Effects of perfusion cell culture method on human osteoblastic cell

Objectives: To better simulate the dental pulp system, perfusion cell culture system was introduced. The purpose of this study is to evaluate the proliferation and differentiation of human osteoblastic MG-63 cells in perfusion cell culture system.

Methods: MG-63 cells were seeded(10,000 cell/cm²) on 12 mm-diameter cover glasses. Cell culture medium were DMEM supplemented with 10% FBS, b-glycerophosphate, dexamethasone, and L-ascorbic acid. The cell seeded-cover glasses were maintained at 37 ℃ in a humidified incubator with 5% CO₂ during 24 hours for cell attachment. Static cell culture system(control group) was the same condition with the cell attachment method. Perfusion cell culture was performed in commercially available cell culture perfusion chamber(Minucells & Minutissue GmbH). Chambers were perfused with 1 mL/hr of medium(control medium plus 25 mM HEPES buffer for maintaining pH between 7.2 and 7.4). Cell proliferation was determined by measuring routine MTT assay after 48h and 72h. And cell differentiation was studied by measuring alkaline phosphatase(ALPase) activity after 3day, 5day with QuantiChrom^TM Alkaline Phosphatase Assay Kit(BioAssay Systems). Reliability was assessed with paired t-tests.

Results:

Table 1. MTT, ALPase activity data

	MTT		ALP
	48 hr	72hr	3day	5day
Perfusion	0.163±0.007	0.268±0.033	9.526±1.316	14.536±2.065
Static	0.160±0.006	0.249±0.022	8.710±4.069	13.619±1.217

Conclusion: The proliferation and differentiation of human osteoblastic MG-63 cells was not influenced by the method(perfusion or static)of cell culture.

Acknowledgement: This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R13-2003-013-02001-0).