I have recently begun working with the HepG2 cell line to induce insulin resistance using a high-glucose environment. I acquired the cells from a -80°C storage where they had been since 2019, and I thawed them in a low-glucose medium (DMEM with 1 g/L glucose). However, the cells are exhibiting slow growth. Since thawing them on February 6th, nearly five days ago, their duplication rate has been slower than expected ( they typically double in number within two days) Could anyone provide recommendations regarding the glucose concentration or growing conditions to improve their growth rate, or shall I wait for more time?
Hello Tasnim Ramadan Matarid
In vitro cell proliferation and survival are highly dependent on experimental conditions such as cell density, stress, and nutrients. HepG2 cell line may be grown in DMEM low glucose (900mg/L; 5mM) medium +10% FBS.
From the image, it seems to me that the cell density is low. What was the percent viability on thawing? For HepG2 cells, the cell density, and the way these cells are dispersed are important. These cells should be cultured at high seeding density. HepG2 cell doubling time is approximately 48 hours. I feel you should wait for some more time. The culture does not look healthy due to low cell density.
Best.
Thank you, Malcolm Nobre for your answer. I didn't calculate the percent viability on thawing. However, the vial contained 2x106 cells. Is it very necessary to count the percent viability?
Yes. You would come to know whether there is a problem in freezing or storage because HepG2 cells would have been much happier to be stored in liquid nitrogen than in - 80 deg C for a period of nearly 5 years which is a long duration.
Having frozen 2 × 10^6 cells, I would have expected a reasonable number of cells in the selected field in the image. You should have obtained at least 85- 90% viability after thawing to get most of the area covered with cells which I do not observe in this case.
Best.
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