Hello

Cell clumps are formed due to the following reasons:

1. Overgrowth of cells in culture causes excessive build-up of cell debris and free DNA from cell lysis. The sticky nature of DNA causes cells and other debris to aggregate into large clumps.

2. Excessive treatment with proteolytic enzymes like trypsin can cause cells to clump.

3. Environmental stress in culture can accelerate cell death which could lead to cell-cell adhesion causing clumping.

4. High centrifugation speed during the process of cell pelleting can also cause cell clumping.

So, when one handles cells in culture one should as far as possible avoid the above mentioned points.

Subculture the cells when they are 80% confluent. Expose cells to trypsin for a maximum of 5 minutes. Avoid environmental stress by replacing old culture medium with fresh medium every second day. Cell clumps reduces the access of cells to important nutrients and as a result hinders the overall cell growth.

I would suggest if they are minor few clumps they can be taken care of by pipetting the cell suspension gently up and down few times.

I hope this is helpful.

Good Luck.

After centrifugation do vigorous pipetting till the clumps became single cells while observing in microscope.

No of passage doesn't play much role in clumping.

I see, thank you both Malcom and Archana. I will follow your recomendations next time I have to subcultivate.

All the best