Various problems arise during cell culture. From the perspective of cell growth, the following analysis addresses the causes of cells failing to adhere, growing slowly, growing poorly or even dying, and proposes corresponding solutions.
I. Cultured Cells Fail to Adhere
Possible Causes:
Excessive trypsin digestion;
Mycoplasma contamination;
Medium pH too alkaline (decomposition of NaHCO3);
Cell aging;
Initial cell seeding density too low or too high.
Solutions:
Shorten the trypsin digestion time or lower the trypsin concentration;
Isolate the culture and test for mycoplasma. Clean the racks or incubator. If mycoplasma contamination is found, discard the culture;
Adjust the pH with sterile acetic acid solution or introduce sterile CO2;
Use a new stock of cryopreserved cells;
Adjust to the optimal cell seeding density.
II. Cultured Cells Grow Slowly
Possible Causes:
Switching to a different medium or serum;
Essential growth components in the medium, such as glutamine or growth factors, are depleted, lacking or degraded;
Minor bacterial or fungal contamination in the culture;
Improper storage of reagents;
Initial cell seeding density too low;
Cells have aged;
Mycoplasma contamination .
Solutions:
Compare the composition of the new and original medium, and compare the new and old serum in cell growth support tests; allow cells to gradually adapt to the new medium;
Replace with freshly prepared medium, or supplement with glutamine and growth factors;
Culture in antibiotic-free medium; if contamination is found, discard the culture;
Serum must be stored below -15 °C. Medium should be stored at 2-8 °C protected from light. Complete medium containing serum should be stored at 2-8 °C and used within 1 week;
Increase the initial cell seeding density;
Switch to a new stock of cryopreserved cells;
Isolate the culture and test for mycoplasma. Clean the racks and incubator. If mycoplasma contamination is found, discard the culture.
III. Cultured Cells Grow Poorly
Possible Causes:
The state of the cells themselves
Too many passages, cells have aged;
Seeding density:large split ratio and low cell density, seeding density too low, cells grow slowly;
Passaging too late: cell intoxication affects cell growth after passaging;
Trypsin digestion too long or too short: if too long, cells die; if too short, cells are not fully separated and form clumps, causing cell death;
Cell cryopreservation and recovery: slow freezing, rapid thawing.
Contamination
Mycoplasma contamination;
Fungal contamination.
Medium or serum
Serum or medium was changed without prior validation;
Whether the selected medium is appropriate;
Whether the medium was prepared accurately.
Culture environment
CO2Whether CO2 supply is normal;
Whether incubator or shaker temperature control is correct.
Solutions:
Develop targeted solutions based on the possible causes in the four areas above
Pay attention to the state of the cells themselves, such as passage number and seeding density;
Avoid contamination (use serum that is legitimate, legally sourced and traceable);
Use appropriate serum or medium, preferably validated;
Pay attention to the laboratory environment.
IV. Cultured Cells Die
Possible Causes:
No CO2;
Temperature fluctuations in the incubator are too large;
Damage during cell cryopreservation or recovery;
Incorrect medium osmolality;
Accumulation of toxic metabolites in the medium.
Solutions:
Detect CO2 in the incubator;
Check the temperature in the incubator;
Take a new stock of cryopreserved cells;
Measure medium osmolality;
Replace with fresh medium.
