3D-printed insert-array and 3D-coculture-array for high-throughput screening of cell migration and application to study molecular and cellular influences.

Collective cell migration refers to the movement of groups of cells and collective cell behavior and relies on cell-cell communication and cell-environment interactions. Collective cell migration plays a fundamental role in many aspects of cell biology and pathology. Current protocols for studying collective cell migration either use destructive methods or are not convenient for liquid handling. Here we present a novel 3D-printed insert-array and a 3D-coculture-array for collective cell migration study in high-throughput. The fabricated insert-array is comprised of 96 cylinder shaped inserts which can be placed in each well of a 96-well plate generating watertight contact with the bottom of each well. The insert-array has high manufacturing tolerance, and the coefficient of variations of the insert diameter and circularity are 0.67% and 0.03%, respectively. Each insert generates a circular cell-free area within the well without cell damage and provides convenient access for both manual and robotic liquid handling. Using the 3D-printed insert-array, we studied the migration of human umbilical vein endothelial cells (HUVECs) under the molecular influences of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and under the cellular influences of human mesenchymal stem cells (hMSCs) using the 3D-coculture-array. Our results show that the migration of HUVECs was dose-dependent on the VEGF and bFGF with different correlation patterns. They also generated a synergic pro-migration effect. When cocultured with hMSCs, the migration rate increased significantly while dependent on the number of hMSCs. The effects were partially blocked by VEGF inhibitor which suggests that VEGF secreted from hMSCs plays an important role in cell-to-cell communication during cell migration. The 3D-coculture-array can be manufactured at very low cost and shows higher biomolecule transport efficiency than the commercially available transwell. The calculated Z-factor is 0.66, which classifies our system as a perfect high-throughput assay. In summary, our newly developed insert-array and 3D-coculture-array provide a versatile platform to study collective cell migration in high-throughput as well as the molecular and cellular influences upon it.