Establishment and characterization of a continuous adipose-derived stem cell line from Epinephelus fuscoguttatus and its application in cell-cultured fish meat

Abstract

Cell-cultured meat, a new cell-based agricultural technology, aims to provide sustainable and safe meat production. Fat is essential for the flavor, texture, and nutritional value of cell-cultured meat. However, research on immortalized fish adipose-derived stem cells remains limited. Here, a continuous marine fish adipose-derived stem cell line, designated EfADS, was successfully established from the visceral adipose tissues of brown-marbled grouper (Epinephelus fuscoguttatus) and sub-cultured beyond 65 times with robust proliferative capacity in L-15 medium supplemented with 20% fetal bovine serum, 10% muscle tissue extract, 5% adipose tissue extract, and 20 ng/mL basic fibroblast growth factor at 28 ℃. The cell lineage origin of EfADS was confirmed by the sustained expression of CD73 and CD105 marker genes from passages 12 to 65. We also developed an efficient adipogenic differentiation medium for EfADS cells, resulting in a 5.3-fold increase in triglyceride content by day 15 post-induction, along with 6.5-fold and 3.3-fold upregulations in the expression of adipogenic marker genes of PPARγ and LPL, respectively. Furthermore, by cultivating EfADS cells on edible microcarriers and subsequently inducing adipogenesis, cell-cultured fish fats were successfully fabricated. The resulting fats exhibited a nutritionally favorable fatty acid profile consisting of ~ 31% saturated fatty acids, ~ 60% monounsaturated fatty acids, and ~ 9% polyunsaturated fatty acids. In conclusion, this study has for the first time established an immortalized fish adipose-derived stem cell line and optimized its growth and differentiation conditions, providing a useful seed cell line for fat-containing cultured fish meat.

Keywords

Fish cell culture / Epinephelus fuscoguttatus / Adipose-derived stem cell line / Adipogenic differentiation / Cell-cultured meat

References

[1] Banaszak M, Dobrzyńska M, Kawka A, Górna I, Woźniak D, Przysławski J, Drzymała-Czyż S. Role of Omega-3 fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) as modulatory and anti-inflammatory agents in noncommunicable diet-related diseases—reports from the last 10 years. Clin Nutr ESPEN, 2024, 63: 240-258

[2] Basto-Silva C, Balbuena-Pecino S, Oliva-Teles A, Riera-Heredia N, Navarro I, Guerreiro I, Capilla E. Gilthead seabream (Sparus aurata) in vitro adipogenesis and its endocrine regulation by leptin, ghrelin, and insulin. Comp Biochem Physiol A Mol Integr Physiol, 2020, 249 110772

[3] Bhat ZF, Fayaz H. Prospectus of cultured meat—advancing meat alternatives. J Food Sci Technol, 2011, 48: 125-140

[4] Bhat ZF, Kumar S, Fayaz H. In vitro meat production: challenges and benefits over conventional meat production. J Integr Agric, 2015, 14: 241-248

[5] Bora P, Majumdar AS. Adipose tissue-derived stromal-vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther, 2017, 8: 145

[6] Bou M, Montfort J, Le Cam A, Rallière C, Lebret V, Gabillard JC, Weil C, Gutiérrez J, Rescan PY, Capilla E, Navarro I. Gene expression profile during proliferation and differentiation of rainbow trout adipocyte precursor cells. BMC Genomics, 2017, 18: 347

[7] Bou M, Todorčević M, Torgersen J, Škugor S, Navarro I, Ruyter B. De novo lipogenesis in Atlantic salmon adipocytes. Biochim Biophys Acta, 2016, 1860: 86-96

[8] Bouraoui L, Gutiérrez J, Navarro I. Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss). J Endocrinol, 2008, 198: 459-69

[9] Chalamaiah M, Dinesh kumar B, Hemalatha R, Jyothirmayi T. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review. Food Chem, 2012, 135: 3020-3038

[10] Chriki S, Hocquette JF. The myth of cultured meat: a review. Front Nutr, 2020, 7: 7

[11] Datsomor AK, Gillard G, Jin Y, Olsen RE, Sandve SR. Molecular regulation of biosynthesis of long-chain polyunsaturated fatty acids in atlantic salmon. Mar Biotechnol, 2022, 24: 661-670

[12] Dyall SC. Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA. Front Aging Neurosci, 2015, 7 52

[13] Feng X, Liu L, Yu BQ, Huang JM, Gu LD, Xu DF. Effect of optimized collagenase digestion on isolated and cultured nucleus pulposus cells in degenerated intervertebral discs. Medicine, 2018, 97 e12977

[14] Foti R, Storti G, Palmesano M, Scioli MG, Fiorelli E, Terriaca S, Cervelli G, Kim BS, Orlandi A, Cervelli V. Senescence in adipose-derived stem cells: biological mechanisms and therapeutic challenges. Int J Mol Sci, 2024, 25 8390

[15] Freshney RI. Subculture and cell lines. Culture of animal cells: a manual of basic technique and specialized application, 20107New York, Wiley187206

[16] Han J, Li X, Liang B, Ma S, Pu Y, Yu F, Lu J, Ma Y, MacHugh DE, Jiang L. Transcriptome profiling of differentiating adipose-derived stem cells across species reveals new genes regulating adipogenesis. Biochim Biophys Acta Mol Cell Biol Lipids, 2023, 1868 159378

[17] Heyman E, Devriendt B, De Vlieghere E, Goethals K, Van Poucke M, Peelman L, De Schauwer C. Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation. NPJ Sci Food, 2024, 8: 70

[18] Huang TS, Todorčević M, Ruyter B, Torstensen BE. Altered expression of CCAAT/enhancer binding protein and FABP11 genes during adipogenesis in vitro in Atlantic salmon (Salmo salar). Aquac Nutr, 2010, 16: 72-80

[19] Jahir NR, Ramakrishna S, Abdullah AAA, Vigneswari S. Cultured meat in cellular agriculture: advantages, applications and challenges. Food Biosci, 2023, 53 102614

[20] Jayaprakash J, Nath LR, Siddabasave SG, Gowda D, Hui SP. Analysis and functions of bioactive lipids in food. Discov Food, 2024, 4 107

[21] Lee-Okada HC, Xue C, Yokomizo T. Recent advances on the physiological and pathophysiological roles of polyunsaturated fatty acids and their biosynthetic pathway. Biochim Biophys Acta Mol Cell Biol Lipids, 2025, 1870 159564

[22] Li X, Zheng S, Wu G. Nutrition and functions of amino acids in fish. Adv Exp Med Biol, 2021, 1285: 133-168

[23] Li Y. Establishment and evaluation of a new model for studying lipogenesis in grass carp (Ctenopharyngodon idella) preadipocytes. In Vitro Cell Dev Biol Anim, 2012, 48: 37-42

[24] Lim PY, Suntornnond R, Choudhury D. The nutritional paradigm of cultivated meat: bridging science and sustainability. Trends Food Sci Tech, 2025, 156 10483

[25] Liu Y, Wang R, Ding S, Deng L, Zhang Y, Li J, Shi Z, Wu Z, Liang K, Yan X, Liu W, Du Y. Engineered meatballs via scalable skeletal muscle cell expansion and modular microtissue assembly using porous gelatin microcarriers. Biomaterials, 2022, 287 121615

[26] Liu P, Ji H, Li C, Tian J, Wang Y, Yu P. Ontogenetic development of adipose tissue in grass carp (Ctenopharyngodon idella). Fish Physiol Biochem, 2015, 41: 867-878

[27] Ljubojević DB, Pelić MM, Novakov NJ, Lazić SM, Lupulović DM, Ćirković MA, Lj Todorĉević M. The establishment of the cell culture of preadipocytes of common carp (Cyprinus carpio L.). Food Feed Res, 2014, 41: 103-108

[28] Martins B, Bister A, Dohmen RGJ, Gouveia MA, Hueber R, Melzener L, Messmer T, Papadopoulos J, Pimenta J, Raina D, Schaeken L, Shirley S, Bouchet BP, Flack JE. Advances and challenges in cell biology for cultured meat. Annu Rev Anim Biosci, 2024, 12: 345-368

[29] Navarro-Perez J, Carobbio S. Adipose tissue-derived stem cells, in vivo and in vitro models for metabolic diseases. Biochem Pharmacol, 2024, 222 116108

[30] Oku H, Tokuda M, Okumura T, Umino T. Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major. Comp Biochem Physiol B Biochem Mol Biol, 2006, 144: 326-333

[31] Post MJ, Levenberg S, Kaplan DL, Genovese N, Fu J, Bryant CJ, Negowetti N, Verzijden K, Moutsatsou P. Scientific, sustainability and regulatory challenges of cultured meat. Nat Food, 2020, 1: 403-415

[32] Raajendiran A, Ooi G, Bayliss J, O’Brien PE, Schittenhelm RB, Clark AK, Taylor RA, Rodeheffer MS, Burton PR, Watt MJ. Identification of metabolically distinct adipocyte progenitor cells in human adipose tissues. Cell Rep, 2019, 27: 1528-1540

[33] Rakhmatullina AR, Mingaleeva RN, Gafurbaeva DU, Glazunova ON, Sagdeeva AR, Bulatov ER, Rizvanov AA, Miftakhova RR. Adipose-derived mesenchymal stem cell (MSC) immortalization by modulation of hTERT and TP53 expression levels. J Pers Med, 2023, 13: 1621

[34] Ramírez-Zacarías JL, Castro-Muñozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O. Histochemistry, 1992, 97: 493-7

[35] Raynaud CM, Rafii A. The necessity of a systematic approach for the use of MSCs in the clinical setting. Stem Cells Int, 2013, 2013 892340

[36] Riera-Heredia N, Lutfi E, Balbuena-Pecino S, Vélez EJ, Dias K, Beaumatin F, Gutiérrez J, Seiliez I, Capilla E, Navarro I. The autophagy response during adipogenesis of primary cultured rainbow trout (Oncorhynchus mykiss) adipocytes. Comp Biochem Physiol B Biochem Mol Biol, 2022, 258 110700

[37] Rizzo G, Baroni L, Lombardo M. Promising sources of plant-derived polyunsaturated fatty acids: a narrative review. Int J Environ Res Public Health, 2023, 20: 1683

[38] Rosen ED, MacDougald OA. Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol, 2006, 7: 885-896

[39] Salmerón C. Adipogenesis in fish. J Exp Biol, 2018, 221(Pt Suppl 1 jeb161588

[40] Salmerón C, Acerete L, Gutiérrez J, Navarro I, Capilla E. Characterization and endocrine regulation of proliferation and differentiation of primary cultured preadipocytes from gilthead sea bream (Sparus aurata). Domest Anim Endocrinol, 2013, 45: 1-10

[41] Salmerón C, Riera-Heredia N, Gutiérrez J, Navarro I, Capilla E. Adipogenic gene expression in gilthead sea bream mesenchymal stem cells from different origin. Front Endocrinol (Lausanne), 2016, 7 113

[42] Seaman SA, Tannan SC, Cao Y, Peirce SM, Lin KY. Differential effects of processing time and duration of collagenase digestion on human and murine fat grafts. Plast Reconstr Surg, 2015, 136: 189e-199e

[43] Skrypnyk M. Current progress and limitations of research regarding the therapeutic use of adipose-derived stem cells: literature review. J Umm Al-Qura Univ Appll Sci, 2024

[44] Song W, Liu P, Li H, Ding S. Large-scale expansion of porcine adipose-derived stem cells based on microcarriers system for cultured meat production. Foods, 2022, 11: 3364

[45] Sugii S, Wong CYQ, Lwin AKO, Chew LJM. Alternative fat: redefining adipocytes for biomanufacturing cultivated meat. Trends Biotechnol, 2023, 41: 686-700

[46] Tang P, Virtue S, Goie JYG, Png CW, Guo J, Li Y, Jiao H, Chua YL, Campbell M, Moreno-Navarrete JM, Shabbir A, Fernández-Real JM, Gasser S, Kemeny DM, Yang H, Vidal-Puig A, Zhang Y. Regulation of adipogenic differentiation and adipose tissue inflammation by interferon regulatory factor 3. Cell Death Differ, 2021, 28: 3022-3035

[47] Thompson CC, Wasielesky WJr, Landuci F, Lima MS, Bacha L, Perazzolo LM, Lourenço-Marques C, Soares F, Pousão-Ferreira P, Hanson L, Gomez-Gil B, Thompson M, Varasteh T, Silva TA, Swings J, Zhang X-H, de Souza W, Thompson FL. Understanding the role of microbes in health and disease of farmed aquatic organisms. Mar Life Sci Technol, 2024, 6: 579-609

[48] Todorčević M, Škugor S, Krasnov A, Ruyter B. Gene expression profiles in Atlantic Salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes. BMC Genomics, 2010, 11: 39

[49] Tsuruwaka Y, Shimada E. Reprocessing seafood waste: challenge to develop aquatic clean meat from fish cells. NPJ Sci Food, 2022, 6: 7

[50] Volz AC, Huber B, Kluger PJ. Adipose-derived stem cell differentiation as a basic tool for vascularized adipose tissue engineering. Differentiation, 2016, 92: 52-64

[51] Wang X, Huang M, Wang Y. The effect of insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of preadipocytes isolated from large Yellow Croaker (Pseudosciaena crocea R.). PLoS One, 2012, 7 48069

[52] Xu D, Gong Y, Xiang X, Liu Y, Mai K, Ai Q. Discovery, characterization, and adipocyte differentiation regulation in perirenal adipose tissue of large yellow croaker (Larimichthys crocea). Fish Physiol Biochem, 2023, 49: 627-639

[53] Xu E, Niu R, Lao J, Zhang S, Li J, Zhu Y, Shi H, Zhu Q, Chen Y, Jiang Y, Wang W, Yin J, Chen Q, Huang X, Chen J, Liu D. Tissue-like cultured fish fillets through a synthetic food pipeline. NPJ Sci Food, 2023, 7: 17

[54] Xue T, Zheng H, Zhao Y, Zhao Z, Wang J, Zhang Y, Li Y, Wang S, Liu Y, Xue C, Guo H. A spontaneously immortalized muscle stem cell line (EfMS) from brown-marbled grouper for cell-cultured fish meat production. Commun Biol, 2024, 7: 1697

[55] Yao T, Asayama Y. Animal-cell culture media: history, characteristics, and current issues. Reprod Med Biol, 2017, 16: 99-117

[56] Yu SE, Liao ML, Dong SL, Dong YW. Mapping the potential for global offshore finfish mariculture. Mar Life Sci Technol, 2024, 6: 651-664

[57] Zagury Y, Ianovici I, Landau S, Lavon N, Levenberg S. Engineered marble-like bovine fat tissue for cultured meat. Commun Biol, 2022, 5: 927

[58] Zhao Y, Guo L, Guo H. Routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp (Metapenaeus ensis) and virus susceptibility. Aquaculture, 2023, 563 739007

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