L ascorbic acid phosphate magnesium salt n hydrate

Dex (Cat: D-4902), recombinant human Ins (Cat: 093-06351), fatty acid-free bovine serum albumin (Cat: 011-07493), streptomycin sulfate (Cat: S6501), penicillin G potassium salt (Cat: PENK-10MU), Dulbecco’s modified Eagle medium (DMEM-HEPES) (Cat: D1152-10L), and Oil red O (Cat: 00625-25G) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Wako Pure Chemical Industries Ltd. (Osaka, Japan) supplied IBMX (Cat: 095-03413) and L-ascorbic acid phosphate magnesium salt n-hydrate (Cat: 013-12061). MP Biomedicals (Solon, OH, USA) supplied the fetal bovine serum (FBS) (Cat: 2910154). Arachidonic acid (AA) (Cat: 506-32-1), Δ¹²-PGJ₂ (Cat: 87893-54-7), H89 (Cat: 130964-39-5), PA (Cat: 57-10-3), SA (Cat: 57-11-4), OA (Cat: 112-80-1), ALA (Cat: 463-40-1), EPA (Cat: 10417-94-4), and forskolin (Cat: 66575-29-9) were acquired from Cayman Chemical (Ann Arbor, MI, USA). Dibutyryl-cAMP (Cat: 16980-89-5) was sourced from Santa Cruz Biotechnology (Dallas, TX, USA). M-MLV reverse transcriptase without ribonuclease H activity (Cat: M3682) was supplied by Promega (Madison, WI, USA). Sigma Genosys Japan (Ishikari, Japan) provided oligonucleotides for the real-time quantitative (RT-q) PCR amplification.

DPSCs were obtained from a patient with CS and healthy donors with approval by the Committee of Ethics, Nippon Dental University School of Life Dentistry, Tokyo. Informed consent was obtained from the patient. The dental pulp tissue was enzymatically digested as described in a previous study (Matsui et al., 2018). The cells were cultured in serum‐based minimum essential medium alpha (MEMα; Gibco/Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (FBS; SAFC Biosciences; Gibco/Thermo Fisher Scientific), 100 µM l‐ascorbic acid phosphate magnesium salt n‐hydrate (Wako Pure Chemical Industries), 2 mM l‐glutamine (Gibco/Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco/Thermo Fisher Scientific) at 37°C with 5% CO₂ until Passage 1. Subsequently, the cells were cultured in STEMPRO® MSC SFM (Gibco/Thermo Fisher Scientific), a serum‐free medium for mesenchymal stem cell culture. The medium was changed every 2 days. At confluency, they were subcultured at a split ratio of 1:2 by gentle separation with TrypLE™ Express solution (Gibco/Thermo Fisher Scientific) at room temperature. To analyze the response to PMA, the cells were cultured in 60‐mm dishes (Corning), treated with 2.5 nM PMA (LC Laboratories), and harvested in centrifuge tubes. Cells cultured without PMA served as controls.

Dulbecco’s modified Eagle medium containing 25 mM HEPES (DMEM-HEPES), penicillin G potassium salt, streptomycin sulfate, dexamethasone, fatty acid-free bovine serum albumin, and recombinant human insulin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). L-Ascorbic acid phosphate magnesium salt n-hydrate, and 3-isobutyl-1-methylxanthine (IBMX) were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Solon, OH, USA). AA, LA, PGE₂, PGF_2α, Δ¹²-PGJ₂, and 6-keto-PGF_1α were from Cayman Chemical (Ann Arbor, MI, USA). We purchased M-MLV reverse transcriptase (point mutation without Ribonuclease H activity) from Promega (Madison, WI, USA). Oligonucleotides for real-time quantitative (RT-q) PCR amplification were provided by Sigma Genosys Japan (Ishikari, Japan).

FAD medium (Biochrom GmbH, Berlin, Germany) consisted of Dulbecco’s modified Eagle’s medium (DMEM)/HAM’s F12 (3.5:1.1), 50 μM calcium chloride (CaCl₂) and 4.5 g/L -glucose and was supplemented with 2.5% Chelex 100-treated fetal bovine serum (FBS), 0.18 mM adenine (Sigma-Aldrich), 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 5 μg/ml insulin (Life Technologies, Carlsbad, CA, USA), 10^–10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Sigma-Aldrich), 2 mM l-glutamine (Nacalai Tesque, Kyoto, Japan) and 1 mM sodium pyruvate (Wako, Osaka, Japan). Hereafter, this culture is referred to as the ‘complete FAD (c-FAD)’ medium. Serum calcium was removed by treating 500 ml FBS (HyClone, South Logan, UT, USA) with 20 g of Chelex 100 (Bio-Rad Laboratories, Hercules, CA, USA) [32 (link)]. For 3D cell cultures [33 (link)], the cells were cultured in the c-FAD medium supplemented with 1.2 mM CaCl₂ (Nacalai Tesque), 10 ng/ml human keratinocyte growth factor (PeproTech, Rocky Hill, NJ, USA) and 0.283 mM l-ascorbic acid phosphate magnesium salt n-hydrate (Wako, Osaka, Japan), a stable derivative of ascorbic acid. Hereafter, this is referred to as the ‘FAD-3D medium’.

Freshly harvested cBMSC sheets were prepared by reviving cryopreserved cells from a P8 working cell bank in a 37 °C water bath more than 20 min, collecting the cells in a pre-warmed cell culture medium, and centrifuging at 210×g for 5 min. P8 cBMSCs were then seeded onto conventional cell culture flasks (CELLTREAT, 229351) and passaged twice prior to seeding harvested cells at P10 onto 35-mm temperature-responsive culture dishes (TRCDs, Thermo Fisher Scientific, 03150025) (Fig. 1). Freeze-thawed cBMSC sheets were prepared by reviving cryopreserved cells from the P10 working cell bank and seeding them directly onto a 35-mm TRCD without prior cultivation (Fig. 1). Both freshly harvested and freeze-thawed cBMSC sheets were prepared on TRCDs at seeding densities of 4 × 10⁵ and 1 × 10⁶ cells/dish and cultured for 24 h at 37 °C, 5% CO₂ incubator in cell culture medium comprising DMEM (Thermo Fisher Scientific, 11885076) supplemented with 10% FBS (Thermo Fisher Scientific, 16000044), 0.05% MycoZap Prophylactic (Lonza, VZA-2023), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140163), and 50 µg/ml of l-ascorbic acid phosphate magnesium salt n-hydrate (Fujifilm Wako Pure Chemical, 013-19641).

ZF4 cells were purchased from ATCC (CRL-2050). The cells were cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 (DMEM/Ham’s F12, Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 28 °C in an atmosphere of 5% CO₂.
For all experiments, the cells were maintained at 18, 23, 28, or 33 °C for one day for acclimation after they became confluent. The medium was replaced with DMEM/Ham’s F12 containing 200 µM L-ascorbic acid phosphate magnesium salt n-hydrate (Wako Pure Chemical Industries, Osaka, Japan), 2% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained at 18, 23, 28, or 33 °C in a 5% CO₂ atmosphere for 3 days and used for the experiments.

Osteoblasts were obtained by differentiating KUSA-A1 cells, a mouse mesenchymal stem cell line derived from the C3H/He mouse strain (Riken Cell Bank, Tsukuba, Japan). KUSA-A1 cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂ in culture medium. The culture medium was alpha minimum essential medium (α-MEM; CM1401-012, Nikken Bio, Kuze, Japan) supplemented with 10% fetal calf serum (FCS; FBS01-500, Biofill Australia Pty. Ltd., Elsternwick, Australia) and 60 mg/L kanamycin sulfate (119-00703, Wako, Osaka, Japan). These cells were plated at about 2,500 cells/cm² on a 100 mm TC-treated culture dish (430167, Corning Life Sciences, Tewksbury, USA). For subculturing, the cultured cells were recovered from the substrate 3 days after the last plating and replated at the same density.
To differentiate the KUSA-A1 cells into osteoblasts, osteogenic differentiation medium, α-MEM supplemented with 10% FCS, 60 mg/L kanamycin, 10 mM β-glycerophosphate disodium salt hydrate (G9422, Sigma-Aldrich, St. Louis, USA), and 50 μg/mL L-ascorbic acid phosphate magnesium salt n-hydrate (013-12061, Wako) was used as the culture medium. The differentiation medium was replaced every 3 days.
In this research, cultured KUSA-A1 cells, a mouse mesenchymal stem cell line, were used and we didn’t use any live vertebrates and/or higher invertebrates.