Optimizing conditions for spheroid formation of dental pulp cells in cell culture

Abstract

Background/Aim: Spheroid formation is a well-known feature of stem/progenitor cells. Dental pulp cells (DPCs) cultured in serum-free medium can also form spheroids. However, the success rate varies largely depending on various factors. This study aimed to explore these factors and optimize the conditions. Materials and Methods: Primary DPCs were obtained from 6 wisdom teeth. Possible influencing factors including donor teeth, concentrations of the KnockOut Serum Replacement (KSR), seeding density (regarding surface and volume), passage and freezing were tested. Results: DPCs from all 6 donor teeth formed spheroids in serum-free medium. Number, size, and total area of spheroids varied among different donor teeth. Optimal concentration of the KSR and seeding densities also varied from tooth to tooth. Generally, high KSR and high cell density lead to better spheroid formation. However, very high KSR and cell density can also lead to cell death and the fusion of spheroids to irregular aggregates. Conclusion: An initial setting can be recommended as: Serum-free MEM plus 10-15% KSR and seeding densities of 1-2×105 cells/ml and 2×105 cells/cm2. These parameters provide a direction for optimizing the condition for obtaining spheroids from human DPCs.