3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons
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 cultur...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-04-01
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| Series: | SLAS Discovery |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555224000054 |
| _version_ | 1797234371815538688 |
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| author | Zhi-Bin Tong Ruili Huang John Braisted Pei-Hsuan Chu Anton Simeonov David L. Gerhold |
| author_facet | Zhi-Bin Tong Ruili Huang John Braisted Pei-Hsuan Chu Anton Simeonov David L. Gerhold |
| author_sort | Zhi-Bin Tong |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T16:31:00Z |
| format | Article |
| id | doaj.art-ee1d3af8859b4431a448185a48cd089e |
| institution | Directory Open Access Journal |
| issn | 2472-5552 |
| language | English |
| last_indexed | 2024-04-24T16:31:00Z |
| publishDate | 2024-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj.art-ee1d3af8859b4431a448185a48cd089e2024-03-30T04:39:48ZengElsevierSLAS Discovery2472-55522024-04-012931001433D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neuronsZhi-Bin Tong0Ruili Huang1John Braisted2Pei-Hsuan Chu3Anton Simeonov4David L. Gerhold5Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesCorresponding author.; Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United StatesThree-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.http://www.sciencedirect.com/science/article/pii/S2472555224000054LUHMESParkinson's diseaseNeurospheroids3D cultureHigh throughput screening |
| spellingShingle | Zhi-Bin Tong Ruili Huang John Braisted Pei-Hsuan Chu Anton Simeonov David L. Gerhold 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons SLAS Discovery LUHMES Parkinson's disease Neurospheroids 3D culture High throughput screening |
| title | 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons |
| title_full | 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons |
| title_fullStr | 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons |
| title_full_unstemmed | 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons |
| title_short | 3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons |
| title_sort | 3d suspension culture platform for high throughput screening of neurotoxic chemicals using luhmes dopaminergic neurons |
| topic | LUHMES Parkinson's disease Neurospheroids 3D culture High throughput screening |
| url | http://www.sciencedirect.com/science/article/pii/S2472555224000054 |
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