Mc3t3 e1 osteoblastic cells

MC3T3-E1 osteoblastic cells were obtained from American Type Culture Collection (ATCC number CRL-2594) and maintained in high glucose Dulbecco’s modified eagle medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), antibiotic-antimycotic (100 units/ml of penicillin, 100 mg/ml of streptomycin, and 0.25 mg/ml of amphotericin B, Gibco, USA), and 2 mM alanyl-L-glutamine (Gibco, USA). Cells were incubated at 37 °C and 5% CO₂, and the culture medium was changed every 2 days. After washing the PDMS substrates with 70% alcohol once and PBS twice, the MC3T3-E1 cells were seeded at a density of 3 × 10⁴ cells/cm². The culture dish was placed in an incubator (37 °C, 5% CO₂) for 6 h to allow the complete attachment of MC3T3-E1 cells onto the designed platforms.

MC3T3-E1 osteoblastic cells were purchased from the American Type Culture Collection (USA) and cultured in α-MEM containing 10% FBS, 100 μg/ml streptomycin, and 100 U/ml penicillin on the polystyrene-coated dishes. Cell densities were maintained so as not to exceed 70-80% confluency. Briefly, MC3T3-E1 cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells/well and cultures for 24 h. And α-MEM-based medium was replaced with fresh differentiation medium containing 10% FBS, 1% penicillin-streptomycin, 10 mM β-glycerophosphate and 100 mg/ml ascorbic acid as osteogenic agents. And the cells were treated with ultrafiltrated-fucoidan fractions at different concentrations and incubated for an additional 7 days. To determine ALP activity using a colorimetric assay, cells were washed with 1x PBS and lysed by incubation in 100 μl of lysis buffer (0.5 M Tris, pH 9.0, 150 mM NaCl, 1% Triton X-100) for 1 h at 4°C. Cell lysates were then treated with 100 μl of 5 mM p-nitrophenyl phosphate solution for 30 min at 37°C and 50 μl of resulting supernatants were transferred to new 96-well plates. The reaction was stopped by adding an equal volume of 0.1 N NaOH and absorbances were measured at 405 nm using a SpectraMax M2e microplate reader (Molecular Devices). All data were expressed as percentages of non-treated group.

MC3T3-E1 osteoblastic cells were obtained from American Type Culture Collection (ATCC numbers CRL-2594) and maintained in the cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), at 37°C and 5% CO₂. The DMEM consisted of high glucose (Invitrogen, CA, U.S.A.) with 10% FBS (Gibco, MD, U.S.A.), antibiotic-antimycotic (100 units/ml of penicillin, 100 mg/ml of streptomycin, and 0.25 mg/ml of Amphotericin B, Gibco, MD, U.S.A.), and 2 mM alanyl-l-glutamine (Gibco, MD, U.S.A.). The cells were kept below full confluence at all times. The MC3T3-E1 cells were trypsinized (0.05% w/v trypsin in EDTA) and seeded at a density of 5 × 10³ cells/ml on the open micropost platforms. For seeding MC3T3-E1 cells in the confined micropost platform with a top cover, cells at a density of 1 × 10⁵ cells/ml in DMEM were delivered into the channel by a syringe. The culture dish was maintained at 37°C and 5% CO₂ for 6 h to allow the complete attachment of cells on to the PDMS platform. The DMEM was then replaced by a CO₂-independent medium (Invitrogen 18045-088, CA, U.S.A.), 10% FBS, antibiotic-antimycotic, and supplemented with 2 mM alanyl-l-glutamine (Gibco, MD, U.S.A.) for time-lapse imaging. The MC3T3 cell passage was controlled in the range of 3–20.