A spontaneously immortalized cell line from the muscle of red sea bream (Pagrus major)

A spontaneously immortalized cell line from the muscle of red sea bream (Pagrus major) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A spontaneously immortalized cell line from the muscle of red sea bream (Pagrus major) Mikinori Ueno, Yu Yamaguchi, Asami Yoshida, Katsuya Hirasaka, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6798589/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 12 You are reading this latest preprint version Abstract Cell culture plays a crucial role in various fields of life science research such as cancer, immunology, and virology. Numerous cell lines have been established in mammals, while fish cell lines remain comparatively limited in number. This study established a spontaneously immortalized cell line from the muscle of red rea bream, Pagrus major , which is a commercially important fish in Japan. Primary cells were isolated from muscle tissue using 0.2% collagenase and, cultured in Leibovitz’s L-15 medium without CO 2 . The cells required fetal bovine serum in a dose-dependent manner at 28°C for optimal growth. The cells were also able to grow in F12 medium, but not in Dulbecco’s modified Eagle’s medium. RNA-sequencing analysis indicated that the isolated cells differentiated into fibroblasts or epithelial-like cells with an increasing number of passages because collagen-related genes were expressed more than in muscle tissue. Upon serum starvation, the cells differentiated into adipocyte-like cells because of slight lipid accumulation. Therefore, the established cells were considered to lack myogenic potential. In conclusion, we established the Nagasaki University Fisheries- P. major 1 (NUF-PM1) cell line, which consisted of fibroblast/epithelial-like cells, but not myosatellite/myoblast cells from the muscle of P. major , and we observed the cells over 80 passages. This study adds to the limited number of fish cell lines available for research, which could help advance fish cellular aquaculture and related fields. fish cell line red sea bream Pagrus major muscle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction According to global economic growth, urban populations with rising incomes are consuming diets that are high in calories, such as refined fats, oils, and meats, which is driving a global dietary transition, so that agriculture increasingly utilizes cropland and pastureland (Tilman and Clark, 2014 ). This will lead to a crisis in which food requirements will exceed food provision by 2030. Recently, seafood, such as finfish, shellfish, and marine algae has received attention because they are rich in nutrients equivalent to livestock and vegetables. In recent decades, the total global production of freshwater and seawater aquaculture has been increasing compared to the total global freshwater and seawater fish catch (FAO, 2020 ). The total global biological production of marine aquaculture has been predicted to increase in the future as it is expanded in scope (Gentry et al., 2017 ). However, there are serious problems associated with aquaculture fields such as infectious diseases and harmful algal blooms, which cause severe economic losses to aquaculture businesses. Therefore, many researchers have been trying to develop vaccines and medicines for infectious diseases and take measures against harmful algal blooms. Cell culture plays a crucial role in various fields of life science research, such as oncology, immunology, virology, vaccines, and medicine development, and numerous cell lines have been established in mammals. Likewise, cell lines established from freshwater and seawater finfish have been well-documented (Thangaraj et al., 2021 ). For instance, Grunt Fins cells (GF cells) established from the fin of Parapristipma trilineatum have been used as amodel for red sea bream ( Pagrus major ) iridovirus infection and vaccine development (Nakajima et al., 1997 ; Kawato et al., 2017 ). Epithelioma Papulosum Cyprini cells (EPC cells) from the skin of the fathead minnow ( Pimephales promelas ) have been used as a model for resistance to cellular senescence (Futami et al., 2021 ; 2022 ). RTgill W-1 (ATCC, CRL-2523) were established from the gill of the rainbow trout ( Oncorhynchus mykiss ) (Bols et al., 1994 ) and have been used to examine the ichthyotoxicity of harmful algal blooms (Dorantes-Aranda et al., 2011 ; Zou et al., 2013 ). However, the number of fish cell lines is small compared to those of mammals. This scarcity of fish cell lines underscores the need for continued efforts to develop and characterize new fish cell cultures to advance our understanding of aquatic biology and to address pressing issues in fisheries and aquaculture. Recently, meat derived from cellular agriculture/aquaculture has attracted attention as a next-generation food that is clean and sustainable with the minimum consumption of livestock and fish. Natural and cultured finfish are exposed to many environmental pollutants such as microplastics, medical waste, and food with the accumulation of poisonous heavy metals and marine toxins (Rubio et al., 2019). To date, several fish cell lines characterized as muscle cells have been established, although they lack myogenic differentiation potential. Recently, two fish myoblast cell lines with myogenic potential from the olive flounder ( Paralichthys olivaceus ) (Krishnan et al., 2023 ) and Atlantic mackerel ( Scomber scombrus ) (Saad et al., 2023 ) have been established. Both cell lines spontaneously immortalized with increasing passage number, indicating that fish myoblasts have a strong proliferative potential and are suitable for cellular aquaculture. As discussed above, we focus on P. major , which is a commercially important fish species in marine aquaculture fisheries in Japan, second only to the yellowtail ( Seriola quinqueradiata ). P. major is cooked in various dishes in Japan, such as sashimi and sushi, and it is important not only as an ingredient but also in Japanese culture on ceremonial occasions. Gilthead sea bream ( Sparus aurata ), a species closely related to P. major , has been used to characterize and differentiate myoblasts (Monstserrat et al., 2007; García de la serrana 2014; Millan-Cubillo et al., 2019 ). Several cell lines from P. major have been reported (Watanabe et al., 1981 ; Ku et al., 2010 ; Luo et al., 2023), however, no cell lines have been established from muscle tissue. In the present study, we tried to isolate and culture myosatellite cells prepared by enzymatic digestion of muscle tissue from P. major . This is the first report of a fibroblast/epithelial-like cell line, Nagasaki University Fisheries- P. major 1 (NUF-PM1), established from the muscle of cultured P. major . Materials and methods Materials Collagenase type I (LS004196) was purchased from Worthington Biochemical Corporation (Lakewood, NJ). Leibovitz’s L-15 (L-15; 11415064) medium, F-12 Nutrient Mixture (F-12; 21700075) medium, and fetal bovine serum (FBS; 12483020) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA). Easy iMatrix-511 was purchased from TAKARA BIO Inc. (Shiga, Japan). Basic fibroblast growth factor (bFGF) was from PeproTech Inc. (Cranbury, NJ). Chicken embryo extract (C3999) was from US Biological (Salem, MA). Nystatin (N6261) was from Sigma-Aldrich Co (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM; 043-300859) medium, Dulbecco’s PBS (D-PBS; 045-29795) Penicillin (021-07732), streptomycin (194–08512), gentamycin (079-02973), and Penicillin-Streptomycin Solution (168-23191) were purchased from FUJIFILM Wako Chemical Corporation (Osaka, Japan). All other reagents were of the highest commercially available grade. Experimental animal Cultured juvenile P. major (15.3 ± 1.0 cm, 55.4 ± 11.7 g) were purchased from Kaneko Sangyo Co. Ltd. (Nagasaki, Japan), and cultured in artificial sea water (Marine Art Hi, Tomita Pharmaceutical Co., Ltd., Naruto, Japan) in 300-L tank at 17°C. This study was reviewed and approved by the Animal Care and Use Committee of the Faculty of Fisheries, Nagasaki University (permit no. NF-0072), in accordance with the Guideline for Animal Experimentation of the Faculty of Fisheries and the Regulations of the Animal Care and Use Committee of Nagasaki University. Cell isolation from muscle tissue Figure 1 shows the scheme for cell isolation from muscle tissue. P. major was anesthetized in 0.05% of 2-phenoxyethanol in sea water and euthanized by brain disruption and exsanguination. The surface of the body was disinfected with 70% ethanol. The muscle tissue was anatomically resected and kept in washing buffer (Table 1 ) on ice for the next procedure. It was disinfected with 0.02% sodium hypochlorite for 1 min, washed with washing buffer, and minced with a blade and scissors. The minced tissue was incubated with 0.2% collagenase, 0.01% trypsin inhibitor, 100 U/ml penicillin, and 100 µg/mL streptomycin in L-15 medium for 2 h at 25°C with stirring. The enzymatic reaction was stopped by the addition of L-15 medium, and the supernatant was recovered after centrifugation at 100 × g for 3 min. The supernatant was passed through 100-µm and 40-µm cell strainers (pluriSelect Life Science UG & Co. KG, Leipzig, Germany) to remove undigested tissues. After centrifugation, cell pellets were resuspended in ACK lysis buffer (0.16 M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA) to hemolyze erythrocytes. The cell suspension was transferred to an iMatrix-coated 6-well plate and cultured in growth medium (Table 2 ) at 28°C without CO 2 . Table 1 Components of the washing buffer. Chemicals Concentration Penicillin 500 U/mL Streptomycin 500 µg/mL Gentamycin 0.05 mg/mL in D-PBS Table 2 Components of the growth medium. Chemicals Concentration FBS 20% NaCl 1% Penicillin 100 U/mL Streptomycin 100 µg/mL Nystatin 20 U/mL bFGF 10 ng/mL Chicken embryo extract 1% in L-15 medium Subculture The isolated cells were cultured in growth medium at 28°C without CO 2 (Table 2 ), detached with 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA, and subculture into T-25 Falcon cell culture flask (Corning, Inc., Corning, NY). Isolated cells from passages 5 to 80 were observed under a BX-X710 microscope (Keyence Corp., Osaka, Japan). Cell growth The effects of FBS concentration, culture temperature, and different culture media on the proliferation of NUF-PM1 cells were examined, respectively. Adherent NUF-PM1 cells (1.0 × 10 4 cells/well) in a 6-well plate were cultured growth medium containing 5, 10, or 20% FBS and 10 ng/mL bFGF at 28°C for 7 days. Adherent NUF-PM1 cells (1.0 × 10 4 cells/well) in a 6-well plate were cultured in growth medium containing 20% FBS at 19, 28, or 37°C for 7 days. Adherent NUF-PM1 cells (1.0 × 10 4 cells/well) in a 6-well plate were cultured in three different growth media (L-15, F-12, and DMEM) containing 20% FBS and 10 ng/mL bFGF at 28°C for 7 days. The cells were detached with 0.25% trypsin-1 mM EDTA and cell number was counted using a hemocytometer. RNA sequencing analysis Total RNA was prepared from muscle tissue, NUF-PM1 passage 5 (P5), and NUF-PM1 passage 20 (P20) cells using TRIzol™ Reagent (15596026; Thermo Fisher Scientific Inc.). Libraries were generated using a NEBNext Ultra II RNA Library Prep Kit for Illumina (7770, New England Biolabs, Ipswich, MA) and analyzed using DNBSEQ T7 (MGI, Shenzhen, China). The draft genome of P. major ( https://figshare.com/articles/dataset/First_Draft_genome_for_Pagrus_major/6962867/1)(Shin et al., 2018) was used for sequence mapping. This study was performed using the “RIAS Omics Analysis System” provided by Rhelixa Inc (Tokyo, Japan). Heatmaps were created from the Z-scores of the normalized counts using the stats (version 3.6.1) and gplots (version 3.0.1.1) R packages. Differentiation Adherent NUF-PM1 cells (confluent cells/in a 6-well plate) were cultured in slightly modified differentiation medium (Rosenblatt et al., 1995 ) containing 2% FBS, 10% horse serum (HS), and 10 ng/mL bFGF (Table 3 ) at 28°C for 7 days. The well-known mouse fibroblast 3T3-L1 cell line can differentiate into adipocytes in differentiation medium containing 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin (MDI induction medium). This medium was also used for NUF-PM1 cells differentiation. Adherent NUF-PM1 cells were incubated with 0.5 mM of IBMX, 1 mM dexamethasone, and 10 µg/mL insulin in growth medium. On day 3, the differentiation medium was replaced with growth medium containing 10 µg/mL insulin and the cells were incubated until day 7 (Fig. 5 A). After incubation, the cells were fixed with 3.7% formalin for 30 min at room temperature. After washing with phosphate buffered saline, the fixed cells were stained with Oil Red O for 20 min at room temperature with gentle shaking. After washing, the stained cells were observed under a BX-X710 microscope. In addition, Oil Red O in the cells were extracted with 2-propanol for 5 min at room temperature with slight shaking, and absorbance was measured at 492 nm (Multiskan GO; Thermo Fisher Scientific, Inc.). Table 3 Components of the differentiation media. Differentiation medium MDI induction medium Insulin medium 2% FBS 20% FBS 20% FBS 10% HS 0.5mM IBMX 10 µg/mL Insulin 10 ng/mL bFGF 1 mM Dexamethasone 10 ng/mL bFGF 10 µg/mL Insulin 10 ng/mL bFGF in L-15 medium in L-15 medium in L-15 medium Results Primary culture and subculture Since finfish are constitutively exposed to bacteria, viruses, and parasites in freshwater and seawater, it is important to prevent their contamination of cell culture. To avoid this, we disinfected the surface of P. major using a combination of sodium hypochlorite and 70% ethanol. Additional sodium chloride was added and osmolarity was adjusted to a final sodium chloride concentration of 1% in L-15 medium, as described previously (Watanabe et al., 1997 ). Next, we tried to isolate myosatellites/myoblasts from muscle tissue of P. major using collagenase digestion (Fig. 1 ). We used iMatrix-511, which is a human laminin-511 E8 fragment, to allow primary cells to adhere to the culture flask and observed primary cell adhesions at 5 days after the start of culture (Fig. 2 ). The isolated cells were subcultured using 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA twice per week and cultured the cells for over 80 passages. Following this approach, we established the NUF-PM1 cell line. The cells could be stored at -80°C using conventional cryopreservation solutions, such as CELLBANKER I (TAKARA BIO Inc.) and Bambanker (Nippon Genetics Co., Ltd., Tokyo, Japan). Optimal cell culture conditions Next, we determined the optimal culture conditions for NUF-PM1 cells. As shown in Fig. 3 A, NUF-PM1 cells grew in an FBS dose-dependent manner and doubling times of 20%, 10%, and 5% were observed at 42.0 ± 7.8 h, 95.1 ± 27.1 h, and 120.3 ± 33.0 h, respectively. NUF-PM1 cells showed the best growth curve at 28°C. Although they grew slightly at 19°C, cell death was not induced. No cell growth was observed at 37°C (Fig. 3 B). L-15 and F-12 media promoted NUF-PM1 cells growth, although the cells grew slowly in DMEM (Fig. 3 C). Since minimum essential medium (MEM) is often used for fish cell culture, we examined its effect of MEM on the growth of NUF-PM1 cells. The cells grew better in L-15 medium than in MEM at 28°C (Supplementary Data 1). bFGF is an essential growth factor that promotes the proliferation and differentiation of stem cells, such as induced pluripotent stem (iPS) cells and myosatellite cells. NUF-PM1 cells showed a spindle shape in the presence of 10 ng/mL of bFGF, even though they developed into a large and blurred shape in the absence of bFGF. In addition, the number of NUF-PM1 cells in medium containing 10 ng/mL bFGF was twice that in medium without bFGF. Therefore, bFGF is an essential growth factor for the proliferation of NUF-PM1 cells and maintains these cells in an undifferentiated state. Characterization of the NUF-PM1 cell line by RNA sequencing In general, gene expression of Pax7 and MyoD, which are markers for myosatellite cells (Parker et al., 2003 ), is analyzed to identify the presence of cultured fish myosatellite cells (Krishnan et al., 2023 ; Saad et al., 2023 ), although these genes have not been identified in P. major . Therefore, to characterize NUF-PM1 cells, RNA sequencing was performed. Total RNA extracted from muscle tissue and P5 and P20 NUF-PM1 cells was analyzed using DNBSEQ T7. Since we used the draft genome of P. major for sequence mapping (Shin et al., 2018 ), there were various predicted sequences. First, we created a heatmap for muscle-related gene expression. Muscle-related genes such as myogenin-2, troponin T and I, and myosin light chain 2 were highly expressed in muscle tissue, although they were expressed at a low level in P5 and P20 NUF-PM1. Myocilin, tropomyosin 4a, and myotrophin were expressed at the highest levels in P5 NUF-PM1 among the three groups. Fatty acid binding protein 3, keratin, and type I cytoskeletal 18 were expressed at the highest level in P20 NUF-PM1 cells compared with muscle and P5 NUF-PM1 cells. Next, we compared the expression of collagen-related genes because of the decrease in the expression of muscle-related genes with increasing NUF-PM1 cell passage number. They were expressed at low levels in muscle tissue. Collagen type I alpha 3 chain, collagen type VI, and vitronectin showed the highest expression in P5 NUF-PM1 cells, although their expression decreased at P20. Collagen alpha-1(I) chain-like precursor and collagen type VI alpha 2 showed the highest expression in P20 NUF-PM1 cells. These results suggest that myosatellites/myoblasts in NUF-PM1 cells were replaced by fibroblasts and/or epithelial cells with increasing passage number. Interestingly, CD44 in P20 NUF-PM1 cells (Supplemental Fig. 2). CD44 is not only a hyaluronan receptor but is also a marker of human mesenchymal stem cells indicating that NUF-PM1 cells have the potential to differentiate. Adipocyte-like differentiation First, we attempted to differentiate NUF-PM1 cells into a myotube, but failed to observe myotube formation, indicating that this cell line lacked myogenic potential. In contrast, microscopic observation showed lipid droplet-like substances in differentiated NUF-PM1 cells. Therefore, we examined lipid accumulation in differentiated NUF-PM1 cells by Oil Red O staining. NUF-PM1 cells incubated in growth medium showed a thin shape and no lipid accumulation. NUF-PM1 cells incubated in differentiation medium clearly showed lipid accumulation. Conversely, culture in MDI induction medium did not result in the accumulation of lipids in NUF-PM1 cells (Fig. 5 A). As shown in Fig. 5 B, the differentiation medium significantly increased absorbance at 492 nm. MDI induction medium slightly increased lipid accumulation compared to that in control medium. These results suggest that NUF-PM1 cells have adipogenic potential under culture conditions with low FBS concentrations. Discussion In this study, we spontaneously immortalized and established the NUF-PM1 cell line from the muscle of P. major . We used collagenase type I and iMatrix-511 for the enzymatic digestion of muscle tissue and the adhesion of primary cells, respectively. Generally, explanted cell isolation from finfish is often used and is well-documented (Thangaraj et al., 2021 ; Kumar et al., 2024). Since enzymatic digestion with collagenase is widely employed to isolate myosattelites/myoblasts from mammalian muscle, we followed a previously reported method for mammals. In our preliminary study, collagen, fibronectin (a gift from Immuno Probe Co., Ltd., Saitama, Japan), laminin (a gift from Immuno Probe Co., Ltd.), and ε-poly-L-lysine (SPL01, Cosmo Bio Co., Ltd., Tokyo, Japan) were used to coat cell culture plates to allow primary cells to adhere, and iMatrix-511 was found to be an optimal coating for primary cells from the muscle of P. major . iMatrix-511 is the active site of the human laminin-511 E8 fragment and promotes the adhesion and proliferation of iPS cells without feeder cells (Miyazaki et al., 2012 ). Saad et al. ( 2023 ) used iMatrix recombinant laminin-511-E8 for the adhesion of primary mackerel satellites cell isolated from S. scombus . Furthermore, a Japanese eel myoblast cell line was established using iMatrix-511 for the adhesion of primary cells from muscle tissue (Ikeda et al., 2024 ). Hence, these results suggest that laminin is an optimal cellular matrix for the adhesion of primary cells from the muscle tissue of marine finfish. On the other hand, previous studies have established muscle cell lines with myogenic differentiation potential without using a coating from Sebastes schlegelii (Kong et al., 2021 )d olivaceus (Krishnan et al. 2023 ). These reports suggest that it is essential to choose a cellular matrix for the adhesion of primary muscle cells according to the fish species. FBS plays an important role not only in mammalian cell culture, but also in fish cell culture, supplying essential micronutrients and growth factors. NUF-PM1 cells showed FBS-dependent growth (Fig. 3 A), in agreement with previous reports (Goswani et al., 2023; Krishnan et al., 2023 ). However, it is still unclear why fish cells line reached plateaus when cultured in low concentrations of FBS (Goswani et al., 2023; Krishnan et al., 2023 ). It is conceivable that high FBS concentrations may promote the growth of fish cell lines regardless of limited culture area or cell-to-cell contact inhibition and maintain cell shape. NUF-PM1 showed the best growth curve at 28°C (Fig. 3 B), which is reasonable because the optimal water temperature for P. major is in the range of 20 − 28°C. L-15 medium has been used frequently for fish cell culture because it does not require CO 2 (Leibovitz 1963 ), which necessitates the use of an expensive incubator. In addition, fish cells have relatively high amino acid requirements due to amino acid metabolism because their carbohydrate utilization efficiency is low (Wilson, 1986 ). L-15 and F-12 media both promoted proliferation of NUF-PM1 cells (Fig. 3 C). F-12 medium contains rich amino acid components, hypoxanthine, linoleic acid, and lipoic acid, which have the potential to promote the cell proliferation of NUF-PM1 cells. Chicken embryo extract contains important growth factors and nutrients, and is often used for myosatellite culture in mouse studies (Yoshioka et al., 2020 ). We also used Chicken embryo extract to support primary cell culture from the muscle of P. major , however, we found it was not essential. NUF-PM1 cells were subcultured with 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA and passaged more than 80 times. These results indicated that muscle cells from juvenile P. major are capable of growing and undergoing cell division, although we failed to isolate and culture muscle cells from adult P. major . Hence, juvenile or young fish are suitable for cell isolation and muscle cell culture. Recently established myoblast cell lines were spontaneously immortalized by continuous cell culture without cell cloning (Krishnan et al., 2023 ; Saad et al., 2023 ). However, we failed to establish a myoblast cell line. Ikeda et al. ( 2024 ) established an eel myoblast cell line by single cell cloning expressing Pax7, MyoD, and Cdh17. In a preliminary study, we observed myotube differentiation from primary cells suggesting that they contained myoblasts. However, fibroblast and epithelial-like cells isolated from the muscle of P. major showed strong cell proliferation compared to myoblasts. Hence, it would be better to establish a myoblast cell line using the cell cloning approach described above. To characterize NUF-PM1cells, RNA sequencing analysis was performed, resulting in the identification of NUF-PM1 cells as fibroblasts, and not myosatellites. Muscle-related genes were highly expressed in muscle tissue, although their expression was decreased in P5 and P20 NUF-PM1 cells, indicating that myosatellites were replaced by fibroblasts or epitheliocyte-like cells by increasing the passage number. Fibroblasts and epitheliocytes have active cell expansion compared to myosatellites. Myosatellites play an important role in homeostasis such as the growth and repair of muscle fibers in mammals (Mauro et al., 1961; Yoshioka et al., 2020 ). In the case of muscle growth and injury, myosatellites self-renew and differentiate into myofibroblasts and myofibers. Myosatellites and myogenesis are regulated by myogenic regulatory factors such as Pax7, MyoD, and Myf5 (Parker et al., 2003 ). Teleost myosatellites have been studied widely (Gabillard et al., 2010 ; Biacchesi et al., 2016 ; Peng et al., 2016 ; Krishnan et al., 2023 ; Saad et al., 2023 ). They differentiated into myotubes at low FBS concentrations. In contrast, NUF-PM1 cells expressed CD44, which is a marker for mesenchymal stem cells, and were able to differentiate into adipocyte-like cells due to lipid accumulation under low FBS concentrations. NUF-PM1 cells may be a valuable model for in vitro lipid metabolism in fish. How NUF-PM1 cells differentiate into adipocyte-like cells remains to be determined and it should be addressed in the next step of characterization this cell line. Almost all mammalian cell lines have been established as cancer cells isolated from tumor tissues and immortalized cells with deliberate immortalization by lentiviral vector transduction. In contrast, almost all fish cell lines have been established by spontaneous immortalization. Fish are capable of resistance to senescence (Reznick et al., 2002 ; Finch, 2009 ). However, it remains unclear how fish cells are capable of spontaneous immortalization. Interestingly, the fish genome lacks p16, a cyclin-dependent kinase inhibitor 2A, which functions as a cell cycle regulatory protein (Futami et al., 2022 ). Mutations of p16 in higher mammals cause oncogenesis (Liggett and Sidransky, 1998). This indicates that mammalian cells evolutionarily acquired cell cycle regulator genes to inhibit oncogenesis. Clarifying the mechanism underlying senescence resistance in fish will not only contributes to aging research but may also enable the development of anti-aging drugs. Therefore, further study is required to elucidate how fish cells undergo spontaneous immortalization. Conclusion We spontaneously immortalized and established NUF-PM1 cell line from the muscle of juvenile P. major , which was characterized as fibroblast/epithelial cells lacking myogenic differentiation. NUF-PM1 cells can differentiate into adipocyte-like cells with the accumulation of intracellular lipid droplets so they have the potential to be utilized in basic research for cellular aquaculture. We must further improve the method for myosatellites isolation and culture for cellular aquaculture research. Declarations Author contribution statement M.U. mainly designed the project, performed the experiments, and prepared the manuscript. Y.Y. performed the experiments. A.Y., H.K., N.T., T.K., and T.T. discussed the data. K.Y. supervised this study. All authors reviewed the manuscript. Conflict of interest MU received a research grant from Ichimasa Kamaboko Co., Ltd. Competing Interests MU received a research grant from Ichimasa Kamaboko Co., Ltd. Funding This study was supported in part by Adaptable and Seamless Technology transfer Program through Target-driven R&D (A-STEP) from Japan Science and Technology Agency (JST) Japan Grant JPMJTM22EG. This work was supported in part by a research grant from Kohjin Bio Co., Ltd., Saitama, Japan. This study was supported in part by a research grant from G-7 Scholarship Foundation. This work was supported by a tenure-track system, CHODAI Kyoso Grant, and State of the Art Research (STAR) Program from Nagasaki University. Author Contribution M.U. mainly designed the project, performed the experiments, and prepared the manuscript. Y.Y. performed the experiments. A.Y., H.K., N.T., T.K., and T.T. discussed the data. K.Y. supervised this study. All authors reviewed the manuscript. Acknowledgement We thank Mr. Yoshimune Onoda (Kaneko Sangyo Co. Ltd.) for valuable comments on red sea bream rearing. We are deeply grateful to Mr. Hiroshi Nomura (Immuno Probe Co., Ltd.) for providing the fibronectin and laminin. We appreciate Mr. Manato Morimoto (Rhelixa, Inc., Tokyo, Japan) for valuable comments on the RNA sequence analysis. Data Availability The data underlying this article will be shared on reasonable request to the corresponding author. References Biacchesi S, Jouvion G, Mérour E, Boukadiri A, Desdouits M, Ozden S, Huerre M, Ceccaldi PE, Brémont M (2016) Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection. Vet Res 47:9. https://doi.org/10.1186/s13567-015-0301-1 Bols NC, Barlian A, Chirino-Trejo M, Caldwell SJ, Goegan P, Lee LE (1994) Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. 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J Clin Oncol 16(3):1197–1206. https://doi.org/10.1200/JCO.1998.16.3.1197 Luo X, Fu X, Liang H, Niu Y, Lin Q, Ma B, Liu L, Li N. Development of a new marine fish continuous cell line derived from brain of red sea bream ( Pagrosomus major ) and its application of fish virology and heavy metal toxicology. Animals 13(22):3524. https://doi.org/10.3390/ani13223524 Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495 https://doi.org/10.1083/jcb.9.2.493 Millan-Cubillo AF, Martin-Perez M, Ibraz A, Fernandez-Borras J, Gutierrez J, Blasco J (2019) Proteomic characterization of primary cultured myocytes in a fish model at different myogenesis stages. Sci Rep 9:14126. https://doi.org/10.1038/s41598-019-50651-w Miyazaki T, Futaki S, Suemori H, Taniguchi Y, Yamada M, Kawasaki M, Hayashi M, Kumagai H, Nakatsuji N, Sekiguchi K, Kawase E (2012) Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat Commun 3:1236. https://doi.org/10.1038/ncomms2231 Nakajima K, Maeno Y, Kurita J, Inui Y (1997) Vaccination against red sea bream iridoviral disease in red sea bream. Fish Pathog 32(4):205–209. https://doi.org/10.3147/jsfp.32.205 Parker MH, Seale P, Rudnicki MA (2003) Looking back to the embryo: defining transcriptional networks in adult myogeneis. Nat Rev Genet 4:497–507. https://doi.org/10.1038/nrg1109 Peng LM, Zheng Y, You F, Wu ZH, Tan X, Jiao S, Zhang PJ (2016) Comparison of growth characteristics between skeletal muscle satellite cell lines from diploid and triploid olive founder Paralichthys olivacues . PeerJ 4:e1519. https://doi.org/10.7717/peerj.1519 Reznick D, Ghalambor C, Nunney L (2002) The evolution of senescence in fish. Mech Ageing Dev 123(7):773–789. https://doi.org/10.1016/S0047-6374(01)00423-7 Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA (1995) Culturing satellite ells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10):773–779. https://doi.org/10.1007/BF02634119 Saad MK, Yuen Jr. JSK, Joyce CM, Lim T, Wolfson TL, Wu J, Laird J, Vissaparagada S, Calkins OP, Ali A, Kaplan DL (2023) Continuous fish muscle cell line with capacity for myogenic and adipogenic-like phenotypes. Sci Rep 13:5098. https://doi.org/10.1038/s41598-023-31822-2 Shin HG, Shin Y, Jung M, Hong JM, Lee S, Subramaniyam S, Noh ES, Shin EH, Park EH, Park JY, Kim YO, Choi KM, Nam BH, Park CI (2018) First draft genome for red sea bream of family sparidae. Front Genet 9:643. https://doi.org/10.3389/fgene.2018.00643 Thangaraj RS, Narendrakumar L, Geetha PP, Shamuganathan AR, Dharmaratnam A, Nithianantham SR (2021) Comprehensive update on inventory of finfish cell lines developed during the last decade (2010–2020). Rev Aquacult 13:2248–2288. https://doi.org/10.1111/raq.12566 Tilman D, Clark M (2014) Global diets link environmental sustainability and human health. Nature 515:518–522. https://doi.org/10.1038/nature13959 Watanabe T, Shoho T, Ohta H, Kubo N, Kono M, Furukawa K (1997) Long-term cell culture of resident peritoneal macrophage from red sea bream Pagrus major . Fish Sci 63(6):862–866. https://doi.org/10.2331/fishsci.63.862 Watanabe Y, Hanada H, Uchiyama M (1981) Monolayer cell cultures from marine fishes. Fish Pathog 15:201205. https://doi.org/10.3147/jsfp.15.201 Wilson RP (1986) Protein and amino acid requirements of fishes. Ann Rev Nutr 6:225–244. https://doi.org/10.1146/annurev.nu.06.070186.001301 Yoshioka K, Kitajima Y, Okazaki N, Chiba K, Yonekura A, Ono Y (2020) A modified pre-plating method for high-yield and high-purity muscle stem cell isolation from human/mouse skeletal muscle tissues. Front Cell Dev Biol 8:793. https://doi.org/10.3389/fcell.2020.00793 Zou Y, Kim D, Yagi M, Yamasaki Y, Kurita J, Iida T, Matsuyama Y, Yamaguchi K, Oda T. (2013) Application of LDH-release assay to cellular-level evaluation of the toxic potential of harmful algal species. Biosci Biotechnol Biochem 77(2):345–352. https://doi.org/10.1271/bbb.120764 Additional Declarations Competing interest reported. MU received a research grant from Ichimasa Kamaboko Co., Ltd. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6798589","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489586735,"identity":"8867aec9-0dc8-4937-84c8-d7db7ca29377","order_by":0,"name":"Mikinori Ueno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYDACZjaGAyCaH8o3YINJENQi2UC0FgaoEoMDUJqgu3Tb2RIP81TckTO+3fz4xY8/dsZ8DDwGDD9qGNjNcWgxO8x24DDPmWfGZneOmVn2tiWbsQG1MPYcY2C2bMClhb3hMG/b4cRtNxLMDHgbmG3Y5N8YMPA2MDDDnIpdy7/DiZtnpH8z/POn3gZsy1+8WoAO4204nLhBIsf4MQ/bYbDDmPHbwpZwcM6xw8YSN3LKmGXbjhuzMbAVHJY5JoHbL+ePGX94U3NYjn9G+uaPb/5UG85vYN748E2NTTKuEEMGbBIwFtBJEsmEYwgY3x+QeXbEaBkFo2AUjIIRAQBkyla0++YUEwAAAABJRU5ErkJggg==","orcid":"","institution":"Nagasaki University","correspondingAuthor":true,"prefix":"","firstName":"Mikinori","middleName":"","lastName":"Ueno","suffix":""},{"id":489586736,"identity":"dde3df48-b1e0-4f26-8c9d-3a7d525c2257","order_by":1,"name":"Yu Yamaguchi","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":489586737,"identity":"35a2e73c-cf1f-47f1-9d01-a642fc8de2de","order_by":2,"name":"Asami Yoshida","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Asami","middleName":"","lastName":"Yoshida","suffix":""},{"id":489586738,"identity":"4dd5ad91-f3bc-4e53-883d-0bcdcb65f0c3","order_by":3,"name":"Katsuya Hirasaka","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Katsuya","middleName":"","lastName":"Hirasaka","suffix":""},{"id":489586739,"identity":"db9b88d1-1b04-4052-9592-a0a31af47826","order_by":4,"name":"Natsumi Takahashi","email":"","orcid":"","institution":"Ichimasa Kamaboko Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Natsumi","middleName":"","lastName":"Takahashi","suffix":""},{"id":489586740,"identity":"ef7dd0c6-afb6-49a4-8507-63f039265342","order_by":5,"name":"Tomoko Kadowaki","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Kadowaki","suffix":""},{"id":489586741,"identity":"a0a7325d-8ea4-49b0-bb78-5b1cd2427fea","order_by":6,"name":"Takayuki Tsukuba","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Tsukuba","suffix":""},{"id":489586742,"identity":"c92ef826-f683-4d70-9c17-6ee638bcfaab","order_by":7,"name":"Kenichi Yamaguchi","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Yamaguchi","suffix":""}],"badges":[],"createdAt":"2025-06-02 04:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6798589/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6798589/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10695-025-01603-x","type":"published","date":"2025-11-08T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87517246,"identity":"ece96ddc-2645-4a4c-ad62-0f6a0614b7a6","added_by":"auto","created_at":"2025-07-24 16:53:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60594,"visible":true,"origin":"","legend":"\u003cp\u003eScheme for cell isolation.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/08bfa76d8a3d0d75b0b45f52.jpg"},{"id":87517248,"identity":"2e7603f0-8cdf-4e3d-964d-11c0f7371a03","added_by":"auto","created_at":"2025-07-24 16:53:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83824,"visible":true,"origin":"","legend":"\u003cp\u003eThe isolated cells were cultured in growth medium at 28°C, and passaged by 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA. Images of the cells were taken at the indicated passage number. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/b42daa23d9346ef11b4cbb7a.jpg"},{"id":87518184,"identity":"8e7ed8ff-97fd-4f89-98b0-089d76402dd7","added_by":"auto","created_at":"2025-07-24 17:01:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e NUF-PM1 cells (1.0 × 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 6-well plate) were cultured in the indicated concentration of FBS for 7 days. (\u003cstrong\u003eB)\u003c/strong\u003e NUF-PM1 (1 × 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 6-well plate) were cultured in indicated temperature for 7 days. (\u003cstrong\u003eC)\u003c/strong\u003e NUF-PM1 cells (1.0 × 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 6-well plate) were cultured in indicated medium for 7 days. (\u003cstrong\u003eD)\u003c/strong\u003e NUF-PM1 cells (1.0 × 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 6-well plate) were cultured in the presence or absence of bFGF for 7 days. The cells were detached with 0.25%-1 mM EDTA, and cell number was counted using a hemocytometer. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/72d70bfc0a6497a5ff9480cf.jpg"},{"id":87517250,"identity":"929aa8b6-1cdc-4ae4-8da3-0c87bd6ae8fe","added_by":"auto","created_at":"2025-07-24 16:53:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46848,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-sequencing analysis of \u003cstrong\u003e(A)\u003c/strong\u003e muscle-related gene and \u003cstrong\u003e(B)\u003c/strong\u003e collagen-related genes in muscle tissue, and NUF-PM1 cells at P5 and P20.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/1effd28404137723195ee3e2.jpg"},{"id":87517254,"identity":"53471553-2e88-4bfa-bff8-e98387becc11","added_by":"auto","created_at":"2025-07-24 16:53:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e NUF-PM1 cells were cultured in growth medium, differentiation medium, or MDI induction medium for 7 days at 28°C. The cells were stained with Oil Red O and observed under a microscope. Scale bar, 100 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Oil Red O was extracted from the treated cells by 2-propanol for 5 min at room temperature, and absorbance was measured at 492 nm. \u003cstrong\u003e(C)\u003c/strong\u003e The components of each differentiation and induction medium.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/ef963c8095e905c8b0af7d8c.jpg"},{"id":95564813,"identity":"7c351ddb-94e5-4620-b179-190436bcc3d7","added_by":"auto","created_at":"2025-11-10 16:10:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1022574,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/11d9f80e-b12d-4c45-9b04-fcb3850ab7e3.pdf"},{"id":87517247,"identity":"5dfe17cf-edb1-4682-8228-044b1c7e6782","added_by":"auto","created_at":"2025-07-24 16:53:05","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29896,"visible":true,"origin":"","legend":"","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/b2e4a48ae4bf3a67cc1d0eba.jpg"},{"id":87517249,"identity":"f472b6c3-0571-4492-8c7e-2f918550d32d","added_by":"auto","created_at":"2025-07-24 16:53:05","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41626,"visible":true,"origin":"","legend":"","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6798589/v1/716742573fe63fbd2e528577.jpg"}],"financialInterests":"Competing interest reported. MU received a research grant from Ichimasa Kamaboko Co., Ltd.","formattedTitle":"A spontaneously immortalized cell line from the muscle of red sea bream (Pagrus major)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to global economic growth, urban populations with rising incomes are consuming diets that are high in calories, such as refined fats, oils, and meats, which is driving a global dietary transition, so that agriculture increasingly utilizes cropland and pastureland (Tilman and Clark, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This will lead to a crisis in which food requirements will exceed food provision by 2030. Recently, seafood, such as finfish, shellfish, and marine algae has received attention because they are rich in nutrients equivalent to livestock and vegetables. In recent decades, the total global production of freshwater and seawater aquaculture has been increasing compared to the total global freshwater and seawater fish catch (FAO, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The total global biological production of marine aquaculture has been predicted to increase in the future as it is expanded in scope (Gentry et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, there are serious problems associated with aquaculture fields such as infectious diseases and harmful algal blooms, which cause severe economic losses to aquaculture businesses. Therefore, many researchers have been trying to develop vaccines and medicines for infectious diseases and take measures against harmful algal blooms.\u003c/p\u003e\u003cp\u003eCell culture plays a crucial role in various fields of life science research, such as oncology, immunology, virology, vaccines, and medicine development, and numerous cell lines have been established in mammals. Likewise, cell lines established from freshwater and seawater finfish have been well-documented (Thangaraj et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, Grunt Fins cells (GF cells) established from the fin of \u003cem\u003eParapristipma trilineatum\u003c/em\u003e have been used as amodel for red sea bream (\u003cem\u003ePagrus major\u003c/em\u003e) iridovirus infection and vaccine development (Nakajima et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kawato et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eEpithelioma Papulosum Cyprini\u003c/em\u003e cells (EPC cells) from the skin of the fathead minnow (\u003cem\u003ePimephales promelas\u003c/em\u003e) have been used as a model for resistance to cellular senescence (Futami et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). RTgill W-1 (ATCC, CRL-2523) were established from the gill of the rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) (Bols et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and have been used to examine the ichthyotoxicity of harmful algal blooms (Dorantes-Aranda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, the number of fish cell lines is small compared to those of mammals. This scarcity of fish cell lines underscores the need for continued efforts to develop and characterize new fish cell cultures to advance our understanding of aquatic biology and to address pressing issues in fisheries and aquaculture.\u003c/p\u003e\u003cp\u003eRecently, meat derived from cellular agriculture/aquaculture has attracted attention as a next-generation food that is clean and sustainable with the minimum consumption of livestock and fish. Natural and cultured finfish are exposed to many environmental pollutants such as microplastics, medical waste, and food with the accumulation of poisonous heavy metals and marine toxins (Rubio et al., 2019). To date, several fish cell lines characterized as muscle cells have been established, although they lack myogenic differentiation potential. Recently, two fish myoblast cell lines with myogenic potential from the olive flounder (\u003cem\u003eParalichthys olivaceus\u003c/em\u003e) (Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Atlantic mackerel (\u003cem\u003eScomber scombrus\u003c/em\u003e) (Saad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) have been established. Both cell lines spontaneously immortalized with increasing passage number, indicating that fish myoblasts have a strong proliferative potential and are suitable for cellular aquaculture.\u003c/p\u003e\u003cp\u003eAs discussed above, we focus on \u003cem\u003eP. major\u003c/em\u003e, which is a commercially important fish species in marine aquaculture fisheries in Japan, second only to the yellowtail (\u003cem\u003eSeriola quinqueradiata\u003c/em\u003e). \u003cem\u003eP. major\u003c/em\u003e is cooked in various dishes in Japan, such as sashimi and sushi, and it is important not only as an ingredient but also in Japanese culture on ceremonial occasions. Gilthead sea bream (\u003cem\u003eSparus aurata\u003c/em\u003e), a species closely related to \u003cem\u003eP. major\u003c/em\u003e, has been used to characterize and differentiate myoblasts (Monstserrat et al., 2007; Garc\u0026iacute;a de la serrana 2014; Millan-Cubillo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several cell lines from \u003cem\u003eP. major\u003c/em\u003e have been reported (Watanabe et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Ku et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Luo et al., 2023), however, no cell lines have been established from muscle tissue. In the present study, we tried to isolate and culture myosatellite cells prepared by enzymatic digestion of muscle tissue from \u003cem\u003eP. major\u003c/em\u003e. This is the first report of a fibroblast/epithelial-like cell line, Nagasaki University Fisheries-\u003cem\u003eP. major\u003c/em\u003e 1 (NUF-PM1), established from the muscle of cultured \u003cem\u003eP. major\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eCollagenase type I (LS004196) was purchased from Worthington Biochemical Corporation (Lakewood, NJ). Leibovitz\u0026rsquo;s L-15 (L-15; 11415064) medium, F-12 Nutrient Mixture (F-12; 21700075) medium, and fetal bovine serum (FBS; 12483020) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA). Easy iMatrix-511 was purchased from TAKARA BIO Inc. (Shiga, Japan). Basic fibroblast growth factor (bFGF) was from PeproTech Inc. (Cranbury, NJ). Chicken embryo extract (C3999) was from US Biological (Salem, MA). Nystatin (N6261) was from Sigma-Aldrich Co (St. Louis, MO). Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; 043-300859) medium, Dulbecco\u0026rsquo;s PBS (D-PBS; 045-29795) Penicillin (021-07732), streptomycin (194\u0026ndash;08512), gentamycin (079-02973), and Penicillin-Streptomycin Solution (168-23191) were purchased from FUJIFILM Wako Chemical Corporation (Osaka, Japan). All other reagents were of the highest commercially available grade.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental animal\u003c/h3\u003e\n\u003cp\u003eCultured juvenile \u003cem\u003eP. major\u003c/em\u003e (15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 cm, 55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7 g) were purchased from Kaneko Sangyo Co. Ltd. (Nagasaki, Japan), and cultured in artificial sea water (Marine Art Hi, Tomita Pharmaceutical Co., Ltd., Naruto, Japan) in 300-L tank at 17\u0026deg;C. This study was reviewed and approved by the Animal Care and Use Committee of the Faculty of Fisheries, Nagasaki University (permit no. NF-0072), in accordance with the Guideline for Animal Experimentation of the Faculty of Fisheries and the Regulations of the Animal Care and Use Committee of Nagasaki University.\u003c/p\u003e\n\u003ch3\u003eCell isolation from muscle tissue\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the scheme for cell isolation from muscle tissue. \u003cem\u003eP. major\u003c/em\u003e was anesthetized in 0.05% of 2-phenoxyethanol in sea water and euthanized by brain disruption and exsanguination. The surface of the body was disinfected with 70% ethanol. The muscle tissue was anatomically resected and kept in washing buffer (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) on ice for the next procedure. It was disinfected with 0.02% sodium hypochlorite for 1 min, washed with washing buffer, and minced with a blade and scissors. The minced tissue was incubated with 0.2% collagenase, 0.01% trypsin inhibitor, 100 U/ml penicillin, and 100 \u0026micro;g/mL streptomycin in L-15 medium for 2 h at 25\u0026deg;C with stirring. The enzymatic reaction was stopped by the addition of L-15 medium, and the supernatant was recovered after centrifugation at 100 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 min. The supernatant was passed through 100-\u0026micro;m and 40-\u0026micro;m cell strainers (pluriSelect Life Science UG \u0026amp; Co. KG, Leipzig, Germany) to remove undigested tissues. After centrifugation, cell pellets were resuspended in ACK lysis buffer (0.16 M NH\u003csub\u003e4\u003c/sub\u003eCl, 10 mM KHCO\u003csub\u003e3\u003c/sub\u003e, 0.1 mM EDTA) to hemolyze erythrocytes. The cell suspension was transferred to an iMatrix-coated 6-well plate and cultured in growth medium (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) at 28\u0026deg;C without CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComponents of the washing buffer.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChemicals\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePenicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500 U/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGentamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.05 mg/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ein D-PBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComponents of the growth medium.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChemicals\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePenicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100 U/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNystatin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 U/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebFGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 ng/mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChicken embryo extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ein L-15 medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eSubculture\u003c/h3\u003e\n\u003cp\u003eThe isolated cells were cultured in growth medium at 28\u0026deg;C without CO\u003csub\u003e2\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), detached with 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA, and subculture into T-25 Falcon cell culture flask (Corning, Inc., Corning, NY). Isolated cells from passages 5 to 80 were observed under a BX-X710 microscope (Keyence Corp., Osaka, Japan).\u003c/p\u003e\n\u003ch3\u003eCell growth\u003c/h3\u003e\n\u003cp\u003eThe effects of FBS concentration, culture temperature, and different culture media on the proliferation of NUF-PM1 cells were examined, respectively. Adherent NUF-PM1 cells (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) in a 6-well plate were cultured growth medium containing 5, 10, or 20% FBS and 10 ng/mL bFGF at 28\u0026deg;C for 7 days. Adherent NUF-PM1 cells (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) in a 6-well plate were cultured in growth medium containing 20% FBS at 19, 28, or 37\u0026deg;C for 7 days. Adherent NUF-PM1 cells (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) in a 6-well plate were cultured in three different growth media (L-15, F-12, and DMEM) containing 20% FBS and 10 ng/mL bFGF at 28\u0026deg;C for 7 days. The cells were detached with 0.25% trypsin-1 mM EDTA and cell number was counted using a hemocytometer.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was prepared from muscle tissue, NUF-PM1 passage 5 (P5), and NUF-PM1 passage 20 (P20) cells using TRIzol\u0026trade; Reagent (15596026; Thermo Fisher Scientific Inc.). Libraries were generated using a NEBNext Ultra II RNA Library Prep Kit for Illumina (7770, New England Biolabs, Ipswich, MA) and analyzed using DNBSEQ T7 (MGI, Shenzhen, China). The draft genome of \u003cem\u003eP. major\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/articles/dataset/First_Draft_genome_for_Pagrus_major/6962867/1)(Shin et al., 2018)\u003c/span\u003e\u003cspan address=\"https://figshare.com/articles/dataset/First_Draft_genome_for_Pagrus_major/6962867/1)(Shin et al., 2018)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e was used for sequence mapping. This study was performed using the \u0026ldquo;RIAS Omics Analysis System\u0026rdquo; provided by Rhelixa Inc (Tokyo, Japan). Heatmaps were created from the Z-scores of the normalized counts using the stats (version 3.6.1) and gplots (version 3.0.1.1) R packages.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDifferentiation\u003c/h3\u003e\n\u003cp\u003eAdherent NUF-PM1 cells (confluent cells/in a 6-well plate) were cultured in slightly modified differentiation medium (Rosenblatt et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) containing 2% FBS, 10% horse serum (HS), and 10 ng/mL bFGF (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) at 28\u0026deg;C for 7 days. The well-known mouse fibroblast 3T3-L1 cell line can differentiate into adipocytes in differentiation medium containing 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin (MDI induction medium). This medium was also used for NUF-PM1 cells differentiation. Adherent NUF-PM1 cells were incubated with 0.5 mM of IBMX, 1 mM dexamethasone, and 10 \u0026micro;g/mL insulin in growth medium. On day 3, the differentiation medium was replaced with growth medium containing 10 \u0026micro;g/mL insulin and the cells were incubated until day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After incubation, the cells were fixed with 3.7% formalin for 30 min at room temperature. After washing with phosphate buffered saline, the fixed cells were stained with Oil Red O for 20 min at room temperature with gentle shaking. After washing, the stained cells were observed under a BX-X710 microscope. In addition, Oil Red O in the cells were extracted with 2-propanol for 5 min at room temperature with slight shaking, and absorbance was measured at 492 nm (Multiskan GO; Thermo Fisher Scientific, Inc.).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComponents of the differentiation media.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDifferentiation medium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMDI induction medium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInsulin medium\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2% FBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20% FBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20% FBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10% HS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5mM IBMX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 \u0026micro;g/mL Insulin\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 ng/mL bFGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 mM Dexamethasone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/mL bFGF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 \u0026micro;g/mL Insulin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 ng/mL bFGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ein L-15 medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ein L-15 medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ein L-15 medium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePrimary culture and subculture\u003c/h2\u003e\u003cp\u003eSince finfish are constitutively exposed to bacteria, viruses, and parasites in freshwater and seawater, it is important to prevent their contamination of cell culture. To avoid this, we disinfected the surface of \u003cem\u003eP. major\u003c/em\u003e using a combination of sodium hypochlorite and 70% ethanol. Additional sodium chloride was added and osmolarity was adjusted to a final sodium chloride concentration of 1% in L-15 medium, as described previously (Watanabe et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Next, we tried to isolate myosatellites/myoblasts from muscle tissue of \u003cem\u003eP. major\u003c/em\u003e using collagenase digestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We used iMatrix-511, which is a human laminin-511 E8 fragment, to allow primary cells to adhere to the culture flask and observed primary cell adhesions at 5 days after the start of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The isolated cells were subcultured using 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA twice per week and cultured the cells for over 80 passages. Following this approach, we established the NUF-PM1 cell line. The cells could be stored at -80\u0026deg;C using conventional cryopreservation solutions, such as CELLBANKER I (TAKARA BIO Inc.) and Bambanker (Nippon Genetics Co., Ltd., Tokyo, Japan).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eOptimal cell culture conditions\u003c/h2\u003e\u003cp\u003eNext, we determined the optimal culture conditions for NUF-PM1 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, NUF-PM1 cells grew in an FBS dose-dependent manner and doubling times of 20%, 10%, and 5% were observed at 42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 h, 95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;27.1 h, and 120.3\u0026thinsp;\u0026plusmn;\u0026thinsp;33.0 h, respectively. NUF-PM1 cells showed the best growth curve at 28\u0026deg;C. Although they grew slightly at 19\u0026deg;C, cell death was not induced. No cell growth was observed at 37\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). L-15 and F-12 media promoted NUF-PM1 cells growth, although the cells grew slowly in DMEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Since minimum essential medium (MEM) is often used for fish cell culture, we examined its effect of MEM on the growth of NUF-PM1 cells. The cells grew better in L-15 medium than in MEM at 28\u0026deg;C (Supplementary Data 1). bFGF is an essential growth factor that promotes the proliferation and differentiation of stem cells, such as induced pluripotent stem (iPS) cells and myosatellite cells. NUF-PM1 cells showed a spindle shape in the presence of 10 ng/mL of bFGF, even though they developed into a large and blurred shape in the absence of bFGF. In addition, the number of NUF-PM1 cells in medium containing 10 ng/mL bFGF was twice that in medium without bFGF. Therefore, bFGF is an essential growth factor for the proliferation of NUF-PM1 cells and maintains these cells in an undifferentiated state.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of the NUF-PM1 cell line by RNA sequencing\u003c/h2\u003e\u003cp\u003eIn general, gene expression of Pax7 and MyoD, which are markers for myosatellite cells (Parker et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), is analyzed to identify the presence of cultured fish myosatellite cells (Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), although these genes have not been identified in \u003cem\u003eP. major\u003c/em\u003e. Therefore, to characterize NUF-PM1 cells, RNA sequencing was performed. Total RNA extracted from muscle tissue and P5 and P20 NUF-PM1 cells was analyzed using DNBSEQ T7. Since we used the draft genome of \u003cem\u003eP. major\u003c/em\u003e for sequence mapping (Shin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), there were various predicted sequences. First, we created a heatmap for muscle-related gene expression. Muscle-related genes such as myogenin-2, troponin T and I, and myosin light chain 2 were highly expressed in muscle tissue, although they were expressed at a low level in P5 and P20 NUF-PM1. Myocilin, tropomyosin 4a, and myotrophin were expressed at the highest levels in P5 NUF-PM1 among the three groups. Fatty acid binding protein 3, keratin, and type I cytoskeletal 18 were expressed at the highest level in P20 NUF-PM1 cells compared with muscle and P5 NUF-PM1 cells.\u003c/p\u003e\u003cp\u003eNext, we compared the expression of collagen-related genes because of the decrease in the expression of muscle-related genes with increasing NUF-PM1 cell passage number. They were expressed at low levels in muscle tissue. Collagen type I alpha 3 chain, collagen type VI, and vitronectin showed the highest expression in P5 NUF-PM1 cells, although their expression decreased at P20. Collagen alpha-1(I) chain-like precursor and collagen type VI alpha 2 showed the highest expression in P20 NUF-PM1 cells. These results suggest that myosatellites/myoblasts in NUF-PM1 cells were replaced by fibroblasts and/or epithelial cells with increasing passage number. Interestingly, CD44 in P20 NUF-PM1 cells (Supplemental Fig.\u0026nbsp;2). CD44 is not only a hyaluronan receptor but is also a marker of human mesenchymal stem cells indicating that NUF-PM1 cells have the potential to differentiate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAdipocyte-like differentiation\u003c/h2\u003e\u003cp\u003eFirst, we attempted to differentiate NUF-PM1 cells into a myotube, but failed to observe myotube formation, indicating that this cell line lacked myogenic potential. In contrast, microscopic observation showed lipid droplet-like substances in differentiated NUF-PM1 cells. Therefore, we examined lipid accumulation in differentiated NUF-PM1 cells by Oil Red O staining. NUF-PM1 cells incubated in growth medium showed a thin shape and no lipid accumulation. NUF-PM1 cells incubated in differentiation medium clearly showed lipid accumulation. Conversely, culture in MDI induction medium did not result in the accumulation of lipids in NUF-PM1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the differentiation medium significantly increased absorbance at 492 nm. MDI induction medium slightly increased lipid accumulation compared to that in control medium. These results suggest that NUF-PM1 cells have adipogenic potential under culture conditions with low FBS concentrations.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we spontaneously immortalized and established the NUF-PM1 cell line from the muscle of \u003cem\u003eP. major\u003c/em\u003e. We used collagenase type I and iMatrix-511 for the enzymatic digestion of muscle tissue and the adhesion of primary cells, respectively. Generally, explanted cell isolation from finfish is often used and is well-documented (Thangaraj et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar et al., 2024). Since enzymatic digestion with collagenase is widely employed to isolate myosattelites/myoblasts from mammalian muscle, we followed a previously reported method for mammals. In our preliminary study, collagen, fibronectin (a gift from Immuno Probe Co., Ltd., Saitama, Japan), laminin (a gift from Immuno Probe Co., Ltd.), and ε-poly-L-lysine (SPL01, Cosmo Bio Co., Ltd., Tokyo, Japan) were used to coat cell culture plates to allow primary cells to adhere, and iMatrix-511 was found to be an optimal coating for primary cells from the muscle of \u003cem\u003eP. major\u003c/em\u003e. iMatrix-511 is the active site of the human laminin-511 E8 fragment and promotes the adhesion and proliferation of iPS cells without feeder cells (Miyazaki et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Saad et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) used iMatrix recombinant laminin-511-E8 for the adhesion of primary mackerel satellites cell isolated from \u003cem\u003eS. scombus\u003c/em\u003e. Furthermore, a Japanese eel myoblast cell line was established using iMatrix-511 for the adhesion of primary cells from muscle tissue (Ikeda et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Hence, these results suggest that laminin is an optimal cellular matrix for the adhesion of primary cells from the muscle tissue of marine finfish. On the other hand, previous studies have established muscle cell lines with myogenic differentiation potential without using a coating from \u003cem\u003eSebastes schlegelii\u003c/em\u003e (Kong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)d \u003cem\u003eolivaceus\u003c/em\u003e (Krishnan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These reports suggest that it is essential to choose a cellular matrix for the adhesion of primary muscle cells according to the fish species.\u003c/p\u003e\u003cp\u003eFBS plays an important role not only in mammalian cell culture, but also in fish cell culture, supplying essential micronutrients and growth factors. NUF-PM1 cells showed FBS-dependent growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), in agreement with previous reports (Goswani et al., 2023; Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, it is still unclear why fish cells line reached plateaus when cultured in low concentrations of FBS (Goswani et al., 2023; Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is conceivable that high FBS concentrations may promote the growth of fish cell lines regardless of limited culture area or cell-to-cell contact inhibition and maintain cell shape. NUF-PM1 showed the best growth curve at 28\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which is reasonable because the optimal water temperature for \u003cem\u003eP. major\u003c/em\u003e is in the range of 20\u0026thinsp;\u0026minus;\u0026thinsp;28\u0026deg;C.\u003c/p\u003e\u003cp\u003eL-15 medium has been used frequently for fish cell culture because it does not require CO\u003csub\u003e2\u003c/sub\u003e (Leibovitz \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1963\u003c/span\u003e), which necessitates the use of an expensive incubator. In addition, fish cells have relatively high amino acid requirements due to amino acid metabolism because their carbohydrate utilization efficiency is low (Wilson, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). L-15 and F-12 media both promoted proliferation of NUF-PM1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). F-12 medium contains rich amino acid components, hypoxanthine, linoleic acid, and lipoic acid, which have the potential to promote the cell proliferation of NUF-PM1 cells. Chicken embryo extract contains important growth factors and nutrients, and is often used for myosatellite culture in mouse studies (Yoshioka et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We also used Chicken embryo extract to support primary cell culture from the muscle of \u003cem\u003eP. major\u003c/em\u003e, however, we found it was not essential. NUF-PM1 cells were subcultured with 0.05% trypsin-0.53 mM EDTA or 0.25% trypsin-1 mM EDTA and passaged more than 80 times. These results indicated that muscle cells from juvenile \u003cem\u003eP. major\u003c/em\u003e are capable of growing and undergoing cell division, although we failed to isolate and culture muscle cells from adult \u003cem\u003eP. major\u003c/em\u003e. Hence, juvenile or young fish are suitable for cell isolation and muscle cell culture. Recently established myoblast cell lines were spontaneously immortalized by continuous cell culture without cell cloning (Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, we failed to establish a myoblast cell line. Ikeda et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) established an eel myoblast cell line by single cell cloning expressing Pax7, MyoD, and Cdh17. In a preliminary study, we observed myotube differentiation from primary cells suggesting that they contained myoblasts. However, fibroblast and epithelial-like cells isolated from the muscle of \u003cem\u003eP. major\u003c/em\u003e showed strong cell proliferation compared to myoblasts. Hence, it would be better to establish a myoblast cell line using the cell cloning approach described above.\u003c/p\u003e\u003cp\u003eTo characterize NUF-PM1cells, RNA sequencing analysis was performed, resulting in the identification of NUF-PM1 cells as fibroblasts, and not myosatellites. Muscle-related genes were highly expressed in muscle tissue, although their expression was decreased in P5 and P20 NUF-PM1 cells, indicating that myosatellites were replaced by fibroblasts or epitheliocyte-like cells by increasing the passage number. Fibroblasts and epitheliocytes have active cell expansion compared to myosatellites. Myosatellites play an important role in homeostasis such as the growth and repair of muscle fibers in mammals (Mauro et al., 1961; Yoshioka et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the case of muscle growth and injury, myosatellites self-renew and differentiate into myofibroblasts and myofibers. Myosatellites and myogenesis are regulated by myogenic regulatory factors such as Pax7, MyoD, and Myf5 (Parker et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Teleost myosatellites have been studied widely (Gabillard et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Biacchesi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Krishnan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They differentiated into myotubes at low FBS concentrations. In contrast, NUF-PM1 cells expressed CD44, which is a marker for mesenchymal stem cells, and were able to differentiate into adipocyte-like cells due to lipid accumulation under low FBS concentrations. NUF-PM1 cells may be a valuable model for \u003cem\u003ein vitro\u003c/em\u003e lipid metabolism in fish. How NUF-PM1 cells differentiate into adipocyte-like cells remains to be determined and it should be addressed in the next step of characterization this cell line.\u003c/p\u003e\u003cp\u003eAlmost all mammalian cell lines have been established as cancer cells isolated from tumor tissues and immortalized cells with deliberate immortalization by lentiviral vector transduction. In contrast, almost all fish cell lines have been established by spontaneous immortalization. Fish are capable of resistance to senescence (Reznick et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Finch, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, it remains unclear how fish cells are capable of spontaneous immortalization. Interestingly, the fish genome lacks p16, a cyclin-dependent kinase inhibitor 2A, which functions as a cell cycle regulatory protein (Futami et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mutations of p16 in higher mammals cause oncogenesis (Liggett and Sidransky, 1998). This indicates that mammalian cells evolutionarily acquired cell cycle regulator genes to inhibit oncogenesis. Clarifying the mechanism underlying senescence resistance in fish will not only contributes to aging research but may also enable the development of anti-aging drugs. Therefore, further study is required to elucidate how fish cells undergo spontaneous immortalization.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe spontaneously immortalized and established NUF-PM1 cell line from the muscle of juvenile \u003cem\u003eP. major\u003c/em\u003e, which was characterized as fibroblast/epithelial cells lacking myogenic differentiation. NUF-PM1 cells can differentiate into adipocyte-like cells with the accumulation of intracellular lipid droplets so they have the potential to be utilized in basic research for cellular aquaculture. We must further improve the method for myosatellites isolation and culture for cellular aquaculture research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor contribution statement\u003c/h2\u003e\u003cp\u003eM.U. mainly designed the project, performed the experiments, and prepared the manuscript. Y.Y. performed the experiments. A.Y., H.K., N.T., T.K., and T.T. discussed the data. K.Y. supervised this study. All authors reviewed the manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eMU received a research grant from Ichimasa Kamaboko Co., Ltd.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eMU received a research grant from Ichimasa Kamaboko Co., Ltd.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported in part by Adaptable and Seamless Technology transfer Program through Target-driven R\u0026amp;D (A-STEP) from Japan Science and Technology Agency (JST) Japan Grant JPMJTM22EG. This work was supported in part by a research grant from Kohjin Bio Co., Ltd., Saitama, Japan. This study was supported in part by a research grant from G-7 Scholarship Foundation. This work was supported by a tenure-track system, CHODAI Kyoso Grant, and State of the Art Research (STAR) Program from Nagasaki University.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.U. mainly designed the project, performed the experiments, and prepared the manuscript. Y.Y. performed the experiments. A.Y., H.K., N.T., T.K., and T.T. discussed the data. K.Y. supervised this study. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Mr. Yoshimune Onoda (Kaneko Sangyo Co. Ltd.) for valuable comments on red sea bream rearing. We are deeply grateful to Mr. Hiroshi Nomura (Immuno Probe Co., Ltd.) for providing the fibronectin and laminin. We appreciate Mr. Manato Morimoto (Rhelixa, Inc., Tokyo, Japan) for valuable comments on the RNA sequence analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBiacchesi S, Jouvion G, M\u0026eacute;rour E, Boukadiri A, Desdouits M, Ozden S, Huerre M, Ceccaldi PE, Br\u0026eacute;mont M (2016) Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection. 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Biosci Biotechnol Biochem 77(2):345\u0026ndash;352. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1271/bbb.120764\u003c/span\u003e\u003cspan address=\"10.1271/bbb.120764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fish cell line, red sea bream, Pagrus major, muscle","lastPublishedDoi":"10.21203/rs.3.rs-6798589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6798589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell culture plays a crucial role in various fields of life science research such as cancer, immunology, and virology. Numerous cell lines have been established in mammals, while fish cell lines remain comparatively limited in number. This study established a spontaneously immortalized cell line from the muscle of red rea bream, \u003cem\u003ePagrus major\u003c/em\u003e, which is a commercially important fish in Japan. Primary cells were isolated from muscle tissue using 0.2% collagenase and, cultured in Leibovitz\u0026rsquo;s L-15 medium without CO\u003csub\u003e2\u003c/sub\u003e. The cells required fetal bovine serum in a dose-dependent manner at 28\u0026deg;C for optimal growth. The cells were also able to grow in F12 medium, but not in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium. RNA-sequencing analysis indicated that the isolated cells differentiated into fibroblasts or epithelial-like cells with an increasing number of passages because collagen-related genes were expressed more than in muscle tissue. Upon serum starvation, the cells differentiated into adipocyte-like cells because of slight lipid accumulation. Therefore, the established cells were considered to lack myogenic potential. In conclusion, we established the Nagasaki University Fisheries-\u003cem\u003eP. major\u003c/em\u003e 1 (NUF-PM1) cell line, which consisted of fibroblast/epithelial-like cells, but not myosatellite/myoblast cells from the muscle of \u003cem\u003eP. major\u003c/em\u003e, and we observed the cells over 80 passages. This study adds to the limited number of fish cell lines available for research, which could help advance fish cellular aquaculture and related fields.\u003c/p\u003e","manuscriptTitle":"A spontaneously immortalized cell line from the muscle of red sea bream (Pagrus major)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 16:53:01","doi":"10.21203/rs.3.rs-6798589/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-17T04:18:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T20:54:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77128790292810035286915740501377922222","date":"2025-08-13T16:26:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331467263842494347372919538523667464300","date":"2025-08-06T16:29:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T02:53:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222084052129999918572803418013683606226","date":"2025-07-24T07:00:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221614432443115378079328846157461005971","date":"2025-07-24T06:02:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143868558776084495638655403995852151979","date":"2025-07-23T07:39:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T05:59:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-20T20:41:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-04T10:05:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2025-06-02T04:04:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"25674cdb-8004-4a17-b999-9c899d3157fa","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:09:36+00:00","versionOfRecord":{"articleIdentity":"rs-6798589","link":"https://doi.org/10.1007/s10695-025-01603-x","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2025-11-08 15:57:10","publishedOnDateReadable":"November 8th, 2025"},"versionCreatedAt":"2025-07-24 16:53:01","video":"","vorDoi":"10.1007/s10695-025-01603-x","vorDoiUrl":"https://doi.org/10.1007/s10695-025-01603-x","workflowStages":[]},"version":"v1","identity":"rs-6798589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6798589","identity":"rs-6798589","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}