Triiodothyronine

KCM was prepared according to previously described methods [10] (link). Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher, MA, USA) and DMEM with Ham's F-12 medium (Gibco) were mixed at a 1:1 ratio. The medium was supplemented with 5% autologous human serum, 0.3 μM saxizon (Takeda yakuhin kogyo, Osaka, Japan), 140.0 mU/mL humulin (Novo Nordisk, Bagsvaerd, Denmark), 2.0 nM triiodothyronine (MP Biomedicals, CA, USA), 0.2 μM epidermal growth factor (Higeta-Shoyu, Tokyo, Japan), 1.0 nM cholera toxin (Wako Pure Chemicals, Osaka, Japan), 100 U/mL penicillin, 69 μM streptomycin (Wako Pure Chemicals), and 0.4 μg/mL of amphotericin B (Bristol-Myers Squibb, NY, USA).

We used the keratinocyte culture medium (KCM) for cell culture and manufacture of cell sheets. KCM comprised a basal mixture of three parts Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, MA, USA) and one part Ham's F-12 nutrient mixture (Gibco), containing 5% autologous human serum of each patient, insulin (140.0 mU/mL, Humulin, Novo Nordisk, Bagsvaerd, Denmark), triiodothyronine (2.0 nM, MP Biomedicals, CA, USA), saxizon (0.3 μM, Takeda Yakuhin Kogyo, Osaka, Japan), epidermal growth factor (0.2 μM, Higeta-Shoyu, Tokyo, Japan), cholera toxin (1.0 nM, Wako Pure Chemicals, Osaka, Japan), penicillin (100 U/mL, Wako Pure Chemicals), streptomycin (69 μM, Wako Pure Chemicals), and amphotericin B (0.4 μg/mL, Bristol-Myers Squibb, NY, USA).