Establishment and Characterization of a New Marine Fish Cell Line from Ovary of Barfin Flounder (Verasper moseri)

XU Xiaohui, FAN Tingjun, JIANG Guojian, and YANG Xiuxia

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Establishment and Characterization of a New Marine Fish Cell Line from Ovary of Barfin Flounder ()

XU Xiaohui, FAN Tingjun*, JIANG Guojian, and YANG Xiuxia

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A novel continuous ovary cell line from barfin flounder () (BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco’s modified Eagle medium/F12 medium (DMEM/F12, 1:1; pH 7.2) supplemented with 20% fetal bovine serum (FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number (46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein (GFP) positively expressed in these cells after being transformed with pcDNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.

barfin flounder ovary cell; cell line; gene transformation; green fluorescence protein; GFP;

1 Introduction

Teleostean cell lines are important for fish biological and pathological researches. As powerful research tools, fish cell lines contribute significantly to studies on fish immunology (Bols.,2001; Goswami.,2012), toxicology ( Bols.,2001; Kojima.,2011; Kienzler.,2012), endocrinology (Alok.,2000; Aluru and Vijayan,2009; Thomas,2012), virology (Kim.,2012), functional genomics (Laing and Hansen,2011), and biotechnology (Tafalla.,2006). Additionally, they have been used in researches of cytophysiology (Maclean,2011) and genetic regulation (Castro.,2010; Holopainen.,2012). At present, at least 283 fish cell lines have been established since the establishment of rainbow trout () gonad cell line in 1962(Lakra.,2011).

In recent decades, flounder has become one of the most commonly cultured marine economic species in the world, and many researchers have focused on its cell culture and related applications. By now, there have been reports on the establishment of cell lines from flounders (andand), (Table 1) providing helpful hints for us on establishing more cell lines in the future. Thebarfin flounder () is an important pleuronectiformes fish inhabiting cold sea basin near the northeast coast of China and Japan (Li and Li,2006). Recently, this fish is becoming a promising candidate for aquaculture due to its good qualities in nutrients and the stable growth in cold water (Miyata.,2005; Yao.,2011). In this study, we intended to establish a novel ovary cell line and evaluate its feasibility in foreign gene transferring by using pcDNA3.1-GFP recombinant plasmid with green fluorescence protein (GFP) as a reporter.

2 Materials and Methods

2.1 Fish

Four months old and alive barfin flounder, weighting 60–90g, were purchased from Nanshan Fish Market (Qingdao, China).Before experiment, these fish were acclimated in cold sea water containing 1000IUmL?1penicillin and 1000μgmL?1streptomycin (Lukang) for 6h.

2.2 Primary Culture

After anaesthetization with 70% alcohol for 5min, ovary tissues were harvested aseptically and rinsed three times with PBS solution containing 100IUmL?1penicil-lin and 100μgmL?1streptomycin. Ovary tissues, minced into small pieces (about 1mm3in size) in DMEM/F12 (pH 7.2) containing 5% fetal bovine serum (FBS, Hyclone) at 4℃, were seeded into 25cm2culture flasks with about 500μL 5% FBS-DMEM/F12 medium overnight for tissue pieces attachment. Each flask was added with 5mL DMEM/F12 medium containing 20% FBS, further supplemented with 100μgmL?1carboxymethyl chitooligo- saccharide (AK Scientific), 10ngmL?1basic fibroblast growth factor (bFGF) (Peprotech), 40ngmL?1insulin-like growth factor-I (IGF-I) (Peprotech), 100IUmL?1penicillin and 100μgmL?1streptomycin. The flasks were incubated at 22℃, and half of the medium was refreshed every three days.

Table 1 Characteristics of the flounder cell lines established since 1991

Notes: (1) Meguro., 1991; (2) Tong., 1997; (3) Kasai., 2001; (4) Kang., 2003; (5) Chen., 2004; (6) Fan., 2010a; (7) Fan., 2010b.

2.3 Subculture and Maintenance

Once the tissue grew into a confluent monolayer, the ovary tissue cells from barfin flounder (BFO cells) were subcultured following the method developed by Wei. (2010) with minor modifications. BFO cells were de- tached from the flasks by trypsinisation, inoculated into two 25cm2culture flasks equally, and cultured under the conditions described above.

Logarithmic BFO cells from different passages were harvested and centrifuged. Cell pellet was resuspended to 6×106–7×106cellsmL?1 in 1mL cell freezing medium consisting of DMEM/F12, FBS, dimethyl sulphoxide (DMSO) (3:1:1). The cell suspension was pooled into a 2mL sterile cryovial (Corning), placed into a gradient- cooling box (Thermo) at ?80℃ overnight, and finally stored in liquid nitrogen. When BFO cells were needed for experiment, cryopreserved cells were thawed and cultured as described previously (Wei.,2010).

2.4 Growth Studies

Some basic media used for fish cell culture and various temperatures were selected to determine the optimal culture condition for the BFO cells. The cells at a concentration of 1.0×105cellsmL?1were seeded into 24-well culture plates with 20% FBS-DMEM/F12 medium, and incubated at 22℃ for 4h. Then, the medium was removed, the cells were washed twice with PBS, and then cultured in selected medium of Leibovitz’s L-15, DMEM/F12, M-199 supplemented with 20% FBS for 6 days. At an interval of 1d, the cells in three wells were harvested with 0.25% trypsin digestion method, suspended in 1mL PBS, and counted by the cell analysis system (Innovatis, Germany). The average number of three wells was used to plot the growth curve of BFO cells. The cell growth curve at different culture temperatures (18℃, 22℃, 26℃, 30℃) was graphed outthe same procedure.

The BFO cells at Passage 60 at a concentration of 1.0×105cellsmL?1were incubated into 24-well culture plates supplemented with 20% FBS-DMEM/F12 at 22℃. The number of cells at an interval of 12h were counted and graphed. The population doubling time of cells was calculated by the formula of

(Fan., 2007),

wherestands for population doubling time,for ino- culation time, and0,Nfor cell number.

2.5 Karyotype Properties

BFO cells at logarithmic Passage 60 were used for chromosome specimen preparation according to Fan. (2007). Briefly, logarithmic cells were incubated in the medium with 20μgmL–1colchicine for 10h, harvested and suspended in 0.3% KCl hypotonic solution for 30min, fixed with Carnoy’s solution for 20min, stained with Giemsa for 40–50min and finally air dried. Chromosome numbers of 300 metaphase cells were counted under an E200 microscope (Nikon, Japan) and statistically analyzed.

2.6 BFO Cells Transformation with GFP Reporter Gene

Recombinant plasmid pcDNA3.1-GFP carrying transfection reagent X-treme GENE HP (Roche) was used for the transformation of BFO cells at Passage 60 following the method of Sun.(2011). The concentration of BFO cells in antibiotics-free 20% FBS-DMEM/F12 medium was adjusted into 2×105cellsmL?1. The cells were inoculated into 6-well plates and incubated for 24h at 22℃ in a 5% CO2incubator. Before the transformation, 2μg plasmid was suspended in 200μL opti-MEM for a few minutes, and then 6μL transfection reagent was added. The suspension was mixed immediately, incubated for 20min at room temperature, and finally added drop by drop into the medium. The green fluorescence signal was observed under an Eclipse TE2000-U inverted fluorescent microscope (Nikon, Japan) after 12, 24, 48, and 72h successively. The transformation efficiency was evaluated by calculating the ratio of cells with green fluorescence to all cells.

3 Results

3.1Culture of BFO Cells

Tissue explants adhered quickly to the flask bottom, and the fibroblastic cells started to migrate from the edge of seeded tissue explants in a few days (Fig.1a). Confluent cell layers were obtained 2 weeks later, and were subcultured at a 7–8d interval (Fig.1b). After ten passages, the interval was shortened to 3–4d, and the cells grew at a steady rate in 15% FBS-DMEM/F12 medium. To date, the cells have been subcultured up to Passage 120 (Fig.1c) and a continuous barfin flounder ovary (BFO) cell line has been successfully established. During theculture of BFO cells, cryopreservation has been often performed, and the viability of cryopreserved BFO cells was tested at different times of storage (1, 6, and 12 months). It was found that the viability of BFO cells was 95%, 60%, and 30% after cryopreserved for 1, 6, and 12 months, respectively. The survived cells still kept fibroblastic morphology and grew steadily.

Fig.1 In vitro cultured BFO cells.a, primary BFO cells migrated from ovary tissue explants after 48h. b, the confluent monolayer formed with a fibroblastic morphology after cultured for two weeks. c, subcultured BFO cells at Passage 120. Scale bar=100μm.

3.2 Growth Studies

The effects of different media and temperatures on cell growth were assessed (Fig.2). The cells exhibited different growth curve in three different media. However, the optimum growth medium was found to be DMEM/F12. Different cell growth curves were also observed at a tem-perature range from 18℃ to 30℃. The maximum growth of cells was found at 22℃, and then decreased gradually at 26℃ and 18℃, with the minimum growth at 30℃.

The growth curve of Passage 60 BFO cells is shown in Fig.3. The population doubling time of the cells was about 32.7h by calculation, indicating that these cells were in a rapid proliferative status.

Fig.2 Growth curves of BFO cells in different media (a) and at different temperatures (b).

Fig.3 The growth curve of BFO cells at Passage 60. The lag phase (Lag), logarithmic phase (Log), stationary phase (Sta), and decline phase (Dec) are shown.

3.3 Karyotype Characterization of BFO Cells

Chromosome analysis showed that BFO cells at pas-sage 60 exhibited chromosomal aneuploidy with chromosome numbers 28–54, and the ratio of BFO cells with a chromosome number of 46 was about 72% (Fig.4a), suggesting that the modal chromosome number of the established BFO cell line was still 46. The metaphase chromosomes with a normal diploid number displayed the normal karyotype characteristics, consisting of a pair ofsubtelocentric (st) and 22 pairs of telocentrics (t) chromosomes: 2=46, 2st+44t,=48 (Figs.4b, c).

3.4 Gene Transformation of BFO Cells

After Passage 60 BFO cells were transformed with recombinant plasmidcDNA3.1-GFP, green fluorescence signal could be detected after 12h, and the maximum intensity was observed after 24h (Fig.4). This indicated that the BFO cell line had the feasibility for foreign gene transformation by using X-treme GENE HPtransfection reagent. The transformation efficiency was about 10%.

Fig.4 Chromosome analysis of BFO cells at Passage 60. a, chromosomal aneuploidy of BFO cells with chromosome numbers ranging from 28 to 54. About 72 % of BFO cells have a chromosome number of 46. b, chromosomes from a BFO cell with a diploid number of 46. c, the diploid karyotype of BFO cells: 2n=46, 2st+44t, NF=48.

Fig.5 Fluorescent image of BFO cells transformed by pcDNA3.1-GFP at different times. (a) 12h; (b) 24h; (c) 48h. Scale bar=50μm.

4 Discussion

In this study, BFO cell line was established and characterized, and the feasibility of genetic operations was evaluated. In the primary culture of BFO cells, ovary tissue was directly seeded into flask without digestion. Such explant culture method has advantages over free cell culture, including easier operation, reduced operation time, and fewer traumas to cell interactions. In addition, this method can preserve cellviability by avoiding the damages caused by conventional enzyme digesting method (Avella.,1994). Similar methods have been reported in cell line establishment from Atlantic cod,,koi and rockfish grouper (Ku.,2009; Dong.,2011; Jensen.,2013; Taju.,2013).

In order to promote cell attachment and proliferation, carboxymethyl chitooligosaccharide and growth factors including bFGF and IGF-I were supplemented into the FBS-DMEM/F12 medium (Fan.,2003; Jin.,2008; Wei.,2010). Carboxymethyl chitooligosa- ccharide can improve cell attachment through the formation of anchoring junction between cell and extracellularmatrix(Iida.,1998). FGF and IGF-I have positive effects on promoting cell proliferation, probably because they can bind to tyrosine kinase receptor and consequently activate tyrosine kinases RAS and MAPK successively (Boonstra.,1995). Similar results were reported in other fish cell lines (Chen.,2007; Wei.,2010; Sun.,2011).

In this study, BFO cell line was well adapted to grow in DMEM/F12 medium supplemented with FBS, although Leibovitz’s L-15 medium has been the most commonly used in the culture of fish cells(Lakra.,2011). The growth temperature range for this cell line was 18℃–30℃ with the optimum growth at 22℃, identical with other fish cell lines reported (Pombinho., 2004; Servili., 2009). The duplication time of Passage 60 BFO cells was 32.7h, which suggested that these cells grow faster than some of the other fish cell lines,, brown-marbled grouper fin cell line and large yellow croaker spleen cell line (Wei.,2010; Sun.,2011). Karyotype analysis showed that Passage 60 BFO cells still have a modal chromosome number of 46 with normal karyotype morphology, although aneuploidy was observed. The result was in accordance with those reported early (Wang.,2009).

The successful transformation of BFO cells withcDNA3.1-GFP made it possible to apply this fish cell line to gene targeting and expression studies. The similar result can be found in other fish cells transformed with lipofectin. The transformation efficiency (10%) was higher than those previously reported (Qin.,2006; Parameswaran.,2007; Goswami.,2012), but was lower than viral transfection system such as baculovirus system (Huang.,2011; Li.,2012).

In conclusion, a novel BFO cell line was successfully established with its typical characteristics studied. This was the first report in flounder ovary cell line establishment, which provided a useful tool for studies on fish transgenic modification and gene functions in future.

Acknowledgements

This study was supported by grants from the National 863 High Technology Research Foundation of China (2006AA10A401) and the National Natural Science Foundation of China (31001100). We thank Dr. Ai Sun for the technical assistance, and Dr. Yuan Ge for improving the manuscript.

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(Edited by Qiu Yantao)

DOI 10.1007/s11802-015-2920-6

ISSN 1672-5182, 2015 14 (6): 1105-1110

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

(March 31, 2015; revised April 22, 2015; accepted September 1, 2015)

* Corresponding author. Tel: 0086-532-82031637 E-mail: tjfan@ouc.edu.cn