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Updated: Oct 12, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction
Sunao Sugita1,2, Ayumi Hono1, Shoko Fujino1
1RIKEN Center for Biosystems Dynamics Research, Laboratory for Retinal Regeneration, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan.
Abstract:
Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0-75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.
Insights
A new polymerase chain reaction (PCR) method offers sensitive and rapid detection of mycoplasma contamination in cells for transplantation. This test is crucial for ensuring the safety of cell therapies and preventing post-transplant inflammation.
Area of Science:
- Cell Biology
- Microbiology
- Regenerative Medicine
Background:
- Cell contamination by pathogens like mycoplasma is a significant challenge in cell-based transplantation.
- Mycoplasma infections are often difficult to detect using standard methods.
- Ensuring pathogen-free cells is critical to prevent severe intraocular inflammation after transplantation.
Purpose of the Study:
- To develop and evaluate a rapid, simple, and sensitive polymerase chain reaction (PCR) method for detecting mycoplasma contamination.
- To specifically design a PCR system for use in transplantable retinal cells, including induced pluripotent stem cell (iPS cell)-derived retinal pigment epithelium (RPE) cells.
- To assess the prevalence of mycoplasma contamination in commercially available cell lines.
Main Methods:
- A polymerase chain reaction (PCR) system was designed to detect Mycoplasma species listed in the Japanese Pharmacopoeia.
- Spike tests were performed on RPE cells using nine species of Mollicutes bacteria at varying concentrations (1-100 CFU/mL).
- The specificity of the PCR was confirmed by testing against non-Mollicutes bacteria species.
Main Results:
- The PCR method detected mycoplasma contamination with 100% probability at 100 and 10 CFU/mL in spike tests.
- Detection rates for 1 CFU/mL contamination ranged from 0-75%, indicating high sensitivity.
- The PCR demonstrated high specificity, with no detectable DNA from non-Mollicutes species.
- While laboratory-established iPS and iPS-RPE cells were negative, some commercial cell lines tested positive for mycoplasma.
Conclusions:
- The developed PCR method provides a reliable tool for rapid and sensitive detection of mycoplasma contamination in laboratory cultures intended for clinical use.
- This assay is essential for quality control of cells for transplantation, particularly retinal cells, to prevent adverse inflammatory reactions.
- Routine monitoring using this PCR method is imperative for ensuring the safety and efficacy of cell-based therapies.
