Photolithographic micro-structuring of artifcial 3-dimensional hydrogel environments for cell migration studies

The extracellular matrix (ECM) of cells in vivo is a complex mixture of biopolymers that form a network of varying stiffness and mesh sizes, containing soluble and matrix bound signaling molecules. Due to this complexity, it is not fully understood which cues guide cell migration in such networks. To facilitate the analysis of chemotactic or durotactic migration mechanisms, artificial ECM mimics, whose properties and composition can be fine-tuned, are needed. In this thesis photo-polymerizable, synthetic hydrogel micro-structures were developed to mimic a minimal ECM for cell migration studies. 4-armed polyethylene glycol (PEG) monomers were cross-linked with either linear PEG molecules, which results in a non-degradable network, or with a peptide cross-linker which can be cleaved by matrix-metalloproteinases (MMPs) and allows the proteolytic cell migration within the gel. Addition of a small RGD-containing peptide sequence mediates cell adhesion to the otherwise bioinert PEG network. The biocompatible polymerization induced by short illumination with UV light permits the direct encapsulation of living cells and the micro-structuring of small gel strips inside channel systems using photo-lithography. To carefully characterize the hydrogel system and its swelling properties, particle image velocimetry (PIV) was used. The obtained displacement fields show an anisotropic swelling of the gel due to the confinement in a channel. In the longitudinal middle section, the thin strips swell only in the direction of the strip width. The magnitude of the inherent gel swelling can be tuned by changing the solvent and swelling media, the type and amount of cross-linker used for the polymerization, as well as the dimensions of the channel and strip. To study how cells perceive and react to affine deformations in their surroundings, a micro-structured artificial hydrogel was used to form a strained, degradable matrix. The aforementioned anisotropic swelling uniaxially strained the gel strips. Analysis of HT-1080 cell migration in such structures showed a preferred migration parallel to the swelling direction, with a maximal alignment at intermediate strain levels. To understand the relationship between cell migration directionality and strain, a theoretical model was implemented within the framework of a collaboration. With the simulation of a proteolytic, durotactic cell movement on a 2D lattice, the same non-monotonic response to uniaxial strain as seen in the experiments was obtained. Stiffness analysis within the model showed an anisotropic stiffening of the matrix upon strain, which leads to the anisotropic cell migration. The unraveling of this fundamental mechanism as cell guidance cue in strained networks of macroscopically linear elastic materials demonstrates that strain stiffening on the microscopic scale is crucial for the cell behavior. The inherent swelling of the gel micro-structures was furthermore used to create pressurized hydrogel-hydrogel interfaces, so called sponge clamps, to establish an assay for analyzing non-proteolytic cell migration into predefined clefts. The swelling of parallel hydrogel strips fills the space in between the structures and thereby generates pressurized clefts inside a passivated channel system. By varying the gap size as well as the gel composition, the gel compression in the system can be tuned, which influences the invasion efficiency of cancer cells. Initial strip distance, but also matrix stiffness are key regulators of cell migration in the clefts. The results of this thesis illustrate how photo-structurable artificial ECM mimics and their inherent swelling enable the generation of advanced 3D migration assays to unravel fundamental guidance cues in proteolytic and non-proteolytic cancer cell migration.