一种无血清冻存液在猪胎儿成纤维细胞上的应用研究
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摘要
在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。
In the process of cryopreservation of porcine fetal fibroblasts(PFF), serum quality often limits the cryopreservation effect of cells. To solve this problem, the aim of this study was to develop a serum-free freezing medium(SFM) for the cryopreservation of porcine fetal fibroblasts. Porcine fetal fibroblasts were cryopreserved in 3 different cryopreservation solutions, each frozen in 10 tubes. After 30 days of cryopreservation, the cells were resuscitated, and the survival rate of the frozen cells, the cell proliferation activity, and the cell activity after electroporation were measured. The results showed that the self-made serum-free cell cryopreservation solution had a survival rate of 95.33% after cryopreservation of porcine fetal fibroblasts. The cell proliferation activity and cell activity after electroporation were significantly higher than that of standard fetal bovine serum cryopreservation solution(p<0.05), equal with super fetal bovine serum group(p>0.05). Therefore, the frozen cryopreservation of porcine fetal fibroblasts with self-made cryopreservation solution is stable and could replace the cryopreservation solution containing serum, which has a good prospect of popularization and application.
In the process of cryopreservation of porcine fetal fibroblasts(PFF), serum quality often limits the cryopreservation effect of cells. To solve this problem, the aim of this study was to develop a serum-free freezing medium(SFM) for the cryopreservation of porcine fetal fibroblasts. Porcine fetal fibroblasts were cryopreserved in 3 different cryopreservation solutions, each frozen in 10 tubes. After 30 days of cryopreservation, the cells were resuscitated, and the survival rate of the frozen cells, the cell proliferation activity, and the cell activity after electroporation were measured. The results showed that the self-made serum-free cell cryopreservation solution had a survival rate of 95.33% after cryopreservation of porcine fetal fibroblasts. The cell proliferation activity and cell activity after electroporation were significantly higher than that of standard fetal bovine serum cryopreservation solution(p<0.05), equal with super fetal bovine serum group(p>0.05). Therefore, the frozen cryopreservation of porcine fetal fibroblasts with self-made cryopreservation solution is stable and could replace the cryopreservation solution containing serum, which has a good prospect of popularization and application.
引文
Chen F.J.,2015,Generation of B cell-deficie nt pigs by efficient TALENs or CRISPR/Cas9-mediated gene targeting,Dissertation for Ph.D.,School of Basic Medical Sciences Institute,Nanjing Medical University,Supervisor:Dai Y.F.,pp.11-16(陈凤娇,2015,应用TALENs以及CRISPR/Cas9技术构建B细胞缺失免疫缺陷猪模型,博士学位论文,南京医科大学基础医学院,导师:戴一凡,pp.11-16)
Cozzi E.,and White D.J.,1995,The generation of transgenic pigs as potential organ donors for humans,Nature Medicine,1(9):964-970
Fahy G.M.,Wowk B.,Wu J.,and Paynter S.,2004,Improved vitrification solutions based on the predictability of vitrification solution toxicity,Cryobiology,48(1):22-35
Guan J.,Cui Z.F.,Gong C.Y.,Urban J.,and Zhang X.Z.,2004,Study on cryopreservation of cells and tissues,Yiliao Weisheng Zhuangbei(Chinese Medical Equipment Journal),25(3):7-9(关静,崔占峰,龚承元,Urban Jill,张西正,2004,冷冻保存细胞组织的初步研究,医疗卫生装备,25(3):7-9)
Karlsson J.O.M.,and Toner M.,1996,Long-term storage of tissues by cryopreservation:critical issues,Biomaterial,17(3):243-256
Kues W.A.,and Niemann H.,2004,The contribution of farm animals to human health,Trends in Biotechnology,22(6):286-294
Li H.,Wei H.J.,Xu C.S.,Wang X.,Qing Y.B.,and Zeng Y.Z.,2010,Establishment and biological characteristics of fetal fibroblast cell lines in Banna mini pig inbred line,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),36(6):678-682(李红,魏宏江,许成盛,汪霞,卿玉波,曾养志,2010,版纳微型猪近交系胎儿成纤维细胞系的建立及其生物学特征,湖南农业大学学报(自然版),36(6):678-682)
Li S.X.,Chen S.L.,Li J.Q.,Zhang H.,and Zang Y.A.,2012,Study on the biological characteristics of fetal fibroblast of the dual hybrid porcine,Sichuan Nongye Daxue Xuebao(Journal of Sichuan Agricultural university),30(2):220-225(李仕新,陈松玲,李加琪,张豪,臧莹安,2012,长大二元杂交猪胎儿成纤维细胞的生物学特性,四川农业大学学报,30(2):220-225)
Li X.L.,Yu G.H.,and Sun J.H.,2016,Establishment and identification of a fetal fibroblast cell line with double transgenic(pGH/IGF-Ⅰ)porcine,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),43(8):2053-2059(李晓玲,于光辉,孙金海,2016,转双基因(pGH/IGF-Ⅰ)猪胎儿成纤维细胞系的建立及鉴定,中国畜牧兽医,43(8):2053-2059)
Paradis K.,Langford G.,Long Z.F.,Heneine W.,Sandstrom P.,Switzer W.M.,Chapman L.E.,Lockey C.,Onions D.,The XEN 111 Study Group.,and Otto E.,1999,Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue,Science,285(5431):1236-1241
Cozzi E.,and White D.J.,1995,The generation of transgenic pigs as potential organ donors for humans,Nature Medicine,1(9):964-970
Fahy G.M.,Wowk B.,Wu J.,and Paynter S.,2004,Improved vitrification solutions based on the predictability of vitrification solution toxicity,Cryobiology,48(1):22-35
Guan J.,Cui Z.F.,Gong C.Y.,Urban J.,and Zhang X.Z.,2004,Study on cryopreservation of cells and tissues,Yiliao Weisheng Zhuangbei(Chinese Medical Equipment Journal),25(3):7-9(关静,崔占峰,龚承元,Urban Jill,张西正,2004,冷冻保存细胞组织的初步研究,医疗卫生装备,25(3):7-9)
Karlsson J.O.M.,and Toner M.,1996,Long-term storage of tissues by cryopreservation:critical issues,Biomaterial,17(3):243-256
Kues W.A.,and Niemann H.,2004,The contribution of farm animals to human health,Trends in Biotechnology,22(6):286-294
Li H.,Wei H.J.,Xu C.S.,Wang X.,Qing Y.B.,and Zeng Y.Z.,2010,Establishment and biological characteristics of fetal fibroblast cell lines in Banna mini pig inbred line,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),36(6):678-682(李红,魏宏江,许成盛,汪霞,卿玉波,曾养志,2010,版纳微型猪近交系胎儿成纤维细胞系的建立及其生物学特征,湖南农业大学学报(自然版),36(6):678-682)
Li S.X.,Chen S.L.,Li J.Q.,Zhang H.,and Zang Y.A.,2012,Study on the biological characteristics of fetal fibroblast of the dual hybrid porcine,Sichuan Nongye Daxue Xuebao(Journal of Sichuan Agricultural university),30(2):220-225(李仕新,陈松玲,李加琪,张豪,臧莹安,2012,长大二元杂交猪胎儿成纤维细胞的生物学特性,四川农业大学学报,30(2):220-225)
Li X.L.,Yu G.H.,and Sun J.H.,2016,Establishment and identification of a fetal fibroblast cell line with double transgenic(pGH/IGF-Ⅰ)porcine,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),43(8):2053-2059(李晓玲,于光辉,孙金海,2016,转双基因(pGH/IGF-Ⅰ)猪胎儿成纤维细胞系的建立及鉴定,中国畜牧兽医,43(8):2053-2059)
Paradis K.,Langford G.,Long Z.F.,Heneine W.,Sandstrom P.,Switzer W.M.,Chapman L.E.,Lockey C.,Onions D.,The XEN 111 Study Group.,and Otto E.,1999,Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue,Science,285(5431):1236-1241