D Barnes

D Padilla

Eriko Shimada

F Buonocore

H Hashimoto

K Wolf

LE Hightower

MA Ciemerych

Makiko Kikuchi

NC Bols

S Koyama

Y Tsuruwaka

Y Yan

Yuji Hatada

Yusuke Tsuruwaka

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Characteristics of established KSG cells derivedfrom the scorpionfish Sebastiscus marmoratus: what happensunder the hydrostatic pressure like the deep sea?Yusuke Tsuruwaka & Eriko Shimada & Makiko Kikuchi &Yuji HatadaReceived: 3 October 2013 /Accepted: 9 December 2013 /Published online: 8 January 2014 / Editor: T. Okamoto# The Society for In Vitro Biology 2014Abstract Advances in cell biology depend, partly, on thedevelopment of new cell lines and culture methods. Ourresearch focused on a fibroblast-like cell line, “KSG,” whichis derived from scorpionfish fin tissue (Sebastiscusmarmoratus). Cells were grown in Leibovitz’s L-15 mediumwith 10% fetal bovine serum following standard procedures.The optimum growth temperatures for these lines ranged from15°C to 25°C. All cells survived storage for at least 3 yr at−80°C. Subsequently, they were continuously cultured untilthe 78th generation without evident changes in their morphol-ogy. Moreover, we were able to culture KSG cells in theabsence of fetal bovine serum in a culture medium containingthe fish serum “SeaGrow.”Optimum SeaGrow concentrationsfor these cells ranged from 5% to 20%. The growth rate ofKSG cells decreased when the concentration of SeaGrow wasreduced to 1%. However, this decrease could be partiallyreversed by adding 0.5% “Hy-Fish.” In addition, the inclusionof Hy-Fish improved cell adhesion. KSG cells that werecultured in serum-free culture media containing 0.5% and1% Hy-Fish had been added and were able to survive at lowdensities. Furthermore, we successfully transfected this cellline with a commercial plasmid vector coding a fluorescentprotein using the cationic lipid. Finally, the analyses of cellbehavior under hydrostatic pressure showed that some pres-sures (10 MPa) helped the cells to proliferate more.Keywords KSG cell . Serum-free culture . Hydrostaticpressure . SeaGrow . Hy-FishIntroductionAnalyses of cell proliferation using cultured cells provideuseful information for determining the functions of tissuesand organs as well as defining the process of cell differentia-tion and embryo development (Ciemerych et al. 2011).Abnormal cell growth can result in the development of can-cerous cells or programmed cell death (i.e., apoptosis).Cultured cell lines have been widely used to investigate thefactors controlling these processes (Wolf and Quimby 1969).Cultured cells are typically incubated in blood serum contain-ing multiple growth factors or a synthetic medium with puri-fied growth factors. In mammalian cell culture, a range offactors that regulate cell proliferation have been identified andtheir functional mechanisms documented (Barnes and Sato1980). In fish, researchers have used cultured cell lines tostudy fish viruses (Yan et al. 2011). Cells are cultivated fromembryos, gonads, kidneys, and fins and are typically bathed ina commercially available synthetic medium containing 10–20% fetal bovine serum (FBS). Fish serum is thought topromote higher growth in teleost cells than FBS; however,the growth factors associated with this response have not beenidentified (Hashimoto et al. 1997). Although the cells ofectotherms, including fish, are expected to grow in the tem-perature ranges suitable for adult individuals, the regulatorymechanisms remain unknown (Hightower and Renfro 1988;Electronic supplementary material The online version of this article(doi:10.1007/s11626-013-9723-0) contains supplementary material,which is available to authorized users.Y. Tsuruwaka (*) :M. Kikuchi :Y. HatadaMarine Bioresource Exploration Research Team (MBE), MarineBiodiversity Research Program, Institute of Biogeosciences, JapanAgency for Marine-Earth Science and Technology (JAMSTEC),Yokosuka 237-0061, Japane-mail: tsuruwaka@jamstec.go.jpE. ShimadaDivision of Applied Biosciences, Graduate School of Agriculture,Kyoto University, Kyoto 606-8502, JapanM. KikuchiDepartment of Applied Material and Life Science, Kanto GakuinUniversity, Yokohama 236-8501, JapanIn Vitro Cell.Dev.Biol.—Animal (2014) 50:439–444DOI 10.1007/s11626-013-9723-0Bols et al. 1992; Buonocore et al. 2006). At present, allestablished fish cell lines, including the KSG scorpionfish cellline, are cultured in media containing FBS. Since the outbreakof bovine spongiform encephalopathy at the beginning of2000, there has been an emphasis on developing a culturemethod that does not use mammalian-derived components(Padilla et al. 2011). We investigated the usefulness of cellculture using serum derived from fish (Salmonidae) and eval-uated the effectiveness of fish-derived culture medium addi-tives. Furthermore, we examined the unique fish cell linecharacteristics: an essential relation between the hydrostaticpressure and the cell proliferation.Materials and MethodsPreparation of the cell line. The scorpionfish is distributedthroughout the marine areas around Japan at depths of ≈5–200 m. We captured scorpionfish near Fukuura Wharf inYokohama City, Kanagawa Prefecture, Japan and preparedthe KSG cells as described previously (Japan Patent Kokai2008). In brief, we cultured the caudal fin tissue inLeibovitz’s L-15 (MP Biomedicals, Santa Ana, CA) culturemedium, with 10% FBS (MP Biomedicals), 4 g/L NaCl(Wako, Osaka, Japan), and 1% penicillin-streptomycin (MPBiomedicals). The caudal fin tissue was cut into 1-mmsquares, immersed in the medium, and cultured in anincubator at 25°C. After 3 d, we confirmed the migrationof the fibroblasts. Even after the tissue was removed, thefibroblasts continued to multiply. We have confirmed sur-vival of the fibroblasts to the 78th generation. We observedcell morphology using an inverted microscope CKX41(Olympus, Tokyo, Japan) connected to a digital cameraARTCAM-130MI (Armssystem, Tokyo, Japan).Culture method using FBS. The scorpionfish KSG cell linewas seeded into 25-cm2 tissue culture flasks and cultured inLeibovitz’s L-15 medium containing 10% FBS at 25°C. Cellsthat reached the confluent state (500 cells/mm2) were separat-ed with TrypLE Express (Gibco, Carlsbad, CA), seeded intonew 25-cm2 tissue culture flasks, and subcultured. We re-placed the culture medium with new medium every 3 d.Culture method using fish serum. KSG cells in frozen storagewere thawed and cultured in Leibovitz’s L-15 medium con-taining 10% FBS. Then, the culture medium was substitutedfor a medium containing Salmonidae fish serum, SeaGrow(EastCoast Biologics, North Berwick, ME). We adjusted theconcentration of heat-inactivated SeaGrow to 5%, 10%, and20%. The KSG cell line cultured with SeaGrow is hereafterreferred to as KSG-SeaGrow. We evaluated the effect of theconcentration of SeaGrow on growth rate (doubling time) andcell adhesion in the KSG-SeaGrow cell line. The cells werecounted using an automated cell counter TC10 (Bio-Rad,Hercules, CA). The time taken for the number of cells todouble (doubling time, DT) was calculated using the follow-ing formula:Doubling time DTð Þ ¼ T−T 0ð Þlog2= logN−logN0ð ÞT time [h]N cell density [cells/mm2]T0 initial time [h]N0 initial density [cells/mm2]Effect of a fish-derived culture medium additive on growthrate. Hy-Fish (Maruhachi Muramatsu, Shizuoka, Japan) is acomponent extracted from the bony parts of skipjack tuna.The extract was developed as a culture medium additive forculturing mammalian cells. We investigated whether KSG-SeaGrow cells could be cultured in media containing lowconcentrations 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 mediumadditive. We cultured KSG-SeaGrow cells using Leibovitz’sL-15 culture medium without serum and containing 0.1%,0.5%, or 1.5% Hy-Fish. We measured growth rate and mon-itored cell adhesion as stated above.Transfection of KSG cells. KSG-SeaGrow cells weretransfected with the pCDNA3.1/Zeo plasmid (Invitrogen,Carlsbad, CA), which contains a CMV promoter, a SV40polyadenylation 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 aspCDNA3.1-YC2.12. Constructs of YC2.12 were kindly pro-vided by A. Miyawaki (RIKEN, Wako, Japan). Followingtransfection, YC2.12 is expressed as a cyan and a yellowvariant (YFP) of the green fluorescent protein. We transfectedpCDNA3.1-YC2.12 into KSG-SeaGrow cells, which hadattained a 70% confluent state using lipofectamine2000(Invitrogen), following the manufacturer’s instructions. Thecells were then incubated for 24 h at 25°C. Positive cells wereselected by continuous incubation in Leibovitz’s L-15 medi-um containing zeocin (Invitrogen) at 0, 50, 100, 200, 400,600, 800, or 1,000 μg/ml, added 48 h after transfection. Theculture medium containing zeocin was changed every 3 d, andthe experiment was conducted over a period of ≈1 mo. Weobserved YFP expression in the positive cells using a confocalfluorescence microscope FV5-PSU + IX71 (Olympus) con-nected to a digital camera ORCA-ER (Hamamatsu Photonics,440 TSURUWAKA ETAL.Shizuoka, Japan). Methods for quantifying fluorescence havebeen described previously (Tsuruwaka et al. 2007). However,during this experiment, we focused solely on YFP detection.KSG cell proliferation under hydrostatic pressure. KSG cellsattached to the collagen-coated, pressure-resistant glass wereplaced in a high-hydrostatic pressure chamber (ABLEFigure 1. The KSG cell growthrate and morphological features.(A) The growth curves of first-generation KSG cultured in FBSand of KSG-SeaGrow cultured ina range of concentrations ofSeaGrow. (B) The growth curveof the second-generation KSG/KSG-SeaGrow after subculturing.(C) Cell shapes of KSG. Scalebar, 50 μm. (D) Cell shapes ofKSG-SeaGrow. A few cells werepeeled (arrow). Scale bar, 50 μm.(E) The growth curve of KSG-SeaGrow cultured by adding Hy-Fish to SeaGrow serum culturemedia. (F) Cell shapes of KSG-SeaGrow+Hy-Fish. Scale bar,50 μm.Figure 2. The growth curve ofKSG-SeaGrow in serum-freeculture medium with Hy-Fishadded.SCORPIONFISH KSG CELL POTENTIAL 441Corporation, Tokyo, Japan). A detailed chamber system wasreported by Koyama et al. (2001). Amount of pressure isindicated with megapascals (MPa) here. Atmospheric pres-sure is nearly equal to 0.1 MPa=0.9869 atm=1.0197 kg offorce/cm2.When water depth gets deeper by 10m, hydrostaticpressure rises by 0.1 MPa. The cell morphological feature andproliferation were examined under pressures from 0 to40 MPa after 20 min with the differential interference micro-scope IX70 (Olympus) connected to a CCD cameraM-3204C(Olympus). Twenty minutes later, the pressure was released toatmospheric pressure. Then, the cell was cultured at 25°C, andthe proliferation rate was measured as stated above.Results and DiscussionCulture method using fish serum. Because of the effects ofchanging serum, the first-generation KSG-SeaGrow cellspassed through a lag phase of approximately 100 h beforeentering the multiplication phase (Fig. 1A). The doubling timewas equal to that of KSG cells (≈40 h). The second andsubsequent generations of KSG-SeaGrow began multiplyingimmediately after adhesion, and the doubling time was shorterthan KSG and was stable at 30 h (Fig. 1B). We observedmanyinstances where the area around the KSG-SeaGrow cellsappeared white in color, or where the cells were round dueFigure 3. Expression of the YC2.12 gene in KSG-SeaGrow during division. The transfected cells divided normally (upper, fluorescence images; lower,bright field images). Scale bars, 20 μm.Figure 4. KSG cell morphology under the hydrostatic pressure. (A) KSG cells given 0–40 MPa. Arrows: protruded cells; arrow heads: round cells.(B) The growth curve of KSG under 0–10 MPa.442 TSURUWAKA ETAL.to uplifting, and we found that KSG-SeaGrow cells had moreof these uplifted cells compared with KSG cells. Theseuplifted cells were not sufficiently extended, resulting in weakadhesion of the KSG-SeaGrow line (Fig. 1C, D). We wereunable to determine whether the uplifting was due to theinclusion of SeaGrow or whether the cells required more timeto adjust to the substitution of SeaGrow for FBS. Since thesecond and subsequent generations of KSG-SeaGrow multi-plied faster than those of KSG (Fig. 1B), cells that haveundergone more subcultivations in KSG-SeaGrow may ulti-mately adapt to continuous growth in SeaGrow.Effect of a fish-derived culture medium additive on growthrate. The cell doubling time was 57.8 h when KSG-SeaGrowwas cultured with 1% SeaGrow, but decreased to 31.4 h withthe addition of 0.5% Hy-Fish (Fig. 1E). Figure 1F shows aphotograph of cells cultured in 0.5% Hy-Fish and 2.5%SeaGrow. Cell adhesion was improved by adding Hy-Fish tothe SeaGrow serum culture medium.We successfully culturedfish cells using SeaGrow and/or Hy-Fish in the place ofmammalian-derived constituents such as FBS.Serum-free culture using a fish-derived culture mediumadditive. When KSG-SeaGrow cells were cultured in serum-free Leibovitz’s L-15 culture medium to which Hy-Fish hadnot been added, there was no multiplication after adhesionwas confirmed. In addition, we observed that all cells de-tached on the fourth day after seeding. The addition of 0.1%Hy-Fish delayed detachment of all cells until the 12th day. Ata concentration of 0.5% Hy-Fish, the cells survived for 19 dwhile remaining at a low density (20 cells/mm2) (Fig. 2). At1% Hy-Fish, the cell density increased to ≥200 cells/mm2 andthen decreased to 20 cells/mm2. Interestingly, we did notobserve cell adhesion at a concentration of 5% Hy-Fish.According to the exposure experiment in Konishi et al.(2006), low concentration of stimulants such as alcohols andheavy metals demonstrated higher activation temporarily infish. A similar mechanism may have been involved in the cellgrowth with the presence of 1% Hy-Fish in the KSG cells.Taken together, our results suggest that it is feasible to cultureKSG cells in a serum-free environment by supplementing themedia with Hy-Fish.Yellow fluorescent protein expression in KSG-SeaGrow. Thenext question we need to consider is what promoter wouldfunction in KSG-SeaGrow. Whether the CMV promoter canbe worked was then investigated. As a result, KSG-SeaGrowwas successfully transfected with the pCDNA3.1-YC2.12plasmid using lipofectamine2000. We detected YFP expres-sion 48 h after transfection. Fluorescence of yellow cameleonwas observed in ≈40% of KSG-SeaGrow cells at this stage.Following chemical resistance selection using zeocin, the cellswere all fluorescent and normally divided (Fig. 3). Weobtained similar results using KSG cells, which were culturedwith FBS (data not shown). The expression of pCDNA3.1-YC2.12 by KSG/KSG-SeaGrow cells suggests that it is pos-sible to use a widely available, commercial CMV promoter.KSG cell behavior under the hydrostatic pressure. Fish arealways stimulated by the hydrostatic pressures. Here, weexamined the behavior of cell proliferation under hydrostaticpressure. Figure 4A shows that the KSG cells under 5 MPa(equivalent to 500-m depth) showed cell protrusions. Then,less protrusions and a round shape were observed in the cellsunder 10 MPa (equivalent to a 1,000-m depth). In those under20–40MPa, however, each cell aggregated and became roundin shape (Supplemental Fig. 1). Also, some of the cells de-tached from the culture plate. Moreover, the cells subjected tovarious pressures were cultured to investigate the cell density.The cells given 5 and 10MPa showed higher cell density than0 MPa (Fig. 4B). Those under ≥20 MPa did not attach on theculture flasks. These results suggest that 10-MPa hydrostaticpressures help the cells to proliferate more.Results in this study show that KSG scorpionfish cells givea choice of Leibovitz’s L-15 culture media additive:mammalian-derived serum (FBS), fish serum (SeaGrow), orfish bone extraction (Hy-Fish). The KSG cell line is an inter-esting research platform for gene and protein expression stud-ies to elucidate deep-sea fishes.ReferencesBarnes D.; Sato G. Serum-free cell culture: a unifying approach. Cell 22:649–655; 1980.Bols N. C.; Mosser D. D.; Steels G. B. Temperature studies and recentadvanceswith fish cells in vitro.Comp. Biochem. Physiol. A Physiol.103: 1–14; 1992.Buonocore F.; Libertini A.; Prugnoli D.; Mazzini M.; Scapigliati G.Production and characterization of a continuous embryonic cell linefrom sea bass (Dicentrarchus labrax L.). Mar. Biotechnol. (NY) 8:80–85; 2006.Ciemerych M. A.; Archacka K.; Grabowska I.; Przewoźniak M. Cell cycleregulation during proliferation and differentiation of mammalian mus-cle precursor cells. Results Probl. Cell Differ. 53: 473–527; 2011.Hashimoto H.; Toyohara H.; Yokoyama Y.; Sakaguchi M.; Ozato K.;Wakamatsu Y. Effects of carp serum on the growth of goldfish fincells in early passage. J. Fish Biol. 50: 201–207; 1997.Hightower L. E.; Renfro J. L. Recent applications of fish cell culture tobiomedical research. J. Exp. Zool. 248: 290–302; 1988.Japan Patent Kokai 2008-220198 (2008.09.25).Konishi T.; Matsumoto S.; Tsuruwaka Y.; Shiraki K.; Hirata K.;Tamaru Y.; Takagi M. Enhancing the tolerance of zebrafish(Danio rerio) to heavy metal toxicity by the expression ofplant phytochelatin synthase. J. Biotechnol. 122(3): 316–325;2006.Koyama S.; Miwa T.; Aizawa M.; Sato T. Optical chambersystem designed for microscopic observation of living cellsunder extremely high hydrostatic pressure. Extremophiles 5:409–415; 2001.SCORPIONFISH KSG CELL POTENTIAL 443Padilla D.; Béringue V.; Espinosa J. C.; Andreoletti O.; Jaumain E.;Reine F.; Herzog L.; Gutierrez-Adan A.; Pintado B.; Laude H.;Torres J. M. Sheep and goat BSE propagate more efficientlythan cattle BSE in human PrP transgenic mice. PLoS Pathog. 7:e1001319; 2011.Tsuruwaka Y.; Konishi T.;Miyawaki A.; TakagiM. Real-time monitoringof dynamic intracellular Ca(2+) movement during earlyembryogenesis through expression of yellow cameleon. Zebrafish4: 253–260; 2007.Wolf K.; QuimbyM. C. Fish physiology. Academic Press, London; 1969.Yan Y.; Cui H.; Jiang S.; Huang Y.; Huang X.; Wei S.; Xu W.; Qin Q.Identification of a novel marine fish virus, Singapore Grouperiridovirus-encoded microRNAs expressed in grouper cells by solexasequencing. PLoS One 29: e19148; 2011.444 TSURUWAKA ETAL.

In Vitro Cellular & Developmental Biology - Animal

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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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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