Origin and Features of Mouse Cortical Neuronal Cells
Mouse cortical neurons are isolated from the cerebral cortical tissue. Cortical neurons constitute the fundamental structural and functional units of the central nervous system. Neurons are cells with long processes (axons) and are composed of a cell body (soma) and neuronal processes. The long axon is often enveloped by a myelin sheath, forming a nerve fiber, whose fine terminal branches are called nerve endings. The cell body is located within the brain, spinal cord, and ganglia, while the neuronal processes can extend throughout various organs and tissues of the body.
The cell body, the part containing the nucleus, varies greatly in shape and size, with diameters ranging from approximately 4 to 120 micrometers. It contains a large, round, centrally located nucleus with sparse chromatin and a prominent nucleolus. The cytoplasm contains patchy Nissl bodies (rough endoplasmic reticulum) and numerous neurofibrils. Neuronal processes are slender extensions from the cell body and can be further classified into dendrites and axons. Each neuron may possess one or multiple dendrites, which receive stimuli and transmit excitation to the cell body. Each neuron has only one axon, responsible for conveying excitation from the cell body to another neuron or to other tissues, such as muscles or glands.
Cortical neurons are one of the primary cellular components of the cerebral cortex and serve as the basic units for functional regulation in the brain. They are involved in the pathological processes of various central nervous system diseases in animals.
Morphological observations reveal that neurons initially adhere to the substrate and begin to extend processes. The number of processes gradually increases, subsequently forming an extensive network. Concurrently, the cell body enlarges, and a distinct, bright halo often becomes apparent around the soma. The cells appear most robust between 10-12 days in culture. Following this period, neurons begin to degenerate, characterized by a gradual reduction in processes, signs of degradation and degeneration within the cells, blurred contours, disappearance of the halo, and overall morphological distortion.
Morphological Observation and Live Cell Station Detection of Mouse Cortical Neuronal Cells at Different Densities
Figure 1. Mouse cortical neuronal cells cultured on Ucallm® Ultra-Low Attachment Surface forms tumor spheroids. Mouse cortical neuronal cells were planted in 96-well ultra-low attachment plates at concentrations of 500, 1000, 2000, 4000, and 8000 cells per well. Live-cell imaging was conducted at 24, 48, 72, 96, and 120 hours after seeding. Scale bars represent 200 μm.
Method
Culture Conditions
Mouse cortical neuronal cells: Neuronal medium
Cell Thawing and Cell Seeding
1) Frozen vials of mouse cortical neuronal cellswere retrieved from liquid nitrogen and rapidly thawed in a 37 ℃ water bath with gentle agitation.
2) The thawed cell suspension was transferred into a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.
3) The cell pellet was resuspended in complete medium, and cell counting was performed. Cells were seeded into 96 well U bottom ultra‑low attachment plates at densities of 1000, 2000, 4000, and 8000 cells per well (two plates were prepared). Each density was replicated in five wells. The peripheral wells of the plates were filled with 100 μL of sterile PBS. The plates were then transferred to a live cell imaging station for culture and imaging.
Note: The live cell station, pre-installed in a CO₂ incubator, was pre-warmed for 30 minutes and maintained at 37 ℃ with 21 % O₂, 5 % CO₂, and saturated humidity.
Medium Change
The medium for mouse cortical neuronal cells was replaced every 24 hours (including those in the live‑cell station). The frequency of medium changes was adjusted according to cell density: wells seeded with 1000 and 2000 cells per well were changed less frequently than those with 4000 and 8000 cells per well. This regimen was continued for 120 hours, totaling five medium changes. During each change, the old medium was carefully aspirated from all five replicate wells of a given density at once and promptly replaced with 100 μL per well of fresh complete medium.
Materials and Instruments
Table 1 Main equipment
Name | Manufacturer | Catalog Number |
CO2 Incubator | Thermo | 3111 |
Inverted Microscope | OLYMPUS | IX73 |
96-well Ultra-Low Attachment（U-bottom）Cell Culture Plate | Ucallm | L1096UA |
Table 2 Major Reagents
Name | Manufacturer | Catalog Number |
Mouse Neurons-cortical from C57BL/6 | Sciencell | M1520-57 |
Neuronal medium (NM) | Sciencell | 1521 |
FBS | Gibco | 10099141 |
Penicillin-Streptomycin Solution | Gibco | 15140122 |
0.25% Trypsin | Gibco | 25200072 |
PBS Buffer Solution | Gibco | 10010023 |
References
[1] Harris, K.D., and Shepherd, G.M.G. (2015). The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181.
[2] Oh, S.W., Harris, J.A., Ng, L., Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A.M., et al. (2014). A mesoscale connectome of the mouse brain. Nature 508, 207–214.
[3] Zingg, B., Hintiryan, H., Gou, L., Song, M.Y., Bay, M., Bienkowski, M.S., Foster, N.N., Yamashita, S., Bowman, I., Toga, A.W., et al. (2014). Neural networks of the mouse neocortex. Cell 156, 1096–1111.
[4] Harris, J.A., Mihalas, S., Hirokawa, K.E., Whitesell, J.D., Choi, H., Bernard, A., Bohn, P., Caldejon, S., Casal, L., Cho, A., et al. (2019). Hierarchical or ganization of cortical and thalamic connectivity. Nature 575, 195–202.
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