The Bulletin, MDI Biological Laboratory V. 50, 2011
Development and partial characterization of two cell lines derived from pituitaries
of adult Atlantic salmon, Salmo salar
Nguyen T.K. Vo, Michael S. Mikhaeil, and Lucy E.J. Lee
Department of Biology, Wilfrid Laurier University, ON, CANADA N2L 3C5
Atlantic salmon, Salmo salar, are highly valued fish for both fisheries and aquaculture in Canada and world-wide. Growth and
reproduction in this species have been widely studied and factors regulating their life cycle including somatic growth, sexual
maturation and reproduction, spawning, smoltification have been extensively investigated. However, the molecular
mechanisms controlling expression of the hormones involved in these processes are largely unknown. Pituitary cell cultures
could be valuable for elucidating these mechanisms, and cell lines derived from this teleost‘s pituitary could make significant
impacts in understanding growth and hormonal regulation as well as endocrine disruption by environmental contaminants.
Here we report on the development of two continuous cell lines derived from adult Atlantic salmon pituitary and their
preliminary characteristics.
Atlantic salmon, Salmo salar, is the predominant fish species farmed in Canada as well as in Maine.
Research on these species is of high priority for enhancing their output and minimizing disease, and cell lines
are being sought to assist in this quest. In the past year, we reported on the establishment of an Atlantic salmon
intestinal myofibroblast cell line, ASimf20, that was established in an initial attempt to study inflammatory and
cytotoxic responses caused by plant-derived fish feeds in the gastrointestinal tract8. In this article, we report on
the development and initial characterization of two Atlantic salmon pituitary cell lines.
Primary cell cultures were initiated from pituitaries of several mature female Atlantic salmon that were
obtained from the National Cold Water Marine Aquaculture Center in Franklin, ME. The pituitary tissues were
aseptically removed, minced into smaller pieces, and rinsed twice in sterile Hank’s Buffered Salt Solution with
1% Penicillin/Streptomycin. The minced tissues were placed in 12.5-cm2 tissue culture flasks and allowed to
attach to the culture surface in Leibovitz’ L-15 media supplemented with 10% fetal bovine serum (FBS) at
15°C. Cell outgrowth directly from the pituitary explants was observed after 7-10 days in culture. However,
confluent monolayers of cells were obtained after more than 4 months in culture only for two out of four
attempted cultures, resulting in the establishment of two cell lines, designated as ASP309 and ASP409.
ASP309
ASP409
A
B
C
D
E
F
G
H
Figure 1. Phase-contrast morphologies and some culture properties of ASP309 and ASP409 cell lines. Mitotic
figures (arrow) are seen in confluent monolayers of ASP309 at passage 12 (Panel A) and ASP409 at passage 5 (Panel
C). ASP309 readily attach and spread out to the culture surface after treatment with TryplE and being re-plated onto a
new 24-well microplate within an hour (Panel B) whereas ASP409 cells or cell clumps are still in suspension (Panel D).
Pituitary cells were incubated in either serum-free L-15 (Panels E and G) media or L15/ex (Panels F and H). Unlike
ASP309, ASP409 experience morphological stresses (presence of apoptotic bodies) in 24-h exposure to L15/ex. Scale
bars = 100 µm.
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The Bulletin, MDI Biological Laboratory V. 50, 2011
Table 1. Comparison of two Atlantic salmon pituitary-derived ASP309 and ASP409 cell lines
Pituitary cell line
Properties
ASP309
ASP409
Morphology
Fibroblastic (predominant)
Epithelial
Cell sizea (µm)
20.5 ± 0.5
9.5 ± 0.5
Enzymatic dissociation
Cells readily separate (within 1 min) into Cells take longer to dissociate and form
individual cells
small clumps/sheets
Culture properties after
Cells readily attach and spread out within Cells take longer (more than 4 hours) to
dissociation
the hour on cell culture surfaces
spread out
Doubling timeb (estimated)
9 days
3 days
Current passage
10
14
18-20
18-20
Toptimal growth (°C)
Cells show clear signs of stresses even
Acute tolerance in minimal salt
No morphological sign of stresses
only after 24 hours (viability decreases to
solution L15/exc
70% compared to cells exposed to L15
alone)
Phagocytotic capacityd
Yes
Yes
!-galatosidase activity
16.89%
1.82%
Spheroid body formatione
Tightly packed
Loosely packed
a
calculated from two independent counts by the TC10TM automated cell counter (BioRad)
cell proliferation was assayed by using fluorometric dye Alamar Blue® (InVitrogen)
c
L15/ex is a simplified solution of L-15 media that excludes all the amino acids, vitamins, and phenol red
d
determined by the ability of cells to uptake fluorescent carboxylate-modified latex beads of 2 µm in diameter after 24 hrs
e
Hanging drop method employed to form cell aggregates called spheroid bodies
b
These cell lines have been maintained in L-15 media supplemented with 10% FBS at 18-20°C, have been
routinely subcultured for over 15 generations with TryplE (a type of recombinant trypsin trademarked by
InVitrogen) in a span of a year, and successfully cryopreserved at passages 3-14. ASP309 are predominantly
fibroblastic surrounding small islands of polygonal-like large epithelial cells, whereas the ASP409 cell line
consists of mostly smaller-sized epithelial cells containing dark condensed nuclei with several nucleoli (Fig. 1A&C, Table 1). Interestingly, in contrast to ASP309, ASP409 cell proliferation is relatively fast. DAPI staining
revealed that a small population of cells underwent triploid and tetraploid mitotic divisions (Fig. 2). Moreover,
by the hanging drop method9, ASP409 formed loosely-packed spheroid bodies, an in vitro characteristic of
tumor-derived cell cultures, whereasASP309 formed well-defirned spheroid bodies, a stem cell characteristic
(Fig. 3). Whether ASP409 arose from a neoplastic or malignant pituitary, or was a result of oncogenic
transformation was not known. Karyotyping is currently underway to determine the chromosome distribution of
ASP409 cell line.
In addition, about 17% of ASP309 cells within a monolayer stained positive for !-galatosidase activity
whereas only 2% of ASP409 stained positive (Table 1). This result indicates that ASP309 has not yet been
completely spontaneously immortalized (a common phenomenon in fish cell cultures10) or continues to develop
terminally differentiated specialized cells such as gonadotrophs from progenitor cell populations. On the other
hand, low senescence detection in ASP409 suggests this cell line has become an immortal cell line, which could
account for the high mitotic activities and proliferation rate.
A
B
C
D
Figure 2. Phase-contrast and fluorescence pictures of DAPI staining of confluent ASP409 cultures at passage 13.
Triploid (Panels A and B) and tetraploid (Panels C and D) mitotic divisions were randomly distributed in cultures. Scale
bars = 50 µm.
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The Bulletin, MDI Biological Laboratory V. 50, 2011
ASP309
ASP409
Figure 3. Formation of spheroid bodies by the hanging drop method. Pituitary cell suspensions were allowed to “hang”
in tiny droplets on non-adherence culture surface for 3 days. ASP309 form nicely compact spheroid bodies whereas
ASP409 form loosely packed cell aggregates and failed to form well-defined spheroid bodies.
The pituitary gland produces growth hormones, endocrine-related peptides, and neuropeptides that can have
immunogenic roles. Thus the effects of conditioned media (CM) of ASP309 and ASP409 cultures on the
immune cells such as professional macrophages were also investigated. Preliminary results suggested that
seven-day-old CM from ASP309 cultures caused the round rainbow trout monocyte/macrophage cells, RTS114
to enhance adherence onto culture surfaces and become more spindle-like only after 24 h (Fig. 4). In contrast,
CM from ASP409 cultures did not induce any noticeable morphological changes. Neuropeptides such as
pituitary adenylate cyclase-activating polypeptides (PACAP-38) have been shown to enhance adherence of
peritoneal rat macrophages5. Since PACAP-38 is highly conserved in verterbrates including fish7, it is possible
that ASP309 are capable of producing and releasing PACAP-38 into the culture media in vitro, thus accounting
for previously mentioned observations. Moreover, growth hormones and prolactin have been reported to
stimulate the activation of mammalian and teleost professional phagocytes2,6. Thus, both enhancement in
adherence and the quick change in cell morphologies could also imply that RTS11 cells had differentiated into
more mature macrophages. Molecular characterization is currently underway to understand which factors in
ASP309 CM induce these observations with RTS11.
Further molecular and biochemical characterizations are needed to clearly identify the cell type(s) present
within these Atlantic salmon pituitary cell lines. Whether these cells produce pituitary hormones needs to be
further investigated. Subsequently, studies of how environmental contaminants affects endocrine functions and
induce cytotoxic insults would be promising. Pituitary is one of the tissues that have been reported to express
cytochrome P450 1A1 in fish through EROD activity both in vivo and in vitro1,11,12. In particular, gonadotrophs
in the anterior pituitary is believed to serve as “target cells” for CYP450-inducing aromatic hydrocarbon
compounds (AHCs) 1. Thus we are currently investigating whether these two pituitary cell lines are inducible by
a variety of AHCs by measuring EROD activities.
Figure 4. Effect of conditioned media (CM) from ASP309 and ASP409 cultures on rainbow trout macrophage
(RTS11) cells. RTS11 were exposed to 10% FBS as a control (panel A), ASP309 CM (panel B), or ASP409 CM (panel C).
Morphological change was observed 24-h post-exposure. Scale bar = 50 µm.
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The Bulletin, MDI Biological Laboratory V. 50, 2011
ASP309 and ASP409 join a very modest list of pituitary cell lines originating from poikilothermic
vertebrates3, and could become invaluable tools for mechanistic, endocrinological, toxicological and fish
nutrition studies in Atlantic salmon.
This research was supported by a 2009 New Investigator Award from MDIBL and Discovery and Strategic
grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to LEJL. We would
like to acknowledge Dr. William R. Wolters from the USDA, ARS National Cold Water Marine Aquaculture
Center, Franklin, ME, for providing the fish used in this study. Assistance from Brooke Beggs, Neil McCaffrey
and Krista Schleicher for maintaining the cell lines is acknowledged. The authors are also grateful to Wilfrid
Laurier University for a STEP travel award to NTKV that facilitated his travel and accommodation at MDIBL
where this study was initiated.
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