Formation of Tumor Spheroids of MKN-74(Gastric cancer cell line)

Culture Conditions

MKN-74 Cells: 89%RPMI 1640 Cell Culture Medium+10% FBS+1% P/S

Cell recovery

1)  Remove the MKN-74 cells from liquid nitrogen and quickly place them in a 37℃ water bath. Gently shake the cryovial to thaw the freezing medium.

2)  After thawing, transfer the cells to a centrifuge tube containing 3 mL of culture medium. Centrifuge to collect the cells at 1000 rpm for 5 minutes at room temperature, then discard the supernatant.

3)  Suspend the cell pellet in complete medium, seed them into a culture dish, gently pipette to mix, and culture in a standard incubator at 37℃, 21% O₂, and 5% CO₂.

Cell Passaging

1)  When the cell density reaches 80%, perform subculturing.

2)  Discard the culture medium and wash once with PBS.

3)  Add 1-2 mL of 0.25% trypsin to digest the cells. Incubate at 37℃ for 2-5 minutes. Observe under a microscope; digestion is complete when the cells detach and become rounded.

4)  Quickly discard the trypsin, add complete medium, and pipette to create a single-cell suspension. Subculture at a ratio of 1:2 to 1:4, and expand the culture in a standard incubator at 37℃, 21% O₂, and 5% CO₂.

Cell Seeding

Take MKN-74 cells in the logarithmic growth phase with good growth condition. Seed them into a Ultra-Low Attachment Surface 96U Plate at densities of 500, 1000, 2000, 4000, and 8000 cells per well respectively (for one plate), with 6 replicate wells per density. Add 100 μL of sterile PBS to the peripheral wells of the plate. Place the Ultra-Low Attachment Surface 96U Plate in the live cell station for cultivation and imaging.

*Note: The live cell station is installed in a CO₂ constant temperature incubator , pre-warmed for 30 minutes, and maintained at 37℃, 21% O₂, 5% CO₂., and saturated humidity.

Medium Change

For the first 3 days, the initial number of MKN-74 cells is relatively low, and their metabolism is slow; the medium can be changed every 24 hours for 2-3 days. From days 4 to 7, as cell proliferation increases, the density gradually rises and metabolism accelerates, requiring medium changes every 1-2 days.

*Note: The plate in the live cell station is also changed simultaneously. The medium change frequency for the 500, 1000, and 2000 cells/well densities is lower than that for the 4000 and 8000 cells/well densities. During medium change, carefully transfer the cell clusters gently using a 200 μL yellow pipette tip to a 35 mm cell culture dish containing 1 mL of complete medium. Transfer the cell clusters from all 6 replicate wells of one density at once. Then, aspirate and discard the old medium from the wells, rinse the well plate 2-3 times with 100 μL/well of PBS. Subsequently, use the 200 μL yellow pipette tip again to transfer the cell clusters back into the wells one by one, maintaining a medium volume of 100 μL per well.