Abstract
Understanding the phenomena for habitat isolation of a heterogeneous cell population in a three-dimensional constructs will be useful for developing biomimetic and functional engineered tissues. To examine the habitation of co-cultured cells, a five-layered human skeletal muscle myoblast (HSMM) sheet, regarded as a plate-shaped aggregate, was overlaid onto target cells, namely human skeletal muscle fibroblasts (HSMFs) and human umbilical vein endothelial cells (HUVECs), with different initial densities on the bottom surface to investigate the behaviors of target cells in the plate-shaped aggregate. Under low-density conditions, HSMFs rapidly and linearly migrated in the vertical direction after detaching from the bottom surface; cells reached the upper layers, resulting in habitation of both the top and bottom of the plate-shaped aggregate. HSMFs under high-density conditions maintained a horizontally spread aggregation, and remained in the lower layers of the five-layered sheet. HUVECs show a horizontally elongated shape to maintain their horizontal connections with neighboring cells; this led to slow migration in the vertical direction through the formation of net-shaped aggregates in the middle of the five-layered sheet, regardless of initial density. In addition, measurement of local fluidity in the plate-shaped aggregate by evaluating target cell migration revealed the highest fluidity on the top layer, suggesting that fast-moving co-cultured cells migrate towards the top of the plate-shaped aggregate. Thus, the heterogeneous cell population made habitation inside a plate-shaped aggregate due to differences in their behaviors of migration and cell-cell connection, as well as their environment of sheet fluidity.
Keywords: cell sheet, cell-cell connection, habitation, image processing, migration, skeletal muscle myoblasts, skeletal muscle fibroblasts, sheet fluidity.
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