Mcdb 201 medium

Primary lung stem cell (LSC) culture was performed as previously described [14 (link)]. LSCs were isolated from neonatal CD-1 mice by FACS sorting using phycoerythrin- (PE-) conjugated anti-CD157 (BioLegend, CA, USA), fluorescein isothiocyanate- (FITC-) conjugated anti-CD54 (BD Biosciences, CA, USA), and allophycocyanin- (APC-) conjugated anti-CD45 (eBioscience, CA, USA) antibodies. Isolated CD45⁻CD54⁺CD157⁺ cells or irradiated cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 1% insulin–transferrin–selenium (ITS), and 1 ng/ml epidermal growth factors (EGF) (all obtained from Thermo Fisher Scientific, CA, USA) through several passages in a collagen I-coated plate. To conduct differentiation studies, the attached LSCs were incubated in MCDB-201 medium (Sigma-Aldrich, MO, USA) supplemented with 1% FBS, 1% ITS, and 10 ng/ml EGF for 7 or 14 days to induce AECII or AECI cells.
To determine the fibrogenic effect of transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) (PeproTech, NJ, USA), LSCs (2.5 × 10⁴ cells/cm²) in 12-well culture plates were treated with TGF-β (5 ng/ml) or CTGF (50 ng/ml) for 3 days.

The human prostate cancer cell line PC3 was obtained from American Type Culture Collection and maintained in 10% fetal bovine serum (FBS, Fisher) in Roswell Park Memorial Institute medium 1640 (RPMI, Corning). ASCs from two male Caucasian donors, generously provided by J. Gimble (Tulane University), were collected as previously described (Zolochevska et al. 2014 (link)) and cultured on fibronectin-coated plasticware in modified media conditions that have been described previously (Ruiz et al. 2010 (link)). ASCs from Donor 1 were isolated from midsection liposuction, and Donor 2 ASCs were isolated from liposuction samples from breast tissue. Briefly, “ASC media” was prepared to a final concentration of 60% Dulbecco’s Modified Eagle Medium (DMEM, Corning), 40% MCDB-201 medium (Sigma), 5% FBS, 1× insulin–transferrin–selenium (Corning), 1 nM dexamethasone (Acros Organics), 10 ng/mL epidermal growth factor (eBioscience), 0.1 μM ascorbic acid (Acros Organics), and 1× antibiotic–antimycotic (Gibco). For iAs exposure, culture medium with 0, 1, 10, or 75 ppb iAs (sodium arsenite, RICCA) was used. All ASC experiments used cells with a passage number < 10.

Human umbilical cord mesenchymal stem cells were acquired from the Bioresource Collection and Research Center, Hsinchu, Taiwan. Cells were cultured in medium which contain 56% Low-glucose Dulbecco's Modified Eagle Medium (DMEM-LG; Life technology, NY, USA), 37% MCDB201 Medium (Sigma, MO, USA), 2% fetal bovine serum (Thermo, Logan, UT), 0.5 mg/mL of bovine serum albumin fraction V (Sigma, MO, USA), 10 ng/mL epidermal growth factor (PeproTech, NJ, USA), 1 ng/mL of platelet-derived growth factor-BB (PeproTech, NJ, USA), 50 μM l-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma, MO, USA), 10 nM Dexamethasone (Sigma, MO, USA), 1 × antibiotic–antimycotic solution (Corning, NY, USA), 1 × Insulin-Transferrin-Selenium-A (Life technology, NY, USA), and sterilized secondary water to total volume. The cells were incubated at 37 °C and 5% CO₂. Cells were separated by Accutase ® (Innovative Cell Technologies, SD, USA) when cells reached confluency of 80% density and reseeded 8 × 10⁵ cells per 100 mm cell culture dish.

Human mesenchymal stem cells (hMSCs) were purchased from the Bioresource Collection and Research Center (BCRC, No. RM60596), Hsinchu, Taiwan. ATCC (PCS-500-010). Cells were grown in culture dishes containing 56% Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) and 37% MCDB-201 medium (Sigma Aldrich, St Louis, MO, USA), supplemented with 2% fetal bovine serum (Thermo, Logan, UT, USA), 1× Insulin-Transferrin-Selenium-A (Invitrogen, Carlsbad, CA, USA), 0.5 mg/mL of AlbuMax I (Invitrogen, Carlsbad, CA, USA), 10 ng/mL of epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 1 ng/mL of platelet-derived growth factor (PeproTech, Rocky Hill, NJ, USA), 10 nM dexamethasone (Sigma Aldrich, St Louis, MO, USA), and 50 μM L-ascorbic acid-2-phosphate (Sigma Aldrich, St Louis, MO, USA) [29 (link)]. The cells were maintained at 37 °C in a 5% CO₂ incubator. When cells reached 70–80% confluence, they were detached with HyQtase (Thermo-Fisher Scientific, Waltham, MA, USA), centrifuged, and resuspended for subculture or further experiments. Cells between Passages 6 and 10 were used in the experiments. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma Aldrich, St Louis, MO, USA) was dissolved in DMSO as a stock solution at 10 mM, further diluted in hMSC cultured medium, and then used at various concentrations of 0.1, 0.3, 1, 2, 3, 10, and 30 μM.

The sphere-forming assay was conducted as previously described 18 (link) as follows: cells (800 cells/well) were seeded in 24-well ultralow plates (Corning; 3473) in MCDB201 medium (Sigma; M6770) (pH=7.1) supplemented with EGF (Sigma; E9644; 20 ng/mL), bFGF (PeproTech; 100-18b; 20 ng/mL), NaHCO₃ (Sigma; S5761; 1.2 g/L) and L-glutamine (2 mM); growth factors were added every 2 days, and the spheres were observed after 1-4 weeks.

Total 308000 MSCs and 308000 SaOS-2 cells were used. Adhesion assays were carried out on days 3 and 7. Control groups were MSCs and Saos type 2 cells cultured in the appropriate medium without samples. Tests were carried out in triplicate for each material and cell group. The cells were cultured with MSC or SaOS-2 medium, according to cell type. MSC culture medium consisted of 52.8% Dulbecco’s Modified Eagle Medium (DMEM) with 1 g/L glucose (Lonza, Basel, Switzerland), 35.2% MCDB-201 medium (Sigma Aldrich, St. Louis, MO, USA), 10% heat inactivated fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin solution (Biochrom AG, Berlin, Germany), and 1% l-glutamine (Biochrom AG, Berlin, Germany), while SaOS-2 culture medium consisted of 89% DMEM with 4.5 g/L glucose (Sigma Aldrich), 10% heat inactivated FBS, and 1% penicillin/streptomycin solution. Every three to four days, the medium was replaced. The plates were incubated at 37 °C with relative humidity under an atmosphere of 5% CO₂. On the third and seventh days, the wells were washed with PBS (Oxoid, Hampshire, United Kingdom) and 1 mL Trypsin/EDTA (Thermo Fisher, Waltham, MA, USA) was applied. A 50 µL cell suspension was mixed with 50 µL Trypan blue (Biological Industries, Beit HaEmek, Israel) in a centrifuge tube and the cells were counted on Thoma lamina.

Each of the cross-linked scaffolds was sterilized with 70% v/v ethanol for 30 min and 40-W ultraviolet light for 20 min. Each scaffold was then washed with DW and 1× PBS (pH 7.4) and subsequently incubated in 1× PBS (pH 7.4) for 1 day. Before cell seeding, the scaffolds were rinsed with conditioned medium consisting of 60% DMEM-LG (GibcoBRL), 40% MCDB-201 medium (Sigma-Aldrich, St. Louis, MO, USA), 10⁻⁴ M ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), and 1% antibiotic/antimycotic solution (GibcoBRL) with 10% fetal bovine serum (FBS, WelGENE, Daegu, Korea). For optical confirmation, ADSCs were stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA), seeded on scaffolds in 24-well culture plates at a final concentration of 1.0 × 10⁵ cells, and allowed to attach for 1 day. Then, the ADSC-seeded scaffolds were transferred to another 24-well culture plate.