Objective To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells. Methods The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry. Results After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs. Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.
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.