【摘要】 目的:比较传统及改良冻存法对人胎肝细胞保护效果,建立一种适合于人胎肝细胞冷冻保存的方法。方法:实验于2006-07/11在广州医学院进行。①人胎肝组织取自中期终止妊娠胎儿(20周,经胎儿亲属知情同意及医院伦理委员会批准)。冷冻保护液包括A液(DMEM培养液含1.5mol/L乙二醇+体积分数为0.2胎牛血清);B液(A液+0.1mol/L蔗糖)两部分。②传统冻存法:用0.025%Ⅰ型胶原酶消化胎肝组织,分离、收集消化后的胎肝细胞。胎肝细胞在A液、B液中分别渗透平衡20min后,在冷冻保护B液中经液氮蒸汽冷平衡5min后迅速入液氮冻存,1周后复苏培养观察。③改良冻存法:胎肝组织在A液、B液中分别渗透平衡20min后,在冷冻保护液B液中经液氮蒸汽冷平衡5min后迅速入液氮冻存,1周后复苏,入DMEM培养液37℃孵育1h,胶原酶消化胎肝组织,分离、收集消化后的胎肝细胞培养观察。④新鲜分离的胎肝细胞做为对照组。⑤复苏后分离所得胎肝细胞用锥虫兰染色评估细胞活率,并记录贴壁时间,观察集落生长情况。结果:①锥虫兰染色结果显示传统冻存法复温后的胎肝细胞活率为(73.8±2.2)%,明显低于新鲜分离的胎肝细胞活率(89.2±3.6)%及改良冻存法所得胎肝细胞活率(82.5±4.2)%(P<0.01)。②贴壁情况观察发现新鲜胎肝细胞与改良冻存法胎肝细胞于培养24h开始贴壁,而传统冻存法胎肝细胞贴壁时间滞后48h。③新鲜胎肝细胞及改良冻存法胎肝细胞于第3天出现生长集落(细胞数>10为一个集落),传统冻存法胎肝细胞在第5天出现生长集落,集落的数量低于改良冻存法及对照组(P<0.01)。结论:改良冻存法可避免冻存前损伤,提高细胞对冷冻的耐受性和冻存复温后细胞的活率。更多还原

【Abstract】 AIM: To compare the protective effects of traditional and reforming cryopreserving methods on human fetal hepatocytes, and find out a suitable method for the preservation. METHODS: The experiment was performed in Guangzhou Medical College from July to November 2006. ①The human fetal hepatocytes, obtained with maternal consent and approval of the Human Ethics Committee of the college, were harvested from a mid-gestation fetus (20 weeks) after elective abortion. Cryoprotectant included cryoprotectant A composed of DMEM + 1.5 mol/L ethylene glycol + 0.2 volume fraction fetal bovine serum, and cryoprotectant B composed of cryoprotectant A + 0.1 mol/L sucrose. ②In the traditional cryopreserving method, the fetal liver tissues were digested with 0.025% collagenase Ⅰ to isolate and collect the digested fetal hepatocytes, which were firstly permeated and equilibrized in cryoprotectant A and B for 20 minutes, respectively, and then put into liquid nitrogen quickly after cold equilibrium at liquid nitrogen steam for 5 minutes. The cryopreserved cells were thawed and observed 1 week later. ③In the reforming cryopreserving method, the fetal liver tissues were permeated and equilibrized in cryoprotectant A and B for 20 minutes, respectively, and then put into liquid nitrogen quickly after cold equilibrium at liquid nitrogen steam for 5 minutes. One week later, the liver tissues were thawed and incubated in DMEM culture medium at 37 ℃ for 1 hour. The tissues were digested to isolate and collect the fetal hepatocytes. ④The fresh isolated fetal hepatocytes were used as control. ⑤The survival rate of thawed cells was evaluated by trypan blue staining, and the adherence time and colony growth were observed. RESULTS: ①The trypan blue staining appealed the survival rate of fetal hepatocytes in the traditional cryopreservation group was (73.8±2.2)%, which was significantly lower than that of the fresh isolated fetal hepatocytes [(89.2±3.6)%] and reforming cryopreservation group [(82.5±4.2)%], respectively (P < 0.01). ②The hepatocytes in the control group and reforming cryopreservation group adhered since the 24 hour after cultivation, while the cells was 48 hours later than the two groups. ③Colony (a colony containing more than 10 cells) appeared in the control group and reforming cryopreserving group on day three, while in the traditional cryopreserving group on day five; moreover, the colony number was lower than that of the control and reforming cryopreservation groups (P < 0.01). CONCLUSION: Reforming cryopreserving method could avoid the lesion of fetal hepatocytes before cryopreservation, improve the toleration to frozen, and elevate the cell survival rate after thawing.更多还原