Micropatterned Adhesion Sites for Spheroid Cultivation Under Flow

Abstract

The focus of cell based assays in recent years switched from 2D to 3D applications using spheroids and organoid cell assemblies due to the higher validity of the experimental outcome concerning in vivo relevance. However, spheroid generation, continues media exchange for good nutrients supply and high quality imaging over a long period of time is often very laborious. We use a perfusable channel system with micropatterned adhesion ligands on a highly passivated coverslip, which enables high resolution fluorescence microscopy, to generate numerous homogenously distributed spheroids in parallel within one channel. In this system spheroids can either be directly generated by simple injection of a large number of cells and a self-sorting process or by addition of preformed spheroids, which will bind to the patterned adhesion sites. Due to the immobilization of spheroids to the adhesion spots, long-term observation of the same spheroids can be performed with many spheroids in parallel. Perfusion of the spheroid chip at low flow rates not only enables the homogenous supply of all spheroids with nutrients, but also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. With our micropatterned channel system we were able to show that by applying a flow the overall spheroid growth increases over time and spheroid size is very homogenous. By applying very high flow rates spheroids can be harvested from the surfaces, collected and further analyzed. The flow also influences the drug response of spheroids towards a more in vivo like reaction, showing that 3D spheroid cultivation under flow is the method of choice for generating relevant experimental results.