Cell culture troubleshooting

Troubleshooting tips and solutions for common cell culture problems.

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Cell Culture Troubleshooting

My cells are not growing

Check that the medium is recommended for the particular cell type. Recommendations can be obtained from the biggest cell banks (ATCC and ECA CC) and fr om previous scientific publications reporting use of the cell line.

Most cell lines require additional supplements for their growth. The most commonly used are serum (e.g., fetal bovine serum, concentration depends on the cell type, typically 5–20% (vol/vol)), glutamine, and non-essential amino acids (NEAA).

My adherent cells are not attaching to cell culture dishes

Check the type of dishes that you use. Many manufacturers provide dishes for suspension cultures with very hydrophobic surfaces that are not suitable for adherent cell culture. In this case, adherent cells do not attach to the surface and die.

Consider whether your cell line requires a special coating to improve cell adherence. Most commonly used coating agents include poly-L-lysine (diluted in water), collagens (diluted in low molar acid solutions such as acetic acid or hydrochloric acid), and fibronectin (diluted in calciumand magnesium-free PBS).

Specific guidelines are provided by manufacturers, but most commonly used protocols involve the preparation of a diluted coating agent, which is applied to dishes, followed by 1 h incubation at room temperature in the tissue culture hood and removal of excess agent by washing surfaces a few times with PBS.

My cells are regularly infected with mycoplasma or other pathogens

Examine your aseptic procedures for possible methods of optimization.

Always work with cells under dedicated cell culture hoods and wear appropriate PPE, including gloves and a lab coat.

Regularly clean and decontaminate the working area. Use sterile tips and pipettes, sterilize any glassware and tools by autoclaving.

If needed, maintain two cell cultures, one with antibiotics and one antibiotic-free and examine both cultures regularly for any signs of infection.

Ideally, the regular use of antibiotics in routine cell culture should be limited because they can mask low-level infections, making them hard to detect, and provide selective pressure for developing antibiotic-resistant pathogens in your working area.

Consider microbiological testing of your working and master stocks to validate they are not contaminated.

I cannot recover any/ many cells from my cell banks

Cells may not have been frozen or thawed properly; please follow our guidelines above.

Ensure that frozen vials are stored at the appropriate temperature (below -130°C) at all times.

Consider freezing more cells per one vial and seed freshly thawed cells at a higher density to allow logarithmic growth from the very beginning.

I have problems achieving efficient transfection of my samples

The efficiency of cell transfection depends on many aspects such as general cell metabolic state and passage number. Also, some medium supplements, such as antibiotics or serum, can decrease transfection efficiency. In principle, transfection protocols should be optimized for every cell line, considering variables such as the selection of a transfection reagent, the ratio of DNA/RNA to transfection reagent, and cell density during transfection.