Characteristics of established KSG cells derived from the scorpionfish Sebastiscus marmoratus: what happens under the hydrostatic pressure like the deep sea?
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Advances in cell biology depend, partly, on the development of new cell lines and culture methods. Our research focused on a fibroblast-like cell line, “KSG,” which is derived from scorpionfish fin tissue (Sebastiscus marmoratus). Cells were grown in Leibovitz’s L-15 medium with 10% fetal bovine serum following standard procedures. The optimum growth temperatures for these lines ranged from 15°C to 25°C. All cells survived storage for at least 3 yr at −80°C. Subsequently, they were continuously cultured until the 78th generation without evident changes in their morphology. Moreover, we were able to culture KSG cells in the absence of fetal bovine serum in a culture medium containing the fish serum “SeaGrow.” Optimum SeaGrow concentrations for these cells ranged from 5% to 20%. The growth rate of KSG cells decreased when the concentration of SeaGrow was reduced to 1%. However, this decrease could be partially reversed by adding 0.5% “Hy-Fish.” In addition, the inclusion of Hy-Fish improved cell adhesion. KSG cells that were cultured in serum-free culture media containing 0.5% and 1% Hy-Fish had been added and were able to survive at low densities. Furthermore, we successfully transfected this cell line with a commercial plasmid vector coding a fluorescent protein using the cationic lipid. Finally, the analyses of cell behavior under hydrostatic pressure showed that some pressures (10 MPa) helped the cells to proliferate more.
KeywordsKSG cell Serum-free culture Hydrostatic pressure SeaGrow Hy-Fish
Analyses of cell proliferation using cultured cells provide useful information for determining the functions of tissues and organs as well as defining the process of cell differentiation and embryo development (Ciemerych et al. 2011). Abnormal cell growth can result in the development of cancerous cells or programmed cell death (i.e., apoptosis). Cultured cell lines have been widely used to investigate the factors controlling these processes (Wolf and Quimby 1969). Cultured cells are typically incubated in blood serum containing multiple growth factors or a synthetic medium with purified growth factors. In mammalian cell culture, a range of factors that regulate cell proliferation have been identified and their functional mechanisms documented (Barnes and Sato 1980). In fish, researchers have used cultured cell lines to study fish viruses (Yan et al. 2011). Cells are cultivated from embryos, gonads, kidneys, and fins and are typically bathed in a commercially available synthetic medium containing 10–20% fetal bovine serum (FBS). Fish serum is thought to promote higher growth in teleost cells than FBS; however, the growth factors associated with this response have not been identified (Hashimoto et al. 1997). Although the cells of ectotherms, including fish, are expected to grow in the temperature ranges suitable for adult individuals, the regulatory mechanisms remain unknown (Hightower and Renfro 1988; Bols et al. 1992; Buonocore et al. 2006). At present, all established fish cell lines, including the KSG scorpionfish cell line, are cultured in media containing FBS. Since the outbreak of bovine spongiform encephalopathy at the beginning of 2000, there has been an emphasis on developing a culture method that does not use mammalian-derived components (Padilla et al. 2011). We investigated the usefulness of cell culture using serum derived from fish (Salmonidae) and evaluated the effectiveness of fish-derived culture medium additives. Furthermore, we examined the unique fish cell line characteristics: an essential relation between the hydrostatic pressure and the cell proliferation.
Materials and Methods
Preparation of the cell line.
The scorpionfish is distributed throughout the marine areas around Japan at depths of ≈5–200 m. We captured scorpionfish near Fukuura Wharf in Yokohama City, Kanagawa Prefecture, Japan and prepared the KSG cells as described previously (Japan Patent Kokai 2008). In brief, we cultured the caudal fin tissue in Leibovitz’s L-15 (MP Biomedicals, Santa Ana, CA) culture medium, with 10% FBS (MP Biomedicals), 4 g/L NaCl (Wako, Osaka, Japan), and 1% penicillin-streptomycin (MP Biomedicals). The caudal fin tissue was cut into 1-mm squares, immersed in the medium, and cultured in an incubator at 25°C. After 3 d, we confirmed the migration of the fibroblasts. Even after the tissue was removed, the fibroblasts continued to multiply. We have confirmed survival of the fibroblasts to the 78th generation. We observed cell morphology using an inverted microscope CKX41 (Olympus, Tokyo, Japan) connected to a digital camera ARTCAM-130MI (Armssystem, Tokyo, Japan).
Culture method using FBS.
The scorpionfish KSG cell line was seeded into 25-cm2 tissue culture flasks and cultured in Leibovitz’s L-15 medium containing 10% FBS at 25°C. Cells that reached the confluent state (500 cells/mm2) were separated with TrypLE Express (Gibco, Carlsbad, CA), seeded into new 25-cm2 tissue culture flasks, and subcultured. We replaced the culture medium with new medium every 3 d.
Culture method using fish serum.
cell density [cells/mm2]
- T 0
initial time [h]
- N 0
initial density [cells/mm2]
Effect of a fish-derived culture medium additive on growth rate.
Hy-Fish (Maruhachi Muramatsu, Shizuoka, Japan) is a component extracted from the bony parts of skipjack tuna. The extract was developed as a culture medium additive for culturing mammalian cells. We investigated whether KSG-SeaGrow cells could be cultured in media containing low concentrations of SeaGrow and Hy-Fish. We added 0.5% Hy-Fish to culture media containing SeaGrow (1% or 2.5%) and measured growth rate and cell adhesion in the KSG-SeaGrow cell line as mentioned above.
Serum-free culture using fish-derived culture medium additive.
We cultured KSG-SeaGrow cells using Leibovitz’s L-15 culture medium without serum and containing 0.1%, 0.5%, or 1.5% Hy-Fish. We measured growth rate and monitored cell adhesion as stated above.
Transfection of KSG cells.
KSG-SeaGrow cells were transfected with the pCDNA3.1/Zeo plasmid (Invitrogen, Carlsbad, CA), which contains a CMV promoter, a SV40 polyadenylation signal, a gene conveying resistance to zeocin, and a multicloning site inserting a yellow cameleon 2.12 (YC2.12) sequence. This plasmid is hereafter referred to as pCDNA3.1-YC2.12. Constructs of YC2.12 were kindly provided by A. Miyawaki (RIKEN, Wako, Japan). Following transfection, YC2.12 is expressed as a cyan and a yellow variant (YFP) of the green fluorescent protein. We transfected pCDNA3.1-YC2.12 into KSG-SeaGrow cells, which had attained a 70% confluent state using lipofectamine2000 (Invitrogen), following the manufacturer’s instructions. The cells were then incubated for 24 h at 25°C. Positive cells were selected by continuous incubation in Leibovitz’s L-15 medium containing zeocin (Invitrogen) at 0, 50, 100, 200, 400, 600, 800, or 1,000 μg/ml, added 48 h after transfection. The culture medium containing zeocin was changed every 3 d, and the experiment was conducted over a period of ≈1 mo. We observed YFP expression in the positive cells using a confocal fluorescence microscope FV5-PSU + IX71 (Olympus) connected to a digital camera ORCA-ER (Hamamatsu Photonics, Shizuoka, Japan). Methods for quantifying fluorescence have been described previously (Tsuruwaka et al. 2007). However, during this experiment, we focused solely on YFP detection.
KSG cell proliferation under hydrostatic pressure.
KSG cells attached to the collagen-coated, pressure-resistant glass were placed in a high-hydrostatic pressure chamber (ABLE Corporation, Tokyo, Japan). A detailed chamber system was reported by Koyama et al. (2001). Amount of pressure is indicated with megapascals (MPa) here. Atmospheric pressure is nearly equal to 0.1 MPa = 0.9869 atm = 1.0197 kg of force/cm2. When water depth gets deeper by 10 m, hydrostatic pressure rises by 0.1 MPa. The cell morphological feature and proliferation were examined under pressures from 0 to 40 MPa after 20 min with the differential interference microscope IX70 (Olympus) connected to a CCD camera M-3204C (Olympus). Twenty minutes later, the pressure was released to atmospheric pressure. Then, the cell was cultured at 25°C, and the proliferation rate was measured as stated above.
Results and Discussion
Culture method using fish serum.
Effect of a fish-derived culture medium additive on growth rate.
The cell doubling time was 57.8 h when KSG-SeaGrow was cultured with 1% SeaGrow, but decreased to 31.4 h with the addition of 0.5% Hy-Fish (Fig. 1E ). Figure 1F shows a photograph of cells cultured in 0.5% Hy-Fish and 2.5% SeaGrow. Cell adhesion was improved by adding Hy-Fish to the SeaGrow serum culture medium. We successfully cultured fish cells using SeaGrow and/or Hy-Fish in the place of mammalian-derived constituents such as FBS.
Serum-free culture using a fish-derived culture medium additive.
Yellow fluorescent protein expression in KSG-SeaGrow.
KSG cell behavior under the hydrostatic pressure.
Results in this study show that KSG scorpionfish cells give a choice of Leibovitz’s L-15 culture media additive: mammalian-derived serum (FBS), fish serum (SeaGrow), or fish bone extraction (Hy-Fish). The KSG cell line is an interesting research platform for gene and protein expression studies to elucidate deep-sea fishes.
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