In Situ Control of Cells and Multicellular Structures by Two-Photon Lithography

In vivo, the tissue environment often provides strong confinement to cells and multicellular assemblies, particularly in cancer. Therefore it is highly important to mimic such confined environments, in order to investigate the impact of confinement on cellular systems. Here, we employed two-photon lithography, which allows to print directly into and next to multicellular assemblies. For example, we printed dome-shaped confinements with micrometer-sized openings to confine cancer spheroids in order to investigate the migration behavior of the cancer cells and also to study the effects of confinement on the spheroid. We have shown that confinement of the spheroids leads to a decreased cell migration speed and affects actin alignment and dynamics. Furthermore, in situ two-photon lithography provides a novel way of analyzing the behavior of specific regions of multicellular structures, by enabling the separation of multicellular structures from a spheroid. This is possible, as the laser used for printing can reach into the almost transparent multicellular structure and the used material is biocompatible. Hence, two-photon lithography is suitable for controlling the growth, migration and morphological cues of live cells, which is highly relevant to study the impact of the physical microenvironment on biosystems.