Halo everyone, I have cell culture from bovine wharton's jelly through explant method. The medium consists of DMEM high glucose, MEM NEAA, ITS, FBS 10%, and antibiotic, antifungal. Medium change every 3 days. After 6 days, the outgrowth seen from explant (fibroblast like cell). On day 12, the cells become floating and unhealthy morphology, no contamination, 25% confluency. Then, I replace medium with 20% FBS medium culture. But, the cells showed no good changes.
What should I do
Thank you for advices
Riris
Biomedical student
My colleague suggested me to use 2-mercaptoethanol in order to trap any toxics that probably arose from cell metabolism
Based on the information provided, it seems like you are facing some challenges with the cell culture from bovine Wharton's jelly. Here are a few suggestions that may help:
1. Optimize the culture medium:
- The medium composition you described (DMEM high glucose, MEM NEAA, ITS, 10% FBS, and antibiotics/antifungals) is generally suitable for culturing cells from Wharton's jelly.
- However, the floating and unhealthy morphology of the cells on day 12 suggests that the culture conditions may not be optimal.
- Consider adjusting the FBS concentration. Try increasing the FBS to 15-20% to provide more growth factors and support the cells.
- Additionally, you can try adding growth factors such as basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the medium, as they can promote the proliferation and survival of mesenchymal stem/stromal cells (MSCs) derived from Wharton's jelly.
2. Adjust the seeding density:
- The 25% confluency on day 12 may indicate that the initial seeding density was too low.
- Try increasing the seeding density, for example, by seeding the cells at a higher density (e.g., 5,000-10,000 cells/cm²) and observe the growth and behavior.
3. Check the cell attachment:
- The floating and unhealthy morphology of the cells on day 12 may suggest poor cell attachment to the culture surface.
- Ensure that the culture vessels are properly coated with an attachment-promoting substrate, such as collagen, fibronectin, or gelatin, to facilitate cell adhesion.
4. Monitor the culture conditions:
- Ensure that the culture environment (temperature, humidity, CO₂ levels) is properly maintained and consistent.
- Check for any potential contamination, even if you haven't observed visible signs, as this can also impact the cell health and behavior.
5. Consider subculturing the cells:
- If the cells continue to exhibit poor growth and unhealthy morphology, you may need to subculture them.
- Gently detach the cells, collect them, and seed them into a new culture vessel with fresh medium.
- This can help remove any potential inhibitory factors or debris that may have accumulated in the original culture.
If the issues persist after implementing these suggestions, you may need to troubleshoot further by adjusting other culture parameters or seeking advice from experts in the field of stem cell culture.
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