PhD Discussions Sessions: Anna Vila and Alexandre Gomila

Engineering of 3D Small Intestinal Mucosa Models

Hydrogels have been used as a scaffold for engineering tissue-like structures due to their biocompatibility and properties similar to the extracellular matrix. There are two main types of hydrogels (i) natural hydrogels, such as gelatin, which are biodegradable and present cell adhesion motifs and (ii) synthetic hydrogels, such as poly(ethylene glycol) diacrylate (PEGDA), which are non-biodegradable and can withstand long-term cell cultures but do not have bioadhesion sequences [El-Sherbiny M.I. et al., 2013]. Both types of hydrogels present complementary benefits.

For this reason, we fabricated a hydrogel co-polymer composed of gelatin methacrylate (GelMA) co-polymerized with PEGDA [Hutson. B.C. et al., 2011]. We employed a single-step, moldless, UV-photolithography-based fabrication technique [Castaño A. et al., submitted] to fabricate scaffolds mimicking the 3D architecture of the small intestinal mucosa. Using this approach, we obtained hydrogel co-polymers with finger-like microstructures, with the roundness and dimensions found in the villi of the native tissue. Mechanical and physicochemical properties such as an approach of the Young’s modulus, degradation rate and swelling ratio of the hydrogels have been characterized. Adding PEGDA to the GelMA hydrogels have provided hydrogels with lower degradation and higher Young’s modulus, which ca be easily tuned by changing the composition of GelMA and PEGDA polymers. Furthermore, we have demonstrated that our scaffolds support the growth and differentiation of intestinal epithelial Caco-2 cells up to 21 days, obtaining a matured epithelial monolayer with effective tissue barrier properties.

Additionally, we increased the complexity of our model of the small intestinal mucosa by incorporating an additional cell compartment to mimic the stroma of the in vivo tissue. To do that, we embedded 3T3-NIH fibroblasts in our scaffolds during the photopolymerization procedure. Then, we cultured Caco-2 cells on top of the 3T3-NIH fibroblast-laden hydrogels up to 21 days. Our preliminary results showed that the co-culture of Caco-2 cells with 3T3-NIH fibroblasts favours the epithelial cell growth and improves their barrier function. Taking all together, we have generated an intestinal mucosa model that allows for the co-culture of different intestinal cell types distributed in compartments, mimicking the spatial-physiological features of the small intestinal mucosa. We believe our model better recapitulates cell-cell crosstalk and cell-matrix interactions found in vivo, being an improved alternative for the cell-base in vitro assays.

In vivo photomodulation of GABA and Glycine receptor channels

Neuronal networks are highly complex interactions, which determine even the finest behaviour. The major inhibitory pathways in the central nervous system (CNS) act through chloride ion flux, which are mostly driven by fast-acting ionotropic GABAA and Glycine receptors (GlyR). All of them share structural similarities and belong to pentameric ligand-gated ion channels of the Cys-loop family. Photoswitchable molecules have become a powerful tool in any applied field of bioscience, and are broadly used in biomedical research due to their capacity for enlightening biological aspects from their very basics, such as molecular level, up to entire neuronal networks.
Photopharmacology has proven to be advantageous for spatial and temporal control of biological processes without interfering the system natural dynamics and outcomes. Physiology can be tuned with photomimetic ligands and naturally occurring complex network responses can be segmented into light dependent activities discerning relevant data from a vast matrix of results. As these new tools are broadly used for biomedical purposes and most of them focused towards medical applications, an interpretative analytical platform is needed to screen and identify potential photoswitchable molecules.

Zebrafish (Danio rerio) larvae constitute an excellent animal model for studying and screening photoswitchable molecules in vivo. Zebrafish present a transparent body during larval stages, and therefore are capable of receiving specific and determined light applications. From the 19th hour post fertilisation they acquire behavioural traits, from spontaneous twisting movements to full swimming capacities, which are easily traceable and measurable. The use of up to 96 animals simultaneously allows a parallel high throughput data recovery system to analyse high complexity movements and behaviours, all of it with the use of photopharmacology. Here, we aimed at introducing an effective and reliable methodology for high throughput screening of photoswitchable compounds, including photopharmacological derivatives or peptidoswitches, for any in vivo possible target, from specific neuronal correlated diseases up to possible toxicological outcomes. We focused on the study of the main inhibitory neuronal pathways and their locomotion outcomes on a reliable and comparable animal model.

Hence, several photoswitchable compounds with a common benzodiazepine core and an azobenzene photoswitchable moiety were tested. A first light dependent activity ratio was applied in order to discern the most promising candidates. We identified the UR-DW290 molecule as a light dependent trigger of activity in larvae zebrafish. Larvae zebrafish treated with UR-DW290 maintained higher activity in terms of swimming distance (mm) during the relaxation period and UV-Blue light cycles in comparison to controls. Whole-cell recordings of GABAA and glycine mediated currents by URDW290 showed a potentiating of inhibition when irradiated with ultraviolet light in comparison to visible light illumination.

We propose the combination of high through-put screening and optopharmacology tools for the study and characterisation of zebrafish larvae behaviour focusing on their swimming activity. We identified a first photoswitchable molecule for glycine receptor modulation in vitro and in vivo, UR-DW290, which increases basal activity in zebrafish larvae. This increase is tuneable with UV and Blue light illumniation.

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