Flip the chip: trapping a low number of cancer and immune cells for spheroid formation of homogeneous size

Drug tests on patient-derived tumor cells enable a personalized therapy and promise early identification of resistant cells. However, most drug tests are performed on cell monolayers that do not resemble the complex tumor microenvironment. Spheroids are advanced models, as cells in 3D configuration exhibit cell-cell interaction, cell-extracellular matrix, and cell environment more similar to a tumor environment. In this study, we show the formation of homogeneous spheroids from a low number of cells, which is crucial for personalized drug tests with cells from tissue biopsies or liquid biopsies. We introduce a novel microfluidic platform that combines microwell technology with hydrodynamic trapping, thereby combining the advantages of both methods. The platform consists of trapping units, each comprising a microwell and a filter. Initially, the microwell is located on top, and the cells are homogeneously clustered by the filters. The chip is then flipped by 180°, allowing the cells to sediment at the bottom of the microwells, where they form spheroids protected from shear stress. We show that cells can be homogeneously captured along the array, while the filter geometry controls how many cells are captured per trapping unit. We explore several designs, each with different microwell and filter dimensions. With 91.5% capture efficiency, the cell loss is minimal. After turning the chip, spheroids of homogeneous size form in the wells and expand at a growth rate of 8 μm per day. We perform drug tests and show that cisplatin affects not only the viability of spheroids, but also their structural integrity. The drug ouabain octahydrate prevents the formation of spheroids by inhibiting cell-cell adhesion. Finally, we co-culture cancer cells and polarized macrophages. Macrophages can influence the susceptibility of tumors to drugs and indeed, we observe that spheroids co-cultured with M1-polarized macrophages have a lower viability after cisplatin treatment than spheroids without macrophages. In contrast, the presence of M2-polarized macrophages reduces the effect of cisplatin with more cells of the spheroids remaining viable. In summary, this platform has great potential for personalized drug tests, when a very low number of cells are available, for example cells derived from tissue biopsy samples, or circulating tumor cells obtained from liquid biopsies.