Antibiotics in Cell Culture

Why use antibiotics in cell culture?

Antibiotics are a type of secondary metabolites or synthetic analogues produced by microorganisms (including bacteria, fungi, actinomycetes) or higher animals and plants during the life process. They can resist pathogens or other activities and interfere with the development of other living cells. It plays an important role in screening stable expression strains, inhibitory microorganisms or tumor cells, regulatory cells, etc.

As one of the most important technologies in life science research, the maintenance of aseptic environment of cell culture is directly related to the accuracy and reliability of experimental results. In this process, the use of antibiotics is particularly critical. Antibiotics can effectively prevent the pollution of microorganisms such as bacteria and ensure the healthy growth of cells. At the same time, antibiotics can also be used to screen and retain specific genetically modified cell populations, playing an important role in transfection, gene editing and other experiments. The rational use of antibiotics not only helps to maintain the stability of cell culture, reduce the loss of expensive reagents and more time, but also ensures the repeatability and accuracy of experimental results, providing a reliable experimental guarantee for researchers.

Antibiotics for cell culture

Both biological and chemical factors can lead to contamination in cell culture, and in most cases, pollution leads to consequences such as slow cell growth, morphological changes, rapid changes in the pH of the medium, and an increased number of dead or floating cells in culture. Contamination can come from a variety of sources, such as improper aseptic practices, contaminated reagents, or airborne microorganisms. The three most common contamination in cell cultures are bacteria, fungi, and mycoplasma, while viral contamination, while rare, adds additional risk. In order to deal with these pollution, targeted prevention and control measures need to be taken, often considering methods such as the rational use of antibiotics.

Antibiotics for cell culture at BOC Sciences

Bacterial and fungal contamination

Bacterial and fungal contamination is the most common problem in cell cultures, mainly due to their ability to colonize rapidly in nutrient-rich media. Once bacteria and fungi are introduced, they multiply rapidly, often leading to obvious signs of contamination including turbidity or changes in pH, which often manifest as a change in the color of the medium. This color change is particularly pronounced in mammalian cell cultures, as it is closely related to carbon dioxide levels and acidification of the medium.

Antibiotic for mycoplasma contamination

Mycoplasma has a limited biosynthetic capacity and needs to enter a suitable host in which to reproduce and survive for a long time. Their tiny size (about 0.1-0.2 microns) makes them unrecognizable under a standard bright field microscope, which is why they are often undetected in many laboratories. They lack cell walls and are resistant to many common antibiotics used in cell cultures, such as penicillin or streptomycin. Most importantly, mycoplasma contamination does not produce the turbidity characteristic of other bacterial or fungal contamination.

Mycoplasma can broadly affect host DNA, RNA, and protein metabolism, affect intracellular amino acid and available ATP levels, alter cell surface antigens, and may cause DNA breaks and other major chromosomal changes. Macrolides, tetracyclines, quinolones, or a combination of these agents may be used to prevent mycoplasma contamination. However, the use of antibiotics to prevent mycoplasma infection may result in significant cytotoxicity and genotoxicity. Minocycline, for example, promotes the expression of the Bcl-2 protein, which aggregates in the cell's mitochondria and interacts with pro-apoptotic molecules such as Bax, Bak, and Bid to protect the cell from death. Similarly, temicosin effectively slowed the growth and metastasis of human breast cancer cell lines MDA-MB-231 and mouse breast cancer cell lines 4T1 by inhibiting the activity of the enzyme CD73, which catalyzes the conversion of purine 5' mononucleotides to nucleosides. Therefore, these potential effects must be fully taken into account when applying compounds to remove mycoplasma infection in experiments.

Viral contamination

Viral contaminants in cell cultures pose a more serious threat than mycoplasma infections because of the difficulty in detecting them and the lack of ways to treat affected cell cultures. In addition, some viruses have the ability to integrate their genomes into host cells, which in some cases leads to the permanent production of new virus particles. The use of serum tested for BVDV significantly reduces the risk of the virus.

Antibiotic for cell selection

In the stable transfection process, resistance marker genes are transported into cells along with the target genes by the carrier, and the target genes are endowed with resistance markers. Thus, the successfully transfected cells can still grow normally in the selected culture medium containing antibiotics, and the cells that are not transfected or not integrated into the genome are killed, and finally the stable resistant cell lines are screened. In conclusion, antibiotic resistance screening is an important step in the construction of stable cells.

Antibiotics for cell selection at BOC Sciences

Puromycin is an amino-nucleoside antibiotic that replaces normal amino acids by mimicking the 3 'terminus of aminoacylated tRNA (aa-tRNA), catalyzing incorporation into the C-terminus of the new chain through the ribosomal peptidyl transferase center (PTC), preventing the normal extension of the amino acid chain and leading to early termination of translation. Interferes with protein synthesis. The Puromycin resistance gene (PuroR or Pac gene) encodes a puromycin n-acetyltransferase (PAC) that acetylates the puromycin molecule to make the cell resistant to puromycin.

G418 (Geneticin) is an aminoglycoside antibiotic that causes cell death by inhibiting ribosome function and blocking protein synthesis. In cell culture, G418 is used to screen and maintain cell populations transfected with either NeoR or G418 resistance genes (NPT II). The resistance gene encodes aminoglycoside phosphotransferase (APRT), which makes cells resistant to G418 and protects ribosomes from inhibition by antibiotics.

Hygromycin B is an aminoglycoside antibiotic that kills prokaryotic (e.g., bacteria), eukaryotic (e.g., yeast, fungi), and mammalian cells by interfering with 70S ribosomal translocation and inducing misreading of the mRNA template, thereby inhibiting protein synthesis. The hygromycin B resistance gene (hph gene) encodes hygromycin B phosphotransferase, an enzyme that deactivates hygromycin B by partially phosphorylating its amino sugars to deactivate them.