Rehabilitation of culture cell lines from mycoplasma infection

Abstract

From the early 50's of last century and to nowadays the cell culture lines are a cornerstone in virological, cytological, biochemical and other biological studies. Cell cultures are essential in studying of cellular regulation mechanisms, virus interaction processes during their replication in sensitive cells, and in production of biologically active materials, including vaccines, enzymes, hormones, and monoclonal antibodies. However, today there is very common and often catastrophic problem that connected with cell line contamination by other microorganisms. Contamination with mycoplasma is of particular concern to scientists around the world. Due to difficulties in its detection, it stays unnoticed in cell cultures, having detrimental effect on cell function and morphological status. Cell cultures contamination with mycoplasmas can lead to degenerative changes in cells and their complete loss and represents a potential source of artifacts in cytological, virological and biochemical studies. Mycoplasma-contaminated cell lines are a major problem in research center laboratories and biotechnology facilities. Mycoplasmas are ultramicroscopic free-living prokaryotes whose sizes range from 200 to 400 nm. They lack a cell wall, which makes it impossible to detect them even with an ordinary microscope. In addition, mycoplasmas do not cause turbidity in cell culture media, which often accompanies bacterial or fungal cell culture contamination. The most important thing is that mycoplasma infection usually does not lead to cell death. Therefore, they can multiply and go unnoticed in a cell culture flask for a long period, becoming a major obstacle to reliable and standard in vitro experiments. Nowadays, the rehabilitation of the cell line from mycoplasmas is a very complex and urgent problem. According to many researchers, their attempts to decontaminate cell culture from mycoplasmas have been ineffective. Due to conclusions from their work, all cell lines infected with mycoplasmas are subject to immediate destruction. At the same time, the purchase of new cell lines requires considerable material costs. Therefore, there is a need to find real approaches in the rehabilitation of cell culture lines in research and production laboratories. The article represents the results of experimental studies focus on the decontamination of 12 cultures cell lines of animal origin from mycoplasmas. The research was conducted during 2019-2020 at the department of biotechnology and viral vaccine control (culture line sector) of State scientific control institute of biotechnology and strains of microorganisms. The cell culture lines with PCR-detected mycoplasma infection were used in the experiments, namely: FLK - sheep embryonic kidney cell line; PO - sheep kidney cell line; RK-13- rabbit kidney cell line; RK-15 - pig kidney cell line; MDBK- bovine kidney embryo cells; Vero- African green monkey kidney cells; Marc-145 - African green monkey kidney-derived MA-104 cells; BGM - African green monkey kidney cells; BHK-21 / clone 13 - Syrian hamster kidney cells; KST (KST) - cells of coronary vessels of cattle; SPEV (original name - SNEV) - pig kidney cells; A-72 - culture of subcutaneous tumor cells in dogs. On the early stages of the study, the optimal allowable concentration of the drug ciprofloxacin, which was used in our laboratory to rehabilitate cell cultures from mycoplasmas, was determined. It has been experimentally established that ciprofloxacin at concentration of 20 μg/cm3 completely inhibits the reproduction and life cycle of mycoplasmas and does not cause a significant effect on the cells themselves. Ciprofloxacin in concentration of 20 μg/cm3 was added into the growth and maintenance medium for five consecutive passages to the flasks with mycoplasma-contaminated cell cultures. After forming a cell monolayer in the flasks, it was always thoroughly washed five times with Hanks' solution, and only then the medium was replaced with a supportive one. Thus, the full cycle of cell culture remediation, from the moment of seeding the cells in the flasks until the next reseeding of cells took 3-4 days. 5 cycles were performed with each cell culture affected by mycoplasmas. At the completion of rehabilitation process, the samples of cell culture supernatant from each cell culture line were collected and examined by PCR for the presence of mycoplasmas. As a result, after remediation process of cell cultures mycoplasma infection was detected in only one sample. The efficiency is 88.9% and it proves that the drug ciprofloxacin can be successfully used for decontamination of cell cultures in scientific and industrial virological laboratories.