cell line, but, sur-
prisingly, there has not been further reports neither about the cell
line origin nor about the state of tumorigenicity. Additional infor-
mation about these both issues underlies the main objective of this
article. Such information can better support the use of this cell line
for future experiments and also for better contextualizing some
results already obtained.
168 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174

Fig. 12. RTL-W1 behavior with long term in culture. (a) Focus of cell proliferation. (b) Cells forming three dimensional aggregates that co-existed with a monolayer of cells
attached to the bottom of culture flask. (c) Neighboring cell aggregates contacting with each other. (d) Cellular bridges connecting neighboring aggregates. Images (e) and (f)
show aggregates with reinforced cellular bridges. The difference of colors among pictures is because some cells were in PBS and others were in culture medium (reddish).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

4.1. Cell origin and morphology
The simplest and most direct technique for identifying cells
is the observation of their morphology (Freshney, 2005). When
observed in phase contrast microscopy, the RTL-W1 has typical
epitheliod morphology, as earlier described by Lee et al. (1993).
The epithelial character is revealed by the attachment to the culture
vessels and was confirmed by the presence of junctional complexes.
Moreover, the formation of some projections and blebs into the
space among cells in the cell aggregates was described in this work.

Fig. 13. Merging aggregates of RTL-W1. (a) First aggregates establish bridges which become progressively reinforced and within 16 weeks it was possible to observe the
aggregates merging (circles). (b) In some wells the number of aggregates was scarce while the bridges of cells were prominent (arrows).
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174 169

Fig. 14. RTL-W1 aggregates cytospins – cells aggregates dispersed for cytospins with 8–14 weeks in culture. (a) Mitotic figure (arrow). (b) Typical apoptotic cell (arrow).

These features resemble plasma membrane specializations of liver
epithelial cells (McIntyre, 2007; Rocha et al., 1994). Lee et al. (1993)
had already referred some microvillus-like projections in the sur-
face of a few cells; but this feature became much more evident in
the 3D arrangement.
The primary epithelial cells in liver parenchyma are the hepa-
tocytes and biliary epithelial cells (BECs) (Nishikawa et al., 1996;
Rocha et al., 2002). Thus, the major probability of the cell origin of
the RTL-W1 line would be one of those cell types. Both cell types
display plasma membrane modifications: (a) short microvilli pro-
jections from BECs in initial biliary passageways; and (b) microvilli
into the perisinusoidal space, in the case of hepatocytes (McIntyre,
2007; Rocha et al., 1994). The emergence of projections and blebs
seen in cell aggregates suggests a restoration of cell polarity, which
is well known in primary cultures of hepatocytes, from rodent
(Nishikawa et al., 1996) and fish (Braunbeck et al., 1998).
Generally, two morphologically distinct cell types (named B and
S) were observed in RTL-W1. Type B, which appeared in low num-
ber, displayed a morphology compatible with senescent cells (Cho
et al., 2004; White, 2009). Despite this, a specific senescence assay
seems needed for testing this hypothesis. Importantly, some cells
revealed a transitional morphology between the two cell types.
This fact is compatible with the notion that a transitory stage exists
between the two types, and that one type would generate the other.
Less likely, the fact also fits into the hypothesis that a common pre-
cursor would originate two differently differentiated cells types.
Yet, at this point it would be speculative to definitely conclude if B
or S cell types are indeed different in nature or not.
The ultrastructural morphology of RTL-W1 in monolayer was
generally similar to that described by Lee et al. (1993), where,
the presence of dilated cisterns of RER, lysosomes and Golgi com-
plexes was described. On the contrary, in the present report some
mitochondria and a great amount of free ribosomes were observed;
but RER profiles were rare, and Golgi complexes were even rarer.
Peroxisomes were not detected by catalase cytochemistry, prob-
ably because either these organelles are few in number and/or
they do not have significant catalase activity. Also, the filamentous
bodies found in many cells have never been described. These differ-
ences suggest that the cell line does change within time in culture
(passages).

Fig. 15. Immunocytochemistry of cell aggregates – cells aggregates with 8–14 weeks in culture. Images (a) and (b) AE1/AE3 is throughout positive in the cytoplasm.
Nonetheless, it is possible to see some areas with stronger positivity. Images (c) and (d) illustrate the vimentin positive marking of only a set of cells.
170 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174
Fig. 16. RER arrangement in RTL-W1 aggregates – cells aggregates with 8–14 weeks
in culture. In both (a) and (b) the RER is displayed in parallel rows. Filaments and
lamellar bodies were also commonly seen in the cytoplasm. Dense bodies (Db), fil-
aments (Fil), lamellar bodies (Lb), mitochondria (Mi), nucleus (Nu), nucleolus (Ncl),
polyribosome (Prib), rough endoplasmic reticulum (RER).
Fig. 17. Membrane specializations in RTL-W1 aggregates – cells aggregates with
8–14 weeks in culture. (a) Detail of cell-to-cell interdigitations (arrows). (b) Desmo-
somes and the formation of membrane blebs (stars) in the space between cells.
Desmosomes (Desm).
Fig. 18. Membrane specializations in intercellular spaces in RTLW-1 aggregates –
cells aggregates with 8–14 weeks in culture. In the intercellular spaces there are
thin membrane projections (arrows) and blebs (stars). Some blebs can correspond to
tangential sections of larger membrane projections. Dense body (Db), mitochondria
(Mi).
Continuing with the attempt to clarify the cell line origin, the
ultrastructure of the cells may be compared with normal liver
epithelial cells, looking for similarities and differences. In trout,
hepatocytes ultrastructure is characterized by a well-developed
RER matching an exuberant Golgi apparatus, by many mitochon-
dria and by a large number of catalase rich peroxisomes (Rocha
et al., 1994, 1999). Overall, the cell line does not currently share
morphological aspects with differentiated hepatocytes.
On the other hand, rainbow trout BECs typically have a high
nuclear/cytoplasmatic ratio, lack glycogen, and display sparse pro-
files of RER (Blair et al., 1990). More specifically, and in the very
first bile passageways with BECs, bile preductular epithelial cells
(BPDECs) are characterized by scanty cytoplasm, rare profiles of
RER, abundance of free ribosomes and visible intermediate fil-
aments (Okihiro and Hinton, 2000). The poor organelle content
of RTL-W1 cells monolayers was very similar to what has been
described for BPDECs (Howarth et al., 2010; Okihiro and Hinton,
2000). These are viewed as equivalent of the oval cells from mam-
mals. The similarity between BPDECs and oval cells has been well
illustrated under both light and electron microscopy (Di Giulio and
Hinton, 2008; Okihiro and Hinton, 2000). Despite the cited poor-
ness in organelles have also been described for other fish cell lines
(Scholz et al., 1998; Zahn et al., 1996), we can additionally think
that the high ratio of biliary epithelial cells to hepatocytes, 1:7
(Hampton et al., 1989), agrees with a high likelihood that RTL-W1
is a liver stem cell population. Indeed, in vivo such stem cells are
located in the smallest branches of the biliary tree (Braunbeck et al.,
1998).
On a purely morphological perspective, and despite the neces-
sary caution, it is possible to hypothesize about the best potential
targets for which the RTL-W1 cell line could be useful. For exam-
ple, from our observations, the number of mitochondria in the
line is relatively abundant; therefore, studies related to mitochon-
drial genesis, kinetics or activity, the cell line could be appropriate.
However, when looking for mechanisms that require endoplasmic
reticulum (RER or REL), like drug metabolism, this cell line does not
seem a priori the best choice, as the content is so reduced in mono-
layers. An option could be the use of RTL-W1 cells in aggregates,
where reticulum is more abundant.
In the immunocytochemistry study, the great majority of cells
were not positive for Cam5.2 (an antibody commonly used for
immunomarking hepatocytes in humans). Herein, specificity for
Cam5.2 was revealed neither in RTL-W1 cells, nor in hepatocytes,
nor in any other cell type in the intact trout liver. So, that antibody
seems to be unsuitable for marking hepatocytes in trout, at least
under the tested conditions, and the non-tagging of RTL-W1 cells
 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174
cannot be valorized in particular considering that the used antibod-
ies were not developed for trout and negative results can be related
with lack of specificity. In great contrast, in the trout liver control,
biliary ducts and smaller branches of the biliary tree were markedly
positive for the pan cytokeratin AE1/AE3. Our data agree with a pre-
vious study in which normal trout liver hepatocytes were negative
for AE1/AE3, whereas bile ducts were strongly positive (Okihiro and
Hinton, 2000).
With respect to the positive staining for vimentin, it has been
described that many normal and neoplastic epithelial cell lines,
including hepatoma lines, can express not only epithelial spe-
cific cytokeratins but also mesenchymal filaments, like vimentin
(Bannash et al., 1980; Tokiwa et al., 2008), not normally found
in their tissue of origin (Ben-ze’ev, 1984); thus revealing that the
expression of specific markers in vitro can differ from what happens
in vivo. Moreover, both hepatocytes and BECs can express vimentin
in culture (Gebhardt, 1998).
The correlation between electron microscopy and immunocy-
tochemistry data support the idea that RTL-W1, at least at passage
numbers 100–120, seems more representative of BPDECs phe-
notype. BPDECs are considered bipolar progenitor cells capable
of differentiating into both hepatocytes and biliary cell lineages
(Alison et al., 2001; Factor et al., 1994; Michalopoulos et al., 2002).
Our structural and expression marker studies evidenced that
RTL-W1 cells in monolayers resemble BPDECs and thus offers cru-
cial clues of the possible cell origin. The organelle paucity offers a
structural pillar to better understand the often very limited func-
tional performance as observed in biochemical and/or toxicological
tests. However, to achieve a final conclusion further biochemical,
functional and expression data and additional studies should be
equated. An immediate problem is that, as far as we know, cur-
rently there is essentially no knowledge about BPDECs metabolic
or expression profiles. The only study that tried to evaluate the
enzymatic profile of BPDECs was not capable of detection due to
masking by hepatocellular activity (Okihiro and Hinton, 2000).
4.2. 3D cell aggregates
The studies with the RTL-W1 cell line did not report high
metabolic (e.g., enzyme) activities (Thibaut et al., 2009; Whyte et al.,
2000). Considering our data from the monolayers – which to this
date have been the target status of all the assays with this cell line
– the relatively poor level of biochemical responses of the cells is
well in line with the generalized organelle paucity of the cells and
apparent lack of differentiation.
It is common knowledge that, in order to maintain the nor-
mal physiology or to respond to pathological conditions, cells in
any tissue or organ interact with the neighboring cells by chemical
and mechanical signals. As most cell cultures grow in monolayer,
they lose part of this complex network of stimuli. Several studies
have concentrated on developing 3D cell culture models in order to
overcome this lack of “neighboring effect”, necessary to most cell
mechanisms (Flouriot, 1993, 1995). Within the last decades several
types of tissue explants and 3D models have been largely used in
biomedical research. The main aim of 3D models is two reduce the
gap between cell-based assays and animal studies (Lin and Chang,
2008).
Three dimensional hepatocyte aggregates have already been
described for rat (Tokiwa, 1997) and fish primary cultures (Flouriot,
1993, 1995; Ostrander et al., 1995; Tokiwa, 1997), and also in
human cell lines (Chang and Hughes-Fulford, 2009). Hepatocyte
aggregates, also called spheroids, can be self–assembled sponta-
neously, as it happens in rat and fish primary cultures, or can
be induced (Lin and Chang, 2008; van Zijl and Mikulits, 2010).
Similarly to hepatocytes, BECs were also described to form 3D
171
aggregates in primary human culture of cells derived from bile
atresia (Ochiai et al., 2004).
Works made in 3D hepatocyte cultures have reported better
liver-specific functions than when in conventional 2D monolayer
cultures (Flouriot, 1993, 1995). It is now well known that hepa-
tocyte spheroids differ from monolayer cultures in many aspects,
e.g., in phenotype and gene expression (Lin and Chang, 2008). In
spheroids of HepG2 cells genes related to metabolism and synthe-
sis were up-regulated when compared to monolayers (Chang and
Hughes-Fulford, 2009).
The formation of aggregates of RTL-W1 cells was already been
described; however, it was exclusively related to exposure to aflo-
toxin B1 (Bechtel and Lee, 1994). After this exposure, cells displayed
a loss of contact inhibition and managed to grow in agar. We had
very similar results, but with the normal cultivation of the same
cell line. More detailed studies are necessary to elucidate those dif-
ferences in results, but maybe the passage number (greater in our
study) could be a reasonable explanation. It would be fundamen-
tally interesting and useful now to compare our results with those
from initial passages of the cell line.
The RTL-W1 aggregates revealed signs of hepatocytic differen-
tiation including (Factor et al., 1994) a well-developed RER (often
arranged in parallel rows) and organization of bile canalicular-
like structures. Other striking alteration was the disappearance of
the filamentous bodies. The presence of an increased number of
autophagic vacuoles and myelinated bodies, as seen in the present
study, had already been described in trout hepatocytes cultured for
long time (Braunbeck et al., 1998). As far as we know, the referred
above is the first description of this kind of morphological differ-
entiation in fish liver cell lines.
The reestablishment of structural relationships resembling the
tissue of origin suggests that the 3D aggregates could have a more
significant value in metabolic studies about (cooperative) cell–cell
interaction. Our data correlates well with that from other study
that reported higher levels of liver specific mRNA in aggregates in
relation to monolayer primary culture systems of trout hepatocytes
(Flouriot, 1993); in the sense that in aggregates the cell organelle
machinery seems to be more developed. Still, even if aggregates
seem promising for working with more differentiated organelle-
rich cells, eventually apt to offer stronger functional responses
under testing than monolayers, the cut-off size from which noise
from cell death will jeopardize the advantages, and the working-
frame suggested (9–14 weeks) should be considered.
4.3. State of tumorigenicity
The general recognized criteria for identifying malignancy at
cellular level include: pleomorphism, anisocytosis, anisokaryosis,
multinucleation, nuclear molding, and abnormal mitoses with
tripolar and quadripolar mitotic figures (Cowel et al., 2008; Gilvarry
et al., 1990; Kumar et al., 2010). All the general criteria for
malignancy were observed in the cytospins of the RTL-W1 line.
Micronuclei were also found, and this has been described to occur
as a result of numerical and structural chromosome aberrations
(Miller et al., 1995; Strunjak-Perovic et al., 2003). However, some of
the above mentioned characteristics, particularly multinucleation,
were found in rainbow trout hepatocytes after partial hepatectomy
and bile duct ligation (Okihiro and Hinton, 2000). The aspects of
malignancy found in the RTL-W1 were coincident with those found
herein in the human breast cancer epithelial cell line T47D, which is
a proven malignant cell line. Also RTL-W1 revealed altered growth
behaviors such as the loss of contact inhibition, revealed in multi-
layer and 3D growth and the growth in agar. This is in contradiction
to the study by Lee et al. (1993) in which the line was able to grow
in agar but only after exposure to the carcinogen agent aflotoxin
B1.
172 F. Malhão et al. / Tissue and Cell 45 (2013) 159–174
In spite of the enumerated reasons, under our point of view
there is not enough evidence to state that the cell line is not nor-
mal. Taking into account our findings regarding the liver cell origin,
and considering the hypothesis that this cell line can represent a
liver stem cell line, it is well known the narrow difference between
stemness and tumorigenicity (Knoepfler, 2009). Furthermore, for
fish cell lines the criteria of neoplastic transformation are not well
established, and thus care should be taken when classifying a cell
line as malignant if it was not derived from a tumor. It is our under-
standing that more detailed studies should be made with fish cell
lines for establishing criteria to specifically characterize the malig-
nancy status. There are arguments backing our cautious opinion,
as fish cell lines special characteristics have been unexplored. For
instance, the high expression of telomerases in trout (Au et al.,
2009; McChesney et al., 2005) is a relevant difference when com-
paring with the mammalian counterparts. Consequently, the use
of the exact same criteria used for mammalian cell lines does not
seem appropriate.
4.4. Final remarks
Our results support the possibility of RTL-W1 cell line could con-
sist of a population of adult trout liver stem cells, which resemble
BPDECs while showing some typical changes expected from such
long term cultures (is this case for almost 30 years). For future
experiments it would be very interesting to give to this cell line
appropriate culture conditions that could promote its differenti-
ation into hepatocytes and/or bile epithelial cells or even in liver
stromal cells. This work should call the attention about the need to
obtain a better understanding of fish cell lines, including the state
of tumorigenicity (that we could not definitely conclude about), in
relation to which data are still very scarce, in order to improve its
applications and avoid misinterpretations of the results.
Acknowledgments
This work was financially supported by FEDER funds through the
Competitiveness and Trade Expansion Program – COMPETE and by
National Funds provided by Fundação para a Ciência e a Tecnologia
(FCT), via the project PTDC/CVT/115618/2009. This work would not
be possible without the consent of Dr. Lucy Lee from the Depart-
ment of Biology, Wilfrid Laurier University, Waterloo, Canada, who
developed RTL-W1 cell line.
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