High-Content Assay for 3D Neurons
High-Content Assay for Morphological Characterization of 3D Neuronal Networks in the OrganoPlate
- Establish a high-throughput 3D neurite outgrowth assay using iPSC-derived neurons
- Generate more in vivo-like results using the microfludic OrganoPlate® platform
- Optimize high-content imaging for evaluation of treatment effects on neuronal networks
Establishment of physiologically-relevant in vitro models is crucial to further understanding of the mechanisms of neurological diseases as well as targeted drug development. While iPSC-derived neurons show great promise for compound screening and disease modeling, use of three-dimensional (3D) cultures is emerging as a valid approach for neuronal cell based assay development. 3D cultures are recognized as more closely recapitulating aspects of the human tissues including the architecture, cell organization, as well as cell-cell and cell-matrix interactions.
The focus of the present study was to develop a high-throughput 3D neurite outgrowth assay using iPSC-derived neurons developed in the microfluidic OrganoPlate® platform, with the goal of establishing 3D models for neurodegenerative diseases and neurotoxicology screens3. The OrganoPlate® is a high-throughput platform that combines the most recent advances in 3D cell culture, Phaseguides™ and microfluidics. The OrganoPlate contains 96 tissue chips suitable for long-term culture of live cells, is amenable for screening purposes, and is compatible with standard laboratory equipment or automated systems, like the ImageXpress® Micro Confocal High-Content Imaging System.
- OrganoPlate® platform
- Human iPSC-derived iCell® Neurons (Cellular Dynamics International)
- Neuronal media (Cellular Dynamics International)
- Matrigel (Corning)
- Calcein AM (Life Technologies)
- MitoTracker Orange (Life Technologies)
- Hoechst (Life Technologies)
- Saponin (Sigma)
- PBS (Sigma)
- Anti-β-tubulin III (TUJ-1) antibodies (BD Biosciences)
- ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices)
- MetaXpress High-Content Image Acquisition and Analysis Software, version 6.2 (Molecular Devices)
Phenotypic analysis of 3D cultures
High-content imaging and analysis were utilized for evaluation of treatment effects on neuronal networks. We optimized confocal imaging and analysis protocols for assessing morphology and viability of neurons in this 3D matrix. Images were acquired using ImageXpress Micro Confocal system with 10x, 20x, or 40x objectives. A series of images was acquired at different planes along the focal axis (Z-stack). A stack of 17-30 planes separated by 3-10 µm was acquired, covering approximately 150-300 µm in depth. All individual images were saved and used for 3D analysis, as well as 2D projection (Maximum Projection or Best Focus) images.
Neurotoxicity assay in 3D using OrganoPlates
Phenotypic readouts included quantitative characterization of the extent and complexity of neural networks in 3D by multiplexed measurements. In this neuronal model system, we have evaluated assay reproducibility, characterized multiple measurements, and tested several known neurotoxic compounds. Via these analysis methods, we have accurately measured concentration-dependent inhibitory effects of these compounds on the complexity of neurite networks. Therefore, this proposed method can be used for high-throughput, high-content compound screening for prediction of neurotoxicity.
Conclusion and full text
We have developed a quantitative, confocal high-content imaging method that enables high-throughput phenotypic assessment of treatment effects upon the viability and morphology of 3D neuronal cultures using MIMETAS OrganoPlates®. Confocal imaging and multi-parametric 3D analysis allows for counting and characterization of neurites and also provides statistical characterization of neurite development and branching in 3D. Analysis methods enable characterization of neuronal networks and provide quantitative measurements that can be used for defining EC50 values and comparing toxic effects of selected compounds.