Liu Hong-Ling, Wang Ying-Jie, Guo Hai-Tao, Wang Yu-Ming, Liu Jun, Yu Yue-Cheng
Research Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
World J Gastroenterol. 2004 Apr 1;10(7):1010-4. doi: 10.3748/wjg.v10.i7.1010.
To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation.
Hepatocytes, isolated from Chinese experimental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel.
Viability of 150 mL/L DMSO group thawed hepatocytes was (83+/-4)%, but after purification, its viability was (90+/-5)%, attachment efficiency was (86+/-7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatocytes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days' culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups.
Storage in liquid nitrogen can long keep hepatocytes' activities, the concentration of 150 mL/L DMSO is fit for porcine hepatocytes' cryopreservation. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.
研究猪肝细胞的冷冻保存方法及冻存后凝胶胶原培养措施。
采用体外灌注装置，通过两步胶原酶灌注法从中国实验用哺乳小型猪分离肝细胞，用50 mL/L至200 mL/L二甲基亚砜（DMSO）在液氮中冷冻保存4个月，然后解冻并接种于1层或2层凝胶胶原之间。在凝胶培养过程中检测猪白蛋白信使核糖核酸的表达、细胞形态以及天冬氨酸转氨酶（AST）和尿素氮（UN）的含量。
150 mL/L DMSO组解冻肝细胞活力为（83±4）%，但纯化后活力为（90±5）%，贴壁率为（86±7）%，解冻肝细胞活力接近新鲜细胞。解冻肝细胞在添加表皮生长因子的培养基中于凝胶胶原中培养时，肝细胞在混合胶原凝胶中呈不同层次生长，在夹心结构培养中呈束状生长。培养长达10天时，可观察到培养肝细胞典型的细胞形态特征。凝胶培养过程中AST的漏出量低于普通培养。同时，混合凝胶胶原中培养的细胞合成的UN高于其他组。
液氮保存可长期保持肝细胞活性，150 mL/L DMSO浓度适合猪肝细胞的冷冻保存。解冻后的肝细胞可用胶原基质培养，这提供了更接近体内的环境并维持肝细胞某些肝特异性功能的表达。
