Low-Temperature Culture of Tooth and Submandibular Germ

In the low-temperature culture, the temperature was set at 25 and 4 °C, respectively. Tooth germs were cultured in an incubator at 25 °C in a humidified atmosphere of 5% or 0.03% CO₂ for 7–28 days. Tooth germs were also cultured in a refrigerator at 4 °C with the same CO₂ concentration (0.03%) as in the atmosphere. After low-temperature culture, tooth germs were cultured at 37 °C in a humidified atmosphere of 5% CO₂ for 10 days. Submandibular glands were cultured at 25 or 4 °C for 7 days under the same conditions as the tooth germs and then cultured at 37 °C in a humidified atmosphere of 5% CO₂ for 2 days. We changed to a fresh medium every 3 days and captured the images with the microscope IX71 (Olympus, Tokyo, Japan) daily for record-keeping.