Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Plate coating

1. Coat a 96-well microplate with poly-L-lysine (1 mg/mL, diluted in 0.1 M borate buffer [pH: 8.5]; 100 µL/well) and incubate overnight at 37 °C.
   NOTE: Only coat the wells that need to be used. In the experiments performed here, the middle 60 wells are used. The borate buffer is prepared by mixing 50 mM boric acid and 12 mM borate in sterilized water.
2. Wash the plate twice with sterilized water (250 µL/well).
3. Wash the plate once with fresh culture medium without supplements (250 µL/well).
4. Dry the plate on a clean bench for 20 min.
5. Wrap the plate with aluminum foil and keep it at 4 °C until use (valid for 1 month).

2. Cell seeding

1. Add 50 µL/well of the culture medium to the coated plate and keep it in a 37 °C, 5% CO₂ incubator for 30 min to 1 h. Fill the peripheral wells with sterilized water (200 µL/well). 
   NOTE: The culture medium is prepared by adding 50x B-27, 400x Glutamax, and 100 U/mL of penicillin/streptomycin to the neurobasal medium (refer to the Table of Materials for details).
2. Remove the neuron cryovial from the liquid nitrogen tank. The neurons used here were dimethyl sulfoxide-cryopreserved (DMSO-cryopreserved) neurons.
3. Immerse the cryovial in a 37 °C heat block for up to 3 min and partially thaw the contents. Do not warm up the cryovial for too long. Transfer the contents to a 50 mL tube as soon as it is thawed.
4. Slowly transfer the neuron cryovial contents to a sterile 50 mL tube dropwise (50 µL/s) using a 1 mL pipette with a wide-pore tip.
5. Rinse the empty cryovial with 1 mL of the culture medium (room temperature; RT). Transfer this 1 mL of the culture medium from the cryovial drop-wise (50 µL/s) to the 50 mL tube containing the cell suspension.
6. Add 9 mL of the culture medium (RT) to the 50 mL tube dropwise (0.5 mL/s) and make up the volume to 11 mL. Do not repeat pipetting but mix the cell suspension slowly.
7. Count the cell number (use a cell counter or a hemocytometer).
8. Transfer all the cell suspension to a reservoir and dispense the cell suspension to the 96-well plate using a multichannel pipette with wide-pore tips (1.0 x 10⁴ cells/well). To reduce the culture medium evaporation, fill the peripheral wells with sterilized water (step 2.1).    
   NOTE: This study confirms that the culture medium evaporation is small for a 3-week culture without medium exchange. The reduction rate of the medium is 3.6% (n = 120 wells). Thus, the osmolality change will not be drastic during the 3-week incubation period.
9. Incubate the neurons for 1-2 h in a 37 °C, 5% CO₂ incubator.
10.  Replace the culture medium with 100 µL of preheated culture medium (37 °C) per well and put it back in a 37 °C, 5% CO₂ incubator (no medium change is required during culture).