Primary culture of mouse embryonic spinal cord neurons: cell composition and suitability for axonal regeneration studies

Abstract

Objective: Primary culture is an effective experimental model to study molecular mechanisms that drive axonal regeneration after central nervous system injury. However, the culture of spinal cord (SC) cells remains poorly characterized. Here, we have analyzed the cell composition of a primary SC culture during its maturation.

Methods: Primary cell culture was prepared from mouse embryo spinal cords. After 2, 7, and 14 days of cultivation, the cells were fixed and stained with antibodies against β3-tubulin, nestin, crmp1, SMI-32, DCC or GFAP. We counted percentage of cells positive for the mentioned markers and measured the length of cell processes.

Results: We found that β3-tubulin and nestin were both expressed at day 2 of culture in vitro. Surprisingly (given the use of differentiation-supporting culture medium), the number of nestin+ cells has significantly increased during the first week of cultivation. The GFAP+ cells appeared only at the seventh day in vitro, and their fraction increased during the following cultivation. At 14 day in vitro, SC culture contained cells that expressed the markers typical of commissural and motor neurons. At this age, the neurons had the ability to repair injured neurites after mechanical damage.

Conclusion: Primary culture of SC cells is a dynamically developing cell population that contains all main types of SC cells and is capable of self-repair. Therefore, the culture of mouse embryonic SC cells represents an adequate experimental model for studying cellular and molecular processes taking place in SC neurons after axonal damage in the absence of external inhibitors.

Keywords:

• Axon regeneration
• GFAP
• nestin
• neurite damage
• spinal neurons

Acknowledgements

The authors are grateful to Ekaterina A. Tverie for the help with mouse colony maintenance and pregnancy timing.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The work was supported by the grant from Russian Science Foundation #16-15-00243 and intramural grant from Kurchatov Institute #1386.