Establishment of a Spheroid Culture Model for Mouse Intestinal Smooth Muscle Cells(mISMC)

Culture Conditions

mISMC : 89% DMEM H(High Glucose) + 10% FBS +1%P/S

Cell recovery

1) The cryopreserved vial of mISMC was removed from liquid nitrogen and quickly thawed in a 37℃ water bath with gentle agitation.  

2) After thawing, the cell suspension was transferred to a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.  

3) The cell pellet was resuspended in complete medium, seeded into a culture dish, and gently mixed. Cells were cultured in a standard incubator at 37℃ with 21% O₂ and 5% CO₂.

Cell Passaging

1) Cells were passaged upon reaching 80% confluence.  

2) The culture medium was aspirated, and the cells were washed once with PBS.  

3) 1–2 mL of 0.25% trypsin was added to digest the cells at 37℃ for 3–5 minutes. Digestion was considered complete when cells became rounded and detached under microscopic observation.  

4) Trypsin was quickly removed, and complete medium was added to neutralize the enzyme. The cells were gently pipetted to form a single-cell suspension and subcultured at a split ratio of 1:2 to  1:4. Expanded culture was continued under the same conditions (37℃, 21% O₂, 5% CO₂).

Cell Seeding

mISMC in the logarithmic growth phase with good viability were harvested and seeded into a 96-well U-bottom ultra-low attachment plate at densities of 1000, 2000, 4000, and 8000 cells per well. Each density was replicated in 5 wells. The peripheral wells of the plate were filled with 100 μL of sterile PBS. The plate was 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 mISMC 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.