Isolation and Maintenance of Glial Stem Cell

Glial stem cells (GSCs) play a crucial role in the development, maintenance, and repair of the central nervous system (CNS). These specialized cells possess the remarkable ability to self-renew and differentiate into various glial cell types, such as astrocytes, oligodendrocytes, and Schwann cells. Isolation and maintenance of GSCs are pivotal for studying their fundamental properties, investigating their therapeutic potential, and understanding the underlying mechanisms of neurodevelopmental disorders and CNS injuries.

Creative Biolabs presents a protocol, which includes a step-by-step guide to efficiently isolate and maintain GSCs using cutting-edge technology.

Materials Required

• Sterile phosphate-buffered saline (PBS)
• Cell culture dissociation reagent
• DMEM/F-12 medium
• B27 supplement, epidermal growth factor (EGF), fibroblast growth factor (FGF)
• Laminin-coated cell culture plates
• Poly-L-ornithine
• Phosphate-buffered saline with Tween 20 (PBST)
• Primary/Secondary antibodies for GSC markers

Procedure

Animal Tissue Preparation

First, dissect and prepare the brain tissue of the animal. Place the brain tissue into a fresh Petri dish and cut it into small pieces. Transfer the brain pieces to a tube containing DMEM/F12 medium and dissociative enzymes for incubation. Shake the tissue to obtain a single cell suspension.

Glial Stem Cell Enrichment

First stop the enzymatic reaction, then centrifuge the suspension. Discard the supernatant and resuspend the cell pellets in fresh DMEM/F12 medium supplemented with B27. Spread the cells on cell culture plates coated with laminin and place them in a humidified incubator. Replace half of the medium with fresh growth medium every other day to feed the cells.

Maintenance of Glial Stem Cells

Pre-coat new cell culture plates first with poly-L-ornithine and set aside for use. Aspirate the growth medium from primary cultured GSCs. Wash the cells and add enzymes to dissociate the cells. After dissociation, stop the enzymatic reaction, collect the cell suspension and centrifuge. Resuspend the cell pellets in fresh growth medium and mount the cells in prepared coated plates. Finally, incubate the plates in a humidified incubator.

Characterization of Glial Stem Cells

Researchers can use immunofluorescence methods to characterize glial stem cells. For example, use primary and secondary antibodies against GSC markers to incubate with the cells, and then observe the stained cells with a fluorescence microscope.

Notes

• It is important to maintain strict sterility throughout the procedure.
• It is crucial to handle tissues and cells with care to ensure optimal viability and yield.
• Carefully follow the manufacturer's instructions for the preparation of growth medium and ensure appropriate supplement concentrations.
• Regularly monitor cell culture for contamination and maintain suitable culture conditions to promote cell growth and viability.
• Characterization and differentiation experiments are essential to confirm the identity and functionality of glial stem cells.

This protocol provides a detailed guide for effectively isolating GSCs and maintaining their self-renewal capacity in culture. By following these steps, researchers can obtain a renewable source of GSCs, opening up new avenues for studying glial cell biology, developing novel therapies, and unraveling the mysteries of neurodevelopment and repair processes.

If you have any questions or comments, please do not hesitate to contact us.

References

1. Gage F H and Temple S. Neural stem cells: generating and regenerating the brain. Neuron, 2013, 80(3): 588-601.
2. Pollard S M, et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell stem cell, 2009, 4(6): 568-580.

For Research Use Only. Not For Clinical Use.