Trypsin versene edta

JPCs in culture flasks were trypsinized (Trypsin-Versene EDTA, Lonza, Basel, Switzerland) and pooled JPCs of these three donors were seeded in β-TCP constructs (Curasan AG, Kleinostheim, Germany). The constructs were preincubated in 96-well polypropylene culture plates containing DMEM/F-12 media for 1 h. A starting density of 1 × 10⁵ cells/scaffold was seeded into β-TCP constructs in 50 μL volume; the JPCs-seeded β-TCP scaffolds were incubated for 2 h at 37°C. After 2 h of incubation, additional 150 μL medium were subsequently pipetted to overlay JPCs-seeded β-TCP constructs.
For osteogenic differentiation of JPCs-seeded constructs before coculture experiments, the constructs were cultured in a 96-well plate under osteogenic (Ob) conditions (DMEM/F-12 complete medium containing 4 μM dexamethasone, 100 μM L-ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate, Sigma-Aldrich, Germany) for 7 days. JPCs-seeded β-TCP scaffolds cultured with Co (untreated) medium for the same time period served as undifferentiated controls. The medium was replaced every 2 days. The osteogenesis-relevant genes by JPCs cultured within β-TCP constructs and cocultured with DCs were quantified. Supplemental Figure S1 shows gene expression levels of ALP and RUNX2 after 14 days (7 days monoculture and 7 days coculture) of osteogenic differentiation.

Human osteosarcoma (HOS) and Normal Human Dermal Fibroblasts (NHDF) cell lines were purchased from CLS Cell Lines Service GmbH and PromoCell, Eagle’s minimal essential medium (MEM) from Lonza, fetal bovine serum (FBS) from Biochrom GmbH, Germany and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 µg in 100 ml) from Lonza, CellTiter 96® Aqueous One Solution Cell Proliferation Assay from Promega, Trypsin-Versene (EDTA) mixture from Lonza, phosphate buffered saline (PBS) from Invitrogen.

RPMI 1640 media and fetal bovine serum (S001-01) were purchased from Wellgene (Gyeongsan-si, Gyeongsangbukdo, Korea). Trypsin-Versene (EDTA) and antibiotics (penicillin–streptomycin) were from Lonza (Basel, Switzerland). Recombinant human EGF (PHG0311) was from Invitrogen (Carlsbad, CA, USA). Thiazolyl blue tetrazolium bromide for MTT assays was purchased from Sigma (St. Louis, MO, USA). Pro-prep lysis buffer for protein extraction was obtained from iNtRON Biotechnology (17081, Seongnam-si, Gyeonggi-do, Korea). Western blot reagents, including 30% acrylamide/bis-acrylamide solution, Tris–HCl (pH 6.8), Tris–HCl (pH 8.8), ammonium persulfate, and 10% sodium dodecyl sulfate (SDS), were from Bio-Rad (Hercules, CA, USA). Mouse anti-CD29 and cytosolic anti-integrin β1 antibodies were purchased from BD Biosciences (San Jose, CA, USA) and Abcam, (Cambridge, UK), respectively. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, D16H11, rabbit), anti β-actin (13E5, rabbit), and anti Rab25 (D4P6P, rabbit) antibodies were from Cell Signaling Technology (Danvers, MA, USA). All antibodies were diluted 1:1000, except for those against GAPDH and β-actin, which were used at 1:2000. Anti-mouse and anti-rabbit IgG horse radish peroxidase (HRP)-conjugated secondary antibodies were manufactured by Santa Cruz Biotechnology (Dallas, TX, USA).