I have been working with 3T3 cells almost for a year now. But suddenly from past few weeks they become too slow to grow. Usually a 1/10th Split form a 10 mm dish gets completely confluent within 48 hrs. But now it takes almost 4-6 days. I have checked for mycoplasma but the PCR came negetive. I tried reviving different cryos which were frozen on different days. But all of them are behaving the same. If anyone also faced the same issue before? and if they can suggest some wayout it would be really helpful. Thanks in advance.
Dear Dr. Ayan Modak
There are a few questions you need to ask yourself.
1. Have you changed the lot/brand of FBS recently? The quality of FBS vary between batches/brand, and changes in its composition can affect cell growth.
2. Have you used the 100mm petri dish from another vendor? This could affect cell adhesion and proliferation, especially if the cells are sensitive to the surface properties of the vessel.
3. Are you using NIH3T3 cells of a later passage? Prolonged growth by repeated passaging can lead to cellular aging, characterized by decrease proliferation.
4. Have you cultured these cells for extended periods at high density? This may also be a contributing factor.
5. Have you checked the incubator condition? Verify that the incubator settings (like the temperature, CO2, etc.) are correct.
6. Are you seeding NIH3T3 cells at an optimum density? Because these cells are sensitive to both low and high seeding densities. If the cell density is too low, there might not be sufficient cell-to-cell contact for optimal growth, or if the cells are sub cultured when they are overconfluent over a number of passages, they may not perform well at later passages.
7. Finally, mycoplasma contamination can significantly impact cell growth and viability. It is crucial to rule out mycoplasma contamination which you have already done.
Hope these suggestions help!
Regards,
Malcolm Nobre
The issue may be related to subtle changes in culture conditions or accumulated cellular stress. I recommend checking the following:
- Fetal bovine serum quality, even a new batch, can affect growth
- Medium preparation and storage, ensure no pH drift or contamination
- CO₂ levels and incubator humidity
- Plasticware coating or surface treatment, as 3T3 cells are sensitive to adhesion properties
- Passage number, cells near senescence exhibit reduced proliferation.
If all else fails, try sourcing a fresh low-passage vial from a certified cell bank to reset the system.
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