Possible causes and solutions for cells not adherent, slow growth, poor growth, and even death - Database & Sql Blog Articles

During cell culture, various issues can arise. From a growth perspective, problems may include non-adherent cells, slow growth, poor proliferation, or even cell death.

First, Cultured Cells Are Not Attached

Possible Causes:

• Overdigestion with trypsin
• Mycoplasma contamination
• Medium pH is too alkaline (due to NaHCO3 decomposition)
• Cell aging
• Incorrect initial cell seeding concentration

Solutions:

• Reduce trypsin digestion time or lower its concentration
• Isolate and test for mycoplasma; clean incubator and equipment. If contaminated, discard the culture
• Adjust pH using sterile acetic acid or add CO2
• Use new seed cells
• Optimize the initial cell concentration

Second, Cells Form Clusters in Suspension

Possible Causes:

• Presence of calcium and magnesium ions in the medium
• Mycoplasma contamination
• Excessive protease digestion causing cell lysis
• DNA contamination

Solutions:

• Wash cells with calcium-free, magnesium-balanced salt solution and gently resuspend them
• Isolate the culture and test for mycoplasma
• Treat with DNase I to remove DNA

Third, Slow Cell Growth

Possible Causes:

• Change in culture media or serum
• Depletion of nutrients like glutamine or growth factors
• Low-level bacterial or fungal contamination
• Improper storage of reagents
• Low initial seeding density
• Cell aging
• Mycoplasma contamination

Solutions:

• Compare new vs. old media and serum, allow gradual adaptation
• Replace with fresh medium or add missing components
• Culture without antibiotics, discard if contaminated
• Store serum at -10 to -20°C, culture medium at 2–8°C in dark
• Adjust initial seeding density
• Use new seed cells
• Test for mycoplasma and clean equipment if needed

Fourth, Poor Cell Growth

Possible Causes:

• Cell condition: excessive passage, low seeding, late passage, incorrect trypsin time, improper cryopreservation
• Contamination: mycoplasma, mold
• Media or serum issues: not tested before use, not suitable, improperly prepared
• Environmental factors: CO2 supply, temperature control

Solution:

• Monitor cell status, including passage number and seeding amount
• Use high-quality, traceable serum to prevent contamination
• Choose appropriate media and serum
• Ensure proper lab conditions

Fifth, Cell Death During Culture

Possible Causes:

• No CO2 in the incubator
• Temperature fluctuations in the incubator
• Damage during freezing or thawing
• Incorrect osmotic pressure of the culture medium
• Toxic metabolite accumulation

Solutions:

• Check CO2 levels in the incubator
• Verify incubator temperature stability
• Use freshly preserved cells
• Measure and adjust osmotic pressure
• Replace with fresh culture medium