Contact us :customer@cti-eu.com
Views: 6 Author: vivi Publish Time: 2025-06-26 Origin: Site
Cell culture, also known as cell cloning technology, refers to the growth of cells under in vitro conditions. During culture, cells no longer form tissues; instead, the culture consists of individual cells or cell clusters. Since cultured cells live in an artificial environment, they may be affected by various external factors, resulting in several common problems. What are these issues, and how can we solve them?
Possible Causes:
l Culture medium contains calcium and magnesium ions
l Mycoplasma contamination
l Overdigestion by proteases causing cell lysis
l DNA contamination
Solutions:
l Rinse cells with calcium- and magnesium-free balanced salt solution and gently pipette to obtain a single-cell suspension
l Separate and test cultures for mycoplasma. Discard contaminated cultures
l Treat cells with DNase I
Possible Causes:
l Overdigestion by trypsin
l Mycoplasma contamination
l Culture medium is too alkaline (due to NaHCO₃breakdown)
l Cell aging
l Inappropriate initial cell seeding density (too high or too low)
Solutions:
l Shorten digestion time or lower the concentration of trypsin
l Isolate and test cultures for mycoplasma. Clean incubator and culture equipment. Discard contaminated cultures
l Adjust pH using sterile acetic acid solution or supply sterile CO₂
l Use freshly preserved cell stocks
l Optimize cell seeding density
Possible Causes:
l Switching to a different culture medium or serum
l Essential components such as glutamine or growth factors are depleted, missing, or degraded
l Low-level bacterial or fungal contamination
l Improper reagent storage
l Low initial cell seeding density
l Cell aging
l Mycoplasma contamination
Solutions:
l Compare compositions of old and new media and sera. Gradually adapt cells to the new medium
l Use freshly prepared culture medium or supplement with glutamine and growth factors
l Culture in antibiotic-free medium to check for contamination. Discard contaminated cultures
l Store serum at -10°C to -20°C. Store medium at 2°C–8°C protected from light. Use complete medium with serum within one week when stored at 2°C–8°C
l Increase initial seeding density
l Use fresh cell stocks
l Isolate and test cultures for mycoplasma. Clean incubators and culture equipment. Discard contaminated cultures
Possible Causes:
l Lack of CO₂ in incubator
l Large temperature fluctuations in the incubator
l Damage during freezing or thawing
l Incorrect osmotic pressure in the medium
l Accumulation of toxic metabolic by-products
Solutions:
l Check CO₂ level in the incubator
l Monitor incubator temperature
l Use new cell stocks
l Check medium osmolarity
l Replace with fresh culture medium
