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PhD Discussions Sessions: Ferran Velasco and Fabio Riefolo
Friday, November 29, 2019 @ 10:00 am–12:00 pm
Cellulose-based cryogels for long-term culture of pancreatic islets and skeletal muscle tissue
Ferran Velasco, Biosensors for BioengineeringIslet encapsulation inside traditional hydrogels is one of the most common techniques to study insulin secretion for Diabetes Mellitus studies. However, it’s proved that cells encapsulated in a depth of more than 100 microns die due the lack of nutrient diffusion. As pancreatic islets are spherical aggregations of around 100 microns in diameter, this problem increases exponentially. To solve this problem, in this project we propose the use of new Carboxymethyl cellulose – gelatin biocomposite in combination with cryogelation technique to engineer a new in vitro model to mimic the insulin-mediated skeletal muscle glucose metabolism.
Carboxymethyl cellulose (CMC) is biocompatible, but not mammalian cell-degradable and shows extraordinary elasticity features. Gelatin is able to provide the 3D microenvironment for the proliferation of different cell types and cell-interactive biological activity, very desirable properties for muscle and pancreas tissue scaffold. Cryogelation technique consists in freezing a prepolymer solution at sub-zero temperatures, so water-ice crystals are formed while the material crosslinks. When it’s defrosted, these water-ice crystals lead to “empty” cavities that forms a macroporous and very interconnected scaffold that fits with our needs of morphology and nutrient diffusion.
We first optimize the protocol to achieve the desired morphology; for the pancreatic tissue we achieved a random porosity with high interconnected pores and for the skeletal muscle we fabricate it with an anisotropic structure. We characterize it by stiffness, pore distribution, SEM images and swelling to know its mechanical properties. Then we seed cells in the specific cryogel to characterize its biological behavior depending the cryogel approach used.
Our results are promising for seeding both cell types, as the morphology and pore distributions fits with our needs. These scaffolds show higher nutrient diffusion, good material properties and a better manipulation compared to traditional hydrogels for these tissues.
Photocontrol of Muscarinic Receptors and Applications In Vivo
Fabio Riefolo, Nanoprobes and NanoswitchesRemote control of physiological functions with light offers the promise of unveiling their complex spatiotemporal dynamics in vivo, and enabling highly focalized therapeutic interventions with reduced systemic toxicity. Optogenetic methods have been implemented in the heart, but the need of genetic manipulation jeopardizes clinical applicability. We present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. A new light-regulated drug, named PAI, was designed and synthesized to be active on M2 muscarinic acetylcholine receptor (mAChR). PAI can be reversibly photoisomerized between cis and trans conformations under UV and visible light and is able to photocontrol the activation M2 mAChRs in vitro.
We show that PAI has different light-dependent cardiac effects in a mammalian animal model. Finally, we demonstrate the reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles: PAI induced bradycardia and this effect could be reversibly switched using UV and visible illumination. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination. Such a new approach may enable enhanced spatial and temporal selectivity for cardiovascular drugs.
