The thermosensitive collagen gel culture kit (Nitta-gelatin Co. Ltd., Osaka, Japan) consisted of Cellmatrix Type I-A that was prepared from pig collagen type I, NaHCO3-free 10 × concentrated DMEM, and restructured buffer that consisted of 260 mmol/L NaHCO3, 50 mmol/L NaOH, and 200 mmol/L hydroxyethyl piperazine ethanesulfonic acid. The thermosensitive collagen hydrogel was mixed together on ice at a constituent ratio of 8 parts Cellmatrix Type I-A to 1 part NaHCO3-free concentrated DMEM to 1 part restructured buffer. The thermosensitive collagen hydrogel remained as a liquid when kept at temperatures below 10°C and gelled when the temperature was increased to 37°C in an incubator for 30 minutes. In the control group, the thermosensitive collagen hydrogel was replaced with 10 × concentrated DMEM diluted 10-fold with collagen type I.