Hydrogels and aerogels with tunable stiffness for endothelial cell culture

Blood and lymphatic vessels are lined by endothelial cells (ECs) which constantly interact with their luminal and abluminal extracellular environments. These interactions confer physical forces on the endothelium, such as shear stress, stretch and matrix stiffness, to mediate biological responses. Physical forces are often altered during disease, driving abnormal EC behaviour and pathology. Therefore, it is critical to understand the mechanisms by which ECs respond to physical forces. Traditionally, ECs in culture are grown in the absence of flow on stiff substrates such as plastic or glass. These cells are not subjected to the physiological forces that ECs endure in vivo, thus the results of these experiments often do not mimic those observed in the body. The Frye lab investigates how changes in extracellular matrix (ECM) stiffness regulates endothelial behaviour in development and disease.

Although a wide range of physical and chemical hydrogel gelation methods have been developed, it still remains challenging to combine desired chemistry, pore topology and microstructure, mechanical properties, long-term stability and sterilizability. Furthermore, scalability, i.e. ability to process hydrogels at a pilot and industrial scale, should not be left aside. To circumvent these challenges, the Gurikov lab is developing gentle methods towards gelation using pressurized carbon dioxide as gelation trigger. The use of CO₂ strengthens the hydrogel resulting in self-standing gels even at low polymer concentration and therefore can provide a convincing solution to mimic a more physiological environment for 2D and 3D EC culture.

Our overall aim is to develop a new class of biopolymer-based hydrogel matrices which possess controllable stiffness and can thereby closely mimic physiological conditions for the growth of ECs in culture. We aim to develop an inexpensive and scalable processing of soft hydrogels, which can be utilized for sterile 2D and 3D cell culture applications. Furthermore, we aim to convert hydrogels into their solid form, into so called aerogels, to maintain a long shelf life time. Comprehensively, we will establish structure-performance relationships from comprehensive structural characterization of the hydro- and aerogels to an in-depth characterization of blood and lymphatic EC behaviour on hydrogels and aerogels.

We will generate alginate-based and hybrid polymer hydrogels within physiological and pathological stiffness ranges and achieve homogeneous polymer distribution through carbon dioxide-induced gelation. To control EC adhesion, additional biopolymers and RGD peptides will be integrated. Hydrogels will be processed into aerogels through a established procedure (water is exchanged with ethanol followed by supercritical drying). Hydrogel and aerogels will be subjected to structural characterization using Scanning Electron Microscopy, nitrogen sorption analysis and microscale stiffness measurements via Atomic Force Microscopy and nano-indentation.

We will characterize blood and lymphatic EC behaviour on (2D) and within (3D) our novel hydrogel matrices. To this end, we will study EC morphology and cell-matrix adhesions and cell-cell junctions using confocal and super-resolution microscopy. In parallel, we will analyse mRNA expression in ECs cultured on/in different hydrogels and correlate expression data with our stiffness RNAseq database. To functionally characterize EC behaviour, we will study migration and sprouting capacity of ECs on/in different stiffness matrices and assess the regulation of EC monolayer permeability via the hydrogel microenvironment. If successful, our novel hydrogels could provide a more physiological basis for in vitro cell culture of several other adherent cell types.