University Hospital Wuerzburg

12 ARTICLES PUBLISHED IN JoVE

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Miriam Votteler^1,2, Daniel A. Carvajal Berrio², Marieke Pudlas^2,3, Heike Walles^2,4, Katja Schenke-Layland^1,2

¹Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST), Eberhard Karls University, Tübingen, ²Department of Cell and Tissue Engineering, Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany, ³Department for Medical Interfacial Engineering (IGVT), University of Stuttgart, Germany, ⁴Institute of Tissue Engineering and Regenerative Medicine, Julius-Maximillians University, Würzburg, Germany

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Bioengineering

Tissue Engineering of a Human 3D in vitro Tumor Test System

Corinna Moll^*1, Jenny Reboredo^*1, Thomas Schwarz¹, Antje Appelt¹, Sebastian Schürlein¹, Heike Walles¹, Sarah Nietzer¹

¹Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Bioengineering

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Angela Rossi^*1, Antje Appelt-Menzel^*1, Szymon Kurdyn¹, Heike Walles^1,2, Florian Groeber²

¹Department for Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, ²Translational Center Würzburg, Regenerative Therapies in Oncology and Musculoskelettal Disease, Würzburg Branch of the Fraunhofer-Institute Interfacial Engineering and Biotechnology, IGB

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Neuroscience

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Felix Fluri¹, Micheal Bieber¹, Jens Volkmann¹, Christoph Kleinschnitz¹

¹Department of Neurology, University Hospital Wuerzburg

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Bioengineering

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

Claudia Göttlich^*1, Lena C. Müller^*1, Meik Kunz^*3, Franziska Schmitt¹, Heike Walles^1,4, Thorsten Walles², Thomas Dandekar³, Gudrun Dandekar^1,4, Sarah L. Nietzer¹

¹Department of Tissue Engineering and Regenerative Medicine (TERM), University Hospital Wuerzburg, ²Department of Cardiothoracic Surgery, University Hospital Wuerzburg, ³Department of Bioinformatics, University Wuerzburg, ⁴Translational Center Wuerzburg, Fraunhofer Institute Interfacial Engineering and Biotechnology IGB

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Behavior

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Marta Rattka¹, Felix Fluri¹, Miloš Krstić¹, Esther Asan², Jens Volkmann¹

¹Department of Neurology, University Hospital Wuerzburg, ²Institute of Anatomy and Cell Biology, University Wuerzburg

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Neuroscience

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Chi Wang Ip¹, David Cheong¹, Jens Volkmann¹

¹Department of Neurology, University Hospital of Würzburg

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

Bioengineering

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Claudia Siverino¹, Barbara Tabisz², Tessa Lühmann³, Lorenz Meinel³, Thomas Müller⁴, Heike Walles^1,2, Joachim Nickel^1,2

¹Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik (IGB), Translationszentrum Würzburg 'Regenerative Therapien für Krebs- und Muskuloskelettale Erkrankung', Institutsteil Würzburg, ²Lehrstuhl für Tissue Engineering und Regenerative Medizin, Universitätsklinikum Würzburg, ³Lehrstuhl für Pharmazeutische Technologie und Biopharmazie, Universität Würzburg, ⁴Lehrstuhl für molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut für Biowissenschaften, Universität Würzburg

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Bioengineering

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Melva Suarez Muñoz¹, Davide Confalonieri¹, Heike Walles^1,2, Elisabeth M. W. M. van Dongen³, Gudrun Dandekar^1,2

¹Department Tissue Engineering and Regenerative Medicine, University Hospital Wuerzburg, ²Translational Center Regenerative Therapies (TLC-RT), Fraunhofer Institute for Silicate Research ISC, ³Fujifilm Manufacturing Europe B.V.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Neuroscience

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Lisa Rauschenberger¹, Susanne Knorr¹, Jens Volkmann¹, Chi Wang Ip¹

¹Department of Neurology, University Hospital of Würzburg

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

Neuroscience

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

Susanne Knorr¹, Lisa Rauschenberger¹, Tami Lang¹, Jens Volkmann¹, Chi Wang Ip¹

¹Department of Neurology, University Hospital of Würzburg

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

Neuroscience

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Maximilian Wulf^1,2, Katalin Barkovits-Boeddinghaus^1,2, Paula Sommer^1,2, Karin Schork^1,2, Martin Eisenacher^1,2, Peter Riederer^3,4, Manfred Gerlach⁵, Steffen Kösters^1,2, Britta Eggers^*1,2, Katrin Marcus^*1,2

¹Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum, ²Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, ³Center of Mental Health; Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, University Hospital Wuerzburg, ⁴Psychiatry Department of Clinical Research, University of Southern Denmark Odense University Hospital, ⁵Center of Mental Health, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Wuerzburg, University of Wuerzburg

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.