Hello Arti

There could be many reasons.

1. What is the passage number of the cells? Too many passages could lead to

slow growth.

2. The cell culture medium used or the supplements like FBS must be of good

quality and should not vary.

3. The pH of the culture medium should be maintained at the physiological pH of

7.2 to 7.4. Sometimes in culture the pH may vary.

4. During passaging the culture should not be over confluent. During trypsinization

exposure to trypsin must be kept at the minimum to avoid stress on the cells.

5. Check on contamination, especially mycoplasma.

6. Sometimes due to inaccurate freezing technique the cells thawed from the

frozen vials could behave differently, and grow slow or fail to attach to the

substratum.

Best Wishes.

Hi Arti Govindbhai Patel

In addition to Malcolm Nobre comments, also ensure that you are keeping your cell lines at the correct confluency. Contact inhibition will start occurring above 80% confluency which can inhibit proliferation. Similarly, this will also lead to the media having to be changed more frequently due to change in pH and a build up of metabolites that can become toxic at high concentration.

Conversely, some cells below 30% confluency may not be able to grow efficiently if they are not suspended in conditioned media.

My recommendation is to seed cells at a density of 0.8 x 106 cells/60 mm dish. Replace media every 48 hours and once cells reach 80% confluency, trypsinize and replate. If your cells are supposed to have a doubling time of 36 hours, seed your cells at an appropriate number and culture for approximately 5-6 days, replacing media every 48 hours (you should end up with roughly four times the amount). Then split your cells in a ratio of 1:4 and reseed fresh 60 mm dishes.

Best of luck!