Embryonic Stem-cells based Novel Alternative Testing Strategy

Institutions
  • FB Biologie
Publications
    Krug, Anne K.; Balmer, Nina V.; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel(2013): Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants Archives of Toxicology ; 87 (2013), 12. - S. 2215-2231. - ISSN 0340-5761. - eISSN 1432-0738

Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants

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Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Origin (projects)

  Henn, Anja; Lund, Søren; Hedtjärn, Maj; Schrattenholz, André; Pörzgen, Peter; Leist, Marcel(2009): The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation ALTEX : Alternatives to animal experimentation ; 26 (2009), 2. - S. 83-94

The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation

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The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies.

Origin (projects)

Funding sources
NameProject no.DescriptionPeriod
Europäische Union519/08no information
Further information
Period: 01.04.2008 – 31.03.2013