University of Coimbra 9 articles published in JoVE Bioengineering Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol Miguel M. Lopes*1,2,3, Sara M. Lopes*1,2,3, Rafael Baganha1,2,4, Carina Henriques1,2,5, Ana C. Silva1,2,3, Diana D. Lobo1,2,3, Luísa Cortes1,2,3,6, Luís Pereira de Almeida*1,2,4,5, Rui Jorge Nobre*1,2,3,4 1CNC-UC - Center for Neuroscience and Cell Biology, University of Coimbra, 2CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, 3IIIUC - Institute for Interdisciplinary Research, University of Coimbra, 4ViraVector - Viral Vectors for Gene Transfer Core Facility, University of Coimbra, 5FFUC - Faculty of Pharmacy, University of Coimbra, 6MICC-CNC - Microscopy Imaging Center of Coimbra - CNC, University of Coimbra This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies. Biology Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach Alice Martins1, Filipa R Pinto1, Sónia Barroso1, Tatiana Pereira1, Teresa Mouga1, Clélia Afonso1, Marta V Freitas1,2, Susete Pinteus1, Rui Pedrosa1, Maria M Gil1 1MARE-Marine and Environmental Sciences Centre/ARNET-Aquatic Research Network, ESTM, Polytechnic University of Leiria, 2MARE-Marine and Environmental Sciences Centre/ARNET-Aquatic Research Network, Department of Life Sciences, University of Coimbra Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts' antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments. Biochemistry Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment Karina Garcia1,2,3,4, André Alves1,2,3, Teresa M. Ribeiro-Rodrigues1,2,3, Flávio Reis1,2,3, Sofia Viana1,2,3,4 1Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, 2Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, 3Clinical Academic Center of Coimbra (CACC), 4Polytechnic Institute of Coimbra, Coimbra Health School Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment. Medicine Accessing the Cytotoxicity and Cell Response to Biomaterials Anabela Baptista Paula*1,2,3,4, Mafalda Laranjo*2,3,4,5, Ana Sofia Coelho1,2,3,4, Ana Margarida Abrantes2,3,4,5, Ana Cristina Gonçalves2,3,4,6, Ana Bela Sarmento-Ribeiro2,3,4,6, Manuel Marques Ferreira2,3,4,7, Maria Filomena Botelho2,3,4,5, Carlos Miguel Marto1,2,3,4,8, Eunice Carrilho1,2,3,4 1Institute of Integrated Clinical Practice, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO), University of Coimbra, Coimbra, Portugal, 3Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal, 4Clinical Academic Center of Coimbra, CACC, Portugal, 5Institute of Biophysics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 6Laboratory of Oncobiology and Hematology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 7Institute of Endodontics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 8Institute of Experimental Pathology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques. Neuroscience Isolation and Culture of Chick Ciliary Ganglion Neurons Filipa J. Costa*1, Marta S. Dias*1, Rui O. Costa2, Joana R. Pedro2, Ramiro D. Almeida1,2 1Institute of Biomedicine, Department of Medical Sciences - iBiMED, University of Aveiro, 2CNC - Center for Neuroscience and Cell Biology, University of Coimbra Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos. Cancer Research Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors Mafalda Laranjo*1,2,3,4, Maria João Carvalho*1,2,3,4,5,6, Beatriz Serambeque1,2,3, André Alves2,7, Carlos Miguel Marto1,2,3,4,8, Isabel Silva9, Artur Paiva3,9,10, Maria Filomena Botelho1,2,3,4,8 1Institute of Biophysics, Faculty of Medicine, University of Coimbra, 2Coimbra Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, 3CNC.IBILI/Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, 4Clinical Academic Center of Coimbra (CACC), 5Universitary Clinic of Gynecology, Faculty of Medicine, University of Coimbra, 6Gynecology A Service, Coimbra Hospital and Universitary Center, 7Institute of Pharmacology & Experimental Therapeutics, Faculty of Medicine, University of Coimbra, 8Institute of Experimental Pathology, Faculty of Medicine, University of Coimbra, 9Cytometry Operational Management Unit, Clinical Pathology Service, Coimbra Hospital and Universitary Center, 10Polytechnic Institute of Coimbra, ESTESC-Coimbra Health School, Laboratory Biomedical Sciences The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot. Developmental Biology Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors Rita Silvério-Alves1,2,3, Andreia M. Gomes3, Ilia Kurochkin4, Kateri A. Moore5,6, Carlos-Filipe Pereira1,2,3 1Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, 2Wallenberg Center for Molecular, Lund University, 3Center for Neuroscience and Cell Biology, University of Coimbra, 4Skolkovo Institute of Science and Technology, 5Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 6Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells. Biochemistry Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization Xavier Morató*1,2, Marc López-Cano*1,2, Paula M. Canas3, Rodrigo A. Cunha3, Francisco Ciruela1,2 1Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, 2Institut de Neurociències, Universitat de Barcelona, 3Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. Developmental Biology Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program Michael G. Daniel1,2,3, Carlos-Filipe Pereira4, Jeffrey M. Bernitz1,2,3, Ihor R. Lemischka1,3,5, Kateri Moore1,3 1Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 2The Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, 3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, 4Center for Neuroscience and Cell Biology, University of Coimbra, 5Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.
