零下非结冰UW液成功保存生物人工肝用L-02肝细胞

Storage of L-02 hepatocytes for bioartificial liver in UW solution at non-freezing points below zero

目的比较零下非结冰(-0.8℃)与常规低温(4℃)分别用于人工肝用永生化L-02细胞保存的效果,探索零下非结冰保存肝细胞与细胞凋亡的关系。方法制备好的UW液保存的L-02细胞悬液分为以下2组:-0.8℃组(零下非结冰组),4℃组(对照组)。低温保存24、48h及72h后,分别测定细胞存活率、凋亡率、坏死率(流式细胞术)及ALT释放、尿素合成功能、白蛋白分泌功能。结果 -0.8℃(零下非结冰)较4℃明显提高了长时间低温保存L-02细胞的存活率[72h:(70.17±2.82)%vs(60.05±3.17)%,P<0.01],降低了细胞凋亡率[72h:(5.73±1.68)%vs(9.20±2.35)%,P=0.02],抑制了ALT释放[72h:(6.21±0.65)U/Lvs(8.68±1.18)U/L,P<0.01],更好地维持了L-02细胞的尿素合成功能[72h:(1.01±0.14)mmol/Lvs(0.66±0.09)mmol/L,P<0.01]及白蛋白分泌功能[72h:(9.04±0.53)μg/mlvs(7.70±0.52)μg/ml,P<0.01]。2组间细胞坏死率差异无统计学意义[72h:(18.45±1.88)%vs(21.82±3.86)%,P>0.05]。结论零下非结冰可明显维持L-02细胞存活率,降低低温损伤引起的细胞凋亡,有效保护肝细胞尿素合成和白蛋白分泌功能。

Objective To investigate the effect of subzero nonfreezing storage temperature （ - 0.8℃ ） compared with that of conventional hypothermic storage temperature （4 % ） for L-02 hepatocytes in UW solution in bioartificial liver support system （BLASS） and the relationship of this storage with cells apoptosis. Methods L-02 hepatocytes suspended in UW solution were divided into the following 2 groups, subzero nonfreezing group （ -0.8℃ ） and control group （4 % ）. After 24, 48 and 72 h of hypothermic storage, the cell viability and cell apoptosis were measured with flow cytometry. Alanine transferase （ALT） release, the ability of hepatocytes to synthesize urea and secrete albumin were also studied. Results Significant improvement of cell viability was observed in subzero nonfreezing group compared with that in the control group [ 72 h： （70. 17 ±2. 82） % vs （60.05 ± 3. 17 ） %, P 〈 0. 011. Cell apoptosis was inhibited in subzero nonfreezing group [ 72 h ： （5.73 ±1.68）% vs （9.20±2.35）%, P=0.02], and so did ALT secretion [72 h： （6.21 ±0.65） U/L vs （8.68±1.18） U/L, P 〈0. 01 ]. The ability of hepatocytes to synthesize urea [ 72 h： （1.01± 0. 14） mmol/L vs （0.66±0.09） mmo[/L, P 〈0. 01） and to secrete albumin [72 h： （9.04±0.53） μg/ml vs （7.70 ± 0. 52） p,g/ml, P 〈 0.01 ] were also better maintained in subzero nonfreezing group. There was no difference between the 2 groups in the cellular necrosis rate [72 h： （ 18.45 ± 1.88）% vs （21.82 ± 3.86）%, P 〉 0. 05 ]. Conclusion Subzero nonfreezing storage （ - 0.8 ℃ ） of L-02 hepatocytes in UW solution provides better cell viability and abilities of hepatocytes to synthesize urea and secrete albumin, and lower cell apoptosis. Subzero nonfreezing storage （ - 0.8 ℃ ） of hepatocytes and constructing a ＂ready to use＂ hepatocytes bank will efficiently promote the development of the BLASS.