Matrigel을 이용한 세포주의 기질장벽 침습도 측정에 관한 연구
In Vitro Matrigel Invasion Assay of Cell Lines
김성, 박재갑, 황이숙, 힌다 클라인만, 김진복,
소속 상세정보
김성 ( Kim Song )
한림대학교 의과대학 외과학교실
박재갑 ( Park Jae-Gahb )
서울대학교 의과대학 외과학교실
황이숙 ( Hwang Ee-Sook )
서울대학교 의과대학 외과학교실
힌다 클라인만 ( Hynda K. Kleinman )
National Institute of Health National Institute of Dental Research Laboratory of Developmental Biology and Anomalies
김진복 ( Kim Jin-Pok )
서울대학교 의과대학 외과학교실
Abstract
Considerable progress in tbe study of tumor cell invasion has been made by utilizing in vitro assay systems. Recently, the reconstituted basement membrane systems in blind well chemotaxis chambers have enabled quantitative analyses of the potentiality of the tumor cell invasion. This study was designed to establish the optimal conditions for in vitro invasion assay using matrigel as a basement membrane barrier in blind well chemotaxis chamber and to evaluate the in vitro invasiveness of the established tumor cel1 lines which have been known to be highly invasive in vivo. Two control cell lines(HT-1080, 3T3-Swiss albino) and twenty established cell lines were studied in this investigation. Polyvinylpyrrolidone-free-polycarbonate filters with varying sizes of pores(8pm, 10pm, 12pm) were coated with varying amounts of matrigel(330ug/ml, 500ug/ml, 1,000 ug/ml) and dried under sterile hood for 2 hours. 3T3-Swiss albino-conditioned medium was placed in the lower chamber and each matrigel-coated filter was placed in the blind well chemotaxis chamber. Two hundred thousand cells in the DF-10 media containing 0.1% BSA were added to the upper chamber and assays were carried at 37C in 5% CO,-95g room air for varying durations(2hrs, 4hrs, 6hrs, Shrs). After each incubation period, the number of cells on the lower surface of the matrigel-coated filter were counted as well as the number of cells in the lower chamber. The optimal conditians for in vitro invasion assay were 500 ug/ml of matrigel, 10 pm of filter pore, 4 hrs to 6 hrs of incubation period. No difference was observed in the invasiveness of the cell lines according to the conditioned media of varying cell lines. SUN-Cl and U2OS cell lines had larger number of cells on the matrigel-coated filter and in the lower chamber, but SNV-C4, NCI-H630 cell lines had fewer number af those. Some other cell lines exhibited a few number of matrix barrier- attached cells and larger number of lower chamber invading cells simultaneously or vice versa. This data could provide the possibility of various differences which exists in the tumor cell responsiveness to the conditioned media, the tumor cell attachment to the matrigel-coated filter, and the degree of tumor cell invasiveness. Therefore when comparing the invasiveness of the cell lines using these in vitro matrigel assay systems, it is necessary that the invasiveness to the lower chamber must be cansidered as significant as a matrix barrier-attached invasiveness.
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