Speaker
Description
Introduction
Tissue engineering entails the combination of mechanical, chemical and biological components to process materials that can be engineered towards artificial tissue structures in order to mimic the natural microenvironment of cells; the extracellular matrix (ECM), which provides cells with structural support as well as biochemical, biophysical, and mechanical cues. Biomaterials have been extensively studied as two- or three-dimensional ECM-mimicking substrates for tissue engineering. Cells seeded on these materials indicate how material cues such as stiffness, porosity, and adhesion-ligands influence cellular mechanisms. However, these material cues are usually coupled to each other and effect the cell population as a whole. Additionally, neighbouring cells, soluble factors and biophysical fields also influence cellular behaviour. This complex, heterogeneous and dynamic cell microenvironment makes it challenging to identify the effect of individual material cues on individual cell behaviour.
Engineered cellular micro niches are promising to study cell–material interactions. This could be achieved by the encapsulation of single cells in a micro-hydrogel (microgel) with droplet microfluidics. For this purpose, synthetic microgels were generated based on supramolecular ureidopyrimidinone (UPy)-based molecules. Two of these molecules can self-assemble by the formation of non-covalent complementary quadruple hydrogen bonds.1 Bioactivity can be easily introduced to the hydrogel system by modular approach via the incorporation of UPy-functionalized additives. The modularity of the UPy-hydrogel system provides the opportunity to create a variety of different hydrogel compositions.
Methodology
In this research, supramolecular microgels with different weight percentages were generated using droplet microfluidics according to Sinha et al. (2019).2 Two types of building blocks were designed for the formulation of the microgels: the bifunctional UPy-PEG10K-UPy (BF) and the monofunctional UPy-glycinamide (MF). The BF and MF molecules were both dissolved in 80 mM NaOH at 70 °C 1 hour while stirring and after cooling down neutralized with 1 M HCl, whereafter the droplets were generated. Thereafter, the droplets were observed using confocal microscopy. In addition, induced pluripotent stem cells (iPSCs) were mixed in the BF solution and used with 3 mM of the cell adhesive UPy-cRGD integrin-binding peptide to generate cell encapsulated microgels.
Results
Picolitre-sized supramolecular microgels (diameter = 79.2 ± 6.5 μm, n = 9) were generated using droplet microfluidics. The use of different weight percentages (0.625, 1.25, and 2.25 wt%) result in microgels with different stiffnesses ranging from ~ 100 – 1000 Pa. The incorporation of 1 mM fluorescent UPy-Cyanine 5 additive in the microgels showed a homogeneous distribution of the dye throughout the whole microgel, which is promising for the incorporation of other additives. Ongoing research is conducted on the encapsulation of iPSCs in microgels.
Conclusion
UPy-based supramolecular microgels were successfully generated by droplet microfluidics. This enables to encapsulate single cells in microgels that are easily tunable (e.g. stiffness and bioactivity) due to the modularity of the UPy-building blocks and thereby creates a platform to investigate individual cells in a physiological relevant 3D synthetic microenvironment. For future experiments, UPy-microgels could be enriched with biological relevant UPy-additives.
20941853487
