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

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.

Sık Kullanılan Bazı Hücre Hatları için Kalite Kontrol: Mikoplazma Kontaminasyon Tespiti, Sitokrom B ve Sitokrom Oksidaz Alt Birim I Genlerinin DNA Dizi Analizlerinin Gerçekleştirilmesi

“…Mikoplazma kontaminasyonunun hücre büyümesi, amino asit metabolizması, kromozomal anormalliklerin indüksiyonu, lenfosit aktivasyonunun indüksiyonu veya inhibisyonu, sitokinlerin indüksiyonu veya baskılanması ve hatta sinyal transdüksiyonu üzerinde etkisi vardır ve hücre kültürü çalışmaları açısından önemli bir tehdittir 20,35 . Mikoplazma tespitinde 4′,6-diamidino-2-phenylindole (DAPI) ve Hoechst işaretleme, PCR, q-PCR ya da kitler sıklıkla kullanılmaktadır [21][22][23] 38 . CYTB bölgesi temelinde RFLP tekniği ile gerçekleştirilen çalışmada elde edilen ek bantlarla bazı hatlar için klonaliteden farklılaşmalar olduğu tespit edilirken 1 , aynı bölge için yuvalanmış PCR tekniği ile gerçekleştirilen çalışmada kullanılan hücre hatlarında çapraz kontaminasyon olmadığı bildirilmiştir 39 .…”

“…Bu nedenle hücre kültürlerinin sık ve gizli bir kontaminantı olan hücre duvarı bulunmayan mikoplazma hücre kültür çalışmaları için önemli bir tehdittir 14,15,19 . 4′,6-diamidino-2-phenylindole (DAPI) ve Hoechst işaretleme, Polymerase Chain Reaction-Polimeraz Zincir Reaksiyonu (PCR), q-PCR ya da kit kullanımları mikoplazma tespitinde sıklıkla kullanılmaktadır [19][20][21][22][23][24] .…”

Role of Recent PCR Tests for Infectious Ocular Diseases: From Laboratory-Based Studies to the Clinic

“…PCR, in which primers and probes are immobilized on tubes, is also being developed for use in regenerative medicine. Recent reports indicate a trend toward comprehensive PCR testing to identify mycoplasma infection in transplanted grafts [14]. In the field of regenerative medicine, there is a need for kits that can simultaneously and rapidly detect multiple infectious antigen genomes (e.g., viruses, bacteria, and fungi) in addition to mycoplasma infections.…”

Automated Production at Scale of Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells, Chondrocytes and Extracellular Vehicles: Towards Real-Time Release

“…Mycoplasma (gram-negative bacteria) can infect cells but must be absent in any therapeutic cell product. Some mycoplasma species are human pathogens and are a serious regulatory concern in medicinal products [30]. The mycoplasma assay requires PCR, for which the qTOWER device 3 was chosen.…”