Abstract: Objective　To compare the effects of different methods and explore a simple primary culture technique of human dental pulp cell in vitro. Methods: Pulp tissue was separated from healthy young human teeth extracted for orthodontic purpose. The pulp explants were cultured by the tissue explants method, tissue-explants collagenase /dispase digestion method and modified tissue culture techniques respectively. The cell culture efficiency and cell morphology were observed and evaluated by phase-contract microscopy. Immunocytochemical staining method was used to characterize the dental pulp cell lineage. Results: The cultured cells appeared to be fibroblast-like cells．Anti-vimentin was positive and anti?cytokeratin was negative by immunohistochemical stain. The primary dental pulp cells cultured by the tissue explants method could reach confluence after 4 weeks and the culture successfulness was 26.7%. The culture successfulness of dental pulp cells obtained by collagenase /dispase digestion method was only 13.3%. Dental pulp cells can be obtained by the modified tissue culture without floating tissue fragments and the success rate could reach to 80%. Conclusion: Modified tissue culture techniques solved the difficulty of adherence of tissue fragments, simplified the procedure, and kept a high success rate. The modified tissue culture established in this study is an effective method for the primary culture of human dental pulp cells in vitro.

Key words: Human dental pulp cells, Cell culture, Primary culture, Tissue explants