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Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures
Published on: November 11, 2010
Cryopreservation of adherent neuronal networks
Wu Ma1, Thomas O'Shaughnessy, Eddie Chang
1Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6900, 4555 Overlook Ave. SW, Washington, DC 20375, USA. wma@cbmse.nrl.navy.mil
Neuroscience Letters
|June 9, 2006
Summary
Developing effective cryopreservation for neuronal networks is crucial. This study introduces a novel method combining DMSO, collagen gel, and trehalose for improved storage and viability of mammalian adherent neuronal networks.
Area of Science:
- Neuroscience
- Cell Biology
- Biotechnology
Background:
- Neuronal networks are vital tools in neurophysiology, drug discovery, and toxicity testing.
- Efficient cryopreservation is essential for the widespread application, storage, and transportation of neuronal networks.
- Existing cryopreservation methods are insufficient for adherent mammalian neuronal networks.
Purpose of the Study:
- To develop and report the first efficient cryopreservation protocol for mammalian adherent neuronal networks.
- To optimize cryopreservation by evaluating the combined effects of DMSO, collagen gel entrapment, and trehalose loading.
- To assess the viability, cellular integrity, and functional recovery of cryopreserved neuronal networks.
Main Methods:
- Dissociated spinal cord cells were cultured to form adherent neuronal networks on a poly-d-lysine/laminin surface.
- Networks were embedded in collagen gel, loaded with trehalose, and then transferred to a freezing medium containing DMSO, FBS, and culture medium.
- Slow cooling to -80°C for 24 hours, followed by storage in liquid nitrogen at -196°C for up to 2 months.
Main Results:
- The combination of DMSO, collagen gel, and trehalose loading significantly enhanced post-thaw viability compared to individual or two-component protocols.
- Post-thaw neuronal networks exhibited comparable neuronal and astrocytic markers and morphological structure to unfrozen controls.
- Functional synaptic vesicle recycling was observed in post-thaw networks upon depolarizing stimulation, indicated by FM1-43 staining.
Conclusions:
- A novel cryopreservation protocol utilizing DMSO, collagen gel entrapment, and trehalose loading effectively preserves mammalian adherent neuronal networks.
- This combined approach significantly improves upon conventional slow-cooling methods for neuronal network cryopreservation.
- The developed protocol ensures high post-thaw viability and functional recovery, paving the way for broader applications of neuronal networks.
