Isolation and identification of human odontoblast cells

Objectives: Dentin formation is a consecutive process in whole life of a tooth and odontoblast cells are responsible for it through secretion and mineralization of dentin matrix. Odontoblast cells isolated directly from freshly extracted teeth remain original characteristics, and have intact genetic information. Thus, this study intent to establish a method to isolate odontoblasts cells in vitro from human teeth. Methods: Cells were isolated from normal human permanent teeth sections digested by collagenase±A and protease±. Identification of isolated cells as odontoblasts was confirmed by cell morphology. In addition, cells were identified using immunocytochemistry for expression of specific markers. The spatial structure of F-actin in odontoblast cells was observed through Laser confocal scaning microscope combined with fluorescence double-labeled technique. Results: Isolated odontoblast cells had a columnar or cuboidal cell body with an intact long process, expressing collagen I, dentin matrix protein-1(DMP-1) and dentin sialoprotein (DSP). After repeated trials of different digestion condition and enzyme treatment, 3mg/ml collagenase IA and 0.25 mg/ml protease I about 45 minutes in a water bath at 38-38.5°C was the best condition for human odontoblast cells isolation in vitro. Under LCSM, isolated odontoblast cells possessed unipolar figure, clear outlines of nucleolus, and the filamentous F-actin existed mainly in cytoplasm, distributing in bunches. Conclusion: Mature and intact odontoblast cells were isolated successfully. This provided a source of odontoblast cells for continued study of cell function and characteristics.supported by National Natural Science Foundation of China (code:30471889)