2, Department of Bioengineering, Lehigh University, Bethlehem, Pennsylvania, United States
Introduction: Most of the current epilepsy models use 2D neuronal cultures which cannot correctly mimic the cytoarchitecture of brain. State of the art 3D neuronal cultures use scaffolds which introduce foreign material and are often characterized by a lower cell density than the brain. Here, we present a simple yet novel and high-yield method to create polydimethylsiloxane (PDMS) confined scaffold-free 3D neuronal cultures from dissociated rat cortex and hIPSC derived neurons that show spontaneous epileptic seizure-like activity without any convulsant agent. This method can potentially provide a new and better way for high-throughput anti-epileptic drug (AED) screening. A patient-specific drug development system also becomes feasible with the integration of hIPSC with this technique.
Materials and Methods: We used PDMS to create 100 µm high micro-wells of different diameters (500-1500um). Dense solution of cells from neonatal rat cortex or hIPSC derived neurons was put into these PDMS micro-wells. Diluted solution of cells was also seeded outside the PDMS to create 2D cultures. Ca2+ indicator (R-GECO1) was applied to these cultures. Optical recordings were performed on both 2D and 3D cultures from day in vitro (DIV) 08 to DIV 21 to observe the spontaneous activity. Multi-electrode array was used to measure the extracellular field potential. Cultures were fixed and stained with antibody to NeuN at DIV21 for confocal imaging. tetrodotoxin (TTX), kynurenic acid (KYNA) and different concentrations of AEDs, phenytoin, carbamazepine, levetiracetam and topiramate were applied to 3D cultures created with rat and hIPSC derived neurons and the activity modulations were observed.
Results and Discussion: Optical and electrical recordings showed synchronous activity (bursts) across the whole culture in both 2D and 3D cultures. 3D cultures showed signficantly different burst duration from 2D cultures. Burst duration increased significantly with increasing micro-well diameter for 3D cultures. Application of TTX and KYNA abolished all burst activities, indicating the key-role of neuronal firing and glutamatergic network behind this seizure-like activity. Application of different concentration of carbamazepine and phenytoin showed sigmoidal modulation of total activity time in 3D cultures. 3D cultures from hIPSC derived neurons showed similar seizure-like activity and pharmacological response.
Conclusion: The data presented here proves the presence of epileptic seizure-like activity in the developed 3D cultures from neonatal rat and hIPSC derived neurons. It further shows significant differences of activity patterns between 2D and 3D cultures. Pharmacological experiments on these cultures showed that they can be reliably used as a powerful tool for high-throughput drug screening for AED development.
Acknowledgements: This work was supported in part by NIH/NINDS R33 NS088358.