The influence of two different cultivations on human liver cancer cell line SMMC-7721 cells

Abstract

Objective To observe and compare thechange of human liver cancer cell line SMMC-7721 cells’ related characteristics using adherent cultivation and suspension cultivation. Methods (1) To collect human liver cancer cell line SMMC-7721 cells cultured with ordinary medium (adherent cultivation) and with serum-free medium (suspension cultivation). (2) To observe morphologic patterns of SMMC-7721 tumor cells treated with two kinds of cultivationunder inverted microscope. (3) To measure expressions of tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds of cultivation by the flow cytometry. (4) To detect mRNA expressions of the tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds cultivation by real-time quantitative polymerase chain reaction (Real-time PCR). (5) To observe changes of the tumor volume in nude mice after subcutaneous injection of SMMC-7721 tumor cells treated with two kinds of cultivation and to compare their tumorigenicity and multiplication capacity. Results Growing like typical cobblestone and arraying regularly for SMMC-7721 tumor cells in ordinary medium were observed under inverted microscope, while SMMC-7721 tumor cells in suspensive culture gathering from single cell into spherical or near spherical cells. The expression of CD90 and CD24 by the flow cytometryin suspension culture group was significantly higher than that in adherent culture group. The difference was statistically significant (CD90: P=0.000; CD24: P=0.000). But there was no significant difference between the expressions of CD133 in suspension culture group and adherent culture group. The Real-time PCR results showed that the mRNA expression of CD90 and CD24 in suspension culture group was significantly higher than that in adherent culture group. The difference was statistically significant (CD90: P=0.020, CD24: P=0.000). But there was no significant difference on expressions between suspension culture group and adherent culture group for the CD133. Statistical analysis of the tumorigenic ability and capacity to proliferate in vivo of tumor cells treated with two kinds of cultivation showed the suspension culture group’s capacity to proliferate in vivo was better than the adherent culture group. And tumorigenic ability in the suspension culture groups was higher than that in the adherent culture group. Conclusion For the human liver cancer cell line SMMC-7721 cells, hepatocarcinoma stem-like cells in suspension culture could be obtained more than that in adherent culture. Key words: Liver cancer stem-like cells; Suspension culture; SMMC-7721; CD90; CD133; CD24