Alginate three-dimensional culture of SW1353 human chondrosarcoma cells: Kinetics characteristics and ultramicro location of type Ⅱ collagen / 中国组织工程研究

ABSTRACT

BACKGROUND: Dedifferentiation changes in monolayer culture and limited resource are 2 problems in chondrocyte study. There are few reports concerning the phenotype, biological characteristics, and three-dimensional culture SW1353 human chondrosarcoma cells.OBJECTIVE: To observe the kinetics characteristics and ultramicro location of type Ⅱ collagen in three-dimensional cultured SW1353 human chondrosarcoma cells.DESIGN, TIME AND SETTING: The material-cytology in vitro observation was performed at the Xiangya School of Medicine, Central South University, from August 2004 to January 2006.MATERIALS: SW1353 human chondrosarcoma cells were provided by ATCC Company.METHODS: After resuscitation, the cells were cultured in DMEM containing fetal bovine serum and vitamin C. Then the cell aggregate was suspended in alginate solution, with final concentration of 2×10~9/L, gel solution was dropped to obtain alginate beads. At weeks 8 after three-dimensional culture, citrate sodium was added to breakdown gel, and the chondrocyte was harvested. The growth characteristics of SW1353 between in monolayer and in alginate three-dimensional were compared. MAIN OUTCOME MEASURES: Phenotype shift was observed by Toluidine blue staining. Type Ⅱ collagen expression in cell was directed by immunohistochemistry. The location of type Ⅱ collagen in ultrastructure was observed by Immunoelectron microscope. Isotope 14C-proline pulse chase experiment was performed to find out the kinetics of collagen Ⅱ in synthesis and secretion. RESULTS: There were phenotype changes after 8 weeks monolayer cultured in SW1353 as the articular chondrocytes had done. Compare with the monolayer culture, the good phenotype was maintained and the proliferation was slow when the cell was cultured in alginate culture and the type Ⅱ collagen in suspension and alginate matrix was decreased either. Type Ⅱ collagen expression in cell can be directed both in cells and outside of cells; type Ⅱ collagen locate almost in rough endoplasmic reticulum in Immunoelectron microscope; Pulse chase experiment found that the 180 minute was the peak of synthesis and secretion of collagen Ⅱ after intervention by ~(14)C-proline. ELISA confirmed that the expression of type Ⅱ collagen was obvious increased at 8 weeks than 1 week in the suspension of monolayer culture (P < 0.05), which was greater than that in alginate matrix of three-dimensional. CONCLUSION: Long term monolayer culture in SW1353 may lead to cell phenotype change. Alginate three-dimensional can improve the phenotype changes by decrease cells metabolism and maintaining the dormancy situation of SW1353. Expression of type n collagen in SW1353 was abundant. This cell line can be a good research model of chondrocyte function.