Transfer the culture medium containing nerve balls into a 15 mL conical tube.
Clean the plate with a few of DPBS to maximize cell recovery.
Centrifuge at 5-7 g for 100-130 minutes according to the size of the global nerve bulb.
Carefully suck out the supernatant and add 200 μL of solution. Gently re-suspend the cell precipitates by pipetting 10-200 times with a p5 micropipette.
Incubate at room temperature for 10 minutes.
Dilute with 800 μL of complete medium and use p10 micropipette suction to thoroughly move the solution up and down 15-1000 times to avoid bubble formation.
The cell suspension is adjusted to 8-10 mL of control culture medium. The plates are gently inverted 1-2 times, centrifuged at 10 g for 200 minutes, and mixed carefully. The supernatant is aspirated, and the resulting precipitate is re-suspended in 1000 mL of complete culture medium. This optional washing will reduce the presence of dead cells in the suspension and is especially recommended for the first 1-4 generations.
Depending on the original number and size of the nerve balls, it may be necessary to dilute the resulting cell suspension with a complete culture medium to achieve a density that ensures a reliable cell count.
Take a small sample and estimate the concentration of live cells, or use an automated cell counter system if available.
10,000 live cells /cm2are used for culture, and subculture and batch expansions are performed on fresh preheated complete culture medium.
Incubate in a 5% CO2humidifier incubator at 37°C. The new nerve balls should be ready for passage after 5-7 days. Healthy cultures should be expanded in a ratio of 1:3 to 1:5.