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University of Pisa

12 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry
Olimpia Gamucci 1, Alice Bertero 1,2, Maria Ada Malvindi 3, Stefania Sabella 3, Pier Paolo Pompa 3, Barbara Mazzolai 1, Giuseppe Bardi 1
1Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia, 2Department of Biology, University of Pisa, 3Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

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Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow
Marina Montali *1, Serena Barachini *1, Simone Pacini 1, Francesca M. Panvini 1, Mario Petrini 1
1Department of Clinical and Experimental Medicine, University of Pisa

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

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Medicine

Ultrasound-based Pulse Wave Velocity Evaluation in Mice
Nicole Di Lascio 1,2, Claudia Kusmic 2, Francesco Stea 2,3, Francesco Faita 2
1Institute of Life Science, Scuola Superiore Sant'Anna, 2Institute of Clinical Physiology, National Research Council, 3Department of Clinical and Experimental Medicine, University of Pisa

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

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Cancer Research

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
Rocco Sciarrillo 1,2,3, Anna Wojtuszkiewicz 1, Irsan E. Kooi 4, Valentina E. Gómez 3, Ugo Boggi 5, Gerrit Jansen 6, Gert-Jan Kaspers 1,7, Jacqueline Cloos *1, Elisa Giovannetti *3,8,9
1Department of Pediatric Oncology/Hematology, VU University Medical Center, 2Department of Hematology, VU University Medical Center, 3Department of Medical Oncology, VU University Medical Center, 4Department of Clinical Genetics, VU University Medical Center, 5Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana, Universita’ di Pisa, 6Amsterdam Immunology and Rheumatology Center, VU University Medical Center, 7Princess Máxima Center for Pediatric Oncology, 8Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa, 9Institute of Nanoscience and Nanotechnology, CNR-Nano

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

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Bioengineering

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications
Luisa Trombi 1,2, Serena Danti 2,3, Sara Savelli 4, Stefania Moscato 1, Delfo D'Alessandro 2,5, Claudio Ricci 2, Stefano Giannotti 6, Mario Petrini 1
1Dept. of Clinical and Experimental Medicine, University of Pisa, 2OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP), 3Dept. of Civil and Industrial Engineering, University of Pisa, 4Immunohematology Operative Unit, Azienda Ospedaliero-Universitaria Pisana (AOUP), 5Dept. Of Surgical, Medical, Molecular Pathology and Emergency Medicine, University of Pisa, 6II Orthopedic and Traumatologic Clinic, Azienda Ospedaliero-Universitaria Pisana (AOUP)

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

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Neuroscience

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation
Maria Paola Tramonti Fantozzi *1, Tommaso Banfi *2,3, Vincenzo De Cicco *1, Massimo Barresi 4, Enrico Cataldo 5, Davide De Cicco 1, Luca Bruschini 6, Paola d'Ascanio 1, Gastone Ciuti 2,3, Ugo Faraguna 1,7, Diego Manzoni 1
1Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, 2The BioRobotics Institute, Scuola Superiore Sant'Anna, 3Department of Excellence in Robotics & AI, Scuola Superiore Sant'Anna, 4Institut des Maladie Neurodegeneratives, University of Bordeaux, 5Department of Physics, University of Pisa, 6Department of Surgical, Medical, Molecular Pathology and Critical Care Medicine, University of Pisa, 7Department of Developmental Neuroscience, IRCCS Foundation Stella Maris

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

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Medicine

Robot-Assisted Radical Antegrade Modular Pancreatosplenectomy Including Resection and Reconstruction of the Spleno-Mesenteric Junction
Niccolò Napoli 1, Emanuele F. Kauffmann 1, Francesca Menonna 1, Sara Iacopi 1, Concetta Cacace 1, Ugo Boggi 1
1Division of General and Transplant Surgery, University of Pisa

The robotic technique shown herein aims at faithfully reproducing the open procedure for radical treatment of cancer of the body-tail of the pancreas. The protocol also demonstrates the ability to master involvement of major peripancreatic vessels without conversion to open surgery.

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Cancer Research

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing
Alice Usai 1, Gregorio Di Franco 2, Chiara Gabellini 1, Luca Morelli 2, Vittoria Raffa 1
1Department of Biology, University of Pisa, 2General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

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Medicine

Surgical Treatment of an Endolymphatic Sac Tumor
Luca Bruschini 1,2, Francesca Forli 1,2, Guido Andrea Lazzarotti 3, Antonella Borraccino 1,2, Mirco Cosottini 3,4, Stefano Berrettini 1,2,5, Francesco Lazzerini 1,6
1Otolaryngology, Audiology and Phoniatrics Operative Unit, University of Pisa, 2Department of Surgical, Medical and Molecular Pathology and Critical Care Medicine, University of Pisa, 3Neuroradiology Operative Unit, Pisa University Hospital, 4Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, 5Clinical Science, Intervention and Technology, Karolinska Institute, 6Department of Clinical and Experimental Medicine, University of Pisa

Here, we discuss clinical and radiological features of endolymphatic sac tumors and report the methodology and results of a surgical removal in a case in care at our otologic referral center.

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Genetics

Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes
Aurora Falaschi 1, Anna Chiaramonte 1,2, Serena Testi 1, Roberto Scarpato 1
1Unità di Genetica, Dipartimento di Biologia, University of Pisa, 2Department of Women-Child-Newborn Obstetrics and Gynaecology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

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Neuroscience

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging
Danila Di Meo *1, Josephine Ramazzotti *1, Marina Scardigli *1,2, Franco Cheli 1, Luca Pesce 1,3, Niamh Brady 1, Giacomo Mazzamuto 1,4,5, Irene Costantini 1,4,6, Francesco S. Pavone 1,4,5
1European Laboratory for Non-linear Spectroscopy (LENS), University of Florence, 2Division of Physiology, Department of Experimental and Clinical Medicine, University of Florence, 3Department of Physics, University of Pisa, 4National Research Council - National Institute of Optics (CNR-INO), 5Department of Physics and Astronomy, University of Florence, 6Department of Biology, University of Florence

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

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Neuroscience

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury
Francesca Merighi 1, Sara De Vincentiis 1, Marco Onorati 1, Vittoria Raffa 1
1Department of Biology, University of Pisa

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

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