A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells

doi:10.3969/j.issn.2095-4344.0496

Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (13): 2020-2026.doi: 10.3969/j.issn.2095-4344.0496

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A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells

Zhang Xue-juan^{1, 2}, Liu Gao-mi-yang², Liu Ju-fen², Song Yi-jia^{1, 2}, Lin Qing-keng^{1, 2}, Bai Ying-ying^{1, 2}, Pan Xing-hua²

¹Kunming General Hospital Clinical College of Kunming Medical University, Kunming 650032, Yunnan Province, China; ²Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China

Revised:2018-02-02 Online:2018-05-08 Published:2018-05-08
Contact: Pan Xing-hua, M.D., Chief physician, Professor, Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China
About author:Zhang Xue-juan, Master candidate, Kunming General Hospital Clinical College of Kunming Medical University, Kunming 650032, Yunnan Province, China; Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China. Liu Gao-mi-yang, Master, Physician, Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China. Zhang Xue-juan and Liu Gao-mi-yang contributed equally to this work.
Supported by:
the Project of the Stem Cell Therapy Key Laboratory of Yunnan Province, No. 2015DG034; the Experimental Animal Specific Fund of PLA, No. SYDW(2016)004

Abstract

Abstract:

BACKGROUND: Studies have shown increasing risks and problems in the serum culture system, such as immune rejection, batch differences and virus risk. In addition, with the discovery and application of exosomes, the serum-free culture system is becoming an increasing concern.
OBJECTIVE: To compare the similarities and differences between the serum-free culture system and the traditional serum culture system, which lays the foundation for the clinical transformation of human umbilical cord mesenchymal stem cells (hUCMSCs) and provides experimental data.
METHODS: Umbilical cord was collected from term infants of cesarean section under aseptic condition, and hUCMSCs were isolated and cultured by explant tissue technique. hUCMSCs was cultured with 10% fetal bovine serum (FBS) and 15% serum substitutes (AGS) from the original generation. Then an inverted microscope was used to observe cell morphological changes. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 was used to detect cell proliferation. Induced differentiation experiment was used to detect cell differentiation potential. Western Blot was used to detect the protein levels of oct4, nanog and sox2.
RESULTS AND CONCLUSION: Under the inverted microscope, hUCMSCs cultured with AGS showed more uniform vortex-like growth, and those cultured with FBS gradually appeared with cell differentiation or aging with the increase of cell generations. hUCMSCs cultured by both methods expressed CD73,CD90 and CD105 but lowly expressed CD34 and CD45, and there was no significant difference between the two culture methods. FBS method was superior to AGS method in proliferation ability. Results from the induced differentiation experiments showed that hUCMSCs cultured by both methods had adipogenic, osteogenic and chondrogenic abilities, and there was no significant difference between the two culture methods. hUCMSC cultured by both methods expressed oct4 and nanog but showed no significant difference in level, while the expression of sox2 was significantly higher in the hUCMSCs cultured by AGS than by FBS (P < 0.05). To conclude, the hUCMSCs cultured with AGS are in accordance with the international standards of mesenchymal stem cells. The AGS method as an alternative to the FBS method can become a preferred method for hUCMSCs culture.

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: Umbilical Cord, Mesenchymal Stem Cells, Serum, Tissue Engineering

CLC Number:

R394.2

Zhang Xue-juan, Liu Gao-mi-yang, Liu Ju-fen, Song Yi-jia, Lin Qing-keng, Bai Ying-ying, Pan Xing-hua. A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2020-2026.

Figures/Tables 2

2.1 人脐带间充质干细胞的分离及培养结果采用组织块贴壁法分离培养人脐带间充质干细胞，原代培养10 d后，血清替代物法形态更均一(图1)；随着继续传代培养，血清替代物组细胞呈现较均一的涡漩状生长，而传统胎牛血清组则随着细胞代数的增加逐渐出现细胞分化或者老化现象(图2)。 2.2 人脐带间充质干细胞的表面抗原表达流式细胞仪检测血清替代物组人脐带间充质干细胞表面抗原CD73、CD90和CD105阳性率为100%、99.5%和98.8%，CD34和CD45阳性率为11.6%和0.079%，胎牛血清组人脐带间充质干细胞表面抗原CD73、CD90和CD105阳性率为99.9%、97.9%和93.5%，CD34和CD45阳性率为2.22%和0.209%(图3)，符合人脐带间充质干细胞的表型特征，两种方法培养的人脐带间充质干细胞表面抗原无明显差异。 2.3 人脐带间充质干细胞的诱导分化能力用第5代人脐带间充质干细胞进行体外诱导分化实验(图4)，诱导分化实验用时达14-21 d，阴性对照组细胞100%融合后继续培养细胞逐渐死亡，因此阴性对照的图片未列出。成骨诱导后经茜素红染色，出现红染的钙结节，胎牛血清组和血清替代物组的阳性率分别为90%和80%。成软骨诱导后经阿新蓝染色，出现蓝染的软骨细胞合成的蛋白多糖，胎牛血清组和血清替代物组的阳性率均为90%。成脂诱导后经油红O染色，出现红染的脂滴，胎牛血清组和血清替代物组的阳性率分别为90%和80%。两种培养法无显著差异。 2.4 人脐带间充质干细胞的增殖能力将人脐带间充质干细胞接种于96孔板，条件培养基分别为体积分数为10%胎牛血清、10%血清替代物、15%血清替代物和20%血清替代物，连续培养6 d，绘制生长曲线，见图5。第1-3天体积分数为10%胎牛血清组增殖速率明显高于3个血清替代物组(P < 0.05)，第4天后增殖速率无明显差异(P > 0.05)，血清替代物组随着体积分数增高，增殖速率增快。 2.5 人脐带间充质干细胞oct4、nanog及sox2蛋白的表达 Western blot实验结果显示，血清替代物组与胎牛血清组oct4、nanog的表达水平无显著差异，血清替代物组sox2表达水平高于胎牛血清组(P < 0.05)，见图6。"

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A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells

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