FIGURE 1.

Mesenchymal stem/stromal cell (MSC) spheroids formation process and structure. (A) Cell aggregation and spheroid formation involving three phases. Initially, cells form loose aggregates via the tight binding of extracellular matrix arginine–glycine–aspartate (RGD) motifs with membrane-bound integrin. Due to increased cell–cell interactions, cadherin gene expression levels are upregulated and cadherin protein is accumulated on the cell membrane. In the later phase, homophilic cadherin-to-cadherin binding induce the formation of compact cell spheroids from cell aggregates. (B) Methylcellulose-based technique can be used to generate viable MSC spheroids on low-attachment gas-permeable plates (left panel). Generated MSC spheroids show stable immunophenotypic profile by expressing high levels of the pericytic marker CD146 (green) and MSC-related marker CD90 (red) (middle panel) (unpublished data). Structurally, based on their size and abundance of nutrients and oxygen in vitro, MSC spheroids can be divided into zones (outer and inner). The nutrients, oxygen, and waste concentration gradients within the spheroids should be always taken into consideration when selecting the optimal technique to generate spheroids in vitro in order to achieve increased spheroid functionality in in vivo settings (right panel).