Photolithographic microfabrication of hydrogel clefts for cell invasion studies.

Invasion of migrating cells into surrounding tissue plays a key role in cancer metastasis and immune response. In order to assess invasiveness, most in vitro invasion assays measure the degree to which cells migrate between microchambers that provide a chemoattractant gradient across a polymeric membrane with defined pores. However, in real tissue cells experience soft, mechanically deformable microenvironments. Here we introduce RGD-functionalized hydrogel structures that present pressurized clefts for invasive migration of cells between reservoirs maintaining a chemotactic gradient. Using UV-photolithography, equally spaced blocks of polyethylene glycol-norbornene (PEG-NB) hydrogels are formed, which subsequently swell and close the interjacent gaps. The swelling ratio and final contours of the hydrogel blocks were determined using confocal microscopy confirming a swelling induced closure of the structures. The velocity profile of cancer cells transmigrating through the clefts, which we name 'sponge clamp', is found to depend on the elastic modulus as well as the gap size between the swollen blocks. The 'sponge clamp' discriminates the invasiveness of two distinct cell lines, MDA-MB-231 and HT-1080. The approach provides soft 3D-microstructures mimicking invasion conditions in extracellular matrix.

Sequential and Switchable Patterning for Studying Cellular Processes under Spatiotemporal Control.

Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our "sequential photopatterning" system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science.