A preliminary establishment of the “single-cell-transplant-umorigenic-test”model for identifying liver cancer stem cells

doi:10.3969/j.issn.2095-4344.2017.09.003

Chinese Journal of Tissue Engineering Research ›› 2017, Vol. 21 ›› Issue (9): 1324-1328.doi: 10.3969/j.issn.2095-4344.2017.09.003

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A preliminary establishment of the “single-cell-transplant-umorigenic-test”model for identifying liver cancer stem cells

Jia Wei, Cen Yan-hui, Bao Juan, Yang Rui, He Guo-zhen, Wu Xiao-jun, Li Yi-yi

Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China

Online:2017-03-28 Published:2017-03-31
Contact: Cen Yan-hui, M.D., Associate professor, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China Corresponding author: Yang Rui, Master, Lecturer, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China
About author:Jia Wei, Master, Lecturer, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China Bao Juan, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China Jia Wei and Bao Juan contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81503406; University Research Projects in Guangxi, No. ZD2014068; Project of Improving the Basic Ability of Young Teachers in Universities in Guangxi, No. KY2016YB218, KY2016YB224; Special Scientific Project of Traditional Chinese Medicine supported by Health Department of Guangxi Zhuang Autonomous Region, No. GZPT13-04, GZBZ16-07, GZLC16-23; and Student Research Training Project of Guangxi Medical University, No. 2014DXS02

Abstract

Abstract:

BACKGROUND: There is no effective method to identify liver cancer stem cells until now, which has become one of the major challenges in this field.
OBJECTIVE: To explore a more effective and suitable way for the identification of liver cancer stem cells in hepatocellular carcinoma cell lines.
METHODS: Firstly, the single cell separation technique was used to obtain single cells, which were seeded into 96-well plates one by one. Secondly, cell sublines derived from single cell colonies (tumorigenic colonies) were selected and obtained. At last, the cells from these clones were transplanted into the forelimb armpits of nude mice to observe the tumorigenic ability.
RESULTS AND CONCLUSION: The number of holes of single cells obtained was 371 by the single cell separation technique, and the success rate was 96.4%. According to the growth of cell clone, tumorigenic colonies were selected to be transplanted to the forelimb armpits of nude mice, and the tumor formation rate of the colonies was 100%. The identification model of “single-cell-transplant-tumorigenic-test” for liver cancer stem cells is confirmed to be preliminarily established, which lays the technology and methodological basis for the follow-up research.

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: Hep G2 Cells, Neoplastic Stem Cells, Tissue Engineering

CLC Number:

R394.2

Jia Wei, Cen Yan-hui, Bao Juan, Yang Rui, He Guo-zhen, Wu Xiao-jun, Li Yi-yi. A preliminary establishment of the “single-cell-transplant-umorigenic-test”model for identifying liver cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1324-1328.

Figures/Tables 1

Culture and observation of HepG2 hepatoma cell line After recovery, the HepG2 cells were cultured in the 37 ℃, 5% CO2 incubator. We found that the HepG2 cells in the logarithmic growth phase showed adherent growth and strong proliferation ability, which were able to quickly aggregate and covered about 80% of the bottom. These cells exhibited a "cobblestone"-like appearance (Figure 1). Efficiency of single cell collection Number of single cells yielded per well were 371, with the 96.4% cell yield. Tumorigenicity verification Single cell cloning general observation Twenty-four hours after cell seeding, some cells were found to grow well and began to proliferate, and formed colonies (cell count > 100) 1 week later. As shown in Figure 2A, these single cell clones grew rapidly, developed into cells > 300 after 2 weeks. These cells were good, stereoscopic and full of translucent cytoplasm (Figure 2B). These 151 clones were classified as “suspected tumor cloning”, which would be further amplified to confirm whether they were tumorigenic clones. A few of clones with a flat shape grew slowly, and these clones were eliminated finally. The amplification and determination of tumorigenic colonies In order to select tumorigenic colonies from the four 96-well plates, 151 “suspected tumorigenic colonies” were moved from the 96-well plate to the 6-well plate. By this method, 147 tumorigenic colonies were verified, the success rate of which was 97.3%. Tumor cloning tumorigenicity verification Twenty sub-cell lines derived from tumor clones were randomly amplified to a sufficient amount of cells (about 1×106/well), and then were transplanted into the forelimb armpit of nude mice. Tumor formation rate was 100% after 2 months, and the tumors were irregular, outwardly projected, and lobulated, which were pink in color and had hard texture. Hematoxylin-eosin staining showed that the transplant tumor cells were distributed in a large and polygonal shape; the nuclei were large, some of which were dual nuclei; and there were a lot of blood capillaries between the cells (Figure 3)."

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A preliminary establishment of the “single-cell-transplant-umorigenic-test”model for identifying liver cancer stem cells

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