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Optimisation of a PC12 cell-based in vitro stroke model for screening neuroprotective agents
1Department of Paraclinical Sciences, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, Malaysia.
Scientific Reports
|April 15, 2021
Summary
This study optimizes an in vitro stroke model using PC12 cells. We identified optimal conditions and found serotonin and 8-OH-DPAT show neuroprotective effects in this model.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Stroke remains a leading cause of global death and disability with no approved neuroprotective treatments.
- Existing in vitro stroke models using nerve growth factor (NGF)-differentiated PC12 cells face challenges with variant differentiation and optimal culture conditions.
- PC12 pheochromocytoma cell variants (PC12 Adh, PC12 Riken, Neuroscreen-1) are commonly used but require specific culture optimization.
Purpose of the Study:
- To optimize culture conditions for differentiating PC12 cell variants for an in vitro stroke model.
- To validate a serum-free culture system for PC12 cell differentiation and substrate preference.
- To screen known neuroprotective compounds and identify novel therapeutic candidates for post-stroke neuronal injury.
Main Methods:
- Evaluated three PC12 cell variants (PC12 Adh, PC12 Riken, NS-1) under various culture media and substrate conditions.
- Developed a novel serum-free media approach and identified collagen IV as a preferred adhesive substrate.
- Optimized oxygen-glucose deprivation (OGD) parameters for differentiated NS-1 cells to establish a reliable in vitro stroke model.
Main Results:
- DMEM was identified as the optimal medium for PC12 Riken and NS-1 cells.
- PC12 Adh cells showed poor attachment without serum and failed to differentiate with NGF.
- NS-1 cells achieved maximal differentiation (72.7%) and were optimized for a 3-hour OGD model with ~70% cell viability.
- Serotonin demonstrated antiapoptotic effects, and 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) showed neuroprotection in the PC12 cell stroke model.
Conclusions:
- Established optimized culture conditions and a validated PC12 cell-based in vitro stroke model.
- Demonstrated the neuroprotective potential of serotonin and the 5-HT1A agonist 8-OH-DPAT in a cellular stroke model.
- Provides a foundation for identifying and developing novel neuroprotective agents for stroke treatment.
