Characterization of a new cell line from ornamental fish Amphiprion ocellaris (Cuvier, 1830) and its susceptibility to nervous necrosis virus

Amphiprion ocellaris (ocellaris clownfish) is one of the most commercially important marine ornamental fish. A cell line designated as OCF was developed for the first time from the caudal fin of this fish species. The cell line was maintained in Leibovitz’s—15 medium supplemented with 15% FBS (Fetal Bovine Serum) and was successfully subcultured up to 34 passages. The cell line was authenticated by sequencing mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes. The growth rate of the OCF cell line was maximum in medium containing 20% FBS and 1% of 0.2 M NaCl at 28 °C. Chromosome analysis revealed 48 diploid chromosomes. The OCF cell line was transfected with the pMaxGFP plasmid vector with 7% efficiency and GFP expression was observed. The OCF cell line was used for testing nervous necrosis virus (NNV) susceptibility. Cytopathic effect (CPE) was observed in terms of plaque formation after virus inoculation. Nested PCR confirmed the susceptibility of the OCF cell line to NNV. The cell line was successfully cryopreserved by a slow freezing procedure at − 80 °C with a revival efficiency of 70–75%. The study revealed that the OCF cell line would be useful for virological studies. In addition, the cell line would play an important role as an in vitro tool for carrying out toxicological and biotechnological studies.

Cell lines have been used as vital in vitro tools for performing various studies in life science including their best applications for various studies i.e. studying virology, environmental toxicology, cytobiology, oncology, drug screening and development, gene expression studies, genetics and genomics 1,2 . Fish cells have an advantage over mammalian or avian cells with minimal maintenance requirement, replicate within a broad range of incubation temperatures, and a flexible culture program 3 . These distinctive features of fish cells make them useful tools for a variety of biological requisitions in life science. The first permanent cell line from fish was developed from the gonads of Rainbow trout, Salmo gairdneri (RTG2) 4 . Since then, many fish cell lines have been established using a broad variety of tissues representing marine and freshwater fish. Bairoch has provided more updated details enlisting 883 fish cell lines worldwide in Cellulosaurus; a knowledge resource on cell lines 5 . Characterization of the cultured cells is one of the important parameters for cell line authentication, i.e., to confirm the species of origin and biology of the cultured cell line. Almeida et al. reported the standard methods for authentication of cell lines such as cytochrome c oxidase subunit1 (COI) barcode, karyotyping, short tandem repeat (STR) profiling and single nucleotide polymorphisms (SNP) profiling 6 . Some other properties of cell lines including plating efficiency, which provides the proliferation capacity of the cell line, transfection efficiency of the foreign DNA for the gene expression studies, viability assay after cryopreservation. Cryopreservation of cultured fish cells more often relies on very simple and facile protocols using cryoprotectant DMSO (Dimethyl Sulfoxide). The DMSO is added to the cultured cell suspension in the medium, and short-term cryopreservation is carried out by keeping the cells in − 80 °C freezer 7,8 . Primary cell culture. Normal and healthy live specimens of A. ocellaris (body weight: 1.5 ± 0.25 g; total length: 3.6 cm) originally collected from Reef Aquaria, Mumbai in live condition were transported to the Wet Laboratory of ICAR-Central Institute of Fisheries Education, Mumbai, Maharashtra and maintained in an aquarium with seawater. The donor fish were kept in well-aerated sterile seawater without feeding for 24 to 36 h. The fish was exposed to rapid hypothermic shock in an ice-chilled bath for 1-2 min. The caudal fin, eye, heart, gill, liver, and skin tissues were taken out aseptically and washed with 1 mL PBS containing 500 µg/mL streptomycin and 500 IU/mL penicillin and 2.5 μg/mL fungizone. The tissues were then minced into small pieces using a pair of sterile surgical scissors. The explants of 1 mm 3 size were prepared and washed thrice with PBS (Thermo Fisher Scientific) containing antibiotics. The minced explants were then seeded into 25 cm 2 cell culture flasks. The adherence of explants was accomplished by the addition of 0.2 mL of heat-inactivated Fetal Bovine Serum (FBS) (Gibco); then the flasks were incubated at 28 °C and allowed to attach properly to the surface of the flask by keeping the flask in the incubator. After 18-24 h, L-15 (Leibovitz) (HiMedia) medium supplemented with 20% FBS was added gently. The medium was changed after 3-5 days. The radiations of cells from the caudal fin showed faster compared to other tissues and it was used for the cell line development.

Subculture and maintenance.
Once the cells reached the confluency of 80-90%, the old medium was removed followed by rinsing the monolayer of cells with PBS. The cells were detached by trypsinization with 1-2 mL of trypsin-EDTA (0.25%) until the cells got completely detached from the flask surface. The detached cells were resuspended in 5 mL of L-15 fresh growth medium supplemented with 20% FBS and seeded in 25 cm 2 cell culture flasks from the second passage onwards a split ratio of 1:2 was maintained for subsequent passages. In the initial subcultures, 50% of the culture medium was replaced with a fresh medium. The cells were incubated at optimum pH 7.4 and temperature 28 °C. The cultures were monitored daily and subcultured upon reaching 80% confluency 34,43 . Growth studies. Serum concentration(s). Cells at a concentration of 2 × 10 4 were seeded in 25 cm 2 cell culture flasks having L-15 containing 20% serum and incubated overnight at 28 °C. The succeeding day, the medium was removed and thoroughly washed with PBS, and fresh culture medium containing different concentrations of FBS such as 5, 10, 15 and 20% FBS was added to the different cell culture flask and incubated at 28 °C and observed for seven days 15 . At every 24 h intervals, the relative number of viable and live cells in triplicate flasks in each set was estimated using a hemocytometer under an inverted microscope.
Salt concentration(s). Cells were seeded at a density of 2 × 10 4 in 25 cm 2 cell culture flasks having L-15 medium supplemented with 20% FBS and incubated at 28 °C overnight. From the next day onwards, the medium was Temperature(s). To assess the optimum growth response at different temperature ranges, cells were seeded at a density of 2 × 10 4 in 25 cm 2 cell culture flasks having L-15 containing 20% serum and incubated for overnight at 28 °C 15 . Next day onwards the cells were incubated at different temperature ranges such as 20, 24, 28, and 32 °C in 25 cm 2 cell culture flasks at an initial concentration of 2 × 10 4 cells/mL in culture medium having 20% serum. Every 24 h post-subculture, the relative number of live cells in each set was estimated using a hemocytometer.
Plating efficiency. The plating efficiency of the cells was estimated according to the methods described by

Authentication of cell line using molecular markers. Amplification of COI and 16S rRNA genes and
sequencing. Genomic DNA from the Ocellaris clownfish fin (OCF) cell line was isolated following Sambrook 18 . For amplification of the mitochondrial COI gene, the universal pair of primers FishF1 and FishR1 19 were used, and for amplification of 16S rRNA gene, 16sf1F140 and 16sf1R1524 primers (Xcelris) were used 20 . The details of primers, master mix preparation, and thermal regime of COI and 16S rRNA are mentioned in Tables 1, 2, 3 and 4. PCR products were visualized on 1.0% agarose gel by staining with ethidium bromide (EtBr) and documented using a gel documentation system (OmegaLum G, Aplegen, USA). The amplified products of both COI and 16S rRNA were sequenced and the sequences were analyzed using BLASTn.   www.nature.com/scientificreports/ Immunocytochemistry. Morphology of the OCF cells was checked by immunocytochemistry using monoclonal antibodies against Vimentin (V6630-CLONE 9 Sigma) and Cytokeratin (C2931-Clone C-11 Sigma). Briefly, the cells were grown on cover slips upto 90% confluency in a 12 well tissue culture plates (Nunc). The cells were washed with PBS and fixed in 4% p-formaldhyde (PFA) and again washed twice with PBS. The cells were permeabilized with 0.1% Triton X-100. To prevent the unspecific binding of the antibodies, blocking was done with PBS containing 5% sheep serum for 40 min at 37 °C. The blocking solution was removed and 100 μL of anti-Vimentin (1:40 dilution) along with anti-pan cytokeratin (1:200 dilution) was added and incubated for overnight at 4 °C. The reaction was carried out in duplicate wells. The cover slip was washed with PBS to remove any unbound antibodies and treated with 100 μL of FITC-labelled anti-mouse IgG (1:300 dilution). The cover slip containing stained cells was washed again with PBS and mounted using 50% glycerol (in PBS) and observed under fluorescent microscope 52 .
Chromosome analysis. OCF cells at passage 19th were used for chromosome analysis. The cells were inoculated in a 25 cm 2 culture flask and incubated for 24 h. Subsequently, 10 µL of 0.5% Colchicine (Sigma-Aldrich, St. Louis, MO) was added to the cells and incubated for 2 h in BOD incubator at 28 °C. Cells were removed from the flask by trypsinization and the cell pellet was collected by centrifugation at 1500×g for 5 min. The pellet was gently resuspended in 0.56% KCl and incubated for 20 min at room temperature for swelling. The hypotonic cell pellet was collected by centrifugation at 1500×g for 5 min. The hypotonic action was stopped by gradually adding 1 mL freshly prepared chilled Carnoy's fixative (Methanol: Glacial Acetic acid in 3:1) and mixed gently and the cell pellet was collected by centrifuging at 1500×g for 5 min. The above step of fixing was repeated 2-3 times by fresh fixative till clear transparent cell suspension was obtained. A small quantity of cell suspension was taken in a pasture pipette and dropped onto grease, from a height of 1-1.5 feet on a pre-cleaned glass slide and stained with 4-5% Giemsa (pH 6.8) for 30 min. Then, slide was washed with double distilled water thoroughly. Photomicrographs of metaphase spreads were taken under the oil immersion objective (100x).

Transfection of OCF cell line with pmaxGFP vector.
The OCF cell line with 80-90% confluency at 26th passage was trypsinized from the flask and then seeded at different densities in a six-well plate, each containing 1 mL media with 10% FBS and incubated at 28 °C overnight. The culture medium was aspirated completely and gently rinsed with PBS and 1 mL of fresh serum-free media (SFM) added followed by adding 250 µL of Transfection mixture (Lipofectamine 3000 Reagent, Invitrogen) drop-wise to each well-containing cell and mixed gently. The transfected cells were incubated at 28 °C overnight in BOD incubator. The next day, 1 mL of media containing 10% serum was added to each well and incubated for 48 h at 28 °C and then observed for the expression of GFP under an inverted fluorescence microscope.  Short-term cryopreservation. Shor-term cryopreservation of the OCF cell line was carried out in − 80 °C ultra-freezer, and their stability was assessed according to Freshney 23 . OCF cells of 23rd passage at densities of 4 × 10 6 cells/mL were harvested and concentrated by centrifugation. The cell suspensions were carefully resuspended in a freezing medium embrace of L-15 medium with 10% serum and 10% dimethyl sulphoxide (DMSO). Aliquots of 1 mL were dispensed into 2 mL of sterile cryovials. The cryovials were kept at 4 °C for 2 h followed    www.nature.com/scientificreports/ by − 20 °C for 1 h, and then cryovials were kept at − 80 °C for one month. The recovery of frozen cells after onemonth of post-storage was performed by thawing at 37 °C in a water bath. The cell viability was determined by staining with trypan blue, and the total viable cells were enumerated using a hemocytometer.

Results
Primary and subculture of OCF cells. In the primary culture, the caudal fin explants of A. ocellaris were found to be properly affixed after 20 h of explant preparation and cells start radiating after 72 h of explant preparation. During the first 10 subcultures, a mix of 50% each of the fresh and spent L-15 medium with 20% FBS was utilized for 4 days of interval. In succeeding subcultures, cells were passaged using a fresh medium of L-15 with 15% FBS at 4 days of interval. OCF cell line has been subcultured or passaged and maintained up to 34 passages (Fig. 1).
Growth studies. Serum concentration(s). Cell growth was observed at different concentrations of FBS, i.e., 5, 10, 15, and 20% to find out optimum FBS concentration. OCF cells revealed poor growth at 5% of FBS concentration whereas comparatively better growth was observed at 15% (Table S1, Fig S1), but maximum growth of the cells was observed with 20% FBS concentrations (Fig. 2).
Salt concentration. Cells did not survive beyond 1 or 2 days during initial subcultures. Optimization of salt concentration in the medium was indeed essential since the cells were derived from a marine fish. To estimate  www.nature.com/scientificreports/ the optimum salt concentration to support consistent cell growth, 0.2 M sodium chloride was supplemented with L-15 medium containing 20% serum at different salt concentrations, i.e., 0.5%, 1%, 1.5%, and 2% (Table S2, Fig S2). The cells were found to grow well at 1-1.5% NaCl with maximum growth recorded at 1% (Fig. 3).
Temperature. Cells were incubated at different temperatures i.e., 20, 24, 28, and 32 °C to find out the optimum temperature to support consistent cell growth. The growth of cells increased as the incubation temperature increased from 24 to 28 °C (Table S3, Fig S3), but the maximum growth was recorded at 28 °C (Fig. 4). No significant growth was observed at 20 and 32 °C (Error bar represents the standard deviation (SD) (n = 3)).
Plating efficiency and cell doubling time. The maximum plating efficiency of OCF cells at 16th passage was found to be 2% when the cells were seeded at a density of 1 × 10 3 cells per well in a 6-well plate. The estimated cell doubling time of the OCF cell line at the 25th passage was 40 h.
Authentication of cell line using molecular markers. DNA barcode generated by PCR amplification of COI and 16S rRNA genes of the OCF cell line yielded 655 bp (Fig. 5) and 1380 bp (Fig. 6) respectively. The alignment of COI and 16S rRNA gene sequences obtained from the cell lines with the known gene sequences of A. ocellaris revealed a 99-100% similarity (Table 8). Thus, mitochondrial genes such as 16S rRNA and COI gene sequence derived from the cell lines authenticated the species of origin of the cell line.
Immunocytochemistry. The expression of Vimentin-FITC in OCF cell line was observed which confirmed the fibroblastic morphology of the cell line (Fig. 7).
Chromosome analysis. The chromosome counts of 104 metaphase plates revealed that the diploid number of chromosomes in the OCF cell line at 19th passage ranged from 28 to 58 (Table S4, Fig S4) with a modal value of 48 (Fig. 8).
Transfection efficiency. The OCF cells at 26th passages were successfully transfected with pmaxGFP plasmid Lipofectamine 3000 Reagent from Invitrogen. The expression of pmaxGFP in the OCF cells was detected after 48 h of transfection by the observation of a clear green fluorescent signal under a fluorescent microscope (Fig. 9). The transfection efficiency of OCF cells was estimated at 7%.     was evaluated by the observation of the cytopathic effect (CPE). The advanced CPE was observed at 24 and 48 h of post-infection. Initially, the occurrence of a lot of small foci in the monolayer with cell aggregations and lysis was observed. The virus titre (TCID 50 ) was calculated and it found to be significantly increased from 2.1 log TCID 50 /mL to 3.8 log TCID 50 /mL at 9 days of post infection. As the development of CPE progressed, additional adjoining cells became detached and granular until the entire cell sheet was eventually lysed and affected completely after 7 days of post-infection (Fig. 10A-D). The monolayer of the OCF cell line completely disintegrated after 4 days of post-infection. The infection in OCF cells with NNV was further confirmed by Nested PCR.

Confirmation of NNV by nested PCR.
The amplification of the NNV genome was performed by nested PCR using specific gene primers. The cDNA of infected cells amplified with NNV primers exhibited positive results in the expected amplified size of 570 and 420 bp (Fig. 11) in the first and second steps, respectively. The results confirmed the susceptibility of OCF cells to NNV.

Discussion
The present study was carried out to develop and characterize a new cell line from A. ocellaris. The primary culture developed from caudal fin by explant technique has several advantages over the trypsinization method in terms of speed, ease, and maintenance of cell interactions and the avoidance of enzymatic digestion which can damage the cell surface 24 . Patkaew et al. were the first to report on the cell culture system developed from the ocellaris clownfish. Their study describes only a simple explant method from the vertebra up to 4th passage, using RPMI-1640 supplemented with 20% FBS and they studied its growth characteristics 25 . Nanda et al. studied the comparison of the explant method and trypsinization method and found better attachment of cells by using the explant technique compared to the trypsinization method in Cirrhinus mrigala 26 . The radiation of cells from the primary cultures consisted of a heterogeneous population, containing both epithelial and fibroblast cells. Such type of heterogeneous group of cells during the establishment of primary cultures was reported by many researchers [27][28][29] . The process for the development of cell lines was standardized to promote cell growth as cells did not survive beyond 1 or 2 days during initial subcultures. After the optimization of salt concentration in the  www.nature.com/scientificreports/ medium, it was possible to subculture the cells, which ultimately resulted in stable cell lines. Adjustment of salt concentration to maintain osmolality was quite crucial for the development of cell lines. The osmolality of commercial L-15 varies between 300 and 340 mOsmkg −1 and standard preparations of L-15/ex fell into this range, with a mean of 326 mOsmkg −1 T 9 (n = 8) 30 . In the process of subsequent passaging, the cells of caudal fin were successfully subcultured up to 34 passages and it was designated as OCF. Domination of fibroblast cells over epithelial cells was observed in the OCF cell line from the 10th passage onwards under an inverted microscope. The predominance of fibroblast cells over epithelial cells in cell cultures from fish was reported in previous studies [31][32][33][34][35] . The maximum growth of the OCF cell line was observed in the L-15 medium with 20% FBS, followed by a gradual decrease in the FBS concentration to 15% for subsequent passage after the 34th subculture. About 15-20% FBS was required during the initial stages of development 15,28,[36][37][38] . The osmolality of marine fish blood is generally around 250 to 400 mOsmkg -1 , and the marine fish cell line survives well within this range 39,40 . Osmolality can be adjusted with addition or dilution of salts. During the initial subculture, the cells grew very fast at the beginning of day one, afterward, there was a drastic slow growth and finally, total cell death occurred. To overcome the problem, the culture media L-15 was supplemented with different concentrations of 0.2 M sodium chloride solution, and it was found that OCF cells grew well and formed a complete monolayer at 2 mM NaCl. The fish cell line can grow in a wide range of temperatures from 24 to 32 °C 34,[41][42][43] . In the present study, the most suitable temperature for optimum growth and proliferation of the developed OCF cell line was revealed to be 28 °C which showed conformity with other marine fish cell cultures 44 . Two cell lines from carp have been growing well at 37 °C 45 .
The maximum plating efficiency observed for the OCF cell line at 16th passage was 2% when seeded at 1000 cells. The high proliferation of cells and plating efficiency observed in the cell lines is expressive of a transformed characteristic or genotypic change 46 . Goswami et al. reported 64% of plating efficiency seeded at 1000 cells per flask 43 . The gene sequence of both COI and 16S rRNA obtained from the OCF cells revealed the similarity of 99-100% with the known sequences of A. ocellaris submitted in NCBI, Genbank Database. Hebert 47 has demonstrated the usage of the mitochondrial gene, COI as a universal barcode, designated as-DNA barcoding for the genetic recognition of animal life and it has also been used to specify the species and to study relationships between the species 48 . Other alternatives to COI, such as the 16S rRNA gene sequence can also be used to confirm the origin of fin and muscle cell lines 49 . Chromosomal analysis indicates that the OCF cell line at the 19th subculture possessed a diploid chromosomal number of 2n = 48 which was alike to the modal chromosomal number of A. ocellaris 50 . The immunocytochemistry based on the Vimentin-FITC expression revealed the fibroblastic morphology of the OCF cell line which showed conformity with the previous studies for the confirmation of fibroblast morphology 34,51-54 . However, additional markers like CauVim and CauK8-IIS, CauK49-IE and CauK50-IE, the tight junction protein, zonula occludens-1 (ZO-1), would be useful to support the confirmation of the fibroblast morphology of the cell line 55,56 .
In the current study, the OCF cell of the 26th subculture was successfully transfected with Lipofectamine 3000 reagent to determine the efficiency of transfection from the commercially accessible transfection reagents. The estimated transfection efficiency from the OCF cell line was low, i.e., 7%, which can be comparable to 10% efficiency reported in the PSCF cell line 34 . However, Zhou et al. showed a transfection efficiency of 2% in a CSTF cell line established from Acipenser sinensis 57 . The OCF cell line could be utilized for the production of recombinant proteins and gene expression studies using improved transfection methods e.g. nucleofection other than lipofectamine.
Isolation and propagation of recently emerging nodavirus 16 were reported in clown fish 58,59 . The OCF cell line of the 30th subculture exhibited high susceptibility to nervous necrosis virus (NNV), and the susceptibility of the virus to the OCF cell line was confirmed through VNN-specific nested PCR. The increasing rate of virus titre was found to be similar with Parameswaran et al. 60 . The study validated the potential of the OCF cell line as a robust in vitro tool for the isolation and identification of NNV for studying the pathogenesis in A. ocellaris.
The OCF cell line was successfully cryopreserved by a slow freezing procedure using dimethyl sulphoxide (DMSO) at − 80 °C with 70-75% of cell revival of the initial cell population. Cryopreservation of fin cells derived from glass catfish by 15% DMSO slow-freezing method facilitates the recovery rate of 95% of cells 8

Conclusion
The OCF cell line was successfully developed from the caudal fin of A. ocellaris. The process for the development of cell lines was standardized to promote cell growth as cells did not survive beyond 1 or 2 days during initial subcultures. After the optimization of salt concentration in the medium, it was possible to subculture the cells, which ultimately resulted in stable cell line. The cell line was characterized for species authentication, chromosomal analysis, growth potential at ifferent temperatures, FBS concentrations, and salt concentrations. The revival ability of cells was evaluated by short-term cryopreservation. In vitro transfection efficiency of the cell line was assessed by the expression of green fluorescent protein (GFP). The OCF cell line was tested for virus susceptibility. The study revealed that the OCF cell line would be a useful tool for virological studies. In short, the developed cell line from A. ocellaris would be a useful tool for in vitro research and conservation genetics of this important ornamental fish species. www.nature.com/scientificreports/