Efficient isolation of porcine mesenchymal stem cells and research on food-grade adipogenic inducers

  • WANG Luyi ,
  • XIN Na ,
  • TANG Haohao ,
  • SUN Wanqiang ,
  • GUAN Xin ,
  • DU Guocheng
Expand
  • 1(Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University-School of Biotechnology, Wuxi 214122, China)
    2(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
    3(Jiangsu Basic Research Center of Synthetic Biology, Wuxi 214122, China)

Received date: 2025-01-17

  Revised date: 2025-04-16

  Online published: 2025-12-15

Abstract

Cell cultured meat technology offers innovative solutions for the efficient, stable, and sustainable supply of meat products.The efficient in vitro production of animal adipose tissue is crucial for enhancing the flavor and nutritional value of cell-cultured meat.This study optimized the isolation method for porcine adipose-derived mesenchymal stem cells and investigated the effects of various enzyme types, digestion times, and tissue locations on cell isolation efficiency.Additionally, the biological characteristics and in vitro proliferation capacity of the isolated cells were evaluated.The study further assessed the impact of different fatty acids and vegetable oils on the differentiation of porcine mesenchymal stem cells into adipocytes, comparing these effects to traditional adipogenic induction protocols.The results indicated that using 0.1% type I collagenase to digest subcutaneous fat for 75 min yields the highest tissue dissociation efficiency, with approximately 4×104 cells released per gram of tissue.Immunofluorescence and flow cytometry analyses confirmed that the isolated cells meet the standards for mesenchymal stem cells and possess high purity.Porcine mesenchymal stem cells were cultured in vitro for 35 days, during which they divided a total of (15.24±0.69) times and expanded by (4.15±1.7)×104 times.Furthermore, the application of 10 μmol/L α-linolenic acid or 0.5 μL/mL flaxseed oil significantly induced the differentiation of porcine mesenchymal stem cells into adipocytes, with the area of lipid droplet formation being approximately twice that of traditional methods, and the time required for differentiation shortened by 50%.In summary, this study established an efficient method for isolating porcine mesenchymal stem cells and developed a rapid, efficient, food-grade adipogenic differentiation protocol, thereby laying a crucial foundation for the effective preparation of cell-cultured fat.

Cite this article

WANG Luyi , XIN Na , TANG Haohao , SUN Wanqiang , GUAN Xin , DU Guocheng . Efficient isolation of porcine mesenchymal stem cells and research on food-grade adipogenic inducers[J]. Food and Fermentation Industries, 2025 , 51(22) : 19 -31 . DOI: 10.13995/j.cnki.11-1802/ts.042183

References

[1] POST M J, LEVENBERG S, KAPLAN D L, et al.Scientific, sustainability and regulatory challenges of cultured meat[J].Nature Food, 2020, 1(7):403-415.
[2] JONES N.Lab-grown meat:The science of turning cells into steaks and nuggets[J].Nature, 2023, 619(7968):22-24.
[3] MORITZ M S M, VERBRUGGEN S E L, POST M J.Alternatives for large-scale production of cultured beef:A review[J].Journal of Integrative Agriculture, 2015, 14(2):208-216.
[4] PICARD B, GAGAOUA M. Muscle fiber properties in cattle and their relationships with meat qualities: An overview[J]. Journal of Agricultural and Food Chemistry, 2020, 68(22):6021-6039.
[5] OTHMAN M, KAUR H. Thromboelastography (teg)[M]. New York: Springer New York, 2017:533-543.
[6] SONG W J, LIU P P, MENG Z Q, et al.Identification of porcine adipose progenitor cells by fluorescence-activated cell sorting for the preparation of cultured fat by 3D bioprinting[J].Food Research International, 2022, 162:111952.
[7] 王立斌, 刘婷, 马晓娜, 等.人胎盘间充质干细胞的分离与鉴定[J].宁夏医科大学学报, 2010, 32(8):864-866.
WANG L B, LIU T, MA X N, et al.Isolation and characterization of mesenchymal stem cells from human placenta[J].Journal of Ningxia Medical University, 2010, 32(8):864-866.
[8] MERRICK D, SAKERS A, IRGEBAY Z, et al.Identification of a mesenchymal progenitor cell hierarchy in adipose tissue[J].Science, 2019, 364(6438):eaav2501.
[9] 关欣, 陈坚, 唐好好, 等.应用于细胞培养肉的食品级成脂分化诱导剂:中国, CN118497112A[P].2024-08-16.
GUAN X, CHEN J, TANG H H, et al.Food-grade adipogenic differentiation inducer for cell cultured meat:China, CN118497112A[P].2024-08-16.
[10] KANG D H, LOUIS F, LIU H, et al.Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting[J].Nature Communications, 2021, 12(1):5059.
[11] PASITKA L, COHEN M, EHRLICH A, et al.Spontaneous immortalization of chicken fibroblasts generates stable, high-yield cell lines for serum-free production of cultured meat[J].Nature Food, 2023, 4(1):35-50.
[12] KHAN F, SYEDA P K, NARTEY M N N, et al.Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase[J].Prostaglandins and Other Lipid Mediators, 2016, 123:16-27.
[13] CHEN Y T, YANG Q Y, MA G L, et al.Dose- and type-dependent effects of long-chain fatty acids on adipogenesis and lipogenesis of bovine adipocytes[J].Journal of Dairy Science, 2018, 101(2):1601-1615.
[14] WANG C Z, WANG J Z, CHEN K R, et al.Caprylic acid (C8:0) promotes bone metastasis of prostate cancer by dysregulated adipo-osteogenic balance in bone marrow[J].Cancer Science, 2020, 111(10):3600-3612.
[15] GHNAIMAWI S, SHELBY S, BAUM J, et al.Effects of eicosapentaenoic acid and docosahexaenoic acid on C2C12 cell adipogenesis and inhibition of myotube formation[J].Animal Cells and Systems, 2019, 23(5):355-364.
[16] WANG J H, LIANG Y, JIAN L Y, et al.Linoelaidic acid enhances adipogenic differentiation in adipose tissue-derived stromal cells through suppression of Wnt/β-catenin signaling pathway in vitro[J].Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA), 2016, 110:1-7.
[17] DENKE M A, GRUNDY S M.Comparison of effects of lauric acid and palmitic acid on plasma lipids and lipoproteins[J].The American Journal of Clinical Nutrition, 1992, 56(5):895-898.
[18] MATTSON F H, STRECK J A.Effect of the consumption of glycerides containing behenic acid on the lipid content of the heart of weanling rats[J].The Journal of Nutrition, 1974, 104(4):483-488.
[19] YANG X F, HE X, HE J, et al.High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells[J].Journal of Biomedical Science, 2011, 18(1):59.
[20] 苗轲, 高旭, 曹亚强, 等.不同诱导周期对人白色前脂肪细胞分化水平的影响研究[J].环境卫生学杂志, 2024, 14:378-383;399.
MIAO K, GAO X, CAO Y Q, et al.Effect of different induction stages on the differentiation level of human white preadipocytes[J].Journal of Environmental Hygiene, 2024, 14(5):378-383;399.
[21] YUEN J S K Jr, STOUT A J, KAWECKI N S, et al.Perspectives on scaling production of adipose tissue for food applications[J].Biomaterials, 2022, 280:121273.
[22] LENSCH M, MUISE A, WHITE L, et al.Comparison of synthetic media designed for expansion of adipose-derived mesenchymal stromal cells[J].Biomedicines, 2018, 6(2):54.
[23] RAPOSIO E, SIMONACCI F, PERROTTA R E.Adipose-derived stem cells:Comparison between two methods of isolation for clinical applications[J].Annals of Medicine and Surgery, 2017, 20:87-91.
[24] ARAÚJO A B, FURLAN J M, SALTON G D, et al.Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord:Comparison of four enzymatic protocols[J].Biotechnology Letters, 2018, 40(6):989-998.
[25] KIM M, YEO M, LEE K, et al.Extraction and characterization of human adipose tissue-derived collagen:Toward xeno-free tissue engineering[J].Tissue Engineering and Regenerative Medicine, 2024, 21(1):197-109.
[26] MIERSCH C, STANGE K, RÖNTGEN M.Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles[J].In Vitro Cellular & Developmental Biology Animal, 2018, 54(6):406-412.
[27] TAREEN W A, SABA E, RASHID U, et al.Impact of multiple isolation procedures on the differentiation potential of adipose derived canine mesenchymal stem cells[J].American Journal of Stem Cells, 2024, 13(1):27-36.
[28] QIU L H, SU Y J, ZHANG D L, et al.Identification of the centrifuged lipoaspirate fractions suitable for postgrafting survival[J].Plastic and Reconstructive Surgery, 2016, 137(1):67e-76e.
[29] RITTER A, FRIEMEL A, ROTH S, et al.Subcutaneous and visceral adipose-derived mesenchymal stem cells:Commonality and diversity[J].Cells, 2019, 8(10):1288.
[30] MASTRANGELO F, SCACCO S, BALLINI A, et al.A pilot study of human mesenchymal stem cells from visceral and sub-cutaneous fat tissue and their differentiation to osteogenic phenotype[J].European Review for Medical and Pharmacological Sciences, 2019, 23(7):2924-2934.
[31] HAN I, KWON B S, PARK H K, et al.Differentiation potential of mesenchymal stem cells is related to their intrinsic mechanical properties[J].International Neurourology Journal, 2017, 21(Suppl 1):S24-31.
[32] KIM G Y, CHOI G T, PARK J, et al.Comparative analysis of porcine adipose- and Wharton’s Jelly-derived mesenchymal stem cells[J].Animals, 2023, 13(18):2947.
[33] MALAGUTTI-FERREIRA M J, CRISPIM B A, BARUFATTI A, et al.Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells[J].Brazilian Journal of Medical and Biological Research, 2023, 56:e12713.
[34] EL ATAT O, ANTONIOS D, HILAL G, et al.An evaluation of the stemness, paracrine, and tumorigenic characteristics of highly expanded, minimally passaged adipose-derived stem cells[J].PLoS One, 2016, 11(9):e0162332.
[35] ALMAHASNEH F, ABU-EL-RUB E, KHASAWNEH R R, et al.Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages[J].World Journal of Stem Cells, 2024, 16(4):434-443.
[36] REUMANN M K, LINNEMANN C, ASPERA-WERZ R H, et al.Donor site location is critical for proliferation, stem cell capacity, and osteogenic differentiation of adipose mesenchymal stem/stromal cells:Implications for bone tissue engineering[J].International Journal of Molecular Sciences, 2018, 19(7):1868.
[37] LIN J H, CHANG H H, LEE W S, et al.Palmitic acid methyl ester enhances adipogenic differentiation in rat adipose tissue-derived mesenchymal stem cells through a G protein-coupled receptor-mediated pathway[J].Stem Cells International, 2021, 2021(1):9938649.
[38] MALODOBRA-MAZUR M, CIERZNIAK A, DOBOSZ T.Oleic acid influences the adipogenesis of 3T3-L1 cells via DNA methylation and may predispose to obesity and obesity-related disorders[J].Lipids in Health and Disease, 2019, 18(1):230.
[39] 谢亚萍, 张建平, 王利民, 等.亚麻籽油营养特性、感官评价及主要挥发性物质分析[J].中国油脂, 2023.48:103-108.
XIE Y P, ZHANG J P, WANG L M, et al.Analysis of nutrition property, sensory evaluation and major volatile components of flaxseed oil[J].China Oils and Fats, 2023, 48(7):103-108.
Outlines

/