Origin and Features of Mouse Spinal Cord Neurons
The spinal cord is a slender, tubular nerve structure located within and protected by the vertebral canal of the spine. It is an extension of the central nervous system (CNS) originating from the brain. Cells within the CNS rely on complex connections to process and transmit information. The primary function of the spinal cord is to relay nerve signals between the brain and the periphery. It is a part of the CNS in humans and vertebrates, situated inside the vertebral canal, connecting superiorly to the medulla oblongata and giving off paired nerves that distribute to the limbs, body wall, and visceral organs.
The interior of the spinal cord features an H-shaped (or butterfly-shaped) region of gray matter, composed mainly of nerve cells, surrounded by an area of white matter consisting primarily of myelinated nerve fibers. The spinal cord serves as the center for many simple reflexes. Numerous paired nerves (called spinal nerves) emerge from the sides of the spinal cord and distribute to the skin, muscles, and visceral organs throughout the body. The spinal cord acts as a pathway between the peripheral nerves and the brain, as well as a lower center for many simple reflex activities. The spinal cord can be divided into 31 corresponding segments based on the attachment points of the spinal nerves, which originate from different vertebrae.
The neuron, or nerve cell, is the basic structural and functional unit of the nervous system. The size and appearance of neurons vary greatly within the CNS, but all consist of a cell body (soma), dendrites, and an axon. The cell body, also known as the perikaryon, contains neurofilaments, microtubules, endoplasmic reticulum, free ribosomes, and a nucleus with a prominent nucleolus. In some large neurons, the rough endoplasmic reticulum in the processes can be demonstrated by Nissl staining, appearing as bluish-gray patches under light microscopy, referred to as Nissl bodies. Dendrites and the axon are neuronal processes responsible for transmitting electrical impulses between neurons. The size and morphology of these processes vary considerably and can be difficult to distinguish using conventional microscopy.
Spinal cord tissue contains a large number of glial cells, while neuronal content is sparse, making isolation and purification challenging. Furthermore, spinal cord neurons are highly differentiated terminal cells, unable to divide and proliferate, and thus have high culture requirements. Immediately after plating, spinal cord neurons appear round, small, and translucent, without processes. After 2-3 days in culture, enlargement of the cell bodies and an increase in the number and length of neuronal processes can be observed. By days 6-7, the cell bodies become large and plump, with a significant increase in the number and length of processes that interweave to form a network; a distinct halo is apparent, and the cells exhibit a strong three-dimensional appearance. After 20 days in culture, there is a notable increase in cell death. Cells develop intracellular vacuoles, their processes become irregular in thickness, and they may even detach from the substrate, ultimately undergoing disintegration.
Morphological Observation of Mouse Spinal Cord Neurons at Different Densities
Figure 1. Mouse spinal cord neurons cultured on Ucallm® Ultra-Low Attachment Surface forms tumor spheroids. Mouse spinal cord neurons were planted in 96-well ultra-low attachment plates at concentrations of 500, 1000, 2000, 4000, and 8000 cells per well. Imaging was conducted at 24, 48, 72, 96, and 120 hours after seeding. Scale bars represent 200 μm.
Method
Culture Conditions
mSN Cells: Neuronal medium
Cell Thawing and Plating
Retrieve the Mouse spinal cord neurons from liquid nitrogen and immediately place the cryovial in a 37°C water bath. Gently swirl the vial to thaw the freezing medium.
Once thawed, transfer the cell suspension to a centrifuge tube containing 3 mL of pre-warmed culture medium. Centrifuge to collect the cells: 1000 rpm for 5 minutes at room temperature. Carefully discard the supernatant.
Resuspend the cell pellet in complete medium. Perform a cell count and seed the cells into 96-well U-bottom ultra-low attachment plates at densities of 1000, 2000, 4000, and 8000 cells per well. Plate two such plates, with 5 replicate wells for each density. Add 100 µL of sterile PBS to the peripheral wells of the plate to minimize evaporation. Place the 96-well U-bottom ultra-low attachment plates in the live cell station for culture and imaging.
Note: The live cell station is installed within a CO2 incubator (Thermo, 3111). Pre-warm the station for 30 minutes prior to use, and maintain conditions at 37°C, 21% O2, 5% CO2, and saturated humidity.
Medium Change
Change the medium for Mouse spinal cord neurons every 24 hours (the plates within the live cell station are changed simultaneously). The medium change regimen differs based on cell density: the frequency for the 1000 and 2000 cells/well densities is lower than that for the 4000 and 8000 cells/well densities. This process is continued for 120 hours, resulting in a total of 5 medium changes. During medium changes, carefully aspirate the old medium from all 5 replicate wells of a given density at once, and then promptly replenish with 100 µL/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 Spinal Cord Neurons | Sciencell | M1590-57 |
Neuronal medium | Sciencell | 1521 |
FBS | Gibco | 10099141 |
Penicillin-Streptomycin Solution | Gibco | 15140122 |
0.25% Trypsin | Gibco | 25200072 |
PBS Buffer Solution | Gibco | 10010023 |
References
[1] Russ, D.E., Cross, R.B.P., Li, L. et al. A harmonized atlas of mouse spinal cord cell types and their spatial organization. Nat Commun 12, 5722 (2021).
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