Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test.

Sunao Sugita,Ayumi Hono,Shoko Fujino,Masayo Takahashi,Yoko Futatsugi,Yuta Yunomae,Norio Shimizu

doi:10.3390/ijms222212555

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.

Highlights

Induced pluripotent stem cells can differentiate into several types of cells or tissues, and they are used in clinical studies/trials
The purpose of this study was to examine whether rapid polymerase chain reaction (PCR) could be applied to evaluate mycoplasma contamination in human iPS cell-derived transplantable retinal pigment epithelium (RPE) cells with the following criteria: (1) to serve as a comprehensive test of the genus Mycoplasma listed in the 17th Bureau Act, (2) to have high specificity and sensitivity, and (3) to be a rapid and simple procedure
We obtained the ratios of genomic copy (GC) number and colony-forming units (CFU) of the reference strains

Summary

Introduction

Induced pluripotent stem cells (iPS cells) can differentiate into several types of cells or tissues, and they are used in clinical studies/trials. In 2017, a clinical study was conducted with allogeneic transplants rather than autologous transplants due to cost and other considerations, and HLA homozygote iPS-RPE cells were transplanted into five HLA-matched patients with age-related macular degeneration [2]. These clinical trials were successful and did not accompany any obvious problems, cell transplantation required the utmost care to avoid contamination of the transplants by pathogens, as transplanting infected cells into the eye could cause severe adverse events in patients. We applied a commercially available mycoplasma PCR system, the Myco Finder PCR, for validation on our transplantable RPE cells

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