Fish cell lines provide a useful tool for biological studies in the fields of physiology, toxicology, and virology (Hightower and Renfro 1988; Villena 2003). Establishing cell lines is important and critical, particularly for studying endangered fish species owing to difficulties in the routine collection of cell samples. Although only a few cell lines have been developed from endangered fish species (Lakra et al. 2006; Ciba et al. 2008), hundreds of fish cell lines have been established from a broad range of species (Wolf and Mann 1980; Fryer and Lannan 1994; Lakra et al. 2011).

Honmoroko Gnathopogon caerulescens (Sauvage, 1883) is a critically endangered small cyprinid endemic to ancient Lake Biwa in Japan (Ministry of the Environment, Japan 2014). Recently, we established testicular and ovarian cell lines of G. caerulescens (Higaki et al. 2013b) and demonstrated the antiandrogenic effects of nonylphenol, one of the potent endocrine disruptors, as well as the response to fish-specific hormones, using a testicular cell line (Higaki et al. 2013a). Cell lines derived from various tissues, including early embryos, will be valuable for studying physiological responses to endogenous and exogenous molecules and viral infection at the cellular level. In this study, we focused on early development, establishing a cell line from blastula-stage embryos of G. caerulescens and performing the characterization of the cell line.

The brood stock of G. caerulescens used in this study was reared in outdoor ponds at the Shiga Prefectural Fisheries Experimental Station. Naturally spawned blastula-stage embryos were collected into a 40-μm mesh cell strainer (BD Biosciences, San Jose, CA). A group of about 20 embryos was disinfected by 2-min immersion in sterile phosphate-buffered saline (PBS) supplemented with 0.5% (v/v) commercial bleach solution. Following three washes in sterile PBS for 2 min each, three to four embryos were transferred to one well of a 0.1% (w/v) gelatin-coated four-well plate (Thermo Scientific, Waltham, MA) filled with 300 μl of culture medium. The embryos were then ruptured with stainless steel needles, and scattered blastomeres were cultured at 28°C in humidified air. When the cells reached confluence, they were passaged by trypsinization using PBS supplemented with 0.06% (w/v) trypsin and 1.32 mM ethylene diamine tetraacetic acid. From passage 3, cells were cultured in a gelatin-coated 35-mm plastic tissue culture dish (Asahi Glass, Tokyo, Japan) filled with 1.3 ml of the culture medium. The cells were passaged at 1:2 or 1:3 cell dilutions every 2–3 days.

Culture medium was the same as that used for establishing testicular and ovarian cell lines of G. caerulescens (Higaki et al. 2013b), namely, testicular cell culture medium containing 10% (v/v) fetal bovine serum (TCCM-FBS) (Sakai 2006) supplemented with 10 IU ml−1 human chorionic gonadotropin (ASKA Pharmaceutical, Tokyo, Japan), 10 IU ml−1 pregnant mare’s serum gonadotropin (ASKA Pharmaceutical), 100 ng ml−1 epidermal growth factor (Peprotech, Rocky Hill, NJ), 100 ng ml−1 basic fibroblast growth factor (Peprotech), 10 μM forskolin, and 0.1 mM β-mercaptoethanol. Fish embryo extract (FEE) was prepared from crucian carp (Carassius carassius) embryos according to a previously described procedure (Westerfield 2007).

The expression of genes known to be expressed in the blastula-stage embryos of the closely related zebrafish, such as oct4 (Burgess et al. 2002), sox2 (Okuda et al. 2006), nanog (Xu et al. 2012), and klf4 (Onichtchouk et al. 2010), were examined in the embryonic cell line and embryos of G. caerulescens. For gene expression analyses, total RNA was extracted from the cell cultures at passage 28 and blastula-stage embryos using illustra RNAspin Mini Isolation Kit (GE Healthcare Bio-Sciences, Piscataway, NJ) according to the manufacturer’s instructions. After the DNase treatment of the samples, reverse transcription was carried out with PrimeScript RT regent Kit (Takara Bio, Shiga, Japan) using 500 ng total RNA. The complementary DNA (cDNA) was amplified using primers designed for G. caerulescens zebrafish orthologue oct4, sox2, nanog, and klf4 (Table 1). The tubulin-alpha 1 (tuba1) gene was used as an internal control. PCR was performed using TaKaRa ExTaq DNA Polymerase (Takara Bio) according to the manufacturer’s instructions. The PCR conditions were as follows: initial denaturation for 2 min at 95°C, 35 cycles of denaturation for 20 s at 94°C, annealing for 30 s at the temperatures indicated in Table 1, and extension for 1 min at 72°C, followed by a final extension for 2 min at 72°C. The RT-PCR products were separated by 2% (w/v) agarose gel electrophoresis, and the gel was stained with ethidium bromide.

Table 1. Primers used for reverse transcription polymerase chain reactions (RT-PCR)
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The effects of incubation temperature and concentrations of FBS and FEE on the growth of the embryonic cell line were investigated at passages 30–34. Cells were plated in gelatin-coated 24-well plates in triplicate at 5 × 104 cells per well and incubated at 18°C, 23°C, or 28°C. Cells were cultured for 6 days, and cell number was counted daily using a hemocytometer. Minimum doubling time was calculated from experimental data during the exponential growth phase using the algorithm provided by http://www.doubling-time.com. Growth responses to different concentrations of FBS (1%, 5%, and 10%) and FEE (0, 1, and 2 embryos ml−1) were determined using the procedures described above with the culture medium containing 2 embryos ml−1 FEE and 10% FBS, respectively, at 28°C. The effects of culture temperatures, concentrations of FBS or of FEE, and culture periods on the number of cells were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference as a post hoc test using a computer program (SPSS for Windows, version 12.0, SPSS Inc., IL).

Cells at passage 29 were used for chromosome analysis. Cells undergoing exponential growth were treated with 0.05 μg ml−1 colcemid for 4 h at 28°C. Cells were then harvested and centrifuged at 120×g for 3 min, and the pellet was gently resuspended in 0.75 M KCl and incubated at 28°C for 10 min. Following incubation, cells were centrifuged again, and the pelleted cells were fixed with a mixture of methanol and acetic acid (3:1) for 20 min. A small volume of the cell suspension was dropped on clean glass slides, air dried, and stained with 10% (v/v) Giemsa solution (in 10 mM potassium phosphate, pH 6.8) for 10 min. Chromosome counts were determined in 137 metaphase plates.

In the early passages, cell culture was heterogeneous and epithelium- and fibroblast-like cells were noticed; however, at the later passages, only epithelium-like cells were observed (Fig. 1). Embryonic cells grew rapidly without growth stunting and have been successfully subcultured at a ratio of 1:2 or 1:3 at 2- to 3-d intervals for more than 60 passages since their initiation in June 2011. The cells were cryopreserved at different passages and successfully thawed at high viability with a commercial cell freezing preservation solution (Cell Banker; Nippon Zenyaku Kogyo, Fukushima, Japan) (data not shown). During this period, no phenotypic change was observed, and the cell line was designated as RME1, which did not express the genes detected in blastula-stage embryos, such as oct4, sox2, nanog, and klf4 (Fig. 2). Although many embryonic cell lines have been established from more than 30 fish species (Wolf and Mann 1980; Fryer and Lannan 1994; Lakra et al. 2011), to the best of our knowledge, this is the first embryonic cell line established from an endangered fish species.

Figure 1.
figure 1

The morphology of the cell cultures derived from blastula-stage embryos of Gnathopogon caerulescens. Phase contrast micrographs of embryonic cell cultures were taken at passage 2 (A) and 30 (B). RME1 cells were grown in testicular cell culture medium containing 10% (v/v) fetal bovine serum (TCCM-FBS) supplemented with 10 IU ml−1 human chorionic gonadotropin, 10 IU ml−1 pregnant mare’s serum gonadotropin, 100 ng ml−1 epidermal growth factor, 100 ng ml−1 basic fibroblast growth factor, 10 μM forskolin, and 0.1 mM β-mercaptoethanol. Bars = 500 μm (magnification, ×100).

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Figure 2.
figure 2

Gene expression patterns of embryonic cell line (RME1) and blastula stage embryos of Gnathopogon caerulescens. Total RNAs for RT-PCR were extracted from RME1 cells at passage 28 and G. caerulescens embryos at blastula stage. Following reverse transcription, cDNA was amplified using primers designed for G. caerulescens zebrafish orthologues oct4, sox2, nanog, and klf4. The tubulin-alpha 1 (tuba1) gene was used as an internal control. The RT-PCR products were separated by 2% (w/v) agarose gel electrophoresis, and the gel was stained with ethidium bromide.

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There was an interaction between the effects of the culture temperatures and the culture periods on the number of cells (P < 0.01). Within the tested temperatures, RME1 cells grew optimally at 28°C (Fig. 3A ), which is higher than the upper limit of the water temperature during the spawning season (12–27°C) of G. caerulescens (Nakamura 1949). The cells grew slowly at 23°C, whereas they did not grow at all at 18°C. These results may imply that the cell line was adapted and/or selected to the relatively higher incubation temperature through the establishment and/or subculture at 28°C, as suggested previously (Wolf and Ahne 1982). The growth rate of the cell line increased as the FBS concentrations increased from 1% to 10% (Fig. 3B ) (P < 0.01), indicating that FBS plays an important role in the proliferation of the present cell line, as generally suggested in fish cell culture (Bols and Lee 1991). Conversely, the minimum population doubling times of the RME1 cells cultured with 0, 1, and 2 embryos ml−1 FEE were similar to each other: 18.73 h (days 3–4), 18.78 h (days 3–4), and 20.32 h (days 2–3), respectively (Fig. 3C ). These doubling times were shorter than those of the embryonic cell lines established in other fish species (Collodi et al. 1992; Hong et al. 1996; Ristow et al. 1998; Shimizu et al. 2003; Parameswaran et al. 2007), indicating that the RME1 cells are capable of rapid growth in FEE-reduced or FEE-devoid culture medium.

Figure 3.
figure 3

Growth rate of an embryonic cell line (RME1) of Gnathopogon caerulescens at different incubation temperatures and concentrations of fetal bovine serum (FBS) and fish embryo extract (FEE). Growth rates of RMT1 cells were examined at passages 30–34. Cells were cultured in a medium containing 10% FBS (v/v) and 2 embryos ml−1 FEE at 18°C, 23°C, or 28°C (A). Cells were cultured in a medium containing 1, 5, or 10% FBS and 2 embryos ml−1 FEE at 28°C (B). Cells were cultured in the medium containing 10% FBS and 0, 1, or 2 embryos ml−1 FEE at 28°C (C). Cells were plated in gelatin-coated 24-well plates in triplicate at 5 × 104 cells per well. Cells were trypsinized and counted daily using a hemocytometer. Superscripts a–c Values (mean ± standard error of three replicates) with different letters within the same culture periods differed significantly (P < 0.05).

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Interestingly, RME1 cells showed a normal diploid number (2n = 50; Ueno et al. 1992) in 67.2% (92/137) of the cells (Fig. 4A ). In addition, metaphase with a euploid set of chromosomes displayed the normal diploid karyotype morphology (Fig. 4B ) consisting of seven pairs of metacentric chromosomes and 18 pairs of submetacentric–subtelocentric chromosomes (Fig. 4C ), as reported in G. caerulescens (Ueno et al. 1992). This euploid rate was similar to or even higher than other fish embryonic cell lines at similar passage levels (Ristow and de Avila 1994; Parameswaran et al. 2007; Dash et al. 2010). Accordingly, RME1 cells presumably have less genomic rearrangement, and therefore, the cell line can be a useful tool to study cell biology in G. caerulescens. In addition, the euploidy of RME1 cells imply the potential use for somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation in the future. Many technical obstacles such as poor yields, developmental anomalies, and different nuclear–mitochondrial interactions in SCNT (Loi et al. 2011) remain to be solved, and developmentally competent iPS cells have not yet been accomplished in fish (Rosselló et al. 2013).

Figure 4.
figure 4

Chromosome analysis of embryonic cell line (RME1) of Gnathopogon caerulescens. Chromosome number distribution of RME1 cells was analyzed at passage 29 according to counts from 137 metaphase preparations (A). Metaphase chromosomes were micrographed after Giemsa staining (magnification, ×1000) (B). Giemsa-stained chromosomes attentively arranged into a karyogram (C). Chromosomes grouped in the upper row are metacentric chromosomes; the middle and bottom rows are submetacentric–subtelocentric chromosomes, respectively. An example of a metaphase plate of RME1 cells with euploid (2n = 50) chromosomes is shown. Bar = 10 μm.

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In conclusion, we have succeeded in developing an epithelium-like cell line RME1 from blastula-stage embryos of a critically endangered cyprinid G. caerulescens. This cell line is well adapted to grow at 28°C in the culture medium, which was successfully used for establishing testicular and ovarian cell lines of G. caerulescens, and displayed stable growth over 60 passages with euploidy. RME1 cells can be cultured in the absence of FEE, which simplifies medium preparation and expands its use to any laboratory. In addition, RME1 cells can be a useful tool as a model of the embryonic cell and provides new insights into the research of endemic fish G. caerulescens, particularly emphasizing cell biology.