Optimized Protocols for K7M2-WT Cell Culture and Preservation

K7M2-WT cells (mouse osteosarcoma osteoblasts) form tumors in BALB/c mice and spontaneously metastasize to the lungs in over 90% of the inoculated mice. The antigen expression profile of K7M2-WT cells includes Factor VIII, Bone Sialoprotein (BSP), Dimeric Glycan, Decorin, and Villin. The expression of Bone Sialoprotein, Dimeric Glycan, Decorin, and Osteopontin further confirms the osteoblast lineage characteristics of K7M2-WT cells.

Basic Information

Growth Medium: DMEM + 10% FBS + 1% P/S

Recommended Subculturing Ratio: 1:3 to 1:4

Recommended Medium Change Frequency: 2-3 times per week

Doubling Time: ~31 hours

Cell Morphology: Osteoblast-like

Growth Characteristics: Adherent cells

Cryopreservation Conditions: 55% basal medium + 40% FBS + 5% DMSO

Culture Conditions: Atmosphere: 95% air, 5% CO2; Temperature: 37°C

Cell Recovery

1. Preheat the water bath to 37°C. Prepare clean disposable gloves, and remove the cells from the liquid nitrogen tank. Place the cells in the disposable gloves and quickly immerse the cryovial in the water bath, gently shaking the vial to accelerate thawing. The cells should be completely thawed within one minute.

2. Before opening the cap, wipe the outside of the cryovial with 75% ethanol. Transfer the thawed cells into a centrifuge tube containing fresh culture medium.

3. Centrifuge at approximately 1200 rpm for 3 minutes. After centrifugation, carefully remove the supernatant.

4. Resuspend the cells in an appropriate volume of complete culture medium. Transfer the resuspended cells into a sterile container, add additional culture medium as needed, and place the container in the incubator for further culture.

Cell Cryopreservation

1. Prepare the freezing medium. Since DMSO generates heat when mixed, be sure to allow the freezing medium to cool before use to avoid damaging the cells.

2. After dissociating the cells, terminate the process by adding fresh culture medium to resuspend the cells. Centrifuge at approximately 1200 rpm for 3 minutes, and carefully remove as much of the supernatant as possible.

3. Add the prepared freezing medium to the cells, adjusting the concentration to approximately 1×10⁶ cells/mL. Aliquot the cell suspension into cryovials.

4. Transfer the cryovials into a programmed freezing container and place them in a -80°C freezer overnight.

5. The next day, remove the cryovials from the -80°C freezer and quickly transfer them to liquid nitrogen for long-term storage.

Cell Passaging

1. Aspirate and discard the culture medium, then add 2 mL of PBS to rinse the cells.

2. Aspirate and discard the PBS, then add 2 mL of trypsin to rinse the cells.

3. Place the dish in the incubator for 2-3 minutes to allow the cells to dissociate. Gently tap the side of the culture dish to aid in cell detachment. Once the cells have detached and the clusters have loosened, add 2 mL of complete culture medium to stop the dissociation.

4. Gently pipette the cells to mix them into a uniform suspension. Proceed with seeding the cells according to your experimental needs.

Tips

• Ensure that the cell density is not too low.
• When rinsing, make sure all the cells are fully covered.

Culture Precautions

1. These cells are sensitive to external stimuli, so it is recommended to use fresh high-glucose DMEM medium. If the medium turns purple, it is advisable to stop using it and replace it with fresh medium promptly.

2. The cells do not adhere strongly, so some may remain suspended. When changing the medium, avoid rinsing with PBS. If there are too many suspended cells, they can be collected by centrifugation and transferred back to the original culture flask.

3. When passaging these cells, be cautious about the degree of dissociation; avoid over-dissociating the cells.