Permeable membrane

A human monocyte cell line, THP-1, was placed on the upper layer of cell culture inserts with a permeable membrane (Corning, Glendale, AZ, USA) then incubated in culture media containing NSC14465 and a chemokine, mouse CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA), for 3 h. Two prostate cancer lines (DU145 and PC3) and a mouse pancreatic cancer line, 6606PDA, were also used in a transwell assay for 36 h. The number of migrated cells was counted by Image J software. For a wound-healing assay, TRAMP-C1, DU145, PC3, and 6606PDA cells were seeded into 12-well plates and incubated for 24–48 h. When cells were confluent on the surface of the plate, the assay was performed by using a 200 μL pipette tip to generate a scratch in each well. Cells were incubated in culture media containing different concentrations of NSC14465 for 12 h. Images were collected at the end of the experiment.

In order to determine the effects of different scaffolds on osteogenic differentiation of rBMSCs, we carried out the following experiment. For osteogenic induction, the culture medium was changed to the osteogenic-defined medium. Furthermore, rBMSCs cultured in a non-osteogenic medium (non-OM group) served as a control. The cells were seeded on the bottom of 24-well plates and cultured in the medium. Each scaffold was placed in each transwell insert on the permeable membrane (Corning, United States); thus, BMP-2 and VEGF peptides were released from the scaffold, filtered through the membrane, and allowed to contact the cells (Figure 2B).
Alkaline phosphatase (ALP) staining was used to evaluate the early osteogenesis capacities of rBMSCs in different groups. For ALP staining, rBMSCs were washed with PBS and fixed with 4% paraformaldehyde for 10 min on day 7 after osteoinduction. Next, the cells were incubated with BCIP/NBT ALP Color Development Substrate (Beyotime, China) for 30 min, following the manufacturer’s instruction. Then, each well was imaged after additional washing. Afterward, the ALP activity was assessed by an ALP assay kit (Nanjing Jiancheng, China) after breaking up the cells by ultrasound. The optical density (OD) values at 520 nm were measured using a microplate reader (Infinite M200; Tecan, Switzerland).

The migration assay was performed using a 24-well 8.0 μm polycarbonate transwell chamber consisting of a permeable membrane (Corning Inc., Corning, NY, USA). First, hCPCs were pretreated to 100 nM of DOX with 0.1 and 1 mM of GSH. For the assay, 500 μL of Ham’s F-12 media culture medium was added below the cell permeable membrane whereas 7000 cells/100 μL in serum-free Ham’s F-12 medium were plated on the upper chamber of the permeable membrane. After 24 h of incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet at room temperature. The upper chamber was washed and the top of the membrane was examined for cell migration. After mounting, the cells were observed under an inverted microscope and the number of cells was counted.