STRAHLENTHERAPIE

05

Laboratory of Radiobiology

 

Laboratory of Radiobiology

The associated Laboratory of Radiobiology conducted several projects in the field of nano-technology and tissue engineering funded by the Austrian Nano-Initiative (FFG) or by other peer reviewed governmental grants. The team, headed by Thomas Seppi, is experienced in radiobiology, analytical chemistry, nano-coatings in biomedical applications, laser-optical cell analyses, electron-microscopy protocols, designing and prototyping of advanced cell culture models, as well as in molecular biology and toxicology of cancer cells. Since 1998, 13 doctoral and 11 diploma theses have been supervised in the fields of analytical chemistry, radiobiology, molecular cell biology, and cancer research. Since then, scientific personnel and equipment has mainly been financed by granted fellowships.

Lab-members:

  • Thomas Seppi (Head of Laboratory)
  • Daniel Hekl
  • Oliver Eiter
  • Thomas Schmiedinger

Research areas

Currently, the research group is engaged in projects related to nano-particle applications in vitro, modelling of microfluidics, electro-spinning of nano-fibrous cell culture substrates, as well as in developing cell-to-electrode interfaces (impedance and TEER-analyses) at micron-scales.  

Nano-Particles

A main objective of ongoing projects – performed in collaboration with several local and international partners – is to synthesize advanced nanoparticles (NPs) composed of a coated super-paramagnetic iron oxide core (SPIOs), to accommodate chemotherapeutics on the surface of NPs, and to investigate the potential of inducing drug release by gamma-ray/proton dilation as a trigger modality. NPs made of heavy metals, such as gold, may enhance the efficacy of cancer radiotherapy by increasing the local absorption of photon as well as proton radiation. SPIOs can be detected by magnetic resonance imaging, and thus, could be coupled to gold to enable easily applicable cancer theranostics. In particular, the core magnetic properties of NPs will allow the identification of their bio-distribution in tumour tissues by MRT, whereas gold coating of NPs is used to increase photon energy deposition during radiotherapy of cancer. Investigations to study the effects of concomitant NP-treatment in comparison to conventional RT alone are performed in vitro by using in-house fabricated advanced cell culture models specifically developed to maintain 3D-tumour cell constructs. For this purpose, funded research is focussed on the micro-fluidic and extracellular requirements of three-dimensionally arranged tumour cell aggregates maintained in vitro. Therefore, a special emphasis is placed by the research group on the study of the contribution of functionalised bio-surfaces in enhancing cell adhesion and in protracting culture protocols in order to enable longterm treatment investigations of fractionated radiotherapy in vitro.



Human aortic endothelial cells (HAEC) were loaded with fluorescently labelled nanoparticles (green – Alexa fluor 488) for 24h. The cell membranes are visualized by vGF (pink).


Metabolic activity of epithelial cells (LLC‐PK1) was measured by the Resazurin reduction assay. Cells were exposed to iron oxide nanoparticles for 24 h. No difference in metabolic activity was measured directly (left) and one week (right) after IONP exposure. References: Contrast Media & Molecular Imaging Volume 10, Issue 1, pages 18-27, 21 APR 2014 DOI: 10.1002/cmmi.1595 http://onlinelibrary.wiley.com/doi/10.1002/cmmi.1595/full#cmmi1595-fig-0010  

Modelling of microfluidics

The research project ‘i-scaff’ funded by the ‘Translational research fund – Land Tirol’ investigates the interaction of cell behaviour and fluidics. One aim of the project is to develop and characterise an alternative artificial extra-cellular matrix (ECM) model of defined composition and architecture. Two approaches will be followed to realize ECM-like growth substrates. By applying Electrospinning procedures on one hand, it is possible to fabricate nano- and microscaled filament structures of virtually any kind of synthetic and biopolymer compounds. Thereby obtained (sub-)microfibrous scaffolds are of comparable texture as native ECM structures in vivo. In addition, biochemical modification of the (bio)polymer fibres /meshes will be applied to enhance the in-vivo similarity of the artificial ECM constructs on top of their structural architecture. On the other hand, nanocrystalline diamond (NCD) coated substrates will be used to model nanotopographical features of the ECM. Nanocrystalline diamond is inert, non-cytotoxicand biocompatible in-vivo as well as in-vitro, and thus one of the most advantageous materials for nanoscaled structuring of biointerfaces. The second ambition of the project is to design a novel bioreactor concept primarily based on numerical modelling of the fluidic dynamics. For this purpose, computational fluid dynamic (CFD) simulations will be applied, a technique which enables the integration of all relevant parameters determining the fluidic characteristics of a certain chamber design. Macro- and microfluidic modifications deduced from CFD calculations will be transcribed to prototyping and validated by powerful non-interfering Laser Doppler Anemometry (LDA) and Particle Image Velocimetry (PIV) techniques to accurately record the 3D flow-field inside the novel bioreactor. Especially the combination of both – ECM-like structures and perfusion conditions – may provide a powerful investigative bio-model to scientist, whenever a higher degree of in-vivo-like mimicking under continuously defined culture conditions essentially are requested. This is the case for all metabolism-related studies of cell response like distinct gene expression upon delivery of drugs, irradiation or toxic compounds. Such a novel bio-model has the potential to fill the huge gap between rudimental batch cultures and the ethically controversial animal model. Neither the one nor the other allow for modifying distinct incubation parameters kept constant over the entire period of cell incubation, whereas this is the case for the presented concept to be developed during the proposed project work.


Three dimensional flow field within a modelled bioreactor (streamlines and colour coded velocities).  

Electro-spinning of nano-fibrous cell culture substrates

Electrospinning is a versatile method for the fabrication of nano- and microscaled fibrous meshes. An electrospinning device consists of the components polymer reservoir, pumping device, nozzle, collector, and high-voltage generator.



A huge number of different polymers were tested for their applicability to generate electrospun matrices. In common for all electrospinning materials is that they have to be liquefied prior to the electrospinning process. In general, there are two options for liquefying a polymer: (i) dissolution by a solvent or (ii) melting the polymer. The resulting polymer solution is loaded into the polymer reservoir. A pumping device transports the polymer solution to the nozzle. For a given vertical setup as shown in Figure 1, a conducting collector is positioned beneath the nozzle at a defined distance. Applying a high voltage (several kilovolts) between nozzle and collector, forces the polymer solution towards the collector. During the deposition process, the solvent evaporates resulting in the deposition of fibres on the surface of the collector. The process is controlled by adjusting the parameters voltage, distance between nozzle and collector, feeding rate, and polymer concentration. The team of the Laboratory of Radiobiology (Department of Therapeutic Radiology and Oncology) designed and fabricated an electrospinning apparatus.



In-house made electrospinning apparatus: (A) Scheme of the electrospinning process for gelatine meshes. (B) Nozzle and collector of the electrospinning device (gfm – gelatine fibrous matrice) – scale bar 10 cm. (C) Overview of electrospinning apparatus. The electrospinning device is utilized to fabricate gelatine meshes used as growth substrate for in-vitro experiments. The mesh architecture mimics the structure of native extracellular matrix (ECM) and better reflects the in-vivo situation in comparison to standardized two-dimensional culture dishes. In addition, we could show that the meshes can be used as collecting substrate for cryo-immunoelectron microscopy. (Schmiedinger et al. (2013), Cryo-Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates. Traffic, 14: 886–894. doi: 10.1111/tra.12080).



Scanning-electron microscopy image of glioblastoma cells adhering on a gelatine-fibrous mesh. References: Traffic Volume 14, Issue 8, pages 886-894, 23 MAY 2013 DOI: 10.1111/tra.12080 http://onlinelibrary.wiley.com/doi/10.1111/tra.12080/full#tra12080-fig-0001

Cell-to-electrode interfaces - Biosensors

Together with the ‘Research Centre for Microtechnology’ of the ‘Vorarlberg University of Applied Sciences’ (http://www.fhv.at/fhv-science/microtechnology?set_language=en), we developed interdigitated bio-sensors for assessing cellular behaviour continuous and in real-time. Electric cell-substrate impedance sensing (ECIS) is a novel method for measuring cell behaviour electrically. The great advantage of this technique is its non-invasiveness. Therefore, cell harvesting – as usual for traditional biochemical cell assays – is not necessary. To further increase the significance of this method, we scanned the impedance in a broader frequency range generating impedance spectra. These spectra can be further analysed by equivalent electric circuit modelling and numerical data fitting algorithm to calculate cell-growth specific parameters like cell-adhesion strength, cell-layer tightness and cell proliferation rate. Ongoing work is done focusing on automatic data acquisition and analysis procedures. Especially the design of the electrical cell models is a main topic of our work.  

Selected Publications

  • Paper-Judith Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System. Vogel GF, Ebner HL, de Araujo ME, Schmiedinger T, Eiter O, Pircher H, Gutleben K, Witting B, Teis D, Huber LA, Hess MW. TRAFFIC. 2015; 16:p. 617–34
  • Nucleophilic cross-linked, dextran coated iron oxide nanoparticles as basis for molecular imaging: synthesis, characterization, visualization and comparison with previous product. Borny R, Lechleitner TW, Schmiedinger T, Hermann M, Tessadri R, Redhammer G, Neumüller J, Kerjaschki D, Berzaczy G, Erman G, Popovic M, Lammer J, Funovics M. CONTRAST MEDIA MOL IMAGING. 2014; 10: S.18–27.
  • Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates. Schmiedinger T, Vogel GF, Eiter O, Pfaller K, Kaufmann WA, Flörl A, Gutleben K, Schönherr S, Witting B, Lechleitner TW, Ebner HL, Seppi T, Hess MW. TRAFFIC. 2013; 8: p. 886–93.

Selected Funding

  • NanoDisc-Biomedical application of nano-surfaces on CD-formates, FFG-818050, Seppi T, Lechleitner T (588,700 €)
  • i-Scaff – Intelligent scaffolds of electro-spun nano-fibres in advanced cell culture models, TZS-Translational Research Program, Seppi T, Schmiedinger T (294,000 €)
  • Multiwell-Discs with reference microstructures, FFG-833467, Schmiedinger T (117,000 €)

Collaborations

  • Fachhochschule Vorarlberg, Feldkirch, Austria
  • Unit of Hydraulic Engineering, University of InnsbruckI; Innsbruck,
  • Institute of Physics, Academy of Sciences Prague, Prague, Czech Republic



References:

  • Contrast Media & Molecular Imaging
    Volume 10, Issue 1, pages 18-27, 21 APR 2014 DOI: 10.1002/cmmi.1595
    http://onlinelibrary.wiley.com/doi/10.1002/cmmi.1595/full#cmmi1595-fig-0010
  • Traffic
    Volume 14, Issue 8, pages 886-894, 23 MAY 2013 DOI: 10.1111/tra.12080
    http://onlinelibrary.wiley.com/doi/10.1111/tra.12080/full#tra12080-fig-0001