Synthetic supramolecular hydrogels for the 3D culture of kidney epithelial cells and intestinal organoids

Jun 30, 2022, 12:00 PM
Room: S3 A

Room: S3 A


Rijns, Laura (TU/e )


"Introduction: An abundant and important receptor that regulates numerous ECM-cell interactions is the integrin receptor. To illustrate, integrins influence the cells’ polarity by controlling the apical-basal orientation. Kidney epithelial cells and intestinal organoids cultured in suspension and thus in the absence of matrix, invert the direction of their polarization, with an apical membrane facing outwards. These observations raise the question whether direct control over the integrins via the matrix’ stiffness and degree of bioactivity could possibly control the orientation of polarization, thereby directing epithelial morphogenesis and controlling organoid behavior.
Therefore, we propose synthetic, modular supramolecular hydrogels based on the ureido-pyrimidinone (UPy) motif, which use directional, non-covalent interactions. These assemblies are eminently suitable to study cell-material interactions, because herein we have full and independent control over several different properties, like stiffness and ligand concentration, allowing that the effects of such microenvironment components can be assessed individually. The UPy hydrogels consist of 3 molecules: monofunctional (M), bifunctional (B) and bioactive (RGD) UPys. Herein, the M UPys can form one-dimensional fibers, while the B UPys could act as a crosslinker between the M UPys to create a network with adjustable mechanical properties, by changing the M/B UPy ratio or by varying the hydrogel’s concentration. And finally, RGD UPys could be mixed in as integrin-binding ligands.
Here, we aim to investigate the influence of stiffness on renal epithelial cell and intestinal organoid polarization in 3D using supramolecular hydrogels.

Methodology: UPy supramolecular hydrogels were prepared in a fixed molecular ratio of 80 M to 1 B molecules at the desired concentration (wt%). For kidney cell studies in 3D, 0.5 mM of RGD UPy was incorporated into the hydrogel as part of the M molecules. Rheological experiments were measured at 1 rad/s and 1% strain.

Results: Rheology showed that the mechanical properties of the UPy hydrogels could be varied from ~ 0.1 kPa to 1 kPa to 2 kPa for the 0.6, 1.25 and 2.5 wt% gels, respectively.
Madin-Darby Canine Kidney (MDCKs) were then encapsulated in 3D in UPy hydrogels of different weight percentages and corresponding stiffness. After 10 days, cell encapsulation resulted in cyst formation with apical-basal polarization inside all supramolecular hydrogels. Three different structures were formed, ranging from cell aggregates with inverted polarity to well-polarized cysts with a lumen inside. Quantification of the frequencies of the different morphologies in the different conditions showed that cell aggregates were predominantly formed in the 0.6 wt% gels, whereas the cysts with hollow lumens were mostly observed in the 2.5 wt% gels. This observation indicates that the cell-cell interactions dominate when less hydrogel, and thus less mechanical support, is present.

Conclusions: We showed that the cell-cell interactions dominate in the 0.6 wt% gels, whereas well-polarized cysts are formed in the higher, 2.5 wt% hydrogels. To further investigate the origin of the difference in organization, we will test the influence of ligand concentration. Currently, we are also investigating the influence of mechanical support (i.e. the wt% of UPy gels) on growth and polarity of intestinal organoids."


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