Optimization of Antibiotic and Antimycotic Compositions for the Eradication of Microorganisms from Cell Cultures

Cell cultures underlie the vast majority of in vitro research. Despite strict sterility requirements and the availability of high-tech equipment to prevent contamination, laboratory practice remains associated with a significant risk of contamination of mammalian cell cultures with bacterial and fungal agents. According to the American Type Culture Collection (ATCC), 15-35% of cell lines are contaminated with mycoplasma, posing a high risk of cross-contamination and leading to significant distortions of the experimental data obtained using such cultures. Furthermore, microbial contamination is a major risk factor when working with cell cultures isolated from patient surgical biospecimens. Despite the availability of ready-made solutions for eradicating microorganisms and preventing cell culture contamination, the problem of antibiotic resistance is particularly relevant for such contamination, and commercially available agents are often ineffective. In this study, combinations of antimicrobial agents were selected to address two of the most common practical challenges in cell culture: (i) complete elimination of mycoplasma infection and (ii) preventing contamination of primary cell cultures with bacteria and fungi that are components of the patient microbiome. A combination of tylosin, tetracycline, ciprofloxacin, erythromycin, and chloramphenicol was shown to completely eradicate mycoplasma from infected cell cultures. A combination of levofloxacin, polymyxin B, vancomycin, meropenem, and the antifungals micafungin and voriconazole completely eliminated the risk of microbial contamination during primary culture isolation, including from complex specimens such as surgical resections from colorectal cancer patients. The selected compositions successfully combine low cytotoxicity with a broad spectrum of antimicrobial activity, allowing for their effective use in laboratory practice. Nonetheless, the principle of minimizing the use of antimicrobial agents should be strictly adhered to in order to control the spread of antibiotic resistance.