Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.