Human amniotic epithelial cells: culture technology optimization and biological characteristics

doi:10.3969/j.issn.2095-4344.2017.13.022

Abstract

Abstract:

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 4×10⁸/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×1010/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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Amnion, Epithelial Cells, Cell Culture Techniques, Tissue Engineering

CLC Number:

R394.2

Liu Chao, Xu Zhi-guo, Wang Shao-hong, Cheng Hong-ling, Yang Xu-wei, Gong Bo, Liu Yang, Xu Chun-yan. Human amniotic epithelial cells: culture technology optimization and biological characteristics[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 2100-2107.

Figures/Tables 2

2.1 hAECs的分离极差法分析分离因素正交实验结果，由表1数据得到分离指标——“SSEA-4阳性细胞量”极差最大的因素是消化次数，即消化次数对SSEA-4阳性细胞量影响最大。各因素影响大小排序：消化次数(C)>消化时间(B)>胰酶浓度(A)。比较各因素不同水平的值，获得优化的分离因素组合为A2B2C3，即胰酶浓度0.25%，消化时间20 min，分4次消化。 2.2 hAECs的培养极差法分析培养因素正交实验结果，由表2得到培养指标——“SSEA-4阳性细胞量/细胞接种量”极差最大的因素是细胞接种浓度，即细胞接种浓度对SSEA-4阳性细胞量/细胞接种量影响最大。各因素影响大小排序：细胞接种浓度(A)>血清体积分数(C)>表皮生长因子质量浓度(B)。比较各因素不同水平的T(_)值发现，表皮生长因子质量浓度的最高值(T(_)3与第二T(_)值(T(_)2数值接近，但表皮生长因子质量浓度水平2仅为水平3的一半，从方案经济性考虑，表皮生长因子质量浓度应选择水平2(10 μg/L)更为适宜，故优化的培养因素组合为A3B2C2，即细胞接种浓度4×108 L-1，血清体积分数为5%，表皮生长因子质量浓度10 μg/L。)) 2.3 hAECs的冻存极差法分析冻存因素正交实验结果，由表3得到，细胞冻存浓度与血清体积分数的极差相近，表明两者对冻存指标——“复苏细胞存活率”的影响程度相近。二甲基亚砜浓度的极差最大，表明二甲基亚砜浓度对复苏细胞存活率影响最大。各因素影响大小排序：二甲基亚砜浓度(C)>细胞冻存密度(A)>血清体积分数(B)。通过各因素不同水平的值比较，得到优化的冻存因素组合为A2B3C2，即细胞冻存浓度1×1010 L-1，血清体积分数为80%，二甲基亚砜浓度10%。 2.4 hAECs形态学观察与生长曲线体外原代培养的hAECs在光镜下呈典型上皮细胞形态。细胞接种后二三天内贴壁生长，刚贴壁的细胞呈圆形，折光性较强。完全贴壁后细胞胞质展开，呈不规则多角形，折光性减弱。原代hAECs 7 d左右形成单层贴壁细胞，呈铺路石样生长。传代后第2代细胞增殖速度加快，5 d左右可形成单层贴壁细胞。hAECs培养4代后细胞胞体略有变大，细胞增殖速度减缓(图1A)。苏木精-伊红染色后观察，传代第1代细胞大小较均一，细胞核染蓝色，核膜清晰，核仁明显，胞浆染红色，胞质丰富(图1B)。传代第1代hAECs接种于培养瓶后，0-2 d为细胞生长潜伏期，以细胞贴壁过程为主。3-5 d生长曲线曲率最大，为hAECs快速增殖的指数生长期；6 d后生长速度减缓，细胞基本长满培养瓶底，进入平顶期。传代第1代培养细胞符合典型的细胞生长曲线(图1C)。 2.5 免疫荧光染色检测结果免疫荧光染色显示，原代hAECs表达上皮细胞表面标志CK19和胚胎干细胞表面标志SSEA-4，不表达间充质细胞标志波形蛋白Vimentin和移植排斥发生密切相关的HLA-DR(图2)。表明hAECs免疫原性低，用于细胞治疗不易发生移植排斥现象。 2.6 hAECs流式细胞术检测结果流式细胞仪检测原代hAECs强阳性表达SSEA-4，阳性表达间充质干细胞相关标志物CD73，弱阳性表达CD90，CD105和SSEA-1阳性表达量很低，造血细胞表面标志物CD34、CD45几乎不表达，同样不表达HLA-DR(图3)。实验通过对原代与传代第4代hAECs(DMEM/F12+体积分数5%胎牛血清)相关免疫表型流式检测发现，传代第4代hAECs的SSEA-4和CK19表达较原代显著减少(P < 0.000 1)，Vimentin和CD105表达P4较原代细胞显著增多(P < 0.000 1)，原代与传代第4代hAECs均不表达HLA-DR (图4)。 SSEA-4是细胞全能性表达标志物，CK19是上皮细胞特异性表达标志物，Vimentin和CD105是间充质细胞的特异性表达标志物。实验结果表明，hAECs经过培养传代后，细胞在“分化全能性”上有所减弱。上皮细胞与间充质细胞标志物的变化表明，hAECs培养过程符合上皮间充质转化的特征表现。然而，培养过程中与免疫排斥密切相关的HLA-DR始终不表达，表明hAECs能始终保持免疫原性低的特性，有利于降低细胞治疗过程中排斥反应的发生概率。 2.7 hAECs诱导后的免疫荧光染色与糖原染色成熟肝细胞高表达白蛋白与CK18。诱导后细胞免疫荧光染色显示，诱导3周后hAECs的白蛋白与CK18表达量均较诱导1周明显增加(图5)。 hAECs诱导3周后经糖原PAS染色显示，紫红色物质为反应阳性物质——糖原，蓝色物质为细胞核(图6)。显示hAECs经实验诱导方案诱导后，成功获得具有一定合成功能的肝样细胞。"

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