Vacuum Low-temperature Preservation Combined with bFGF-supplemented Medium Enhances Somatic Cell Line Establishment Efficiency in Apeijiaza Cattle (Bos taurus)
ZHANG Yu-Rong1,2, WANG Jing2, WANG Qing-Wei3, GU Shi-Gang2, HUANG Yong-Ye4, YU Da-Wei2,*, XU Ye-Fen1,*
1 College of Animal Science, Xizang Agricultural and Animal Husbandry University, Linzhi 860000, China; 2 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100080, China; 3 College of Animal Science, Guizhou University, Guiyang 550000, China; 4 College of Life and Health Science, Northeastern University, Shenyang 110000, China
Abstract：The Tibet Autonomous Region boasts abundant and unique animal genetic resources, serving as a critical national reserve for germplasm resources. Restricted by plateau geography and transportation conditions, animal tissue samples collected in the wild cannot be promptly transported to laboratories for processing, which severely impedes the establishment of primary fibroblast cell lines. To improve the efficiency of somatic cell line establishment from endangered livestock breeds in remote plateau regions such as Tibet, a vacuum low-temperature preservation method capable of stably maintaining ear marginal tissue at 0~4 ℃ during transport, as well as a corresponding cell line establishment system, was developed in this study. The feasibility of this approach was first validated in the indigenous Bama pig (Sus scrofa domestica). Ear marginal tissues were collected, subjected to vacuum treatment, and preserved at 4 ℃ for 0, 7, and 14 d prior to cell line initiation, and the success rate was 98%. Subsequently, this vacuum low-temperature preservation method was applied to ear marginal tissues of the Tibet-endangered Apeijiaza cattle (Bos taurus) following long-distance transportation and off-site cell line establishment. Compared with the conventional PBS-immersion method, the vacuum low-temperature preservation protocol yielded a greater number of viable cells at initiation, with cell viability consistently exceeding 85%. The success rate of primary fibroblast establishment from Apeijiaza cattle ear tissues was significantly increased by 37.5% relative to the control group (P<0.05). Furthermore, supplementation of the culture medium with 10 ng/mL basic fibroblast growth factor (bFGF) effectively retarded the senescence progression of Apeijiaza cattle somatic cells during serial passaging in vitro. Under bFGF-supplemented conditions, cells maintained high proliferative activity up to passage 20. There was no obvious apoptosis and senescence morphology, accompanied by significant downregulation of senescence-associated genes, including tumor protein p53 (TP53), insulin-like growth factor-binding protein 2 (IGFBP2), and thrombospondin-1 (THBS1). Collectively, the findings demonstrated that the combined strategy of vacuum low-temperature preservation and bFGF supplementation in culture medium effectively overcame the long-standing bottleneck associated with prolonged sample preservation of livestock genetic resources in remote areas, thereby providing a robust and reliable technological platform for the off-site establishment of somatic cell genetic resources from endangered species.
[1] 刘诗平. 2022. 绝不让雪域高原濒危种质资源灭失——西藏阿沛甲咂牛与樟木牛抢救性保种全面展开[J]. 中国畜牧业, (24): 10-11. (Liu S P. 2022. Rescue conservation of Tibet's ApeiJiaza cattle and Zhangmu cattle underway to prevent the loss of endangered germplasm resources on the Tibetan plateau[J]. China Animal Industry, (24): 10-11.) [2] 刘容麟, 王宁, 李岩异, 等. 2023. 低温保存技术在生物制品中的研究进展[J]. 生物技术进展, 13(04): 547-555. (Liu R L, Wang N, Li Y Y, et al.2023. Research progress of cryopreservation technology in biological products[J]. Current Biotechnology, 13(04): 547-555.) [3] 刘昕, 韩志强, 张淑莹, 等. 2024. 不同品种梅花鹿成纤维细胞系分离培养与保种库的建立[J]. 经济动物学报, 28(03): 173-177. (Liu X, Han Z Q, Zhang S Y, et al.2024. Isolation and culture of different species of sika deer fibroblast cell lines and establishment of seed preservation bank[J]. Journal of Economic Animal, 28(03): 173-177.) [4] 马龙刚, 刘楠, 尼玛群宗, 等. 2024. 基于SNP芯片信息分析西藏四个本地牛种的血统组成[J]. 畜牧兽医学报, 55(10): 4377-4390. (Ma L G, Liu N, Nima Q Z.2024. et al.Study on the genetic composition of four local tibetan cattle breeds based on SNP chip analysis[J]. Acta Veterinaria et Zootechnica Sinica, 55(10): 4377-4390.) [5] 齐浩南, 霍浩楠, 曹磊, 等. 2025. 体细胞核移植克隆西藏濒危樟木牛[J]. 中国畜牧兽医, 52(12): 5740-5748. (Qi H N, Huo H N, Cao L, et al.2025. Cloning of the endangered Zhangmu cattle via somatic cell nuclear transfer in Tibet[J]. China Animal Husbandry & Veterinary Medicine, 52(12): 5740-5748.) [6] 王淑为, 田増民, 郭欣茹, 等. 2013. 4 ℃低温保存对神经干细胞活性的影响[J]. 中华神经外科疾病研究杂志, 12(05): 452-453. (Wang S W, Tian Z M, Guo X R, et al.2013. Effect of cryopreservation at 4 °C on the viability of neural stem cells[J]. Chinese Journal of Neurosurgical Disease Research, 12(05): 452-453.) [7] 周旗, 张亮, 潘雨, 等. 2025. 环黄芪醇对猪供体成纤维细胞衰老、细胞骨架与核移植胚胎早期发育的影响[J]. 中国农业科学, 58(12): 2453-2474. (Zhou Q, Zhang L, Pan Y, et al.2025. Effects of cycloastragenol on cellular senescence of pig doner fibroblast,cytoskeletal dynamic,and early development stage of nuclear transfer embryo[J]. Scientia Agricultura Sinica, 58(12): 2453-2474.) [8] Blagojevic D P, Grubor-Lajsic G N, Spasic M B.2011. Cold defence responses: The role of oxidative stress[J]. Frontiers in Bioscience (Scholar edition), 3(2): 416-427. [9] Cabral-Pacheco G A, Garza-Veloz I, Castruita-De la Rosa C, et al.2020. The roles of matrix metalloproteinases and their inhibitors in human diseases[J]. International Journal of Molecular Sciences, 21(24): 9739-9739. [10] Cordova A, King W A, Mastromonaco G F.2017. Choosing a culture medium for SCNT and iSCNT reconstructed embryos: From domestic to wildlife species[J]. Journal of Animal Science and Technology, 59(1): 1-14. [11] de Araújo J M, de Oliveira R A, Capobianco N E, et al.2021. Effects of refrigeration at 5 ℃ for long periods of time on bovine ear skin as a strategy to transport biological material and isolate fibroblasts to use in the nuclear transfer[J]. Biopreservation and Biobanking, 20(4): 323-330. [12] de Magalhães J P.2024. Cellular senescence in normal physiology[J]. Science, 384(6702): 1300-1301. [13] Dong Z, Luo Y, Yuan Z, et al.2024. Cellular senescence and SASP in tumor progression and therapeutic opportunities[J]. Molecular Cancer, 23(1): 181. [14] Duan X, Chen C, Du C, et al.2025. Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs[J]. Genome Research, 279709: 124. [15] Gouveia C, Huyser C, Egli D, et al.2020. Lessons learned from somatic cell nuclear transfer[J]. International Journal of Molecular Sciences, 21(7): 1-24. [16] Gutiérrez-Fernández A, Soria-Valles C, Osorio F G, et al.2015. Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid[J]. The EMBO journal, 34(14): 1875-1888. [17] Haga M, Iida K, Okada M.2024. Positive and negative feedback regulation of the TGF-β1 explains two equilibrium states in skin aging[J]. iScience, 27(5): 1-31. [18] Hassona Y, Cirillo N, Heesom K, et al.2014. Senescent cancer-associated fibroblasts secrete active MMP-2 that promotes keratinocyte dis-cohesion and invasion[J]. British Journal of Cancer, 111(6): 1230-1237. [19] Itkin T, Kaufmann K B, Gur-Cohen S, et al.2013. Fibroblast growth factor signaling promotes physiological bone remodeling and stem cell self-renewal[J]. Current Opinion in Hematology, 20(3): 237-244. [20] Levi N, Papismadov N, Solomonov I, et al.2020. The ECM path of senescence in aging: Components and modifiers[J]. The FEBS Journal, 287(13): 2636-2646. [21] Li Y, Li L, Chen Z, et al.2017. Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications[J]. Biofabrication, 9(3): 1-20. [22] Mohamad Kamal N S, Safuan S, Shamsuddin S, et al.2020. Aging of the cells: Insight into cellular senescence and detection methods[J]. European Journal of Cell Biology,99(6): 151108-151108. [23] Ma Y W, Zhang L F, Huang X X.2014. Genome modification by CRISPR/Cas9[J]. The FEBS Journal, 281(23): 5186-5193. [24] Nickle A, Ko S, Merrill A E.2023. Fibroblast growth factor 2[J]. Differentiation, 139: 100733. [25] Nishio K, Inoue A, Qiao S, et al.2001. Senescence and cytoskeleton: Overproduction of vimentin induces senescent-like morphology in human fibroblasts[J]. Histochemistry and Cell Biology, 116(4): 321-327. [26] Okonkwo C, Singh M.2015. Recovery of fibroblast-like cells from refrigerated goat skin up to 41 d of animal death[J]. In vitro Cellular & Developmental Biology-Animal,51(5): 463-469. [27] Oyane A, Araki H, Nakamura M, et al.2021. Storable bFGF-releasing membrane allowing continuous human iPSC culture[J]. Materials, 14(3): 651. [28] O'Driscoll S W, Meisami B, Miura Y, et al.1999. Viability of periosteal tissue obtained postmortem[J]. Cell Transplantation, 8(6): 611-616. [29] Praxedes É A, Borges A A, Santos M V O, et al.2018. Use of somatic cell banks in the conservation of wild felids[J]. Zoo Biology, 37(4): 258-263. [30] Regulski M J.2017. Cellular senescence: What, why, and how[J]. Wounds: A Compendium of Clinical Research and Practice, 29(6): 168-174. [31] Riegger J, Brenner R E.2023. Increase of cell surface vimentin is associated with vimentin network disruption and subsequent stress-induced premature senescence in human chondrocytes[J]. eLife, 19(10): 1-20. [32] Salehi P, Spratlin J, Chong T, et al.2004. Beneficial effects of supplemental buffer and substrate on energy metabolism during small bowel storage[J]. Cryobiology, 48(3): 245-253. [33] Shay J W, Wright W E.2000. Hayflick, his limit, and cellular ageing[J]. Nature Reviews. Molecular Cell Biology, 1(1): 72-76. [34] Silvestre M A, Saeed A M, Cervera R P, et al.2003. Rabbit and pig ear skin sample cryobanking: Effects of storage time and temperature of the whole ear extirpated immediately after death[J]. Theriogenology,59(5-6):1469-1477. [35] Sumbal J, Koledova Z.2019. FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis[J]. Development (Cambridge, England), 146(23): 185306-185306. [36] Tenzin J, Chankitisakul V, Boonkum W.2023. Current status and conservation management of farm animal genetic resources in Bhutan[J]. Veterinary Sciences, 10(4): 262-281. [37] Yang J H, Petty C A, Dixon-McDougall T, et al.2023. Chemically induced reprogramming to reverse cellular aging[J]. Aging, 15(13): 5966-5989. [38] Zare R, Abdolsamadi H, Soleimani Asl S, et al.2023. The bFGF can improve angiogenesis in oral mucosa and accelerate wound healing[J]. Reports of Biochemistry & Molecular Biology, 11(4): 547-552. [39] Zhu Y, Liu X, Ding X, et al.2019. Telomere and its role in the aging pathways: Telomere shortening, cell senescence and mitochondria dysfunction[J]. Biogerontology, 20(1): 1-16.
