Isolation and Maintenance of Neural Stem Cell

Neural stem cells (NSCs) are a remarkable type of cells with the potential to differentiate into various neural cell types. The isolation and maintenance of these cells play a critical role in understanding the development, function, and potential therapeutic applications of the nervous system.

Creative Biolabs explores the essential techniques and protocols involved in the isolation and maintenance of neural stem cells.

Materials Required

• Dulbecco's modified eagle's medium (DMEM)
• Fetal bovine serum (FBS)
• Antibiotic-antimycotic solution
• Phosphate-buffered saline (PBS)
• Trypsin-EDTA solution
• Neural stem cell medium

Procedure

Preparation of Culture Medium

Prepare neural stem cell medium by supplementing DMEM with FBS, antibiotic-antimycotic solution, and other required growth factors. Filter sterilize the medium using a 0.22 μm filter.

Neural Tissue Preparation

Obtain neural tissue from the desired source. Dissect the tissue in ice-cold PBS and remove any meninges or blood vessels. Transfer the tissue to a sterile culture dish containing DMEM. Obtain small tissue fragments and transfer the tissue fragments to a tube containing trypsin-EDTA solution and incubate for a specific duration.

Isolation of Neural Stem Cells

After the incubation, add an equal volume of NSC medium to neutralize the trypsin. Triturate the tissue fragments using a sterile pipette to obtain a single-cell suspension. Centrifuge the cell suspension at a low speed to pellet the cells. Resuspend the cell pellet in NSC medium and plate the cells in culture dishes or flasks coated with an appropriate substrate.

Maintenance of Neural Stem Cells

Regularly monitor the cell culture under an inverted microscope to observe the formation of neurospheres or the migration of cells from adherent cultures. Every two to three days, replace half of the medium with fresh NSC medium to provide essential nutrients and growth factors. Subculture the cells when neurospheres reach an optimal size (typically 100-200 μm in diameter) or when adherent cultures reach approximately 70-80% confluency.

Notes

• It is crucial to maintain sterile conditions throughout the isolation and maintenance procedures to prevent contamination and ensure successful culture establishment.
• The choice of growth factors and supplements in the NSC medium can influence the self-renewal and differentiation potential of neural stem cells. Optimization of the medium composition may be required for specific research or therapeutic applications.
• Adherent cultures of neural stem cells may require different substrate coatings to promote attachment and growth.
• Careful monitoring of the cell culture and regular subculturing are essential to maintain the viability and proliferation capacity of neural stem cells.
• Cryopreserved neural stem cells should be thawed using a standardized protocol to ensure high post-thaw viability.

By following appropriate protocols and utilizing the necessary materials, researchers can establish and maintain neural stem cell cultures, enabling a deeper understanding of the central nervous system and the development of innovative therapeutic strategies. Creative Biolabs is committed to providing comprehensive services and customized protocols to assist researchers. Please do not hesitate to contact us.

Reference

1. Sun Y, et al. Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Molecular and Cellular Neuroscience, 2008, 38(2): 245-258.

For Research Use Only. Not For Clinical Use.