• Fisheries and Aquatic Sciences
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• v.16 no.3
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• pp.177-185
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• 2013

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.65-72
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• 2020

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

• Journal of Microbiology
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• v.34 no.3
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• pp.263-263
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• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in CHSE, gradual decrease in $[Ca^{2+}]_i$ in FHM, and no significant change in RTG cells. The degree of $[Ca^{2+}]_i$ increase or decrease was dependent on the amount of infectious virus, and these $[Ca^{2+}]_i$ variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in $[Ca^{2+}]_i$ was not observed. Thus, infectivity of IHNV appears to correlate with changes in $[Ca^{2+}]_i$ in virus-infected cells. These IHNV-induced $[Ca^{2+}]_i$ changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced $Ca^{2+}$ variations were more related with protein synthesis than RNA synthesis. Various $Ca^{2+}$ related drugs were used in search for the mechanisms of the $[Ca^{2+}]_i$, changes following IHNV infection of CHSE cells. Decreasing extracellular $Ca^{2+}$ concentration or blocking $Ca^{2+}$ influx from extracellular media inhibited the IHNV-induced increase in $[Ca^{2+}]_i$, in CHSE cells. Similar results were obtained with intracellular $Ca^{2+}$ blockers. Thus it is suggested that both the extracellular and the intracellular $Ca^{2+}$ sources are important in IHNV-induced $[Ca^{2+}]_i$ increase in CHSE cells.

• Journal of Microbiology
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• v.34 no.3
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• pp.253-269
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• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free Ca$\^$2+/ concentration ([Ca$\^$2+/)] in CHSE, gradual decrease in [Ca$\^$2+/] in FHM, and no significant change in RTG cells. The degree of [Ca$\^$2+/] increase or decrease was dependent on the amount of infectious virus, and these [Ca$\^$2+/] variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in [Ca$\^$2+/] was not observed. Thus, infectivity of IHNV appears to correlate with changes in [Ca$\^$2+/] in virus-infected cells. These IHNV-induced [Ca$\^$2+/] changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced Ca$\^$2+/ variations were more related with protein synthesis than RNA synthesis. Various Ca$\^$2+/ related drugs were used in search for the mechanisms of the [Ca$\^$2+/], changes following IHNV infection of CHSE cells. Decreasing extracellular Ca$\^$2+/ concentration or blocking Ca$\^$2+/ influx from extracellular media inhibited the IHNV-induced increase in [Ca$\^$2+/], in CHSE cells. Similar results were obtained with intracellular Ca$\^$2+/ sources are important in IHNV-induced [Ca$\^$2+/] increase in CHSE cells.

• Journal of fish pathology
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• v.30 no.2
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• pp.71-78
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• 2017

The glycoprotein of novirhabdoviruses is known to play a critical role in the determination of host specificity. Viral hemorrhagic septicemia viruses (VHSVs) in different genotypes have different glycoprotein sequences and show different preferences for specific cell lines. In this study, to know whether the glycoprotein is solely responsible for the host cell preference of VHSV, a recombinant VHSV expressing vesicular stomatitis virus (VSV) glycoprotein instead of VHSV IVa glycoprotein (rVHSV-VSV-G) was generated by reverse genetics and inoculated into several fish cell lines, then, cytopathic effect (CPE) and viral growth caused by rVHSV-VSV-G infection were compared with those caused by rVHSV-wild that was previously generated and has the same genomic sequence with wild-type VHSV except a few nucleotides. The plaque numbers of rVHSV-VSV-G were significantly higher in EPC, BF-2 and GF cells than those of rVHSV-wild. However, in HINAE cells (originated from olive flounder), rVHSV-VSV-G titer was significantly lower than rVHSV-wild titer, and both recombinant VHSVs were not grown well in CHSE-214 cells. Although statistical significances were detected in the titers between rVHSV-wild and rVHSV-VSV-G in several cell lines, the cell line-preference order of rVHSV-VSV-G was not different from that of rVHSV-wild. These results suggest that the replacement of VHSV glycoprotein may not completely change host cell preference, and other regions of VHSV might also involve in the determination of host cell preference.

• Journal of fish pathology
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• v.11 no.2
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• pp.89-96
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• 1998

Marine birnavirus (MABV) has wide host range in marine organisms. To clarify various infection properties of MABV in different host species, in vitro study was performed by subculture for 10 passages in several fish cell lines. In CHSE-214, RTG-2 and RSBK-2 cells, the virus produced high yield of virus. Typical CPE with high protein expression was observed in these cells. On the contrary, the virus grown in EPC, FHM and BF-2 cells exhibited no CPE appearance although virus protein was detected. In EPC and FHM cells, the virus titer increased in later passages. The plaque size was distinctly bigger in CHSE-214, RTG-2 and RSBK-2 cells than in other cell lines. The nucleotide sequence of VP2/NS junction region on genome segment A exhibited one specific nucleotide change at 195. The different infection properties in several cell types performed in the present work might reflect in vivo MABV infection in various host species occurring in natural conditions.

• Korean Journal of Ichthyology
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• v.20 no.1
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• pp.1-6
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• 2008

Three types of clonal cell lines were isolated according to their size and phenotype from the adherent cell populations in long-term liquid cultures from the embryonic fibroblast cells of Zebrafish, Danio rerio. All kind of cell lines were well proliferated. The size and number of clonal cell lines derived colonies from stable embryonic cells were significantly increased in the presence of NAC and A2P conditioned medium from the cell lines. The stable cell lines and clonal cell lines were cap-able of well proliferation in vitro. These cell lines have been maintained in continuous culture without change in characteristics. A majority of the clonal cells (80%) was shown a normal chromosomal complement (50 chromosomes, 2N) in according with FACs analysis. Majority of cells were positive to vimentin staining and none of them were positive for nestin and Oct -4 by immunocytochemistry. These results indicate that the clonal cell lines obtained from cultured cells are fibroblasts and may be extremely useful in genetic manipulation for further nuclear transfer and fish cloning.

• Journal of fish pathology
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• v.15 no.1
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• pp.9-16
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• 2002

The objective of the study was to clarify the mechanism of persistent infection of marine birnavirus (MABV) in various nonpermissive cell lines. It was observed in CHSE-214, RTG-2 and RSBK-2 that the virus produced at high yield with typical cytopathic effect (CPE). On the contrary, the CPE was not produced in EPC, FHM and BF-2 cells. However amount of virus protein in both permissive and nonpermissive cell lines detected by ELISA was almost the same. Electron microscopy showed virions in permissive cells but not in nonpermissive cells. From the results, it is clear that virus protein and RNA were produced in nonpermissive cells as observed in permissive cells; however, assembly of the virus particles did not occur in nonpermissive cells.

• Journal of fish pathology
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• v.6 no.2
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• pp.205-218
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• 1993

On the development of hirame(Paratichtys olivaceus) culture, outbreak of scuticociliata infection was reported to cause severe damage in Japan. To establish effective measures for isolation and cultivation of this ciliate, we tried to culture this pathogenic ciliate using medium for bacteria and fish cell lines in vitro. Scuticociliata from the brain tissues of infected fish was aseptically inoculated to CHSE-214 cells cultured in MEM-10 without antibiotic. Scuticociliata grew well and the number of ciliate reached $10^6\;cells/ml$ at temperatures of $15^{\circ}C$ to $20^{\circ}C$ for 10d. The number of ciliate cultured in the cell lines is 10 times higher than the numbers cultured in the liquid medium alone. This ciliata could be cloned by dilution method. Scuticociliata isolated could grow well on 42 different cell lines that were established from marine fish, warm freshwater fish, and salmonids. This ciliate could be preserved in liquid nitrogen for more than 6 months. Subsequently, we observed the optimal temperature and salinity for growth, and tested the sensitivities of this organism to formaldehyde, flagyl(Metronidazole), Ekuteshin(Combination compound of sulfamonometoxin and ormethoprim), and ozonixation. Optimal temperature for growth was $25^{\circ}C$ and salinity was 1.0 to 1.5%. Washed scuticociliata was killed by formaldehyde at the concentration of 50ppm for 10min, but was not completely killed even at a high concentration of 400ppm for 20min in MEM-5. Flagyl and Ekuteshin can inhibit the growth of scuticociliata at the concentration of 1,000 and $100{\mu}g/ml$ in MEM-10, respectively. More than 99% of this scuticociliata could be killed by ozonization at a dose equivalent to $1.0mg/\ell$ oxidant for 30sec in sea water. Isolated scuticociliata showed the pathogenicity to the cultured hirame by artificial infection(I. P. injection, $10^5\;cells$/fish). The number of scuticociliata in the water could be counted by most probable number(MPN) method using tissue culture, and the minimum detectable number was $1.8\;cells/\ell$. The number in the reservoir tank for water supply to the culture tank was 110 cells/l. After cleaning by elimination of the sediments from of the reservoir tank and disinfected with formaldehyde, number of scuticociliata decreased and was counted less than $1.8\;cells/\ell$ and infection rate of cultured hirame was decreased.

• Journal of Marine Life Science
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• v.8 no.2
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• pp.186-189
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• 2023

Uncoupling protein 1 (UCP1) is a unique mitochondrial membranous protein expressed in brown adipose tissue (BAT) in mammals. While its expression in response to cold temperatures and adipogenic inducers is well-characterized in mammals and human infants, the molecular characterization and expression of UCP1 in fish remain unexplored. To address this gap, we analyzed UCP1 expression in response to adipogenic inducers in a fish cell line, rainbow trout gonadal cells (RTG-2), and compared it with UCP1 expression in three mammalian preadipocytes, 3T3-L1, T37i, and WT1 exposed to the Peroxisome proliferator-activated receptor gamma (PPARγ) agonists, rosiglitazone (Rosi). In mammalian preadipocytes, UCP1 protein was highly expressed by Rosi, with an induction of adipogenesis observed in a time-dependent manner. This suggests that UCP1 plays a significant role in adipogenesis in mammals. However, RTG-2 cells showed no response to adipogenic inducers and exhibited only marginal expressions of UCP1. These results imply that RTG-2 cells may lack crucial responsive mechanisms to adipogenic signals or that the adipogenic response is regulated by other mechanisms. Further studies are needed to confirm these phenomena in fish preadipocytes when an appropriate cell line is established in future research.