A simple and scalable method to generate spheroids from human mesenchymal stem cells for use in tissue engineering

Introduction: Tissue engineering is a field suited for applying stem cells, besides stem cell transplantation. In the current tissue engineering approaches, stem cells are typically seeded onto a suitable scaffold and induced into specific tissues under particular conditions. However, this strategy has faced some limitations, namely that stem cell proliferation on the scaffolds' surface has been inefficient to fill the porous scaffolds to produce solid tissues. Some limitations have been improved by using stem cell spheroids on the scaffold in place of single stem cells. This study aimed to evaluate a simple and feasible method to produce spheroids of mesenchymal stem cells (MSCs) from adipose and umbilical cord tissues for use in tissue engineering. Methods: MSCs from human adipose tissue (adipose-derived stem cells, i.e., ADSCs) and human umbilical cord tissues (umbilical cord-derived mesenchymal stem cells, i.e., UCMSCs) were isolated according to previously published protocols. To produce spheroids, ADSCs and UCMSCs were cultured in non-adherent V-bottom 96-well plate. Three cell densities were evaluated: 250 cells/well, 500 cells/well, and 1,000 cells/well. The generated spheroids were evaluated based on spheroid diameter, necrotic core formation (using propidium iodide (PI) and Hoechst 33342 staining), and spheroid structure (by Hematoxylin & Eosin staining). Results: The results showed that at a density of 250 cells/well, spheroids were formed without necrotic cores from both ADSCs and UCMSCs. However, at a higher density, all spheroids had a necrotic core as part of the three zones (proliferating, quiescent, and necrotic zones). Conclusion: Spheroids from ADSCs and UCMSCs can be easily produced by culturing 250 cells/well in a non-adherent V-bottom 96-well plate. This process can be scaled up by using the liquid handling robot system to load cells into the plates.