Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells

Cells with various structures and proteins naturally come together to cooperate in vivo. This study used cell spheroids cultured in agarose micro-wells as a 3D model to study the movement of cells or spheroids toward other spheroids. The formation dynamics of tumor spheroids and the interactions of two batches of cells in the agarose micro-wells were studied. The results showed that a concave bottom micro-well (diameter: 2 mm, depth: 2 mm) prepared from 3% agarose could be used to study the interaction of two batches of cells. The initial tumor cell numbers from 5 × 10³ cells/well to 6 × 10⁴ cells/well all could form 3D spheroids after 3 days of incubation. Adding the second batch of DU 145 cells to the existing DU 145 spheroid resulted in the formation of satellite cell spheroids around the existing parental tumor spheroid. Complete fusion of two generation cell spheroids was observed when the parental spheroids were formed from 1 × 10⁴ and 2 × 10⁴ cells, and the second batch of cells was 5 × 10³ per well. A higher amount of the second batch of cells (1 × 10⁴ cell/well) led to the formation of independent satellite spheroids after 48 h of co-culture, suggesting the behavior of the second batch of cells towards existing parental spheroids depended on various factors, such as the volume of the parental spheroids and the number of the second batch cells. The interactions between the tumor spheroids and Human Umbilical Vein Endothelial Cells (HUVECs) were modeled on concave agarose micro-wells. The HUVECs (3 × 10³ cell/well) were observed to gather around the parental tumor spheroids formed from 1 × 10⁴, 2 × 10⁴, and 3 × 10⁴ cells per well rather than aggregate on their own to form HUVEC spheroids. This study highlights the importance of analyzing the biological properties of cells before designing experimental procedures for the sequential fusion of cell spheroids. The study further emphasizes the significant roles that cell density and the volume of the spheroids play in determining the location and movement of cells.