Why Use Antibiotics in Cell Culture?

In 1940s, Keilova, Cruikshan and Lowbury first introduced antibiotics in tissue culture as prophylactic agents against contamination. As cell culture evolved and paved the way to disease models, monoclonal antibody production, therapeutic protein production, regenerative medicine and stem cell and cancer research, maintenance of aseptic techniques became imperative. The use antibiotics in cell culture has been debatable since excess use may cause morphological or physiological changes in the cell and continuously mask low levels of contamination. However, it is universally accepted that antibiotics minimize loss of valuable cells, reagents, time and efforts due to contamination.

Types of cell culture contamination

Chemical contamination is caused due to

  • improper weighing of reagents during media preparation
  • lot-to-lot differences in variation in concentration of growth factors and hormones or presence of liposaccharides
  • detergent or disinfectant residues used to wash vessels that  leach into the culture media
  • solvent, stain or organic compound remnants due to improper cleaning of glassware
  • photo activation of media components producing free radicals that are cytotoxic
  • impurities in the CO2 gas cylinders; toxic volatile residues left behind after disinfection

Biological contamination is caused due to

  • lack of good laboratory practices; improper handling of cell culture material
  • non-sterile pipette tips compromising the sterility of media, reagents and supplements
  • aerosol interference during culture, incubation and transportation
  • accident spills during cell culture

Agents of Biological Contamination

  • Bacteria
  • Fungi
  • Mycoplasma
  • Virus

Bacterial and fungal contamination

Bacterial and fungal contaminations are most prominent in cell culture because of quick colonization in enriched media. The medium becomes turbid and changes color (pH change) within few hours/days of contamination. Regular microscopic observations and addition of antibiotics to the culture medium prevents contamination from spreading across cultures (see table below).
 

Antibiotic Bacteria Fungi Yeast Mycoplasma
Gram positive Gram negative
Amphotericin B      
Ampicillin      
Erythromycin      
Gentamycin    
Kanamycin    
Neomycin      
Nystatin      
Penicillin-Streptomycin      
Polymyxin B        
Tetracyclin      
Thiabendazole        
Tylosin        

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Mycoplasma contamination

Mycoplasma are the smallest, free-living (0.3 µM) prokaryotes observed as filamentous or coccal forms under scanning electron microscope. Mycoplasma contamination is cryptic and extremely challenging to handle. They are undetectable under light microscope but result in morphological changes, chromosome aberrations and altered aminoacid and nucleic acid metabolism. Due to the lack of cell wall they are resistant to routinely used antibiotics. While specific antibiotics are effective, the best agents against mycoplasma are proven to be a combination of multiple biological agents.
 

Product # Product
MP0030 LookOut® Mycoplasma Elimination Kit
MP0040A LookOut® Mycoplasma qPCR Detection Kit
MP0025 Venor™ GeM Mycoplasma Detection Kit, PCR-based
L1420 LookOut® Mycoplasma Erase

The best prevention against mycoplasma contamination is to use cell lines from ECACC that are tested for mycoplasma contamination on regular basis.

 

Viral contamination

Viruses are most difficult contaminants to detect in cell culture and are difficult to remove. However, the contamination is self-limiting since viruses are solely dependent on host cellular machinery. Virally infected cells pose health hazards to the laboratory personnel. Use of BVDV-tested serum significantly lowers the risk of viruses. 

When should you use Antibiotics in cell culture

  • Antibiotics safeguard high value stocks of irreplaceable cultures and to produce working stocks. When reproducible results are critical it is recommended that optimal concentrations of suitable antibiotics be added to cultures.
  • Antibiotics like Penicillin-Streptomycin solution at a concentration of 50-100 I.U./ml is recommended for primary cell culture as the chance of contamination is high for the first few weeks. 
  • Antibiotics give extra layer of protection from factors like new researchers under training, particulates and aerosols. Antibiotics are also used to select cells modified by genetic engineering (selection agents). Puromycin and hygromycin B are used to select and establish cells expressing the puromycin-resistance and hygromycin-resistance genes, respectively.

It is wrong to assume that antibiotics are entirely bad for cell culture; the correct approach is to use the optimum concentration in your cell culture experiments to prevent contamination and protect cells in your lab.

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