Antibiotics and selection reagents

Antibiotic selection of mammalian cells

Selective antibiotics or selection antibiotics are used to isolate and establish genetically modified or transfected cells to produce stable cell lines. To select transfected cells, a selection marker is either added to the same nucleic acid sequence as the genetic change, or it’s co-transfected in a separate plasmid. Only the cells that contain the DNA with the selection marker will grow in the presence of the selective antibiotic or reagent.

To select cells that have been modified by recombinant engineering, choose the selection antibiotic or reagent that matches the selection marker. Commonly used options include:

• G418 (geneticin): a selective agent for transfected mammalian, yeast, plant and bacterial cells.
• Puromycin: a selection antibiotic for the pac gene and works broadly in eukaryotes and prokaryotes.
• Hygromycin B: a selection antibiotic for the hph gene that inhibits translocation.

Selection of transformed bacterial cells

In a typical experiment, plasmid DNA delivered to cells includes both an antibiotic resistance gene and the sequence of interest. Ampicillin is a common selection antibiotic used to select genetically modified from cells that didn’t take up the plasmid.

Cell culture contamination

A major challenge faced when doing cell culture in research or biomanufacturing is contamination of culture media by foreign substances. Cell culture contaminants can be grouped into two broad categories:

• Chemical contaminants: These are usually impurities like endotoxins, plasticizers, and detergents in cell culture media, sera, or water.
• Biological contaminants: These are living microorganisms such as bacteria, fungi, viruses, and mycoplasma. These contaminants can enter the culture from a nonsterile reagent or a lack of aseptic technique when handling.

Contaminants often have considerable effects on cell culture health. They also pose challenges in biologic manufacturing, where it’s critical to ensure that the final product does not contain unwanted chemical or biological substances.

Biological contamination by bacteria, mold, and yeast

Found virtually all around our natural environment, bacteria, mold, and yeast can rapidly colonize and proliferate in the nutrient-rich cell culture media. These microbes often have a diameter of a few microns, and varying shapes such as rods and spheres. Their size and fast growth rates make them the most common contaminants with in vitro cell cultures.

Detection of microbial contaminants

With a rapid onset of turbidity, contamination by microbial pathogens may be visible to the naked eye. Bacterial contamination also causes changes in pH. If the cells are grown in a cell culture medium that contains the phenol red indicator, a change in color to yellow also simplifies contaminant detection. Through standard light microscopy, you can easily identify fungal cells and bacteria structures such as E. coli strains.

Prevent contamination using antibiotics

Antibiotics are antimicrobial, antifungal, or other agents that target and kill specific cell types. They either induce death or act as growth inhibitors, which helps reduce the loss of valuable cells and reagents. Bacteriostatic solutions include gentamicin, penicillin-streptomycin or pen-strep-glutamine , which also act on mycobacteria. Other selection antibiotics help to prevent contamination from fungi, mold, and yeast.