Automated neurosphere sorting and plating by the COPAS large particle sorter is a suitable method for high-throughput 3D in vitro applications.

Existing guidelines for testing developmental neurotoxicity (DNT) propose investigations in rodents, which are ethically questionable as well as time and cost intensive. Thus, there is international agreement that predictive in vitro methods are needed to increase efficiency of testing and limit the number of animals used. One of a variety of novel approaches for DNT testing utilizes neurospheres, three-dimensional aggregate cultures of primary normal neural progenitor cells (NPCs). Because sorting and plating of single neurospheres is one of the most time-consuming steps within the assay, the aim of this study was to evaluate if the complex object parametric analyzer and sorter (COPAS PLUS(TM), Union Biometrica Inc.) is a suitable tool for automated sorting and plating of neurospheres. The results of the comparison of NPC viability, proliferation, migration, differentiation and intracellular oxidative stress between manually and COPAS sorted and plated neurospheres of different species show that the automation by the COPAS instrument does not influence the basic performance of neurospheres. Therefore, we consider the COPAS instrument as a useful tool for higher throughput neurosphere research in toxicology, neuroregeneration, brain development, drug development and brain aging research.

[1]  Thomas Dino Rockel,et al.  Human Neurospheres as Three-Dimensional Cellular Systems for Developmental Neurotoxicity Testing , 2009, Environmental health perspectives.

[2]  R. Miller,et al.  Automated method for the isolation of collecting ducts. , 2006, American journal of physiology. Renal physiology.

[3]  C. Colton,et al.  Redox regulation of neuronal migration in a Down Syndrome model. , 2003, Free radical biology & medicine.

[4]  Melvin E Andersen,et al.  Toxicity testing in the 21st century: bringing the vision to life. , 2009, Toxicological sciences : an official journal of the Society of Toxicology.

[5]  Kenneth M. Yamada,et al.  Modeling Tissue Morphogenesis and Cancer in 3D , 2007, Cell.

[6]  Ellen Fritsche,et al.  Neurospheres as a model for developmental neurotoxicity testing. , 2011, Methods in molecular biology.

[7]  A. Goldberg.  Use of animals in research: a science--society controversy? The American perspective: animal welfare issues. , 2002, ALTEX.

[8]  D. Geerts,et al.  The role of the MEIS homeobox genes in neuroblastoma. , 2003, Cancer letters.

[9]  K. Ekdahl,et al.  Acute Antibody-Mediated Complement Activation Mediates Lysis of Pancreatic Islets Cells and May Cause Tissue Loss in Clinical Islet Transplantation , 2008, Transplantation.

[10]  G. Barja.  Minireview: The Quantitative Measurement of H2O2 Generation in Isolated Mitochondria , 2002, Journal of bioenergetics and biomembranes.

[11]  Leonora Buzanska,et al.  Developmental neurotoxicity testing: recommendations for developing alternative methods for the screening and prioritization of chemicals. , 2011, ALTEX.

[12]  J. Bailar,et al.  Toxicity Testing in the 21st Century: A Vision and a Strategy , 2010, Journal of toxicology and environmental health. Part B, Critical reviews.

[13]  Sangeeta N Bhatia,et al.  Multiplexed, high-throughput analysis of 3D microtissue suspensions. , 2010, Integrative biology : quantitative biosciences from nano to macro.

[14]  M. Balls.  The Origins and Early Days of the Three Rs Concept , 2009, Alternatives to laboratory animals : ATLA.

[15]  Ellen Fritsche,et al.  Polychlorinated Biphenyls Disturb Differentiation of Normal Human Neural Progenitor Cells: Clue for Involvement of Thyroid Hormone Receptors , 2005, Environmental health perspectives.

[16]  Thomas Dino Rockel,et al.  Species-Specific Differential AhR Expression Protects Human Neural Progenitor Cells against Developmental Neurotoxicity of PAHs , 2010, Environmental health perspectives.

[17]  D. D. de Groot,et al.  Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science. , 2010, Neurotoxicology and teratology.

[18]  Malin Parmar,et al.  Strengths and limitations of the neurosphere culture system , 2006, Molecular Neurobiology.

[19]  R. Hoskins,et al.  Exploring Strategies for Protein Trapping in Drosophila , 2007, Genetics.

[20]  M. Chevion,et al.  Evidence for production of hydroxyl radicals by pentachlorophenol metabolites and hydrogen peroxide: A metal-independent organic Fenton reaction. , 2000, Biochemical and biophysical research communications.

[21]  F. Wang,et al.  Pipetting causes shear stress and elevation of phosphorylated stress‐activated protein kinase/jun kinase in preimplantation embryos , 2007, Molecular reproduction and development.

[22]  Ellen Fritsche,et al.  Polybrominated Diphenyl Ethers Induce Developmental Neurotoxicity in a Human in Vitro Model: Evidence for Endocrine Disruption , 2009, Environmental health perspectives.

[23]  Clive N Svendsen,et al.  A new method for the rapid and long term growth of human neural precursor cells , 1998, Journal of Neuroscience Methods.

[24]  B. Dietzek,et al.  Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches[S] , 2011, Journal of Lipid Research.

[25]  E. vanBavel,et al.  Shear stress, reactive oxygen species, and arterial structure and function. , 2009, Antioxidants & redox signaling.

[26]  M. Ingram,et al.  Tissue engineered tumor models , 2010, Biotechnic & histochemistry : official publication of the Biological Stain Commission.

[27]  Ellen Fritsche,et al.  ERK-dependent and -independent pathways trigger human neural progenitor cell migration. , 2007, Toxicology and applied pharmacology.

[28]  P. Landrigan,et al.  Developmental neurotoxicity of industrial chemicals , 2006, The Lancet.

[29]  Qihong Zhou,et al.  Effect of oxidative preconditioning on neural progenitor cells , 2008, Brain Research.

[30]  J. Wands,et al.  Aspartyl-(asparaginyl) β-Hydroxylase, Hypoxia-Inducible Factor-1α and Notch Cross-Talk in Regulating Neuronal Motility , 2010, Oxidative medicine and cellular longevity.

[31]  Robert J Kavlock,et al.  Toxicity Testing in the 21st Century: Implications for Human Health Risk Assessment , 2009, Risk analysis : an official publication of the Society for Risk Analysis.