Quality control: mycoplasma
The cell lines are grown in antibiotic free medium for at least two passages in the quarantine area. Three methods are used to detect mycoplasma infection: DNA staining by Hoechst 33258, PCR amplification and MycoTect assay
The fluorochrome bisbenzimide (Hoechst 33258) binds specifically to the Adenine-Thymine (A-T) regions of DNA. Uninfected cell cultures observed under fluorescence microscopy (image on the left) are seen as fluoresceing nuclei against a negative background; cultures contaminated with mycoplasma (image on the right) appear as brightly fluorescent uniformly shaped bodies surrounding the cells, or as small clusters between the cells.
Artifacts may be fluorescent and interfere with the interpretation of results, but they will appear larger in size than mycoplasma and irregular in shape. The main advantages of this staining method are the speed at which results are obtained and the fact that non-cultivable M. hyorhinis strains can be detected.
This mycoplasma detection method has several advantages: it is simple and rapid, highly sensitive and specific. A pool of oligonucleotides is used, able to recognize sequences specific for 25 mycoplasma species, and among these for the five species responsible for most cell culture contaminations (M. arginini, M. fermentans, M. hyorhinis, M. orale e Acholeplasma laidlawii).
Biochemical assay with 6-MPDR
This assay is based on defined biochemical differences between mycoplasmas and their host mammalian cells. Mycoplasmas contain significant amounts of adenosine phophorylase, an enzyme present in small amounts in mammalian cells that converts 6-methylpurine deoxyriboside (6-MPDR), a non toxic analog of adenosine into 6-methylpurine and 6-methylpurine riboside. These products are toxic to mammalian cells. A culture infected with mycoplasma can be detected by incubation with 6-MPDR and subsequent monitoring for mammalian cell toxicity. This method is available in a kit from Gibco BRL, the MycoTect assay.
If a cell line is found to be infected with mycoplasma, the recommended procedure is to discard and replace it with clean cultures, but if the culture cannot be replaced it is possible to attempt to eliminate the infection, even if this is a time-consuming and often unsuccessful exercise, and the risk exists that other cultures become contaminated during the treatment. At Banca Biologica the contaminated cells are treated either with Mycoplasma Removal Agent, or with cyprofloxacin or BM-Cyclin. After treatment, the cells are regularly tested for mycoplasma contamination for at least four weeks, and they are considered cleaned if the results of all assays remain negative along this period.