• 3-D spheroid culture system;
  • epithelial progenitor;
  • mesenchymal stem cell;
  • neuron;
  • rhesus monkey


Background:  Bone mesenchymal stem cells are an attractive source of cells with potential applications in ocular regenerative medicine. However, the low differentiation efficiency in the traditional two-dimensional (2-D) culture system limits their application for clinical therapy. Here, we describe a simple and innovative 3-D culture environment and assess the potential for bone mesenchymal stem cells to differentiate into a variety of cell types in the 3-D system.

Methods:  Bone mesenchymal stem cells of rhesus monkey were isolated and cultured using a density gradient centrifugation and adherence screening method. Cells at passage three were cultured by hanging drop and formed spheres. After 3 days, the spheres were collected and plated onto culture plates and maintained in a floating state by a rotary method for 10 days. Under appropriate induction conditions, the sphere cells were induced into adipocytes, osteoblasts, epithelial progenitors and neuronal cells. Differentiated cells were identified by histochemical staining, immunofluorescence and reverse transcription-polymerase chain reaction.

Results:  Bone mesenchymal stem cells of rhesus monkey in the 3-D spheroid culture system acquired improved efficient multipotency for not only adipogenic and osteogeneic differentiation, but ectodermal epithelial progenitor-like cells and neuron-like cells compared with the 2-D culture system.

Conclusion:  Our 3-D spheroid culture system provides a useful technique to gain insight into the mechanisms of bone mesenchymal stem cells differentiation, and may have potential applications for the treatment of ocular surface and optic nerve disease.