Mycoplasma contaminants have severe negative effects on cellular physiology, and therefore their detection in cell culture and downstream biologicals is vital.
It has long been known that contamination by mycoplasma in continuous animal cell cultures is a serious problem due to the broad range of effects that mycoplasma can induce on cells in culture, ranging from cellular gene expression modifications to activation of the signal transduction pathway (1,2). This leads to data which can be just as compromised as a result of the unintended effects of mycoplasma contamination.
The origin of mycoplasma in biopharmaceuticals can be traced back to the raw materials, cell lines and cell cultures that are utilized in manufacturing. Identifying contamination early on enables quick decision making and corrective actions to regain downstream product integrity.
Being able to quickly analyze cell cultures for microbial contamination, especially Mycoplasma contamination, is vital for commercial suppliers of cell lines. Of paramount importance is monitoring Mycoplasma contamination and ensuring that cell lines are not contaminated.
The new UVP GelStudio has a number of features that make Mycoplasma analysis easier, including automation scripts that allow out-of-the-box capture of a fluorescent PCR gel result with just one click. Using these automation features, it is possible to capture, save and annotate the raw gel image for routine labeling and archiving purposes.
Materials and Methods
A 10 µl cell culture supernatant was boiled for 10 minutes and lightly centrifuged. 2 µl of the supernatant was added to the rehydrated PCR master reaction mixture, after which PCR was run. The bioimaging UVP GelStudio was used to analyze the PCR product after running the agarose gel electrophoresis.
Samples and Reagents
- The PCR mycoplasma test kit (PK-CA91-1024 from Promokine) was used to run the PCR
- Primers, nucleotides, DNAs and hot start Taq polymerase are included in the kit, as needed for PCR
- Rehydration buffer
- Positive controls
- Agarose gel electrophoresis
The UVP GelStudio makes use of customizable action buttons that enable automatic focus, capture, pseudo-color, and image-saving. Moreover, analysis offers automated tools which can identify and quantify the DNA that has been separated by electrophoresis. Routine annotations can be saved and loaded for each gel, which makes labeling and documenting more intuitive.
The system’s low light (Figure 1.2) optical zoom lens allows for a broad range of gel sizes to be analyzed. The zoom settings can also be preset to enable one-click automation. Several fluorescent dyes can be imaged, and the fluorescent emission filters are easy to exchange to accommodate new dyes or multiplexing. Furthermore, there are features that support 21 CFR part 11.
Camera, focus and zoom, exposure, and excitation and emission filters can be manually controlled for experimentation purposes.
Results and Discussion
The UVP work studio offers a very quick and sensitive work studio for imaging and analysis of mycoplasma contamination. The presence of mycoplasma in human and mouse tumor cells which express GFP and RFP fluorescent proteins was analyzed using agarose gel electrophoresis.
Mycoplasma positive samples displayed a clear band at 265-278 bp, whereas mycoplasma negative samples displayed the internal control band and the negative control at 479 bp. RFP fluorescent proteins produced by LLC cells and GFP fluorescent proteins produced by CT-26 cells both tested positive for contamination by mycoplasma. As seen in Figure 1.3, the presence of internal control DNA as a clear band on the gel shows that both the PCR run was successful, and that the obtained data is not erroneous.
Figure 1.3. Agarose Gel of Mycoplasma PCR Test Results - Bioimaging results of agarose gel electrophoresis of PCR products from human and mouse tumor cells that express GFP and RFP fluorescent proteins. LLC (RFP) and C-26 (GFP) have the same number of base pairs as the Mycoplasma positive band DNA.
Promokine’s PCR mycoplasma kit (PK-CA91-1024) detects mycoplasma species such as M. bovis and M. arthritidis but does not detect clinically important species such as M. pneumonia and U. urelyticum.
A reliable method for detecting mycoplasma, even at low concentrations, in cell cultures has been developed. With its innovative design, the UVP GelStudio fits perfectly in the workflow of the laboratory, is easy to use and is accurate when identifying and quantifying mycoplasma in cell cultures. Raw imaging of fluorescence DNA on a gel can be quickly labeled and analyzed. The data obtained from bioimaging makes documentation easy and accessible for future use in referencing and archiving electrophoretic results. The capability of the studio will minimize data loss and facilitate data analysis.
- Drexler, H.G. and Uphoff, C.C., 2002. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39(2), pp.75-90.
- Stanbridge, E.R.I.C., 1971. Mycoplasmas and cell cultures. Bacteriological reviews, 35(2), p.206.
- Chen, T.R., 1977. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Experimental cell research, 104(2), pp.255-262.
This information has been sourced, reviewed and adapted from materials provided by Analytik Jena US.
For more information on this source, please visit Analytik Jena US