cell culture - med uni graz · animal-free culture models are mandatory for translational research...
TRANSCRIPT
Alternative Methods at the Medical University of Graz
EU regulations, the Austrian legislator and innovative research approaches require new dimensions for the implementation of the three Rs (3Rs). The 3Rs „Replacement“,„Reduction“ and „Refinement“ are guiding principles for more ethical use of animals in testing. This folder gives an overview of the alternative methods available at the Medical University of Graz, initiated by the Department for Biomedical Research, Core Facility Alternative Methods and Preclinical Imaging.
The 3Rs at the Medical University of Graz
http://www.reprefred.eu/en/welcome.html
In addition the association “Verein zur Förderung von alternativen Biomodellen” (RepRefRed society) was founded by members of the Medical Universities of Graz, Innsburck and Vienna with the purpose to facilitate alternative means to animal experiments, crit-ically analyze them and be a platform for diploma and PhD students to offer them prospects to learn different techniques. Also, our association is a network for knowledge transfer between scientists and thus enhances animal experiment protocols and enforces the reduction and refinement of these experiments.
cell culture
cell culture
Cell BankThe Cell Bank Graz offers a wide range of services and competent technical support for cell and tissue cultures. The Core Facility’s ISO certification ensures comprehensive quality control of cell lines and a con-trolled standardized storage system.
The Core Facility specializes in the establishment of ad-equate cell culture models for rare tumor diseases and their surrounding tissue. Due to the proximity to the University Hospital Graz, the good cooperation of the different disciplines and the Comprehensive Cancer Center, an efficient transfer of the biopsy tissue, a rapid isolation of the cells, a reduction of the ischemia time and a comprehensive know-how is guaranteed. Furthermore, our facility offers the possibility for High Throughput Screening to test compounds and compound libraries: a cytostatic bench, automated liquid handler and dispenser are available. Continuous monitoring of prolif-eration and migration is possible without labelling of cells with the xCELLigence Real Time Cell Analysis Instrument.
Rare Cancer Cell Line Center (RCCLC)
Assoc. Prof. Beate Rinner, PhDDepartment for Biomedical Research
Phone: +43 316 385 [email protected]
In order to reflect the in vivo state as closely as possi-ble, conventional cell culture systems must be optimally adapted. 3D models such as spheroids, co-cultures and organoids show great potential in many applications, including disease modelling in regenerative medicine.
Furthermore, adequate 3D models offer the possibility to investigate complex biology in a physiologically relevant context. The dynamic cell culture system used at the Core Facility is the Kirkstall system, for the expansion and culti-vation of cells we use bioreactors from Cellab.
COMPLEX CELL BIOLOGY Assoc. Prof. Beate Rinner, PhDDepartment for Biomedical Research
Phone: +43 316 385 [email protected]
BIOBANK GRAZBiobank Graz is a central research infrastructure at the Medical University of Graz responsible for collecting, pro-cessing, storing and delivering high quality biological sam-ples and associated data for research.The biological resources include, among others, FFPE (for-malin-fixed paraffin-embedded) tissues, cryopreserved tis-sues and biological fluids. The catalogue can be accessed via http://catalog.bbmri.at/Special attention is paid to quality. The facility is ISO 9001:2015 certified and prospective fluids’ collection
meets CEN/TS requirements. Samples and data are col-lected and stored according to all ELSI standards.Biobank Graz provides managerial support (e.g. project implementation) as well as technical support (e.g. prepa-ration of derivatives) to academic and industrial research.
Karine Sargsyan, MDBiobank Graz
Phone: +43 316 385 [email protected]
BARRIER MODELS
The body and its organs are protected from uncontrolled infiltration of substances by epithelial barriers. Outer barriers avoid contact with the environment (epidermis, cornea, as well as respiratory, oral and urogenital muco - sae). Inner barriers separate inner organs and blood and exist between lymphatic organs, reproductive organs and brain. Permeation and inflammation at barriers can be assessed by in vitro models.
Respiratory tract
Calu-3 cells on membranes cultured at an air-liquid interface culture
Caco-2 cells on membrane inserts - with and without mucus - overlay
Static: hcMEC/D3+primary astrocytes on membranes Dynamic: CELLMAX® DUO bioreactor
Intestinal tract Blood-brain barrier
Eleonore Fröhlich, MD Center for Medical Research Phone: +43 316 385 73011
ORGANOTYPIC CULTURES
Hippocampus OTC
Cortex in vivo
Colliculus superior
LGNNissl stained Cortex
Cortex OTC
Red-greenstaining10 DIV Hippocampus OTC (LSM)
Pyramidal neuron
Silke Patz, PhD Department of Neurosurgery Phone: +43 316 385 72920
In conventional cell culture cells rapidly lose their tis-sue-specific properties and are thus less useful for drug tests and for studying molecular interactions within a tissue.Therefore it is worthwhile to work with three-di-mensional cultures.These organotypic cultures possess the natural, mechani-cal and chemical environment and thus come very close to the in vivo situation.Here organotypic brain slice cultures have been chosen to illustrate the method. Several organs (kidney, liver, lung, heart), as well as (also human) cancers (glioblastoma mul-tiforme, breast cancer, gastric cancer) can be used as or-ganotypic cultures.Use of organotypic cultures leads to a reduction of ani-mals, because every animal gives rise to several organo-typic cultures that can be treated in different ways (for example cytotoxicity testing). Further biochemical, morphological and physiological changes can be measured in a few organotypic cultures instead instead of sacrificing an animal for every single question.
CELL-BASED TOXICITY ASSAYS
Genotoxicity
Hemocompatibility
Immunotoxicity
Chronic cytotoxicity
Assays for: Mutagenesis, Clastogenic and aneugenic changes, DNA repair mechanism
Assays for: Hemolysis, Plasmatic coagulation, Platelet activation, Complement activation
Assays for: Phagocyte function (NO generation, respiratory burst, chemotaxis, phagocytosis, surface markers), Panels of cytokines
3D culture of cells on: Microbeads in benchtop biorector, Membranes at an air-liquid interface
Eleonore Fröhlich, MD Center for Medical Research Phone: +43 316 385 73011
The Flexcell® FX5K™ Tension System is a computer-regulated bio-reactor that applies cyclic or static tensile strains to cells cultured in vitro. The system uses regulated vacuum pressure to deform flexi-ble-bottomed culture plates producing up to 25% substrate elonga-tion.Multiple frequency, amplitude and waveform changes can be pro-grammed in one regimen to simulate in vivo tissue strains and fre-quencies in cells from muscle, lung, heart, blood vessels, skin, ten-don, ligament, cartilage and bone. Available wave forms are static, sinusoidal, heart stimulation, triangular, square, and custom.Mechanical stimulation is operated by four independent FlexLink re-mote compression and/or tension controllers in 6 well size.BioFlex® Culture Plates are optically clear for direct viewing of cells with inverted or upright microscopes and have a total growth surface area of 57.75 cm2. Available matrix surfaces are Amino, Collagen (Type I or IV), Elastin, Pronectin® (RGD), and Laminin (YIGSR). Ad-ditionally we have the HT BioFlex® 24 well culture plate with a total growth surface area of 34.86 cm2
FLEXCELL® FX5K™ TENSION SYSTEM
Birgit Lohberger, PhDDepartment of Orthopedics and Trauma
Phone: +43 316 385 [email protected]
alternative models
alternative models
CAM ASSAY
The avian chorioallantoic membrane (CAM) assay provides an alternative versatile and ethically less objectionable in vivo model for many applications.
Disadvantages:
• Short observation time• Species-related differences
Advantages:
• Cost effectiveness• Natural
immunodeficiency• Quick results• Easily reproducible• No animal
experiment in Austria/EU
• 3R (replacement, reduction and refinement)
• Alternative drug metabolism/pharmacokinetics when compared to other in vivo (mammalian) models
• Oral drug administration cannot be tested• Non-specific inflammatory reactions may
induce a secondary vaso-proliferative response
Nassim Ghaffari Tabrizi-Wizsy, PhDInstitute of Pathophysiology and Immunology
Phone: +43 316 385 [email protected]
PERFUSION OF THE HUMAN PLACENTA
Spatio-temporal development of pregnancy and placenta in animals differs substantially from humans. Results from animal models to study transfer of substances across the placenta lack significance. The ex vivo perfusion of a single hu-man placental lobule is the most reliable experimental model.
Technology
• Intact human tissue, different biological barriers • To determine uptake, efflux and transfer of substances across
distinct barriers• To study transfer kinetics of substances and metabolites• Easy accessible tissue, no ethical concerns exist
Characteristics
• A reliable model for the transfer of substances from the mother to the fetus in late pregnancy
• To imitate late placental physiology ex vivo over several hours• A dual system with completely separated maternal/fetal circuits• Physiological conditions for different circuits: medium, plasma,
constant body temperature, pressure control and variable O2 concentrations
• Placental tissue from different pathophysiology available
Christian Wadsack, PhDDepartment of Obstetrics & Gynecology
Phone: +43 316 385 81074 [email protected]
PROMETHEUS NETWORK
In cooperation with COREMED and HEALTH (JOANNEUM RESEARCH Forschungsgesellschaft mbH)
Prof. Lars-Peter Kamolz, MD Division of Plastic, Aesthetic and Reconstructive Surgery
Department of Surgery, Medical University GrazPhone: +43 316 385 14685 / [email protected]
Research areas:
Models:
Wound healing and ScarringInflammationSkin aging and Anti-aging
In vitro models
• Monocultures, Co-cultures• 3D Skin models• Wound healing assaysEx vivo models (human)
Complementary to preclinical and clinical studies (Human skin explants for ex vivo testing of different treatment options)• Histologic and molecular characterisation of biopsies• Open flow microperfusion (OFM): Biomarker analysis
(in cooperation with JOANNEUM RESEARCH)
HUMAN PLATELET LYSATE
Petra Krakowitzky, MD Department of Blood Group Serology
and Transfusion Medicine Phone: +43 316 385 82032
Animal-free culture models are mandatory for translational research and the clin-ical application of cell culture products. Consequently, human platelet lysate is replacing the previously used fetal bovine serum in various research fields, espe-cially for mesenchymal stroma cell expansion and in other areas of regenerative medicine. The Department of Blood Group Serology & Transfusion Medicine is offering a pooled human platelet lysate for research only, manufactured from fresh platelet concentrates in ISO 9001:2015 certified laboratories. The “O platelet in AB plasma” human platelet lysate is highly standardized and aliquots are delivered with an extensive quality certificate. On special request, a GMP-grade O/AB HPL is also available.
The rat and mouse dummies (Fig. d) accurate-ly reproduce the anatomy of the animals, and the number of deaths for autopsy can be substantially reduced.
Full Body Skeleton Specimen of a mouse (Fig. e) support demonstration of the animals anatomy.Practicing blood sampling on rabbits itself is a big bur-den on the animal. The silicone model of the ear (Fig f) is a proven replacement method.
We have pursued the following targets:a) to reduce burden (severity level) on the animals by non-experienced students and traineesb) to reduce the number of animals effectively and, at the same time, to improve the quality of education by making frequent repetitions of a technique possibleDummy pig`s head:On the dummy pig’s head, the students can practice the following techniques with unlimited number ofrepetitions:a) Venous blood extraction from the ear (replaceable rub-
ber tube) and placing a venous/arterial catheter in the “ear vein” /”artery of the ear”
b) placing an arterial catheter in the common carotid arteryc) Endotracheal intubation
DUMMIES INSTEAD OF ANIMALS IN EDUCATION
Fig.a Fig.b Fig.c
Fig.d
Fig.fFig.e
Aida Saric, Dipl. vetDepartment for Biomedical Research
Phone: +43 316 385 78020 [email protected]
modeling / in silico
modeling / in silico
Experimental Design• unbiased, adequately powered, having a wide range of
applicability, simple and efficient designs, applicable to to a statistical analysis
• proper sample size calculation in accordance with the principles of 3R’s to ensure that animal studies are suf-ficiently powered to produce reliable and predictive re-sults
• power analysis for given sample sizes and effects of variation in retrospective studies
• consulting: providing project support from the first con-cept until the interpretation of your data
Data Analysis• state-of-the art know how for bioinformatics and statis-
tical data analysis• training: a broad range of postgraduate hands-on
training courses focused on bioinformatics and biosta-tistics (https://www.me-dunigraz.at/medical-re-search-academy-graz)
• a growing number of tools and pipelines for data pro-cessing and analysis
EXPERIMENTAL DESIGN AND DATA ANALYSIS
• access to a web-based platform for accessible, repro-ducible, and transparent computational biological re-search (https://galaxy.medunigraz.at)
• access to a High Performance Computing Cluster for comprehensive data analysis
The facility is ISO 9001:2015 certified to ensure safe, reli-able and excellent quality services and methods. As part-ner of the Austrian Bioinformatics Platform (ATBI http://www.bioinformatik) and as a member of the Austrian Statistical Society (ÖSG http://www.osg.or.at), we are cooperating closely with bioinformatics and biostatistics research groups in the development and establishment of new methods and make them available to all users.
Andrea Groselj-Strele, PhD Core Facility Computational Bioanalytics
Phone: +43 316 385 [email protected]
IN SILICO MODELING AND INTEGRATING OPEN DATA
We are working on the de-sign and development of al-gorithms, methods and tools to support understanding the underlying principles of disease, particularly cancer.We follow the assumption that the key to understand-ing the concepts of cancer lies within an integrative translation and multi-dimensional connection of data sets.Computational approaches to biomedical analysis and simulation involve several techniques such as validation, classification, inference, prediction and modeling. Existing well-maintained databases provide, integrate and anno-tate information on various diseases and are increasingly being used to generate predictive models, which in turn will inform and guide biomedical experiments.
Assoc. Prof. Andreas Holzinger, PhDInstitute for Medical Informatics,
Statistics & DocumentationPhone: +43 316 385 13883
https://hci-kdd.org/project/tumor-growth-machine-learning/
CARDIAC IN SILICO MODELS
Overview
Anatomically accurate and biophysically detailed in silico models of the heart are able to replicate cardiac function under a broad range of experimental or clinical conditions. Models of cardiac tissues or the entire organ are being used to complement or even replace experimental models such as single cell patch clamp, tissue monolayers, Langendorff or working heart models for a wide range of species, from mice to humans. Capabilities
• Advanced model building workflows comprising image based finite element meshing and cardiac navigation systems for local feature control
• Multiphysics simulation engine covering cardiac electro-physiology, biomechanics and hemodynamics
• Cellular dynamics and biomaterial models for various species
• Advanced closed loop hemodynamic afterload models • FSI models for aortic or coronary flow simulations• Data analysis tools for biomarker extraction• Models of pacing, defibrillation, cardiac resynchroniza-
tion and valve therapies• Parameterization and data assimilation techniques to
match simulation setups with experimental or clinical data
Univ.-Prof. Gernot Plank, PhDGottfried Schatz Research Center, Division of Biophysics
Phone: +43 316 385 [email protected]
preclinical imaging
preclinical imaging
3Rs AND PRECLINICAL MRI
In general, MRI is able to enhance the informa-tiveness of all in vivo studies in addition to • Being non invasive
• multiple in vivo readouts (eg. spatial/volumet-ric information, metabolite spectroscopy, blood perfusion) during a single imaging session
• following therapeutic effects over a period of time in the same animal (each animal serves as its own control)
The 7T system is optimized for the use on mice and rats. In vivo MRI protocols cover:
• Cardiovascular imaging
• Quantitative blood flow measurements
• Quantitative perfusion measurements
• Volumetric/quantitative fat imaging
• 1H spectroscopy of brain metabolites
• Water diffusion and diffusion tensor imaging
• Tumor imaging and cancer staging
• Cell tracking
Clemens Diwoky, PhDInstitute of Molecular Biosciences, University of Graz
Phone: +43 316 380 [email protected]
MAGNETIC RESONANCE IMAGING
In close cooperation with the Medical University of Graz the core facility for preclinical MRI operates a 7 Tesla dedicated small animal MRI system at the Institute of Molecular Biosciences, University of Graz.
PRECLINICAL IMAGING
Micro computed tomography is a high-resolution, non-in-vasive radiographic imaging technique that can be used for both pathological and physiological analysis in vivo as well as surface and structural analysis of materials and implants. Thanks to well-developed contrast agents, it is possible not only to visualize bones but also to examine soft tissues.The in vivo resolution with the least value is 15 mikron.Some possible applications are bone densidometry, cancer research and whole body fat measurement.
The ultrasound system Vevo® 3100 by FUJIFILM Visual-Sonics is our newest instrument. Using this non-invasive in vivo imaging technology, the monitoring of long-term studies as well as imaging guided applications help reduc-ing the number of animals necessary.The Vevo® 3100 is the first ultrasound system with a touchscreen panel and every transducer can be used in 3D and 4D mode. For monitoring the important parameters we have a physiological monitoring unit.The main fields of application are cardiology research and cancer research.
The Optical Imaging system effective variant for fluorescence-based in vivo imaging. Features of this system include multispectral data acquisition, data analysis, enhanced autofluorescence sen-sitivity and qualitative analysis of fluoro-phores.
Assoc. Prof. Beate Rinner, PhDDepartment for Biomedical Research
Phone: +43 316 385 [email protected]
