大鼠肝细胞球形体低温保存条件的初步探索  

Primary exploration of conditions for hypothermic preservation of rat hepatocyte spheroids

作　　者：刘鸿凌[1] 游绍莉[1] 李晨[1] 刘婉姝[1] 万志红[1] 臧红[1] 朱冰[1] 荣义辉[1] 柳芳芳[1] Scott L.Nyberg 辛绍杰[1] 

机构地区：[1]解放军302医院肝衰竭诊疗与研究中心,北京100039 [2]Division of Experimental Surgery,Mayo Clinic College of Medicine,Rochester,MN,USA

出　　处：《解放军医学杂志》2014年第12期936-940,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家“十二五”传染病科技重大项目(2012ZX10002004-005)~~

摘　　要：目的探索有效的肝细胞球形体低温保存方法,促进生物人工肝及其他研究的应用。方法采用两步法分离大鼠肝细胞,经无血清培养基(SFM)摇摆培养48h形成肝细胞球形体。将球形体继续培养(对照组),或置于4℃SFM,SFM+1mmol/L去铁胺(Def),SFM+1μmol/L环孢霉素A(Cs A),SFM+1mmol/L Def+1μmol/L Cs A保存液中24h或48h,继续培养4d或5d后观察低温保存后肝细胞球形体的存活率、超微结构变化,以及白蛋白和尿素合成情况。结果Def和Cs A能很好地保护球形体肝细胞,在4℃SFM+Def+Cs A保存液中保存24h,肝细胞球形体功能、结构等变化及尿素合成水平与对照组比较差异无统计学意义,而单独放在SFM中进行低温保存后,细胞活性明显下降。在4℃保存48h后,球形体肝细胞超微结构发生明显变化,死亡细胞增多,但SFM+Def+Cs A组和SFM+Def组细胞结构优于SFM组和SFM+Cs A组,细胞存活率及尿素合成水平明显高于后两组(P<0.05),但合成白蛋白量均处于较低水平,各组间差异无统计学意义(P>0.05)。结论低温状态下肝细胞球形体可保持良好的存活率和功能24h,低温保存能为细胞治疗提供良好的细胞材料。Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium （SFM）. Spheroids were then maintained in rocking culture at 37℃ （control condition）, or cold stored at 4℃ for 24 or 48 hours in four different cold storage solutions： SFM alone; SFM＋1mmol/L deferoxamine （Def）; SFM＋1μmol/L cyclosporin A （CsA）; and SFM＋1mmol/L Def＋1μmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM＋Def＋CsA or SFM＋Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM＋Def＋CsA or SFM＋Def was significantly higher than that stored in SFM or SFM＋CsA（P〈0.05）. The production of albumin was in low level in SFM, SFM＋Def, SFM＋CsA and SFM＋Def＋CsA groups, and there was no difference between them （P〉0.05）. Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases.

关 键 词：肝细胞 低温保存 球形体 细胞 肝 人工 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]