Annual Report 2011
Research Highlights
T Biomolecular Diagnostics Abel Gonzalez Oliva
oliva@itqb.unl.pt
The Biomolecular Diagnostic laboratory is a multidisciplinary research group specialized in the development of diagnostic tools applied in veterinary and biotechnology process. The group is collaborating with the Organic Chemistry lab in the development of nanoparticles (Quantum dots) towards application in veterinary or plant studies. The synthesis of CdS/ZnS nanoparticles, by an optimized laboratory protocol, has been achieved. The functionalization towards making it water soluble for application in biological experiments has been optimized and the conjugation of the functionalized nanoparticles with sugars or larger molecules like antibodies or antigenic proteins has been achieved. These highly stable fluorescence nanoparticles can be size-tuned for specific fluorescence emission. In a collaborative work with the CIN2 (Centre D’Investigació en Nanociencia) of the University of Barcelona, we achieved the deposition of the QDs onto surfaces using a ultraviolet matrix-assisted pulsed laser evaporation (UV-MAPLE) onto SiO2 glass substrates covered by silica thin films. The immobilized materials form self-organized 2D arrays constituted by complex CdSe/ZnS core_shell QDs preserving the functional properties of the base material used for the preparation of the MAPLE targets, which allows us to explore the nanoparticle-coated surface for key applications such as biosensors, lasers, or high performance electronic devices.
T Animal Cell Technology Unit Ana S. Coroadinha avalente@itqb.unl.pt | Paula M. Alves marques@itqb.unl.pt | Manuel J. T. Carrondo mjtc@itqb.unl.pt Human liver cell spheroids in extended perfusion bioreactor culture for repeated dose drug testing. Primary cultures of human hepatocyte spheroids are a promising in vitro model for studies of hepatic metabolism and cytotoxicity. The lack of robust methods to culture cell spheroids, a poor characterization of the human hepatocyte spheroids architecture and of the liver-specific functionality has hampered the widespread adoption of this 3D culture format. At the Animal Cell Technology Unit, we developed long-term automated perfusion bioreactor cultures of primary human hepatocyte spheroids that maintain liver-specific activity. These cultures are suitable for drug testing in a long term, repeated dose format. The spheroids cultured for 3-4 weeks in serum-free conditions sustained phase I enzyme expression and permitted repeated induction cycles; the rate of albumin and urea synthesis, as well as phase I and II drug metabolizing enzymes’ gene expression and activity presented reproducible profiles, despite the basal inter-donor variability. Immunofluorescence microscopy after 3-4 weeks of culture confirmed the presence of liver-specific markers and suggested that the spheroids spontaneously assemble bile canalicular networks extending from the surface to the interior of the spheroids (Figure). Moreover, the excretion of a fluorescent dye by phase III membrane transporter activity was observed by live imaging, proving the functionality of the bile canalicular networks. Tostões R.M. et al. (2012) Hepatology, 55(4)1227
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