Fish Physiol Biochem DOI 10.1007/s10695-015-0036-y
Establishment and characterization of fin cell lines from diploid, triploid, and tetraploid oriental weatherfish (Misgurnus anguillicaudatus) Xia Li • Chen Ma • Yan-Jie Qin • Ya-Juan Li Di Wu • Li-Wen Bai • Ai-Jun Pei
•
Received: 25 July 2014 / Accepted: 15 December 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Continuous fin cell lines from diploid, triploid, and tetraploid oriental weatherfish, Misgurnus anguillicaudatus, were established and characterized. The cell lines, designated DIMF, TRMF, and TEMF, respectively, were subcultured more than 80 times since initiation in October 2012 and were preserved at the China Center for Type Culture Collection as sample numbers C2013109, C2013110, C2013111, respectively. The cell lines consist predominantly of fibroblast-like cells. At the 50th passage, the population doubling times were 48.43 h (DIMF), 36.01 h (TRMF), and 41.45 h (TEMF). Cell survival rate of these three kinds of cells was 80.88 ± 1.38, 84.48 ± 1.13, and 81.57 ± 1.28 %, respectively, when recovered after storage in liquid nitrogen for 60 days at the 40th passage. The chromosome numbers measured from 100 metaphase plates at the 50th passage were 2n = 50 (68 %), 3n = 75 (59 %), and 4n = 100 (54 %) for DIMF, TRMF, and TEMF cells, respectively. At the 60th
passage, the chromosome numbers for DIMF and TRMF cells were still 50 (52 %) and 75 (70 %), but the chromosome number for TEMF cells ranged from 88 to 100; a chromosome number of 96 accounted for 26 % of the cells, and the karyotype analysis showed 4n = 96, 16 m ? 8sm ? 72t, NF = 120; thus, compared with cells at the 50th passage, a group of metacentric chromosomes was missing. Microsatellite marker analysis was conducted using DIMF, TRMF, and TEMF cells and muscle tissue of oriental weatherfish, which confirmed that the three cell lines established in this study were from oriental weatherfish. The cell lines were exposed to two fish viruses to determine their susceptibility to infection; they were susceptible to spring viremia of carp virus but not to piscine nodavirus. Establishment of fin cell lines from different ploidy oriental weatherfish increases the existing number of fish cell lines available for research, and it provides a model for investigating the mechanisms of growth and genetics in polyploid fish.
X. Li (&) C. Ma Y.-J. Qin D. Wu A.-J. Pei Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation in Liaoning Province, Dalian Ocean University, Heishijiao Street 52, Dalian 116023, People’s Republic of China e-mail: [email protected]
Keywords Misgurnus anguillicaudatus Cell line Polyploidy Karyotype Virus susceptibility
Y.-J. Li L.-W. Bai Key Laboratory of Mariculture, Agriculture Ministry, PRC, Dalian Ocean University, Heishijiao Street 52, Dalian 116023, People’s Republic of China
Introduction The oriental weatherfish (Misgurnus anguillicaudatus) is an economically important freshwater fish in
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China. In addition to diploid weatherfish, triploid and tetraploid specimens exist in the natural world (Arm 2003; Itono et al. 2006, 2007). Thus, this species is a good model for biological and genetics research. Previous studies have focused on methods to produce polyploidy, identify polyploidy, and determine growth characteristics of polyploidy fish (Kim et al. 1994; Gao et al. 2007; Wu et al. 2007; Nan et al. 2008). Since the first teleost cell line [rainbow trout (Salmo gairdneri) gonadal cell line RTG-2] was established in 1962 (Wolf and Quimby 1962), 275 other fish cell lines have been established, including 175 freshwater or anadromous migratory fish cell lines and 100 marine fish cell lines (Zhang and Chen 2011; Lakra et al. 2011). These cell lines were derived mainly from tissues such as fin, spleen, and kidney (Ganassin and Bols 1998; Chi et al. 1999; Kang et al. 2003; Imajoh et al. 2007; Xing et al. 2009; Zheng et al. 2012). Among them, fin tissue has proven to be useful for the development of cell lines (Wang et al. 2004; Fan et al. 2007; Zhou et al. 2008). Sterile culture is the foundation of the culturing of fish cells, every aspect of fish cell culture should be performed under sterile conditions, so the first step of culture is the fish or the tissue is rinsed with alcohol, antibiotics, and other disinfectant (Fu et al. 2012; Guo 2010). The use of established fish cell lines has provided much information in studies relating to fish developmental biology (Clem et al. 1996), virology (Ruiz et al. 2009), transgenic applications (Zheng et al. 2012), and others (Hashimoto et al. 2008; Lakra et al. 2011; Zhou et al. 2003). Viral disease is a common problem in fish farming. Some of the freshwater fish cell lines have been shown to be sensitive to certain fish viruses (Yan et al. 2011; Lei et al. 2012; Xiao et al. 2012; Zhang and Gui 2012; Zhu et al. 2013), so they can be used to study disease pathology. However, many types of viruses affect fish, and there are not enough existing cell lines to meet the needs of fish virus research. Development of additional cell lines would be useful for research in the fields of toxicology, genetics, cell physiology, and genomics (Li et al. 2010; Phelps et al. 2012). In the present study, three cell lines were developed from fin tissue from diploid, triploid, and tetraploid oriental weatherfish, and the susceptibilities of these cell lines to two important aquatic viruses were analyzed.
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Materials and methods Initiation of primary cell culture Visually healthy M. anguillicaudatus (10.00–20.24 g in weight) were collected from Hubei Honghu and transported to the laboratory. The fish were maintained in 3-l sterile, aerated water containing 1,000 IU/ml penicillin, and 1,000 lg/ml streptomycin for 24 h at room temperature (23–25 °C). They then were anaesthetized in 1 % benzyl alcohol, and fin tissue was removed. The fin tissue was first dipped in 10 % iodine for 15 min followed by antibiotic medium containing 500 IU/ml penicillin and 500 lg/ml streptomycin for 30 min. The tissue was washed twice in PBS and once in 5 % FBS-DMEM/F12 and then minced into small pieces (approximately 1 mm3 in size). The tissue fragments were inoculated into 25-cm2 cell culture flasks, and the flasks were incubated at 25 °C and 5 % CO2. After 24 h, 5 ml of growth medium was added to each flask, and it was replaced every 5 d. The medium was 20 % FBS-DMEM/F12 containing 10 lg/ml chondroitin sulfate, 10 lg/ml basic fibroblast growth factor (bFGF), and 20 lg/ml insulin-like growth factor I (IGF-I). After the formation of a monolayer, the initiation of primary cell culture was complete. Subculture When the cells formed a monolayer, the old medium was removed and 1 ml 0.25 % trypsin was added. After 30 s, the trypsin was removed, and the old medium was returned to the flask and the flask was knocked lightly. The cell suspension was divided between two new flasks in a volume ratio of 1:1, and new medium was added to bring the volume up to 5 ml. The flasks were incubated at 25 °C and 5 % CO2 until a monolayer of cells formed; then, this method was continued for subsequent passages. From primary culture to 30 generations, the medium contained chondroitin sulfate (10 lg/ml), bFGF (10 mg/ml), and IGF-I (20 ng/ml) in 20 % FBS-DMEM/F12. After 30 generations, the medium consisted of only 20 % FBS-DMEM/F12. Storage method Storage medium containing 20 % FBS, 60 % DMEM/ F12, and 20 % DMSO was prepared and stored
Fish Physiol Biochem
at 4 °C. Every ten generations the fin cells were dispersed with 0.25 % trypsin and resuspended in the old medium, centrifuged for 5 min (1,500 rpm/min), and then resuspended in 1 ml of ice-cold storage medium. The cell density was adjusted to 1.0 °C 9 106 cells/ml, dispensed into 2-ml plastic ampoules, and then kept at 4 °C for 30 min, -20 °C for 2 h, and then -80 °C overnight. They were then transferred into liquid nitrogen (-196 °C). The frozen cells were recovered from storage 60 days poststorage by thawing in water at 40 °C and then in a 25 °C water bath for 3 min. An equal amount of DMEM/F12 medium was added to the cells, and the supernatant was removed by centrifugation for 5 min (1,500 rpm/min). Cells were resuspended in 20 % FBS-DMEM/F12, transferred to a 25-cm2 cell culture flask, and medium was added to attain a volume of 5 ml. Cells were then seeded at 25 °C and 5 % CO2. After 24 h, the full amount of the culture medium was replaced with new medium. Cell viability was tested by counting using a hemocytometer after trypan blue staining, and survival was calculated as follows:
average cell density of three wells was determined using a hemocytometer after trypan blue staining. This procedure was repeated for 7 days. Counts were used to draw the growth curve and to calculate the population doubling time as follows: T ¼ t Lg2=LgðNt =N0 Þ; where Nt is the number of cells after t hours and N0 is the number of inoculated cells. Size of cells and nucleus At the 53rd passage, the three cell lines were trypsinized with 0.25 % trypsin. Cell diameter was measured using the ocular micrometer, and cells were counted using a hemocytometer after trypan blue staining. The nuclei could be clearly observed with inverted microscope, and the diameter of nuclei was directly measured using the ocular micrometer. The diameter of 100 cells and 100 nuclei of every cell line were measured. Microsatellite analysis
Survival ¼ number of live cells=total cells 100%
Chromosome analysis Cells from each cell line at the 50th and 60th passage were used for chromosome analysis. Colchicine (final concentration 1 lg/ml) was added to the cells, which then were incubated for 3–5 h in a 25 °C and 5 % CO2 incubator. The cells were trypsinized with 0.25 % trypsin and centrifuged for 5 min (1,500 rpm/min). The pellets were suspended in 1.5 ml of hypotonic medium (0.075 mol/l KCl) for 40 min, and the cells were fixed by placing them in Carnoy fixative for 15 min at -20 °C three times. The suspension was dropped onto glass slides, air-dried, and stained with Giemsa, then observed, and photographed under the microscope. Chromosome numbers were counted in more than 100 metaphase plates from both passages, and karyotype analysis was performed. Growth At the 50th passage, cells from the three cell lines (3.0 9 104-5.0 9 104 cells/ml) were seeded into 24well plates at 25 °C and 5 % CO2. Every 24 h, the
DNA was extracted using the phenol/chloroform method from the muscle of M. anguillicaudatus and Danio rerio, which were considered to be the positive and negative controls, respectively. DNA concentration was measured with a UV spectrophotometer (Beckman, DU650, USA), and concentrations were adjusted to 50 ng/ll for microsatellite analysis. The cultured cells were collected by centrifugation at 1,500 rpm for 5 min at 4 °C and then immersed in 250 ll DNA digestion buffer (10 mmol/l Tris–Cl, 50 mmol/l KCl, and 0.5 % Tween-20, pH 8.0) with proteinase K at 0.5 mg/ml at 55 °C for 3 h. DNA from the cell lysis solution was extracted with 250 ll of chloroform/isoamyl alcohol (24:1), and the sample was then used as the DNA template for microsatellite analysis. Two microsatellite loci (Mac9 and Mac50) were selected to analyze the genetic divergence of M. anguillicaudatus, D rerio, and the cell lines constructed from fin tissues of diploid, triploid, and tetraploid oriental weatherfish. Primer sequences for microsatellite markers were obtained from Zheng et al. (2012). PCR was performed in a volume of 25 ll containing 1.0 U of Taq DNA polymerase (TaKaRa, Dalian, China), 19 PCR buffer, 0.2 mM dNTP mix, 2 mM
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MgCl2, 1 lM of each primer, and 2.0 ul template DNA. PCR conditions were as follows: 4 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at proper annealing temperatures, 30 s at 72 °C; a final extension of 10 min at 72 °C. PCR products were separated using 2 % agarose gels sized using the DL2000 DNA ladder (TaKaRa).
Data processing Experimental data are given as mean ± standard deviation, and significance tests were conducted using SPSS16.0 (IBM, USA).
Results Viral susceptibility Cell lines The spring viremia of carp virus standard (SVCV-103) and piscine nodavirus were obtained from Fish Disease Laboratory, Dalian Ocean University. Piscine nodavirus was a temporary name which isolated from red sea beam (Pagrosomus major) in the same laboratory. Susceptibilities of the three cell lines to SVCV and piscine nodavirus were tested. The cells used to inoculate the virus were at passage 60–65. After removing the medium, 0.1 ml of virus suspension was inoculated into the cell culture in a culture flask and allowed to adsorb for 2 h. Maintenance medium containing 20 % FBS was then added to reach a volume of 5 ml. The cells were incubated at 25 °C with 5 % CO2. For the control, 0.1 ml DMEM/ F12 medium instead of virus suspension was used. We found that the cells were not susceptible to piscine nodavirus, so subsequent tests involved only SVCV. The three cell lines were seeded into 96-well plates and incubated at 25 °C with 5 % CO2 for 48 h. The SVCV virus suspension was tenfold diluted serially and inoculated into the cells (eight wells per dilution), which then were cultured at 25 °C with 5 %CO2. Cytopathic effects (CPEs) were observed daily under an inverted light microscope. The virus titer that resulted in a 50 % infection rate (TCID50) was calculated using the Reed–Muench formula (Reed and Muench 1938). After the cell lines were infected with the SVCV virus and cells showing CPEs accounted for about 80 % of the bottom area, the virus was collected in a freezing tube. The sample underwent freeze-thawing at -80 °C twice and was centrifuged at 15,000 rpm for 60 min. To test whether SVCV can be propagated in the DIMF, TRMF, and TEMF cell lines, the supernatants obtained from the three cell lines infected with SVCV were used to inoculate virus-free cell cultures in 96-well plates. CPEs then were observed after 5 days, and the virus titers were calculated.
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Primary cells migrating from fin tissue fragments of M. anguillicaudatus were observed at 2 days, and they formed a monolayer within 1 month. The cells were split at a ratio of 1:1. The three cell lines were passaged at intervals of 6–7 days. After ten generations, the speed of growth was high. The time required for a single layer of cells to cover the bottom of the flask was 4–5 days for DIMF, 3–4 days for TRMF, and 4–5 days for TEMF cells. After 30 generations, the medium used no longer contained bFGF, IGF-I, and chondroitin sulfate, and cell growth and division status were not affected. Thus, three fin tissue cell lines (DIMF, TRMF, and TEMF) of M. anguillicaudatus (Fig. 1) were successfully established, and they were preserved at the China Center for Type Culture Collection (CCTCC) numbered C2013109, C2013110, and C2013111. Cell preservation After cells were cryopreserved, morphology of DIMF, TRMF, and TEMF cells was homogeneous and fibroblast-like. Within 3–4 days after thawing, cells were present in a single layer and could continue to be passaged. Survival rates were 83.30 ± 1.12 % (n = 6), 84.85 ± 1.46 %, and 81.79 ± 1.37 %, respectively. Survival rates among the three groups did not differ significantly (P [ 0.05). Chromosome analysis Chromosome analysis of 100 DIMF, TRMF, and TEMF cells at the 50th passage revealed that the DIMF chromosome number ranged from 33 to 55, and cells with 50 chromosomes accounted for 68 % of the total number of cells (Fig. 2a). The TRMF chromosome number ranged from 54 to 77, and cells with 75 chromosomes accounted for 59 % of the total number
Fish Physiol Biochem
Fig. 1 Culture of DIMF (a), TRMF (b), and TEMF (c) cells of oriental weatherfish at passage 60. Scale bar 100 lm
number of cells
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(Fig. 3a, d); TRMF, 3n = 75, 15 m ? 6sm ? 54t, NF = 96 (Fig. 3b, e); and TEMF, 4n = 100, 20 m ? 8sm ? 72t, NF = 128 (Fig. 3c, f). At the 60th passage, the chromosome numbers of DIMF and TRMF cells were still predominantly 50 (52 %) and 75 (70 %). However, the chromosome number of TEMF cells ranged from 88 to 100, and cells with 96 chromosomes accounted for 26 % of the total number of cells (Fig. 4c). The karyotype analysis showed 4n = 96, 16 m ? 8sm ? 72t, NF = 120 (Fig. 5). Thus, compared with the cells at the 50th passage, a group of metacentric chromosomes (m) was missing.
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Fig. 2 Number distribution of chromosome in DIMF, TRMF, and TEMF cells at passage 50. a Number distribution of chromosomes in DIMF cells; b number distribution of chromosomes in TRMF cells; c number distribution of chromosomes in TEMF cells
of cells (Fig. 2b). The TEMF chromosome number ranged from 87 to 101, and cells with 100 chromosomes accounted for 54 % of the total number of cells (Fig. 2c). The karyotype analysis showed the following: DIMF, 2n = 50, 10 m ? 4sm ? 36t, NF = 64
Figure 6 shows the growth curve of DIMF, TRMF, and TEMF cells at passage 50 when cultured at 25 °C and 5 % CO2 in 20 % FBS-DMEM/F12. The DIMF cells’ incubation phase was 0–1 days, the logarithmic phase was 1–1.5 days, the stationary phase was 4–4.5 days, the decline phase was 5 days, and the population doubling time was 48.43 h. The TRMF cells’ incubation phase was 0–1 days, the logarithmic phase was 1–1.5 days, the stationary phase was 3–3.5 days, the decline phase was 5 days, and the population doubling time was 36.01 h. The TEMF cells’ incubation phase was 0–2 days, the logarithmic phase was 2–2.5 days, the stationary phase was 4–4.5 days, the decline phase was 5 days, and the population doubling time was 41.45 h. Size of cells and nucleus The cell nucleus diameters of 100 cells were measured, and volume of the cell nucleus was calculated using the equation of V = (4/3)pR3. The ratio of cell
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Fish Physiol Biochem Fig. 3 Chromosome and karyotype analysis of DIMF, TRMF, and TEMF cells at passage 50. a Chromosomes in DIMF cells; b chromosomes in TRMF cells; c chromosomes in TEMF cells. Scale bar 10 lm
nucleus diameter of DIMF, TRMF, and TEMF cells at passage 53 was 1:1.15:1.25. The ratio of nucleus volume was 1:1.53:1.97 (Table 1). Thus, these values differed significantly among the three cell line types (P \ 0.01). The diameters of 100 cells were measured, and the cell volume was calculated with the equation of V = (4/3)pR3. The ratio of cell diameter of DIMF, TRMF, and TEMF cells at passage 53 was 1:1.11:1.33. The ratio of volume was 1:1.37:2.37 (Table 2). Thus, these values differed significantly among the three cell line types (P \ 0.01). Microsatellite verification No products were amplified using DNA templates from Danio rerio at either microsatellite locus. Clear
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and single bands at *100 bp were detected from samples of M. anguillicaudatus muscle and the DIMF, TRMF, and TEMF cell lines at both loci. The band patterns and sizes for the three cell lines were the same as those in M. anguillicaudatus at the Mac9 and Mac50 loci (Figs. 7, 8). Viral susceptibility The susceptibility of the three cell lines to infection by two viruses, SVCV and piscine nodavirus, was evaluated based on morphological changes and CPEs. Significant CPEs were observed in the three cell lines infected with SVCV but not with piscine nodavirus. No CPEs were observed in uninfected lines (Fig. 9a, b, c). Significant CPEs caused by SVCV infection were observed in DIMF cells at 48 h post-inoculation
Fish Physiol Biochem
Fig. 4 Number distribution of chromosomes in TEMF cells at passage 60
Fig. 5 Chromosome and karyotype analysis of TEMF cells at passage 60. Scale bar 10 lm
cell number/(×10 4 )
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Fig. 6 Growth curve of DIMF, TRMF, and TEMF cells at passage 50 (incubated in FBS-DMEM/F12 at 25 °C and 5 % CO2). Cell number was determined using a hemocytometer after trypan blue staining
(Fig. (Fig. (Fig. cells
9d), in TRMF cells at 96 h post-inoculation 9e), and in TEMF cells at 24 h post-inoculation 9f). The 80 % monolayer of DIMF and TEMF was completely disintegrated after 4 days,
whereas this occurred after 7 days for TRMF cells. The SVCV virus titer was measured daily in the three cell lines, and the values are showed in Fig. 10. The viral titers in the three cell lines were increasing with the virus multiply, and there were no differences among three cell lines. The virus titer obtained using DRMF, TRMF, and TEMF cells reached 105.1, 104.3, and 105.4 TCID50/ml, respectively, within 5 days. These results showed that the SVCV virus was generated by all three cell lines and that the virus generated in these cells can infect normal cells.
Discussion Fin tissue is exposed to the external environment, and large amounts of bacteria adhere to the fin surface.
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Fish Physiol Biochem Table 1 Nucleus size in DIMF, TRMF, and TEMF cells at passage 53 Parameter
DIMF
Nuclear diameter (lm) 3
Nuclear volume (lm )
TRMF
9.99 ± 0.08A 521.97 ± 12.20
A
TEMF
11.5 ± 0.12B
DIMF:RMF:EMF
12.52 ± 0.14C B
800.11 ± 27.91
1,026.36 ± 37.83C
1:1.15:1.25 1:1.53:1.97
The different capital letters after each value in the same row indicate significant differences at P \ 0.01
Table 2 Size of DIMF, TRMF, and TEMF cells at passage 53 Parameter
Diploid
Cell diameter (lm) 3
Cell volume (lm )
11.2 ± 0.06
Triploid A
743.64 ± 10.94
A
Tetraploid B
DIMF:TRMF:TEMF C
12.4 ± 0.07
15.00 ± 0.04 B
1,020.31 ± 16.30
1:1.11:1.33 C
1,764.64 ± 14.44
1:1.37:2.37
The different capital letters after each value in the same row indicate significant differences at P \ 0.01
Fig. 7 SSR verification of species origination at locus Mac9 of three cell lines in M. anguillicaudatus. M, molecular weight marker; lanes 1, 4, and 7 negative control (muscle tissue of D.
rerio); lanes 2, 5, and 8 positive control (muscle tissue of M. anguillicaudatus); lane 3 DIMF; lane 6 TRMF; lane 9 TEMF
Fig. 8 SSR verification of species origination at locus Mac50 of three cell lines in M. anguillicaudatus. M, molecular weight marker; lanes 1, 4, and 7 negative control (muscle tissue of D.
rerio); lanes 2, 5, and 8 positive control (muscle tissue of M. anguillicaudatus); lane 3 DIMF; lane 6 TRMF; lane 9 TEMF
However, sterile culture is the key to successful culturing of cells. Thus, it is crucial that material used for primary culture is processed aseptically. In many studies, the sample tissue is rinsed with alcoholsoaked paper tissue or a double antibody solution (Fan
et al. 2009; Li et al. 2013), but this may not achieve complete sterilization. In this study, we used 10 % iodine to sterilize the samples. Povidone-iodine is a kind of amorphous iodine carried by surfactant solubilization. It is easy to use, allows slow and
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Fig. 9 Three kinds of culture cells and CPE induced by SVCV at 25 °C a DIMF, b TRMF, c TEMF, d DIMF at 48 h post-inoculation, e TRMF at 96 h post-inoculation, f TEMF cells at 24 h post-inoculation. Scale bar 100 lm
Fig. 10 Multiplication of SVCV in DIMF, TRMF, and TEMF cells at 25 °C (TCID50/mL)
sustained release of the active iodine, has long-lasting bactericidal characteristics, and is used in aquaculture widely. In our study, treatment with iodine and double antibiotic medium reduced the possibility of contamination in the primary culture, and it appeared to have no negative effect on the cells. In fish cell culture experiments, DMEM/F12, DMEM, or L-15 often is used as the culture medium. Based on the methods for Hexagrammidae’s fin, snout, and kidney (Li et al., 2013), we used DMEM/F12 medium containing 20 % FBS. Fan et al. (2009) used the same medium to
establish three cell lines from the Epinephlus fuscoguttatus, and Imajoh et al. (2007) used it to establish Pagrus major cell lines. In splenic cells from Epinephelus coioides and fin cells from Scophthalmus maximus, L-15 medium was successfully used for cell culture (Qin et al. 2006; Fan et al. 2007). Results from specific microsatellite amplification of two loci combined with positive and negative controls showed that the DIMF, TRMF, and TEMF cell lines originated from M. anguillicaudatus cells. At the 50th passage, the chromosome numbers were 50 for DIMF cells, 75 for TRMF cells, and 100 for TEMF cells, and the karyotype analysis revealed that they all had five groups of metacentric chromosomes, two groups of submetacentric chromosomes (sm), and 18 groups of telocentric chromosomes. These karyotype results agree with those previously reported for diploid, triploid, and tetraploid oriental weatherfish cells (Li et al. 2009). At the 60th passage, these characteristics of the diploid and triploid cell lines did not differ from those at the 50th passage. In contrast, the characteristic chromosome number of the tetraploid cells decreased to 96, and the chromosome inversion could be discovered; however, cell
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morphology did not change. Previous reports have shown that aneuploidy is a characteristic of cancer cells and that the formation of aneuploidy is associated with tetraploidy. Generally, tetraploidy develops first and then progresses to aneuploidy through unstable tetraploidy (Nguyen and Ravid 2006; Olaharski et al. 2006). This finding may prove useful for the future exploration of tumorigenesis mechanisms and for studies of why aneuploidy occurs. In many human carcinomas, cells with tetraploid DNA content arise as an early step in tumorigenesis and precede the formation of aneuploid cells (Margolis et al. 2003). Aneuploidy and chromosomal instability in turn are characteristics of the great majority of human cancers (Cahill et al. 1999) and are linked to the progressive development of high-grade, invasive tumors. Research has shown that the size of the fish erythrocyte nucleus is proportional to the chromosome number, and the cell diameter increases proportionally with the size of the cell nucleus (Gao et al. 2007). Therefore, cell and nucleus size can be used as an index for ploidy assessment (Gao et al. 2007). For example, the volume of the nucleus of triploid silver carp erythrocytes is 1.63 times greater than that of diploid silver carp erythrocytes (Zhu et al. 1992), and the volume of the nucleus of triploid large yellow croaker erythrocytes is 1.70 times greater than that of diploid ells (Lin and Wu 2004). The volume of the nucleus of triploid loach erythrocytes is 1.77 times greater than that of diploid erythrocytes (Kim et al. 1994), and the volume of the nucleus of tetraploid steelhead erythrocytes is 2.05 times greater than that of diploid cells (Refstie 1981). The ratio of cell volume is another useful proxy. The volume of triploid loach erythrocytes is 1.52 times greater than that of diploid erythrocytes (Kim et al. 1994), and the volume of tetraploid steelhead erythrocytes is 1.91 times greater than that of diploid cells (Refstie 1981). In our experiment, the ratio of the volume of diploid, triploid, and tetraploid nuclei from oriental weatherfish was 1:1.53:1.97, and the ratio of cell volume was 1:1.37:2.37. We found that triploid cells had the shorter population doubling time and diploid cells had the longer population doubling time. Our results indicate that triploid cells grow better than diploid or tetraploid cells. Kim et al. (1994) reported that triploid M. anguillicaudatus cells had higher average weight and growth speed than diploid cells, but tetraploid cells were not analyzed in this study. These characteristics
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appear to be related to the size of triploid cells and the population doubling time. Cytological studies would be helpful to identify the mechanisms responsible for the superior growth and disease resistance observed in polyploid fishes (Gui and Zhou 2010). The polyploid cell line established in this research will provide a platform for cytological studies aimed at understanding growth trends in fish with different ploidy values. The cell lines established from the oriental weatherfish will be useful for SVCV but not viral nervous necrosis virus (VNNC). Cultured cells are used mainly for the isolation and identification of pathogens, propagation of viruses, vaccine preparation, and epidemiological studies. The continuous culture of cells used to isolate fish viruses has become an important application that has allowed the field of fish virology to develop rapidly (Lai et al. 2003; Nishizawa et al. 2008). Spring viremia of carpis is a serious hemorrhagic disease of carp and certain other fish species that is caused by the SVCV. SVCV is a rhabdovirus (Ahne et al. 2002) that was isolated by Fijan et al. (1971). Viral nervous necrosis (VNN) is a virus that affects popular marine fish throughout the world, and it is caused by piscine nodavirus; however, fewer cell lines sensitive to this pathogen are available (Yoshikoshi and Inoue 1990; Munday et al. 1992). Under adverse environmental conditions, triploid and diploid have similar resistance (Kusuda et al. 1991; Svobodova et al. 2001). In addition, triploid fish is no less than their diploid parents in resistance to infectious hematopoietic necrosis virus and susceptible to vaccine terms (Maxime 2008). In our study, we tested the susceptibility of DIMF, TRMF, and TEMF cell lines to two different pathogens. The three cell lines were susceptible to SVCV but not to piscine nodavirus. This may be because most hosts of piscine nodavirus are marine fish. Significantly, CPEs were observed in all three cell lines, and over time, many of the cells disintegrated. TEMF cells were more easily infected by SVCV than TRMF cells. The CPE results also indicated that SVCV can be propagated in the DIMF, TRMF, and TEMF cell lines, which confirms their potential as a powerful tool for isolating and identifying infectious viruses. Acknowledgments This work was funded by the National Natural Science Foundation of China (31272650). The authors thank Dr. Li Qiang and Jiang Zedong for providing the SVC virus and piscine nodavirus.
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Establishment and characterization of fin cell lines from diploid, triploid, and tetraploid oriental weatherfish (Misgurnus anguillicaudatus) Xia Li • Chen Ma • Yan-Jie Qin • Ya-Juan Li Di Wu • Li-Wen Bai • Ai-Jun Pei
•
Received: 25 July 2014 / Accepted: 15 December 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Continuous fin cell lines from diploid, triploid, and tetraploid oriental weatherfish, Misgurnus anguillicaudatus, were established and characterized. The cell lines, designated DIMF, TRMF, and TEMF, respectively, were subcultured more than 80 times since initiation in October 2012 and were preserved at the China Center for Type Culture Collection as sample numbers C2013109, C2013110, C2013111, respectively. The cell lines consist predominantly of fibroblast-like cells. At the 50th passage, the population doubling times were 48.43 h (DIMF), 36.01 h (TRMF), and 41.45 h (TEMF). Cell survival rate of these three kinds of cells was 80.88 ± 1.38, 84.48 ± 1.13, and 81.57 ± 1.28 %, respectively, when recovered after storage in liquid nitrogen for 60 days at the 40th passage. The chromosome numbers measured from 100 metaphase plates at the 50th passage were 2n = 50 (68 %), 3n = 75 (59 %), and 4n = 100 (54 %) for DIMF, TRMF, and TEMF cells, respectively. At the 60th
passage, the chromosome numbers for DIMF and TRMF cells were still 50 (52 %) and 75 (70 %), but the chromosome number for TEMF cells ranged from 88 to 100; a chromosome number of 96 accounted for 26 % of the cells, and the karyotype analysis showed 4n = 96, 16 m ? 8sm ? 72t, NF = 120; thus, compared with cells at the 50th passage, a group of metacentric chromosomes was missing. Microsatellite marker analysis was conducted using DIMF, TRMF, and TEMF cells and muscle tissue of oriental weatherfish, which confirmed that the three cell lines established in this study were from oriental weatherfish. The cell lines were exposed to two fish viruses to determine their susceptibility to infection; they were susceptible to spring viremia of carp virus but not to piscine nodavirus. Establishment of fin cell lines from different ploidy oriental weatherfish increases the existing number of fish cell lines available for research, and it provides a model for investigating the mechanisms of growth and genetics in polyploid fish.
X. Li (&) C. Ma Y.-J. Qin D. Wu A.-J. Pei Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation in Liaoning Province, Dalian Ocean University, Heishijiao Street 52, Dalian 116023, People’s Republic of China e-mail: [email protected]
Keywords Misgurnus anguillicaudatus Cell line Polyploidy Karyotype Virus susceptibility
Y.-J. Li L.-W. Bai Key Laboratory of Mariculture, Agriculture Ministry, PRC, Dalian Ocean University, Heishijiao Street 52, Dalian 116023, People’s Republic of China
Introduction The oriental weatherfish (Misgurnus anguillicaudatus) is an economically important freshwater fish in
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China. In addition to diploid weatherfish, triploid and tetraploid specimens exist in the natural world (Arm 2003; Itono et al. 2006, 2007). Thus, this species is a good model for biological and genetics research. Previous studies have focused on methods to produce polyploidy, identify polyploidy, and determine growth characteristics of polyploidy fish (Kim et al. 1994; Gao et al. 2007; Wu et al. 2007; Nan et al. 2008). Since the first teleost cell line [rainbow trout (Salmo gairdneri) gonadal cell line RTG-2] was established in 1962 (Wolf and Quimby 1962), 275 other fish cell lines have been established, including 175 freshwater or anadromous migratory fish cell lines and 100 marine fish cell lines (Zhang and Chen 2011; Lakra et al. 2011). These cell lines were derived mainly from tissues such as fin, spleen, and kidney (Ganassin and Bols 1998; Chi et al. 1999; Kang et al. 2003; Imajoh et al. 2007; Xing et al. 2009; Zheng et al. 2012). Among them, fin tissue has proven to be useful for the development of cell lines (Wang et al. 2004; Fan et al. 2007; Zhou et al. 2008). Sterile culture is the foundation of the culturing of fish cells, every aspect of fish cell culture should be performed under sterile conditions, so the first step of culture is the fish or the tissue is rinsed with alcohol, antibiotics, and other disinfectant (Fu et al. 2012; Guo 2010). The use of established fish cell lines has provided much information in studies relating to fish developmental biology (Clem et al. 1996), virology (Ruiz et al. 2009), transgenic applications (Zheng et al. 2012), and others (Hashimoto et al. 2008; Lakra et al. 2011; Zhou et al. 2003). Viral disease is a common problem in fish farming. Some of the freshwater fish cell lines have been shown to be sensitive to certain fish viruses (Yan et al. 2011; Lei et al. 2012; Xiao et al. 2012; Zhang and Gui 2012; Zhu et al. 2013), so they can be used to study disease pathology. However, many types of viruses affect fish, and there are not enough existing cell lines to meet the needs of fish virus research. Development of additional cell lines would be useful for research in the fields of toxicology, genetics, cell physiology, and genomics (Li et al. 2010; Phelps et al. 2012). In the present study, three cell lines were developed from fin tissue from diploid, triploid, and tetraploid oriental weatherfish, and the susceptibilities of these cell lines to two important aquatic viruses were analyzed.
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Materials and methods Initiation of primary cell culture Visually healthy M. anguillicaudatus (10.00–20.24 g in weight) were collected from Hubei Honghu and transported to the laboratory. The fish were maintained in 3-l sterile, aerated water containing 1,000 IU/ml penicillin, and 1,000 lg/ml streptomycin for 24 h at room temperature (23–25 °C). They then were anaesthetized in 1 % benzyl alcohol, and fin tissue was removed. The fin tissue was first dipped in 10 % iodine for 15 min followed by antibiotic medium containing 500 IU/ml penicillin and 500 lg/ml streptomycin for 30 min. The tissue was washed twice in PBS and once in 5 % FBS-DMEM/F12 and then minced into small pieces (approximately 1 mm3 in size). The tissue fragments were inoculated into 25-cm2 cell culture flasks, and the flasks were incubated at 25 °C and 5 % CO2. After 24 h, 5 ml of growth medium was added to each flask, and it was replaced every 5 d. The medium was 20 % FBS-DMEM/F12 containing 10 lg/ml chondroitin sulfate, 10 lg/ml basic fibroblast growth factor (bFGF), and 20 lg/ml insulin-like growth factor I (IGF-I). After the formation of a monolayer, the initiation of primary cell culture was complete. Subculture When the cells formed a monolayer, the old medium was removed and 1 ml 0.25 % trypsin was added. After 30 s, the trypsin was removed, and the old medium was returned to the flask and the flask was knocked lightly. The cell suspension was divided between two new flasks in a volume ratio of 1:1, and new medium was added to bring the volume up to 5 ml. The flasks were incubated at 25 °C and 5 % CO2 until a monolayer of cells formed; then, this method was continued for subsequent passages. From primary culture to 30 generations, the medium contained chondroitin sulfate (10 lg/ml), bFGF (10 mg/ml), and IGF-I (20 ng/ml) in 20 % FBS-DMEM/F12. After 30 generations, the medium consisted of only 20 % FBS-DMEM/F12. Storage method Storage medium containing 20 % FBS, 60 % DMEM/ F12, and 20 % DMSO was prepared and stored
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at 4 °C. Every ten generations the fin cells were dispersed with 0.25 % trypsin and resuspended in the old medium, centrifuged for 5 min (1,500 rpm/min), and then resuspended in 1 ml of ice-cold storage medium. The cell density was adjusted to 1.0 °C 9 106 cells/ml, dispensed into 2-ml plastic ampoules, and then kept at 4 °C for 30 min, -20 °C for 2 h, and then -80 °C overnight. They were then transferred into liquid nitrogen (-196 °C). The frozen cells were recovered from storage 60 days poststorage by thawing in water at 40 °C and then in a 25 °C water bath for 3 min. An equal amount of DMEM/F12 medium was added to the cells, and the supernatant was removed by centrifugation for 5 min (1,500 rpm/min). Cells were resuspended in 20 % FBS-DMEM/F12, transferred to a 25-cm2 cell culture flask, and medium was added to attain a volume of 5 ml. Cells were then seeded at 25 °C and 5 % CO2. After 24 h, the full amount of the culture medium was replaced with new medium. Cell viability was tested by counting using a hemocytometer after trypan blue staining, and survival was calculated as follows:
average cell density of three wells was determined using a hemocytometer after trypan blue staining. This procedure was repeated for 7 days. Counts were used to draw the growth curve and to calculate the population doubling time as follows: T ¼ t Lg2=LgðNt =N0 Þ; where Nt is the number of cells after t hours and N0 is the number of inoculated cells. Size of cells and nucleus At the 53rd passage, the three cell lines were trypsinized with 0.25 % trypsin. Cell diameter was measured using the ocular micrometer, and cells were counted using a hemocytometer after trypan blue staining. The nuclei could be clearly observed with inverted microscope, and the diameter of nuclei was directly measured using the ocular micrometer. The diameter of 100 cells and 100 nuclei of every cell line were measured. Microsatellite analysis
Survival ¼ number of live cells=total cells 100%
Chromosome analysis Cells from each cell line at the 50th and 60th passage were used for chromosome analysis. Colchicine (final concentration 1 lg/ml) was added to the cells, which then were incubated for 3–5 h in a 25 °C and 5 % CO2 incubator. The cells were trypsinized with 0.25 % trypsin and centrifuged for 5 min (1,500 rpm/min). The pellets were suspended in 1.5 ml of hypotonic medium (0.075 mol/l KCl) for 40 min, and the cells were fixed by placing them in Carnoy fixative for 15 min at -20 °C three times. The suspension was dropped onto glass slides, air-dried, and stained with Giemsa, then observed, and photographed under the microscope. Chromosome numbers were counted in more than 100 metaphase plates from both passages, and karyotype analysis was performed. Growth At the 50th passage, cells from the three cell lines (3.0 9 104-5.0 9 104 cells/ml) were seeded into 24well plates at 25 °C and 5 % CO2. Every 24 h, the
DNA was extracted using the phenol/chloroform method from the muscle of M. anguillicaudatus and Danio rerio, which were considered to be the positive and negative controls, respectively. DNA concentration was measured with a UV spectrophotometer (Beckman, DU650, USA), and concentrations were adjusted to 50 ng/ll for microsatellite analysis. The cultured cells were collected by centrifugation at 1,500 rpm for 5 min at 4 °C and then immersed in 250 ll DNA digestion buffer (10 mmol/l Tris–Cl, 50 mmol/l KCl, and 0.5 % Tween-20, pH 8.0) with proteinase K at 0.5 mg/ml at 55 °C for 3 h. DNA from the cell lysis solution was extracted with 250 ll of chloroform/isoamyl alcohol (24:1), and the sample was then used as the DNA template for microsatellite analysis. Two microsatellite loci (Mac9 and Mac50) were selected to analyze the genetic divergence of M. anguillicaudatus, D rerio, and the cell lines constructed from fin tissues of diploid, triploid, and tetraploid oriental weatherfish. Primer sequences for microsatellite markers were obtained from Zheng et al. (2012). PCR was performed in a volume of 25 ll containing 1.0 U of Taq DNA polymerase (TaKaRa, Dalian, China), 19 PCR buffer, 0.2 mM dNTP mix, 2 mM
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MgCl2, 1 lM of each primer, and 2.0 ul template DNA. PCR conditions were as follows: 4 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at proper annealing temperatures, 30 s at 72 °C; a final extension of 10 min at 72 °C. PCR products were separated using 2 % agarose gels sized using the DL2000 DNA ladder (TaKaRa).
Data processing Experimental data are given as mean ± standard deviation, and significance tests were conducted using SPSS16.0 (IBM, USA).
Results Viral susceptibility Cell lines The spring viremia of carp virus standard (SVCV-103) and piscine nodavirus were obtained from Fish Disease Laboratory, Dalian Ocean University. Piscine nodavirus was a temporary name which isolated from red sea beam (Pagrosomus major) in the same laboratory. Susceptibilities of the three cell lines to SVCV and piscine nodavirus were tested. The cells used to inoculate the virus were at passage 60–65. After removing the medium, 0.1 ml of virus suspension was inoculated into the cell culture in a culture flask and allowed to adsorb for 2 h. Maintenance medium containing 20 % FBS was then added to reach a volume of 5 ml. The cells were incubated at 25 °C with 5 % CO2. For the control, 0.1 ml DMEM/ F12 medium instead of virus suspension was used. We found that the cells were not susceptible to piscine nodavirus, so subsequent tests involved only SVCV. The three cell lines were seeded into 96-well plates and incubated at 25 °C with 5 % CO2 for 48 h. The SVCV virus suspension was tenfold diluted serially and inoculated into the cells (eight wells per dilution), which then were cultured at 25 °C with 5 %CO2. Cytopathic effects (CPEs) were observed daily under an inverted light microscope. The virus titer that resulted in a 50 % infection rate (TCID50) was calculated using the Reed–Muench formula (Reed and Muench 1938). After the cell lines were infected with the SVCV virus and cells showing CPEs accounted for about 80 % of the bottom area, the virus was collected in a freezing tube. The sample underwent freeze-thawing at -80 °C twice and was centrifuged at 15,000 rpm for 60 min. To test whether SVCV can be propagated in the DIMF, TRMF, and TEMF cell lines, the supernatants obtained from the three cell lines infected with SVCV were used to inoculate virus-free cell cultures in 96-well plates. CPEs then were observed after 5 days, and the virus titers were calculated.
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Primary cells migrating from fin tissue fragments of M. anguillicaudatus were observed at 2 days, and they formed a monolayer within 1 month. The cells were split at a ratio of 1:1. The three cell lines were passaged at intervals of 6–7 days. After ten generations, the speed of growth was high. The time required for a single layer of cells to cover the bottom of the flask was 4–5 days for DIMF, 3–4 days for TRMF, and 4–5 days for TEMF cells. After 30 generations, the medium used no longer contained bFGF, IGF-I, and chondroitin sulfate, and cell growth and division status were not affected. Thus, three fin tissue cell lines (DIMF, TRMF, and TEMF) of M. anguillicaudatus (Fig. 1) were successfully established, and they were preserved at the China Center for Type Culture Collection (CCTCC) numbered C2013109, C2013110, and C2013111. Cell preservation After cells were cryopreserved, morphology of DIMF, TRMF, and TEMF cells was homogeneous and fibroblast-like. Within 3–4 days after thawing, cells were present in a single layer and could continue to be passaged. Survival rates were 83.30 ± 1.12 % (n = 6), 84.85 ± 1.46 %, and 81.79 ± 1.37 %, respectively. Survival rates among the three groups did not differ significantly (P [ 0.05). Chromosome analysis Chromosome analysis of 100 DIMF, TRMF, and TEMF cells at the 50th passage revealed that the DIMF chromosome number ranged from 33 to 55, and cells with 50 chromosomes accounted for 68 % of the total number of cells (Fig. 2a). The TRMF chromosome number ranged from 54 to 77, and cells with 75 chromosomes accounted for 59 % of the total number
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Fig. 1 Culture of DIMF (a), TRMF (b), and TEMF (c) cells of oriental weatherfish at passage 60. Scale bar 100 lm
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(Fig. 3a, d); TRMF, 3n = 75, 15 m ? 6sm ? 54t, NF = 96 (Fig. 3b, e); and TEMF, 4n = 100, 20 m ? 8sm ? 72t, NF = 128 (Fig. 3c, f). At the 60th passage, the chromosome numbers of DIMF and TRMF cells were still predominantly 50 (52 %) and 75 (70 %). However, the chromosome number of TEMF cells ranged from 88 to 100, and cells with 96 chromosomes accounted for 26 % of the total number of cells (Fig. 4c). The karyotype analysis showed 4n = 96, 16 m ? 8sm ? 72t, NF = 120 (Fig. 5). Thus, compared with the cells at the 50th passage, a group of metacentric chromosomes (m) was missing.
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Fig. 2 Number distribution of chromosome in DIMF, TRMF, and TEMF cells at passage 50. a Number distribution of chromosomes in DIMF cells; b number distribution of chromosomes in TRMF cells; c number distribution of chromosomes in TEMF cells
of cells (Fig. 2b). The TEMF chromosome number ranged from 87 to 101, and cells with 100 chromosomes accounted for 54 % of the total number of cells (Fig. 2c). The karyotype analysis showed the following: DIMF, 2n = 50, 10 m ? 4sm ? 36t, NF = 64
Figure 6 shows the growth curve of DIMF, TRMF, and TEMF cells at passage 50 when cultured at 25 °C and 5 % CO2 in 20 % FBS-DMEM/F12. The DIMF cells’ incubation phase was 0–1 days, the logarithmic phase was 1–1.5 days, the stationary phase was 4–4.5 days, the decline phase was 5 days, and the population doubling time was 48.43 h. The TRMF cells’ incubation phase was 0–1 days, the logarithmic phase was 1–1.5 days, the stationary phase was 3–3.5 days, the decline phase was 5 days, and the population doubling time was 36.01 h. The TEMF cells’ incubation phase was 0–2 days, the logarithmic phase was 2–2.5 days, the stationary phase was 4–4.5 days, the decline phase was 5 days, and the population doubling time was 41.45 h. Size of cells and nucleus The cell nucleus diameters of 100 cells were measured, and volume of the cell nucleus was calculated using the equation of V = (4/3)pR3. The ratio of cell
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Fish Physiol Biochem Fig. 3 Chromosome and karyotype analysis of DIMF, TRMF, and TEMF cells at passage 50. a Chromosomes in DIMF cells; b chromosomes in TRMF cells; c chromosomes in TEMF cells. Scale bar 10 lm
nucleus diameter of DIMF, TRMF, and TEMF cells at passage 53 was 1:1.15:1.25. The ratio of nucleus volume was 1:1.53:1.97 (Table 1). Thus, these values differed significantly among the three cell line types (P \ 0.01). The diameters of 100 cells were measured, and the cell volume was calculated with the equation of V = (4/3)pR3. The ratio of cell diameter of DIMF, TRMF, and TEMF cells at passage 53 was 1:1.11:1.33. The ratio of volume was 1:1.37:2.37 (Table 2). Thus, these values differed significantly among the three cell line types (P \ 0.01). Microsatellite verification No products were amplified using DNA templates from Danio rerio at either microsatellite locus. Clear
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and single bands at *100 bp were detected from samples of M. anguillicaudatus muscle and the DIMF, TRMF, and TEMF cell lines at both loci. The band patterns and sizes for the three cell lines were the same as those in M. anguillicaudatus at the Mac9 and Mac50 loci (Figs. 7, 8). Viral susceptibility The susceptibility of the three cell lines to infection by two viruses, SVCV and piscine nodavirus, was evaluated based on morphological changes and CPEs. Significant CPEs were observed in the three cell lines infected with SVCV but not with piscine nodavirus. No CPEs were observed in uninfected lines (Fig. 9a, b, c). Significant CPEs caused by SVCV infection were observed in DIMF cells at 48 h post-inoculation
Fish Physiol Biochem
Fig. 4 Number distribution of chromosomes in TEMF cells at passage 60
Fig. 5 Chromosome and karyotype analysis of TEMF cells at passage 60. Scale bar 10 lm
cell number/(×10 4 )
20
diploid triploid
15
tetraploid
10 5 0
0
1
2
3
4
5
6
7
time/d
Fig. 6 Growth curve of DIMF, TRMF, and TEMF cells at passage 50 (incubated in FBS-DMEM/F12 at 25 °C and 5 % CO2). Cell number was determined using a hemocytometer after trypan blue staining
(Fig. (Fig. (Fig. cells
9d), in TRMF cells at 96 h post-inoculation 9e), and in TEMF cells at 24 h post-inoculation 9f). The 80 % monolayer of DIMF and TEMF was completely disintegrated after 4 days,
whereas this occurred after 7 days for TRMF cells. The SVCV virus titer was measured daily in the three cell lines, and the values are showed in Fig. 10. The viral titers in the three cell lines were increasing with the virus multiply, and there were no differences among three cell lines. The virus titer obtained using DRMF, TRMF, and TEMF cells reached 105.1, 104.3, and 105.4 TCID50/ml, respectively, within 5 days. These results showed that the SVCV virus was generated by all three cell lines and that the virus generated in these cells can infect normal cells.
Discussion Fin tissue is exposed to the external environment, and large amounts of bacteria adhere to the fin surface.
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Fish Physiol Biochem Table 1 Nucleus size in DIMF, TRMF, and TEMF cells at passage 53 Parameter
DIMF
Nuclear diameter (lm) 3
Nuclear volume (lm )
TRMF
9.99 ± 0.08A 521.97 ± 12.20
A
TEMF
11.5 ± 0.12B
DIMF:RMF:EMF
12.52 ± 0.14C B
800.11 ± 27.91
1,026.36 ± 37.83C
1:1.15:1.25 1:1.53:1.97
The different capital letters after each value in the same row indicate significant differences at P \ 0.01
Table 2 Size of DIMF, TRMF, and TEMF cells at passage 53 Parameter
Diploid
Cell diameter (lm) 3
Cell volume (lm )
11.2 ± 0.06
Triploid A
743.64 ± 10.94
A
Tetraploid B
DIMF:TRMF:TEMF C
12.4 ± 0.07
15.00 ± 0.04 B
1,020.31 ± 16.30
1:1.11:1.33 C
1,764.64 ± 14.44
1:1.37:2.37
The different capital letters after each value in the same row indicate significant differences at P \ 0.01
Fig. 7 SSR verification of species origination at locus Mac9 of three cell lines in M. anguillicaudatus. M, molecular weight marker; lanes 1, 4, and 7 negative control (muscle tissue of D.
rerio); lanes 2, 5, and 8 positive control (muscle tissue of M. anguillicaudatus); lane 3 DIMF; lane 6 TRMF; lane 9 TEMF
Fig. 8 SSR verification of species origination at locus Mac50 of three cell lines in M. anguillicaudatus. M, molecular weight marker; lanes 1, 4, and 7 negative control (muscle tissue of D.
rerio); lanes 2, 5, and 8 positive control (muscle tissue of M. anguillicaudatus); lane 3 DIMF; lane 6 TRMF; lane 9 TEMF
However, sterile culture is the key to successful culturing of cells. Thus, it is crucial that material used for primary culture is processed aseptically. In many studies, the sample tissue is rinsed with alcoholsoaked paper tissue or a double antibody solution (Fan
et al. 2009; Li et al. 2013), but this may not achieve complete sterilization. In this study, we used 10 % iodine to sterilize the samples. Povidone-iodine is a kind of amorphous iodine carried by surfactant solubilization. It is easy to use, allows slow and
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Fish Physiol Biochem
Fig. 9 Three kinds of culture cells and CPE induced by SVCV at 25 °C a DIMF, b TRMF, c TEMF, d DIMF at 48 h post-inoculation, e TRMF at 96 h post-inoculation, f TEMF cells at 24 h post-inoculation. Scale bar 100 lm
Fig. 10 Multiplication of SVCV in DIMF, TRMF, and TEMF cells at 25 °C (TCID50/mL)
sustained release of the active iodine, has long-lasting bactericidal characteristics, and is used in aquaculture widely. In our study, treatment with iodine and double antibiotic medium reduced the possibility of contamination in the primary culture, and it appeared to have no negative effect on the cells. In fish cell culture experiments, DMEM/F12, DMEM, or L-15 often is used as the culture medium. Based on the methods for Hexagrammidae’s fin, snout, and kidney (Li et al., 2013), we used DMEM/F12 medium containing 20 % FBS. Fan et al. (2009) used the same medium to
establish three cell lines from the Epinephlus fuscoguttatus, and Imajoh et al. (2007) used it to establish Pagrus major cell lines. In splenic cells from Epinephelus coioides and fin cells from Scophthalmus maximus, L-15 medium was successfully used for cell culture (Qin et al. 2006; Fan et al. 2007). Results from specific microsatellite amplification of two loci combined with positive and negative controls showed that the DIMF, TRMF, and TEMF cell lines originated from M. anguillicaudatus cells. At the 50th passage, the chromosome numbers were 50 for DIMF cells, 75 for TRMF cells, and 100 for TEMF cells, and the karyotype analysis revealed that they all had five groups of metacentric chromosomes, two groups of submetacentric chromosomes (sm), and 18 groups of telocentric chromosomes. These karyotype results agree with those previously reported for diploid, triploid, and tetraploid oriental weatherfish cells (Li et al. 2009). At the 60th passage, these characteristics of the diploid and triploid cell lines did not differ from those at the 50th passage. In contrast, the characteristic chromosome number of the tetraploid cells decreased to 96, and the chromosome inversion could be discovered; however, cell
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Fish Physiol Biochem
morphology did not change. Previous reports have shown that aneuploidy is a characteristic of cancer cells and that the formation of aneuploidy is associated with tetraploidy. Generally, tetraploidy develops first and then progresses to aneuploidy through unstable tetraploidy (Nguyen and Ravid 2006; Olaharski et al. 2006). This finding may prove useful for the future exploration of tumorigenesis mechanisms and for studies of why aneuploidy occurs. In many human carcinomas, cells with tetraploid DNA content arise as an early step in tumorigenesis and precede the formation of aneuploid cells (Margolis et al. 2003). Aneuploidy and chromosomal instability in turn are characteristics of the great majority of human cancers (Cahill et al. 1999) and are linked to the progressive development of high-grade, invasive tumors. Research has shown that the size of the fish erythrocyte nucleus is proportional to the chromosome number, and the cell diameter increases proportionally with the size of the cell nucleus (Gao et al. 2007). Therefore, cell and nucleus size can be used as an index for ploidy assessment (Gao et al. 2007). For example, the volume of the nucleus of triploid silver carp erythrocytes is 1.63 times greater than that of diploid silver carp erythrocytes (Zhu et al. 1992), and the volume of the nucleus of triploid large yellow croaker erythrocytes is 1.70 times greater than that of diploid ells (Lin and Wu 2004). The volume of the nucleus of triploid loach erythrocytes is 1.77 times greater than that of diploid erythrocytes (Kim et al. 1994), and the volume of the nucleus of tetraploid steelhead erythrocytes is 2.05 times greater than that of diploid cells (Refstie 1981). The ratio of cell volume is another useful proxy. The volume of triploid loach erythrocytes is 1.52 times greater than that of diploid erythrocytes (Kim et al. 1994), and the volume of tetraploid steelhead erythrocytes is 1.91 times greater than that of diploid cells (Refstie 1981). In our experiment, the ratio of the volume of diploid, triploid, and tetraploid nuclei from oriental weatherfish was 1:1.53:1.97, and the ratio of cell volume was 1:1.37:2.37. We found that triploid cells had the shorter population doubling time and diploid cells had the longer population doubling time. Our results indicate that triploid cells grow better than diploid or tetraploid cells. Kim et al. (1994) reported that triploid M. anguillicaudatus cells had higher average weight and growth speed than diploid cells, but tetraploid cells were not analyzed in this study. These characteristics
123
appear to be related to the size of triploid cells and the population doubling time. Cytological studies would be helpful to identify the mechanisms responsible for the superior growth and disease resistance observed in polyploid fishes (Gui and Zhou 2010). The polyploid cell line established in this research will provide a platform for cytological studies aimed at understanding growth trends in fish with different ploidy values. The cell lines established from the oriental weatherfish will be useful for SVCV but not viral nervous necrosis virus (VNNC). Cultured cells are used mainly for the isolation and identification of pathogens, propagation of viruses, vaccine preparation, and epidemiological studies. The continuous culture of cells used to isolate fish viruses has become an important application that has allowed the field of fish virology to develop rapidly (Lai et al. 2003; Nishizawa et al. 2008). Spring viremia of carpis is a serious hemorrhagic disease of carp and certain other fish species that is caused by the SVCV. SVCV is a rhabdovirus (Ahne et al. 2002) that was isolated by Fijan et al. (1971). Viral nervous necrosis (VNN) is a virus that affects popular marine fish throughout the world, and it is caused by piscine nodavirus; however, fewer cell lines sensitive to this pathogen are available (Yoshikoshi and Inoue 1990; Munday et al. 1992). Under adverse environmental conditions, triploid and diploid have similar resistance (Kusuda et al. 1991; Svobodova et al. 2001). In addition, triploid fish is no less than their diploid parents in resistance to infectious hematopoietic necrosis virus and susceptible to vaccine terms (Maxime 2008). In our study, we tested the susceptibility of DIMF, TRMF, and TEMF cell lines to two different pathogens. The three cell lines were susceptible to SVCV but not to piscine nodavirus. This may be because most hosts of piscine nodavirus are marine fish. Significantly, CPEs were observed in all three cell lines, and over time, many of the cells disintegrated. TEMF cells were more easily infected by SVCV than TRMF cells. The CPE results also indicated that SVCV can be propagated in the DIMF, TRMF, and TEMF cell lines, which confirms their potential as a powerful tool for isolating and identifying infectious viruses. Acknowledgments This work was funded by the National Natural Science Foundation of China (31272650). The authors thank Dr. Li Qiang and Jiang Zedong for providing the SVC virus and piscine nodavirus.
Fish Physiol Biochem
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