Objective  The aim is to sort CD90⁺ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting (MACS) on sorting the liver cancer stem cells. Methods  ①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group (5.5×10⁵ cells per hole, 1 hole), cells of the experimental group were added with 5 μL CD90^–PE while cells of the blank control group were treated with 5 μL CD90^–PE non fluorescent antibody. Determined the proportion of CD90⁺ cells in the 2 groups by flow cytometry (FCM). ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting (MS) was collected as CD90^– group, and 1 mL PBS after MS wash was collected as CD90⁺ group, as well as blank control group and experimental group. Determined the proportion of CD90⁺ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group (1 hole), and proportions of CD90⁺ cells were determined by FCM at 1 week after culture. Results  ①The positive rate of CD90 was 0 (0/8), 65.5% (38/58), and 20.7% (12/58) in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues (χ²=6.78, P<0.05) and adjacent liver cancer tissues (χ²=20.83, P<0.05). ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90⁺ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90⁺ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90⁺ cells in the blank control group, experimental group, CD90^– group, and CD90⁺ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90⁺ cells in the blank control group, experimental group, CD90^– group, and CD90⁺ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively. Conclusions  The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90⁺ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90⁺ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90⁺ cells.

Citation： CHEN Kefei, LIU Jun, LI Bo. Isolation and enrichment of liver cancer stem cells by magnetic cell sorting and serum-free suspension culture. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2017, 24(8): 946-952. doi: 10.7507/1007-9424.201611088 Copy