The in vitro choice ellen iuf 2013


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The in vitro choice ellen iuf 2013

  1. 1. NEUROSPHERES AS 3D IN VITRO MODELS FOR ASSESSING DEVELOPMENTAL NEUROTOXICITY Prof. Ellen Fritsche IUF- Leibniz Research Institute for Environmental Medicine
  2. 2. IUF Mission: Molecular Preventive Medicine Düsseldorf, NRW, Germany Gottfried Wilhelm Leibniz Scientific Community
  3. 3. Group of Sphere Models and Risk Assessment Risk Assessment Molecular Mechanism Application Safety Testing Drug Development
  4. 4. The Neurosphere Method Neural Stem/Progenitor Cells (NPCs) Fetal Brain NPC Proliferation: -Increase in size/time -BrdU incorporation Culture as Neurospheres NPC Migration: -Total migration distance -number of migrated cells -neuronal migration NPC Differentiation: -Neurons -Astrocytes -Oligodendrocytes
  5. 5. Proliferating Human Neurospheres ß(III)Tubulin, Cx43, Nuclei
  6. 6. Migration Moors et al. Environ Health Perspect 2009
  7. 7. Differentiating Human Neurospheres ß(III)Tubulin, Cx43, Nuclei Coop: Ole Klotz
  8. 8. Neurosphere Proliferation HUMAN DAY 0 DAY 7 RAT DAY 0 HUMAN DAY 7 RAT Each sphere in a single well of a 96-well plate
  9. 9. Neurosphere Migration HUMAN HUMAN RAT RAT
  10. 10. Neurosphere Differentiation: Neurons HUMAN Hoechst ßIII-Tubulin HUMAN RAT Hoechst ßIII-Tubulin RAT
  11. 11. Neurosphere Differentiation: Oligodendrocytes RAT Oligodendrocytes (%) Oligodendrocytes (%) HUMAN
  12. 12. ß(III)tubulin/GFAP/DAPI RAT Synapsin/ß(III)tubulin/DAPI
  13. 13. The ‘Neurosphere-Assay’ Apoptosis Fritsche et al. Environ Health Perspect 2005 Moors et al. Toxicol Appl Pharmacol 2007 Moors et al. Environ Health Perspect 2009 Moors et al. Genes & Immunity 2010 Tegenge et al. Cell. Mol. Life Sci. 2010 Schreiber et al. Environ Health Perspect 2010 Gassmann et al. Environ Health Perspect 2010 Verner et al. Toxicol in Vitro 2011 BrdU Fritsche et al. Methods Mol Biol 2011 Gassmann et al. Toxicol in Vitro 2012 Bal-Price et al. ALTEX 2012 From Breier…Fritsche.. et al. Neurotoxicol Teratol 2009
  14. 14. The Neurosphere Method in Medium Throughput Medium Throughput: • Migration • Neuronal Differentiation • Neuronal Migration Cellomics ArrayScan: Nuclei and beta(III)tubulin stainings of a differentiated Human Neurosphere
  15. 15. Summary & Conclusion • Neurospheres of different species mimic processes of brain development in vitro. • Application domains of neurospheres include safety testing and drug development. • Species-specific VPA effects on rat and human neurospheres. • Medium throughput is possible for compound testing in human and rodent neurospheres. The 3D neurosphere assay can be used to study species-specific pathway-to-function effects (positive/negative) of compounds in a medium throughput fashion by automated plating and usage of ‘High Content Image Analyses’.
  16. 16. Acknowledgements Dr. Marta Barenys Dr. Kathrin Gassmann Dr. Julia Tigges Dr. Susanne Giersiefer Dr. Janette Schuwald Jenny Baumann Katharina Dach Martin Schmuck Maxi Hofrichter Katerina Kefalaki Stefan Masjosthusmann Thomas Temme Denise de Boer Ulrike Hübenthal Cooperations: High Content Imaging: Axel Mosig, University of Bochum Proteomics: Prof. Kai Stühler, HHU Düsseldorf LRI Innovative Science Award 2006 German Alternative Methods Award 2007 Leibniz DAAD Thank you for your attention!