人羊膜上皮细胞培养工艺的优化及生物学特性

Human amniotic epithelial cells:culture technology optimization and biological characteristics

背景:目前人羊膜上皮细胞分离、培养及冻存的研究较为分散,难以形成全面有效的方案以满足未来对移植用干细胞的临床需求。目的:建立人羊膜上皮细胞优化的分离、培养与冻存工艺,并对其生物学特性进行研究。方法:运用正交法研究各因素对人羊膜上皮细胞分离、培养及冻存指标的影响,极差法分析数据得到优化的分离、培养与冻存工艺条件。对人羊膜上皮细胞进行原代与传代培养,通过镜下形态学观察,绘制细胞生长曲线,进行流式细胞术检测、免疫荧光染色及向肝样细胞分化等实验,观察人羊膜上皮细胞的生物学特性。结果与结论:(1)获得优化的人羊膜上皮细胞分离条件为:胰酶浓度0.25%,分4次消化,每次消化时间20 min;优化培养条件为:细胞接种浓度为4×108 L-1,表皮生长因子质量浓度为10μg/L,血清体积分数为5%;优化冻存条件为:细胞冻存浓度为1×1010 L-1,二甲基亚砜浓度为10%,血清体积分数为80%;(2)原代细胞在接种二三天内贴壁生长,贴壁后细胞呈不规则多角形,以铺路石样生长,传代后细胞贴壁与生长速度加快,冻存复苏的传代第2代细胞生长与扩增能力无明显下降;(3)免疫荧光染色显示,原代人羊膜上皮细胞强表达CK19、SSEA-4,不表达Vimentin、CD45和HLA-DR;原代与传代第4代免疫表型检测显示,人羊膜上皮细胞在培养传代过程中有上皮间充质转化现象发生;(4)向肝样细胞分化实验中,免疫荧光染色显示,诱导后的人羊膜上皮细胞肝细胞标志物白蛋白、CK18表达量明显上升,糖原染色显示诱导3周后的人羊膜上皮细胞有糖原合成;(5)结果表明,人羊膜上皮细胞易于获得且体外增殖能力强,表达胚胎干细胞保持未分化状态的表面标志物。

BACKGROUND：As current studies on isolation, culture and cryopreservation of human amniotic epithelial cells （hAECs） are relatively scattered, it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE：To establish the technology of isolation, culture and cryopreservation of hAECs, and to study the biological characteristics of hAECs.METHODS：Orthogonal method was used to study the effects of different factors on the separation, culture and cryopreservation, and range method was adopted to analyze the data to optimize the separation, culture and cryopreservation. We performed cell primary and passage cultures, morphology observed by microscope, drawn cell growth curve and flow cytometry assay, immunofluorescence staining, hepatocyte like cell differentiation to study the biological characteristics of hAECs.RESULTS AND CONCLUSION：（1） The optimal hAECs separation conditions were as follows：trypsin digestions were conducted at a concentration of 0.25%, four times, once for 20 minutes digestion; optimal conditions of culture were 4×10^8/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×10^10/L cell cryopreservation density, 10% dimethyl sulfoxide, 80% serum. （2） The primary cells were adhered to the wall in 2-3 days, exhibiting irregular polygon, paving stone-like growth. Cell adherence and growth rate were accelerated after subculture, and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation. （3） Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19, but did not express Vimentin, CD45 and HLA-DR. The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of hAECs in culture process. （4） Immunofluorescence staining showed that the liver cell marker expression of ALB, CK18 was significantly increased after hAECs were induced to differentiate into hepatocyte-like cells. Glycogen staining revealed glycogen synthesis in hAECs after 3 weeks of induction. To conclude, hAECs are easy to obtain and have strong proliferation ability in vitro, and express surface markers for undifferentiated embryonic stem cells.