In general, healthy cells take up nucleic acids better than poorly maintained cells. Many cells undergo expression profile changes that can adversely affect your experiments when they are stressed by culture conditions. Routinely subculturing cells before they become overcrowded or unhealthy will minimize instability in continuous cell lines. To learn more, watch this free cell health webinar or continue reading. 

How to Keep Cells Healthy with Optimal Culture Conditions
Overly crowded or sparse cultures are not conducive for healthy cells. As a rule, cells should be replated before the medium becomes depleted. As cell cultures approach confluence, they typically contain some number of either unhealthy or dead cells, which make cell counts inaccurate.

In addition, cells that have grown in depleted medium between subculturing events have been deprived of nutrients and may have experienced pH shifts that are detrimental to health and viability. Therefore, avoid overgrowing cells and subjecting them to frequent pH and temperature shifts.

Some adherent cell lines are sensitive to trypsin exposure and to shear forces from vigorous pipetting or high-speed centrifugation. (For most cell lines, trypsinization should be kept shorter than 10 minutes.)

In addition to treating cells gently to protect their health, maintaining strict protocols, including harvesting cells for experiments at similar confluencies and maintaining consistent time intervals between plating and transfecting cells, will improve experimental reproducibility.

Mycoplasma contamination is another common stress to cells in culture that can threaten cell health and deleteriously effect experimental results. What is Mycoplasma? They are small, free-living prokaryotes that are not observable by light microscopy. Because they grow as filamentous or coccal forms without cell walls,they are not sensitive to antibiotics that interfere with cell wall production. Mycoplasmas can alter cell growth characteristics, inhibit cell metabolism,and disrupt nucleic acid synthesis, causing chromosomal abnormalities, and altering transfection or infection rates. In most situations, mycoplasmas from an infected cell line spread to other cultures in a laboratory via aerosolization during routine pipetting and handling or from shared reagents (e.g., medium, serum) that become contaminated.

The best prevention and control of cell health requires good aseptic technique (including working with cultures in order of clean to untested to infected during the work day or week) and routine testing. Many commercial kits (PCR-, ELISA-, fluorescence-,luminescence-, and culture-based assays) are available to test cultures for mycoplasma contamination

Cultures infected with mycoplasmas are usually discarded and replaced, but for irreplaceable cultures, treatment options are available to inhibit or eliminate mycoplasmas.


  • Let freshly thawed cells recover for at least 48 hours. Do not perform analyses on freshly thawed cells within 48 hours of plating.

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