The intention of this study was to investigate the effect of modified 3D culture conditions on dental pulp cells (DPCs). DPCs were isolated from extracted primary molar, premolar, and wisdom teeth. Tooth samples were divided into three groups as control group; plated into methyl cellulose medium without any supplementation, growth factor (GF) group; supplemented with bone morphogenetic proteins (BMP2, BMP4), transforming growth factor-beta 1 (TGF-beta 1) and growth factor+conditioned medium (GF+CM) group; supplemented with both growth factors and pulp conditioned medium. The DPCs were tested for colony forming ability, proliferation capacity and morphology. The highest colony forming ability was detected in the GF and GF+CM groups of DPCs isolated from wisdom teeth. The proliferation capacity was higher in GF+CM group of DPCs isolated from primary molars, and in GF and GF+CM groups of DPCs isolated from wisdom teeth. Scanning electron microscope (SEM) observation of the wisdom teeth samples showed cell-cell interactions in the GF and GF+CM groups. Our results indicate that growth factors and pulp conditioned medium in methyl cellulose culture created proper environment to follow the behavior of dental cells three-dimensionally.