In Vitro Tooth and Salivary Gland Development

The experimental animal protocol was approved by the Ethics Committee of the Kyushu University Animal Experiment Center (protocol number; A20-281-1). All procedures were performed in accordance with the relevant guidelines and regulations of the Kyushu University, and the study was performed in accordance with ARRIVE (Animal Research: Reporting of In Vivo experiments) guidelines. Pregnant mice were euthanized by medetomidine, midazolam, and butorphanol intraperitoneal administration and the embryos were dissected immediately. Tooth germs of mandibular molars were dissected from E14.5 mice embryos and placed on cell culture inserts (BD Falcon, BD Biosciences, Franklin Lakes, NJ, USA), and grown using an air–liquid interface culture technique in Dulbecco’s modified Eagle’s medium (DMEM)/F-12, supplemented with 20% fetal bovine serum (Gibco/Life Technologies, Waltham, MS, USA), 180 g/mL ascorbic acid, 2 mM l-glutamine, and 50 units/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂, as described previously^{31 (link)–34 (link)}. Submandibular glands were dissected from E13.5 mice embryos, placed on cell culture inserts, and grown in the same condition as tooth germs. To record the development of the cultured samples, images were captured daily under the microscope IX71 (Olympus, Tokyo, Japan) during the culture period. The development processes of cultured tooth germs were classified into five scores for evaluation: score 0, no noticeable change from the starting point; score 1, epithelium thickening; score 2, epithelial invagination into the mesenchyme; score 3, multiple cusp formation; score 4, final morphogenesis; score 5, differentiation. E13.5 submandibular glands were cultured for 2 days, and the size was determined using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MA, USA).