Advances in Biological Research 9 (3): 133-139, 2015

ISSN 1992-0067
© IDOSI Publications, 2015
DOI: 10.5829/idosi.abr.2015.9.3.9385

Cytotoxic Effect of Venom Produced in Vitro from Primary Culture

of Conusbiliosus Venom Duct Cells on HEK 293t Cells
1
M.S. Viswanathan, 1S. Arularasan, 2Amrutlal K. Patel, 1, 2M. Chandra Shekar,
2
Neelam M. Nathani, 2Ravi K. Shah, 3D.N. Rank, 2C.G. Joshi and 1T. Balasubramanian

1
Centre of Advanced Study in Marine Biology,
Annamalai University, Parangipettai-608 502, Tamilnadu, India
2
Department of Animal Biotechnology, College of Veterinary Science
and A.H., Anand Agricultural University, Anand, India
3
Department of Animal Genetics and Breeding, College of Veterinary Science
and A.H. Anand Agricultural University, Anand, India

Abstract: Bioweapon synthesis through animal cell culture technique is gaining significant interest for the
development of new drugs related to human diseases. Marine cone snail’s venom is a potential source of
unique bioactive neurotoxic peptides which can be utilized for treating cancer. Here, we describe establishment
of primary culture of venom duct epithelial cells from Conusbiliosusand demonstrate that secretory cells of
venom duct can be potential in vitro source for venom production. The venom duct cells remained in
suspension in the primary culture with no signs of adhesion and continued slow but sustained proliferation.
Cytotoxic effects of culture supernatant on HEK 293T cells showed inhibitory effect on cell growth providing
probable evidence for cono-peptides production.

Key words: Marine Cone Snail Primary Culture Secretory Cells Venom Duct Cytotoxicity

INTRODUCTION well establiehed cell line available till date is that

Conotoxins from marine cone snail have been
explored with great interest by neuroscientists since long
due to their potential therapeutic applications.
Conopeptides have been found to act as highly specific
ligand for ion channel and receptors in the central
nervous system. The cone snail venom comprises a
cocktail of highly complex structured and small sized
neuro peptides which are produced by venom duct [1].
Earlier anatomical studies on venom apparatus of genus
Conus also revealed the well-organized venom duct
responsible for venom production [2, 3]. The columnar
epithelial cells with basal nucleus present in inner layer of
venom duct synthesize these peptides [4, 5]. Till date
concerted efforts have been undertaken to evaluate the
molluscan using various approaches including molecular
techniques and development of cell line by culture based
approach [6]. However, due to lack of relevant knowledge
on mollusc cell physiology and biochemistry, the only
ofBiomphalariaglabrata embryonic (Bge). With the
advancements in animal tissue culture technology this
problem can be overcome by establishment of primary cell
culture from marine invertebrate [7, 8]. Conus venom can
be obtained by several approaches, such as milking,
dissection, recombinant production and chemical
synthesis [9, 10]. However, remarkable changes in the
production of cono-peptides have not yet been achieved.
In contrast, plenty of cone snails are sacrificed for
research purposes [11]. Previously, researchers have
developed the primary culture of venom secretory cells
from snake venom gland to synthesize the venom in
animal cell culture medium [12]. The crude toxin from
venom ducts of Conusbetulinus has shown its potential
as cytotoxic agent on HeLa cell line [13]. Therefore,
In vitro culture of venom duct cells and synthesis of
cono-peptides in animal cell culture medium can be a new
biotechnological approach not only for conservation of
marine cone snail but also for production of conotoxin.

Corresponding Author: M.S. Viswanathan, CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University,
Parangipettai-608 502, Tamil Nadu, India. Tel: +91-9952406206, E-mail: vtpviswa@gmail.com.
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 Advan. Biol. Res., 9 (3): 133-139, 2015

In the present study, we have developed the long-term

primary culture of venom secretory cells from venom duct
of Conusbiliosusand studied its cytotoxic properties
in vitro.
MATERIALS AND METHODS

Collection and processing of snails: Live specimens

of Conusspecieswere collected randomly from Porbandar
coastal line of Gujarat, India (Fig.1). The live specimens
were transported to laboratory and maintained in sea
water before being processed for further purpose.
The collected samples were rinsed with distilled water to
remove salt and other debris for identification based on
shell morphology and colour pattern to confirm its
taxonomy as Conusbiliosus species [14]. Venom glands
were dissected out from the identified Conusbiliosus
specimens in sterile laboratory conditions. Fig. 1: Live marine cone snail Conusbiliosus on sandy
shore of Porbandar beac
Morphology of Venom Secretory Cells: Initially,
histology study of resected Conus venom duct was
carried out by fixing it in 10% formaldehyde to understand
the organization of venom secretory cells in live cone
snail. After fixation, water molecules present in the tissue
was removed using isopropyl alcohol and then tissue
block was infiltrated by paraffin, allowed to solidify in a
mold and embedded with a small cube of paraffin.
Tissue sectioning was accomplished using microtome and
each section was mounted on slides. Clearing process
was applied to remove the paraffin and then histological
section was stained using hematoxylin and eosin (H and
E). Finally, stained slides were observed under
microscope (Lieca).
Fig. 2: Procedure for isolation of venom secretory cells
Cell Culture and Maintenance: Primary culture of venom from venom duct of Conusbiliosus
secretory cells from Conusbiliosus venom duct was
developed using the protocol described previously for
mammalian salivary glands with few modifications (Fig. 2)
[15, 16]. Briefly, live cone snail was wiped with 70%
ethanol. The shell was broken with the help of hammer.
The venom gland was freed from connective tissue and
their venom duct was excised (Fig. 3). Venom duct was
washed thoroughly with a PBS followed by Medium 199
(M-199). The venom duct tissues were minced to slurry in
a basal medium M-199 supplemented with antibiotics (100
IU/mL penicillin, 100µg/mL streptomycin, 5 µg/mL
gentamycin) and subsequently digested with 0.25%
trypsin (Sigma) for 30 min at 24°C to 28°C temperature
with inversion every 10 min. Samples were triturated
by pipetting for 10-15 times and enzyme action was Fig. 3: Venom apparatus of Conusbiliosus (a) Venom
stopped by adding 5% fetal bovine serum (Invitrogen). bulb (b) Venom duct

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 Advan. Biol. Res., 9 (3): 133-139, 2015
The cells were filtered through 70µm filter and pelleted
by centrifugation (1500 rpm for 10 min) at room
temperature. The supernatant was discarded and cell
pellet was washed twice with complete growth
medium (M-199 medium supplemented with 5% fetal
bovine serum) and seeded in a T 25 flask (Corning)
and incubated at 24°C to 28°C temperature. Primary
explant culture cells were observed and photographed
using inverted microscope. Crude supernatant of
venom duct cell culture medium was collected at
regular intervals of 2-3 days and centrifuged at 7500 rpm
for 10 min to clear the supernatant of cell debris or
remaining intact cells. The supernatant was further
preceded for confirmation about venom production by
cytotoxicity analysis. The cultured cells were further
studied by H and E staining to compare these cells with
tissue histology.
In parallel, Human Embryonic Kidney 293T cells
(HEK 293T) were cultivated in DMEM F-12 supplemented
with 10% fetal bovine serum (FBS), penicillin and
streptomycin for cytotoxicity analysis of venom duct cell
culture supernatant to confirm the production of venom
from cultured venom gland cells. Cultured cells from
venom duct and HEK 293T were maintained at 37°C in
5% carbon dioxide (CO2).
Assessment of Cell Viability by MTT Assay: In order to
determine the cytotoxic effects of culture supernatant
of primary venom duct cell culture on HEK 293T
cells, the viability was measured by MTT [3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]
Fig. 4: Conusbiliosus venom duct primary culture. Desegregated venom duct cells in culture medium on day 0 (a),
day 3 (b), day 14 (c) and day 27 (d)
135
assay. The cells were detached by trypsinization,
seeded at a density of 1.0X105 cells/ well into 96-well
culture plates and subsequently, incubated in a
humidified 5% CO2 incubator for overnight. The growth
medium was removed without disturbing the cell sheet.
The monolayer of cells was washed with PBS to remove
dead cells and excess fetal bovine serum. The culture
medium (M-199) and venom bulb extract served as
negative and positive control respectively. Undiluted
crude supernatant and each dilution of the crude sample
ranged from 1:10 to 1:1000 were added to respective wells
of 96 well plates in triplicate. The cells were observed
under inverted microscope for significant changes in their
morphology. After 48 hours of incubation, cell viability
was analyzed by MTT assay. 20µl of MTT (5mg/ml) was
added to each well and incubated at 37°C for 3- 4 hours.
Then, the medium was replaced with 150µl of dimethyl
sulfoxide and incubated for 45 minutes in CO2
incubator. A colored formazon crystal was assayed
spectrophotometrically at 570nm using a plate readerand
theresults are presented as the percentage of cell viability.
RESULTS
In the present study, live specimens of
Conusbiliosus were confirmed through conventional
taxonomical approach. The morphological key characters
observed were cone shaped and less number of spires on
shell, a wide aperture, usually elongated body whorl, the
presence of numerous axial and spiral growth threads and
lines. The colour of the body whorl was pale brown.
 Advan. Biol. Res., 9 (3): 133-139, 2015

Fig. 5: Microscopic observations of venom duct cells. Histology of venom duct (A), Cells in the culture medium (B) and
H and E staining of cultured cells (C).

Fig. 6: Photomicrograph of primary cell culture from the venom duct of Conusbiliosusshowing two different cell types
in the culture medium

They were moderate 34mm to 48mm in size, occurred in
sandy shore of the sea. Venom duct was used as a
starting material for the primary culture. The primary
venom secretory epithelial cell culture experiments were
performed with marine cone snails whose venom duct
length ranged between 5.6cm and 6.4cm. The venom
secretory cells grew well in the culture medium M-199
supplemented with FBS also suitable for primary culture.
The optimum temperature for venom secretory cell culture
was 24°C to 28°C. The cells in primary culture remained in
suspension with no signs of adherence throughout the
culture period. During the initial culture period, limited
numbers of cells were observed in the culture flask.
On 3rd day, cells started proliferating and high numbers of
venom duct cells were observed in the culture medium at
the end of two weeks (Fig.4). Under the sterile conditions,
cell viability was noted as 65% to 70% in culture medium.
From the third week onwards, signs of cell death were
progressively detected. Histological study and
morphological analysis of the primary venom secretory

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 Advan. Biol. Res., 9 (3): 133-139, 2015

Fig. 7: HEK 293T cells after treating with culture supernatant of venom duct cells in different dilutions (A) Cells with
medium as negative control B) Cells with venom bulb crude extract as positive control (C) Undiluted culture
supernatant (D) Dilution 1:10 (E) Dilution 1:100 (F) Dilution 1:1000.

Fig. 8: Cytotoxic effect of culture supernatant (Undiluted; 1:10; 1:100 and 1:1000) on HEK 293T cells after 48-hours
incubation period. Culture medium was taken as negative control with 100% viability.

cell culture, as well as H and E staining confirmed two DISCUSSION

groups of cells existing in the venom duct (Fig.5).
These venom secretory cells were cuboidal and
column-like shaped cells (Fig.6). The secretion of venom
substance in the culture medium was confirmed by
analyzing the effect of culture supernatant on cell viability
using MTT assay. The HEK 293T cells were treated with
primary venom duct cell culture supernatant of different
concentrations for 48 h. The survival rate of the cells
was found to be 40 % with undiluted culture supernatant
as compared to control cells but there was no significant
effect with diluted culture supernatant. However, in-vitro
result evidenced toxin production in the medium (Figs.7
and 8) as suggested by considerable reduction of cell
viability.
Development of marine invertebrate cell culture is
still at preliminary stages. It is necessary to apply the
animal cell culture technology to establish the primary cell
culture of marine mollusc species for investigating
their potential therapeutic applications [8]. To the best
of our knowledge this is the first attempt to culture
Conus venom duct cells. Previously, the ant,
Pseudomyrmextriplarinus were cultured and 0.25%
trypsin was used for dissociation of venom gland cells
[17]. Venom gland cells from the snake, Bothropsjararaca
were cultured in in-vitro condition as a long- term primary
culture to produce venom. These cells were accumulated
to form clusters and considered as functionally viable

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 Advan. Biol. Res., 9 (3): 133-139, 2015
cells during the 21st day to secret the venom in the culture
medium. Above all, secretory cells of the snake venom
gland were non-adherent in nature [12].Conus venom duct
cells during this study were observed to be non-
adhesive in nature similar to the snake venom gland. As
per the comparison of histological studies, both cone
snail and snake venom gland secretory cells were
observed to be similarin appearance [18]. Previously,
crude toxin from the venom ducts of C. betulinus isolated
by extraction method showed anticancer properties
against HeLa cell lines but the mode of action behind
cytotoxic effect remained unclear [13, 19]. Most of the
conopeptides are known to target specific neuronal
receptors and ion-channels of central nervous
system [20]. Omega conotoxin MVIIA (Ziconotide) used
as analgesic for the treatment of cancer and chronic
neuropathic pain and conotoxin GVIA (SNX-124) is also
used as ananalgesic as well as N-type calcium channel
blocker. Apart from this, some conopeptides have
potential applications in a few diseases like Alzheimer,
Parkinson and epilepsy [20]. In case of primary culture
supernatant from secretory cells of venom gland of
B.jararaca, cytotoxic activities upon cancer cells have not
yet been studied, but southern copperhead snake venom
proteins isolated by milking approach have been
investigated that showed the highest cytotoxicity through
arresting metastasis on various cancer cells.
CONCLUSION
In conclusion, we used venom duct as an
ideal model to produce and secrete venom in the
animal cell culture medium in terms of initiating a
functional long-term primary secretory cell culture,
further the cytotoxicity analysis with culture
supernatant on HEK 293T cells suggested the
production of conotoxin in the culture medium and
found that the primary culture supernatant could lead to
loss of viable cells. Despite the evidence of secreted
venom components in the culture medium, their exact
mechanism of action still remains unclear and require
further investigation.
ACKNOWLEDGEMENT
We thank University Grants Commission-Gandhi
National Fellowship (UGC-RGNF), Government of India,
for their financial support during this study.
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