Low glucose dulbecco s modified eagle medium

All the studies using nasal septal chondrocytes (NSCs) were conducted after written approval (HC13TISI0038) obtained from the Institutional Review Board of the Catholic Medical Center Clinical Research Coordinating Center. Human nasal septum tissue was harvested from five patients who were undergoing septoplasty [21 (link)]. Those tissues were cut into 1 mm³ pieces and they were enzymatically digested using 0.2 % protease solution (Gibco) for 60 min, followed by incubation in 0.3 % collagenase (Sigma-Aldrich) for 12 h at 37 °C. The isolated cells were then seeded in a 75-mm² cell culture flask (NUNC) and cultivated in low-glucose Dulbecco’s Modified Eagle Medium (Gibco-BRL) with 10 % fetal bovine serum (FBS; Gibco) and antibiotics at 37 °C in 5 % CO₂ incubator. The confluent chondrocytes were subcultured following a standard protocol using 0.05 % trypsin/EDTA solution (Gibco).

Bovine mesenchymal stem cells (bMSCs) were isolated, cultured, and characterized based on generally accepted criteria [69 (link),70 (link)] as in [71 (link)]. The bMSC-SV40-hTERT cell line immortalization was achieved by introducing simian virus 40 large T antigen (SV40T, a kind gift from Prof. Sara Selig) by transient transfection, and the human telomerase gene hTERT [72 ] by retrovirus-mediated transduction. The cells were grown in low-glucose Dulbecco’s modified Eagle medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and a penicillin–streptomycin mixture (3%) and cryopreserved in fetal bovine serum containing 10% DMSO. GFP-expressing cells were acquired using a pLKO_047 (Broad Institute) lentiviral vector and puromycin selection. Cell counts were determined with a cell counter (TC20 automated cell counter, (Bio Rad, Hercules, CA, USA).

Human bone marrow-derived MSCs (passage 2) were acquired from PromoCell (Germany) and RIKEN BRC (Japan). In this study, we utilized several lines obtained from 5 different donors (PromoCell: C-12974; RIKEN RBC: MSC-R37, MSC-R43, MSC-R44, and MSC-R50.). The cells were maintained in the basal medium which is low glucose-Dulbecco’s Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotic–antimycotic (Gibco, USA) solution in a humidified incubator at 37℃ under 5% CO₂ condition. We carried out the cell passaging every 2–3 days when the cell confluency became 80–90%. For experiments, the cells from passage 4 up to 12 were used. To prepare an osteogenic induction (OI) medium, we utilized high glucose-Dulbecco’s Modified Eagle Medium (Gibco, USA) containing 50 μM ascorbic acid (Wako, Japan), 10 mM β-glycerophosphate (Sigma, USA), and 100 nM dexamethasone (Nacalai Tesque, Japan). To prepare for 2D monolayer samples, 200,000 cells were subcultured on a 35 mm diameter culture dish to become confluent after 2-day incubation. This study was approved by the Ethics Committee of Institute for Frontier Life and Medical Sciences, Kyoto University (Clinical Protocol No. 89).

Curcumin (purity > 99%,), Tween-80, span 80, cholesterol and PEG 6000 (PEG6000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (≥99.5%) which was used as a solvent and dimethyl sulfoxide (DMSO) for cell culture were obtained from Merck (Shuchardt, Germany). Cell culture materials including low glucose Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, penicillin, streptomycin, phosphate buffer solution (PBS) and MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) were bought from Gibco (AG, basel, Switzerland). Also, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was provided from Sigma-Aldrich (St. Louis, MO, USA). A dialysis bag for release measurements with the molecular mass cut-off between 12 kDa and 14 kDa, was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Human promyelocytic leukemia cell line HL-60 (a kind gift from Dr. Abroun, Tarbiat Modares University, Tehran, Iran) was cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma, St. Louis, MO, USA). The BM-MSCs (Stem Cell Technology, Tehran, Iran) were cultured in low-glucose Dulbecco’s modified Eagle medium (GIBCO BRL, Gaithersburg MD, USA) containing 10% fetal bovine serum.