A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

Three-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids.

hDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components.

Compared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions.

The present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.