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3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons.
Tong, Zhi-Bin; Huang, Ruili; Braisted, John; Chu, Pei-Hsuan; Simeonov, Anton; Gerhold, David L.
Afiliación
  • Tong ZB; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
  • Huang R; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
  • Braisted J; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
  • Chu PH; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
  • Simeonov A; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
  • Gerhold DL; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States. Electronic address: david.gerhold@nih.gov.
SLAS Discov ; 29(3): 100143, 2024 Apr.
Article en En | MEDLINE | ID: mdl-38280460
ABSTRACT
Three-dimensional (3D) cell culture in vitro promises to improve representation of neuron physiology in vivo. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, DCX, DRD2, ENO2, NEUROD4, SYN1, TH, and TUBB3. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes MT1G, MT1E and MT2A at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC50 values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC50 values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z' score of 0.68.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ensayos Analíticos de Alto Rendimiento / Neuronas Dopaminérgicas Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Humans Idioma: En Revista: SLAS Discov Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ensayos Analíticos de Alto Rendimiento / Neuronas Dopaminérgicas Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Humans Idioma: En Revista: SLAS Discov Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos