Stemfit ak02n medium

This study was approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (approval number #R0091) and was conducted according to the guidelines of the Declaration of Helsinki. Informed consent was obtained from both the patients and their guardians.
PC12 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% heat-inactivated fetal calf serum supplemented with 50 μg/ml penicillin–streptomycin at 37°C and 5% CO₂ conditions. For the observation of neurites, 50 ng/ml nerve growth factor (Nacalai Tesque, Kyoto, Japan; 24246-02) was added to the medium 48 h prior to the evaluation. Skin biopsy specimens were obtained from the P563L patient. The fibroblasts were expanded in DMEM containing 10% heat-inactivated fetal bovine serum and 50 μg/ml penicillin and streptomycin. iPS cells were generated as described previously (51 (link)). The control iPS cell line was kindly provided by Dr Takayuki Tanaka (Kyoto University). The pluripotency of control iPSC line was evaluated as previously described (52 (link)). Each iPSC line was cultured in Stem Fit AK02N medium (Takara Bio, Shiga, Japan; AJ100) on Matrigel (BD Biosciences, San Diego, CA, USA; 354230)-coated plate at 37°C and 5% CO₂ conditions.

We purchased induced pluripotent human stem cells (409B2 human iPS cell line) from RIKEN BioResource Research Center (Ibaraki, Japan). 409B2 cells were maintained by single-cell passaging in 60 mm plates (Corning, NY, USA) coated with Easy iMatrix-511 (Takara Bio, Shiga, Japan). For passaging, 409B2 cells were harvested with EDTA/PBS (−) (Nacarai, Kyoto, Japan) diluted to 0.5 mM, and then replated at a density of 4000 cells/cm². 409B2 cells were maintained for 22–24 h in StemFit AK02N medium (Takara Bio), which contained Y-27632 (Wako, Osaka, Japan), a Rock inhibitor. Next, the medium was changed to StemFit AK02N without Y-27632, and replenished every 2 days. The cells were passaged again on the 7th day. Then, the cells were divided into control and exposure groups. The control group was maintained in a CO₂ gas incubator maintained at 37 °C. The ClO₂ gas exposure group was maintained in a CO₂ gas incubator continuously filled with ClO₂ gas. We tested two different ClO₂ concentrations: 0.05 ppmv and 0.1 ppmv.