Establishment of a Spheroid Culture Model for Mouse Hippocampal Neurons (mHN)

Culture Conditions

mHN cells: Mouse hippocampal neuron-specific complete growth medium

Cell recovery

• Frozen vials of mHN cells were retrieved from liquid nitrogen and rapidly thawed in a 37℃ water bath with gentle agitation.
• The thawed cell suspension was transferred into a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.
• The cell pellet was resuspended in complete medium, and cell counting was performed. Cells were seeded into 96-well U-bottom ultra-low attachment plates at densities of 1000, 2000, 4000, and 8000 cells per well (two plates were prepared). Each density was replicated in 5 wells. The peripheral wells of the plates were filled with 100 μL of sterile PBS. The plates were then transferred to a live-cell imaging station for culture and imaging. Note: The live-cell station, pre-installed in a CO₂ incubator, was pre-warmed for 30 minutes and maintained at 37℃ with 21% O₂, 5% CO₂, and saturated humidity.

Medium Change

The medium for mHN cells was replaced every 24 hours (including those in the live-cell station). The frequency of medium changes varied with cell density: wells with 1000 and 2000 cells per well were changed less frequently than those with 4000 and 8000 cells per well. This process was continued for 120 hours, totaling 5 medium changes. During each change, the old medium was carefully aspirated from all 5 replicate wells of a given density at once, and promptly replaced with 100 μL per well of fresh complete medium.