Neuron Culture

Preparation of a dispersed neuron culture of rat suprachiasmatic nucleus (SCN)
Sato Honma, M.D., Ph.D.
Associate Professor
Department of Physiology
Hokkaido University Graduate School of Medicine
Sapporo, Japan
E-mail: sathonma@med.hokudai.ac.jp


Rat pups were anesthetized by hypothermia and decapitated. Frontal sections of the hypothalamus (700 µm thick) were obtained using a tissue chopper (McIlwain). Bilateral SCNs were dissected out from the slice using scalpel blades. The SCNs of about 15 pups were incubated together in isotonic salt solution containing 0.03% trypsin for 15 min at 37°C. After rinsing with isotonic salt solution containing 0.02% trypsin inhibitor and 0.01% deoxyribonuclease (Sigma), the cells were dissociated by pipetting gently through a fire-polished Pasture pipette 10 times in the same medium. Single cells were obtained by filtering through a No.200 stainless-steel mesh. Cell suspensions were centrifuged for 5 min at 1300 r.p.m., and SCN cells were resuspended at 106 vial cells/ml in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing 15 mM NaHCO3 , 10 mM HEPES and 20 mg/l Kanamycin (Gibco). Cell viability was between 85% and 95%.

Dispersed SCN cells were cultured on 2 to 4 MED Probes at one time. Cell suspension including 1.5–1.8 x 105 vial cells were plated in the central area (5 mm diameter) of each MED Probe. Eight hours after plating the cells, 1 ml of culture medium supplemented with 2% fetal calf serum and hormones were overlaid. Culture medium was exchanged every other day until recording was started. Spontaneous single unit neuronal activities of the cultured SCN neurons appeared between 4 and 10 days after the plating and lasted continuously for 2–6 weeks. Action potentials were recorded simultaneously from 4–8 neurons in each MED Probe.
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Tips and tricks

1. Cell viability should be over 90%
I recommend cell viability over 90 % (hopefully 93-98%) for long-term recording in a dispersed SCN cell culture on a MED probe. The dissociation step seems to be critical for the viability of the cells. I first cut slices into cubic blocks of about 0.8 x 0.8 x 0.5 mm and incubate them with 0.03% trypsin at 37°C for 15 min in a buffer that only contains NaCl, KCl, HEPES, NaHCO3 and Kanamycin. After the treatment with trypsin inhibitor and DNase I dissociate cells by pipetting gently through a fire polished Pasture pipette. The pipette tip should not be too small. Cells are dissociated usually by pipetting 3~5 times. Do not flush strongly or do not pipette more than 10 times.

2. Surface of the MED Probe is hydrophobic and the special treatment is necessary.
On the day before preparation of the culture, the MED probe must be treated with 0.1N HCl for 6 hrs, rinsed with sterilized distilled water 3x, followed by 70% ethanol for 15~30min. Rinse the MED Probe with water again and dry it under UV for 15 min. Expose it to blue flame very briefly (longer exposure will damage the insulation layer). Exposure can be repeated until the surface becomes hydrophilic. Coat it with poly-L-ornithine overnight. On the day of the culture, rinse the dish with water and treat with serum containing DMEM for at least 2 hrs.

3. Cell density and serum supplement.
I usually reconstruct the cells with DMEM supplemented with 5 % FCS. The cells can be densely cultured only at the center of the MED Probe by placing a cloning ring at the center of the MED Probe. Density is 100,000~150,000 cells in the ring of 7mm diameter. Usually 6~8 hrs after the plating, the ring should be removed and the medium containing 2 % FCS is overlaid. You may reduce the concentration of FCS to 1-2 % depending on your culture system and glia growth (see item 4).

4. Suppress overgrowth of glia
Overgrowth can be suppressed through a combination of the reduction of serum concentration and the application of Ala C.

References
Honma, S., Katsuno, Y., Tanahashi, Y., Abe, H. and Honma, K.,
Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus
Neuroscience 86 No.3. pp.967-976 (1998)

Honma, S., Shirakawa, T., Katsuno, Y., Namihira, M. and Honma, K.,
Circadian periods of single suprachiasmatic neurons in rats.
Neuroscience Letters 250, pp. 157-160 (1998)

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