Nutrient mixture f 12 dmem f12

This study was approved by the Research Ethics Committee of the University of Tokyo Hospital. Auricular cartilages were provided as excised remnant auricular cartilage tissue from the surgery of microtia patients in NAGATA Microtia and Reconstructive Plastic Surgery Clinic. We obtained informed consent from all patients. After the excision of soft tissues and perichondria by scalpel and scissors, auricular cartilage was minced, and digested by shaking with 0.3% collagenase solution for 18 h at 37 °C. The solution was filtered with a cell strainer (100 μm pore size, BD Falcon), centrifuged at 1500 rpm for 5 min and the supernatant was removed to obtain human auricular chondrocytes. Cells were seeded at 2.0 × 10⁵ cells/dish to φ100 mm collagen Type I Coated dich (AGC Techno Glass Co., Ltd.), and cultured in the cartilage growth medium (HFI; Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Sigma–Aldrich Co.) supplemented with 5% Human Serum (Sigma–Aldrich Co.), 100 ng/mL FGF-2 (Kaken Pharmaceutical Co, Ltd.), 5 μg/mL Insulin (Novo Nordisk Pharma Ltd.), 1% Penicillin/Streptomycin (Sigma–Aldrich Co.)) at 37°C in 5% CO₂. After 10 days, cells reached the confluence, and they were detached by Trypsin-EDTA (Sigma–Aldrich Co.), recovered by centrifugation, and stored frozen at −80°C in CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.).

All procedures were approved by the Research Ethics Committee of The University of Tokyo Hospital (ethical permission number 622). ASCs were isolated from 6- or 7-week-old EGFP transgenic mice (C57BL/6-Tg(CAG-EGFP)C14–Y01-FM131Osb; RIKEN BioResource Center, Japan). Adipose tissue was harvested from the inguinal fat pads, minced, and digested by shaking with 0.1% collagenase solution for 30 min at 37 °C. The solution was filtered through a cell strainer (70-μm pore size; BD Falcon, Japan) and centrifuged at 250G for 5 min, and the supernatant was removed to obtain the stromal vascular fraction. Cells were seeded at 1.0 × 10⁶ cells/dish in 100-mm dishes, and cultured in medium consisting of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Sigma–Aldrich,USA), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Sigma–Aldrich) at 37 °C in 5% CO₂. The medium was changed every 3–4 days and cells were passaged using trypsin–EDTA (Sigma–Aldrich), when they reached 80–90% confluence. After 2 passages, ASCs were used for transplantation.