Culture and identification of neonatal rat brain-derived neural stem cells

BACKGROUND

Timing of passaging, passage number, passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells (NSCs) culture. How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.

AIM

To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.

METHODS

First, curved tip operating scissors were used to dissect brain tissues from new born rats (2 to 3 d) and the brain tissues were cut into approximately 1 mm³ sections. Filter the single cell suspension through a nylon mesh (200-mesh) and culture the sections in suspensions. Passaging was conducted with TrypL^TM Express combined with mechanical tapping and pipetting techniques. Second, identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.

RESULTS

Brain derived cells from newborn rats (2 to 3 d) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5^th generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining.

CONCLUSION

This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

Key Words: Neonatal rats, Brain-derived neural stem cells, Culture, Identification

Core Tip: How to harvest sufficient neural stem cells (NSCs) is a basic requirement for the study and clinical application of NSCs. This study describes a simplified and efficient method for neonatal rat brain-derived NSC culture and identification comprehensively considering the influencing factors including timing of passaging, passage number, passaging approaches and methods for cell identification. It demonstrates that combination of TrypL^TM Express and mechanical tapping and pipetting techniques makes a more efficient way of passaging. The optimal timing for NSC passage is on the fourth to fifth day of primary or passage NSC culture.

• Citation: Zhou QZ, Feng XL, Jia XF, Mohd Nor NHB, Harun MHB, Feng DX, Wan Sulaiman WA. Culture and identification of neonatal rat brain-derived neural stem cells. World J Stem Cells 2023; 15(6): 607-616
• URL: https://www.wjgnet.com/1948-0210/full/v15/i6/607.htm
• DOI: https://dx.doi.org/10.4252/wjsc.v15.i6.607