Peptone nerve cells, specifically neurons, are notoriously difficult to culture in vitro. Unlike other cell types, neurons require very specific conditions to survive and differentiate. Only under optimized circumstances—such as culturing on a collagen-coated surface or supplementing the medium with nerve growth factors (NGF) and glial-derived cytokines—can some degree of neuronal differentiation and maturation be achieved. However, even under these conditions, the process remains challenging and often yields limited results.
In contrast, glial cells from nervous tissue are much easier to culture and maintain. They grow more rapidly, adhere better to surfaces, and are more resilient to changes in environmental conditions. This makes them a more practical choice for many laboratory experiments involving neural cell cultures.
The peptone-based method for culturing glial cells is as follows:
1. After obtaining brain gray matter or white matter from the operating room, it should be handled aseptically. Carefully remove any surrounding meninges, blood vessels, and fibrous tissues. Rinse the tissue one or two times in Hanks’ solution to remove debris and contaminants.
2. Place the tissue in a container containing 30–50 times its volume of Hanks’ solution. The brain tissue will become softer, making it easier to break down. Use a pipette to gently triturate the tissue repeatedly, creating a single-cell suspension.
3. Transfer the suspension into a centrifuge tube and centrifuge at room temperature for 5–10 minutes. During this time, cells and cell clusters will settle at the bottom, while fat and other impurities will float to the top. Carefully remove the supernatant and repeat this process 2–3 times to further purify the cell sample.
4. Once the pellet is obtained, resuspend the cells in an appropriate volume of culture medium. Filter the suspension through gauze to remove any remaining clumps or large debris. Count the cells and adjust the density to the desired concentration.
5. Inoculate the cells into a culture flask or dish and incubate them in a 5% CO₂ incubator at 37°C. Glial cells typically take some time to adapt to the new environment and may not show immediate adhesion after seeding. However, once they begin to adhere, they can proliferate quickly. Passage of the cells can be performed using 0.25% trypsinization when the culture reaches confluence.
This method provides a reliable way to establish a glial cell culture, which can be used for various applications such as studying neuroinflammation, drug screening, or co-culture experiments with neurons. With proper maintenance, these cultures can remain viable for extended periods, making them a valuable resource in neuroscience research.
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