Comparison of 2D and 3D culture methods on morphology,viability,surface markers,and pluripotency gene of umbilical cord mesenchymal stem cells

Abstract

Background

Three-dimensional (3D) cell culture is gaining attention for replicating in vivo conditions better than traditional two-dimensional (2D) cultures. The hanging drop method is a simple 3D culture technique with significant potential due to its ease of use. This study compares mesenchymal stem cells’ morphology, viability, surface markers and pluripotency gene of Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) from the umbilical cord in 2D and 3D cultures.

Methods

Umbilical cord MSCs were cultured using the hanging drop technique for 3D culture and 2D culture. Morphology and viability were observed while surface marker analysis was performed using flow cytometry. The harvested cells were then extracted and underwent RNA isolation, cDNA synthesis, and examination of the expression of the OCT4 and SOX2.

Results

Significant differences between 2D and 3D cultures regarding morphology, viability, and CD expression were observed. Spheroids in 3D culture exhibited a significant increase in diameter over time (p<0.0001), while CD73 expression was higher in 2D cultures compared to 3D (p = 0.0225). In 2D culture cells, the OCT4 gene was expressed more than in 3D culture (p = 0.0286).

Conclusions

These findings suggest that 3D culture provides valuable insights into stem cell behavior, with important implications for regenerative medicine.