Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations. Therefore, it is crucial to detect and eliminate Mycoplasma contamination in cell culture. This step-by-step protocol presents a comprehensive approach to prevent Mycoplasma contamination in cell culture, as well as to detect and eradicate Mycoplasma to ensure accurate experimental and sequencing results.
Keywords: Mycoplasmas, contamination, diagnostic, eradication, cell cultureMycoplasmas, which measure between 0.3 and 0.8 μm in diameter, are unique prokaryotic microorganisms that lack a cell wall. They are one of the smallest microorganisms capable of surviving independently, residing between bacteria and viruses. More than 200 species of mycoplasma have been identified so far, and new species continue to be discovered annually[1]. Mycoplasma can infect various parts of the body such as lungs, skin, urinary tract, and bloodstream, resulting in a range of symptoms[2].
Apart from the detrimental effects of mycoplasmas on human health, other aspects, especially their impact on cell culture, are also targeted by biological research [3]. Mycoplasmas can infect a wide array of eukaryotic cells, including mammalian, avian, and insect cell lines. Mycoplasma contamination in cell culture can occur through various routes, including contaminated cell lines, reagents, equipment, or personnel. Mycoplasmas have the ability to pass through the filter (0.22 μm) used for decontamination of bacteria, thus posing a high risk of contamination in laboratory cell culture. The contamination of Mycoplasmas can persist and is difficult to detect by means of visual inspection using a conventional microscope. Moreover, while antibiotics like penicillin and streptomycin kill many kinds of bacteria to prevent bacterial contamination, Mycoplasmas are highly resistant to them. As a result, preventing mycoplasma infections via antibiotic use turns out to be a tough challenge.
It has been well-known that the presence of mycoplasma contamination significantly impacts virtually all aspects of cell biology and pathogenesis[4]. The insidious nature of mycoplasma contaminants tends to make contamination go undetected and unnoticed, consequently jeopardizing the reliability of research results and conclusions. For instance, (1) Mycoplasma orale impedes host cell growth by competing for arginine in culture media. This can lead to inconsistencies and less convincing interpretations of experimental findings. This is particularly true of long-term experiments or those requiring precise cell counting [5]; (2) Mycoplasma-related endonucleases can degrade internucleosomal DNA in cell culture, thus altering intracellular signaling pathways, enzymatic activities, and metabolic fluxes [6]; (3) Mycoplasma infection can contaminate genomic DNA, leading to sequencing failure or misalignment in genomic DNA sequencing[7]; (4) Mycoplasma contamination has been shown to interfere with the immune response of infected cells[8]. The contaminations can modulate host immune signaling pathways, thereby potentially confounding studies investigating immune-related processes or therapeutic interventions[9]. (5) Infection of Mycoplasma can potentially dysregulate hundreds of host genes and thus affect the interpretation of gene expression studies and disrupt the reproducibility of experiments. Differential gene expression between infected and uninfected cells may inadvertently be attributed to experimental conditions or treatment effects, leading to incorrect conclusions [10].
Since the infection of mycoplasmas triggers global changes in gene expression and chromatin state in host cells, it is crucial to place greater emphasis on the RNA sequence-related studies. Currently, high-throughput sequencing techniques are growing fast, such as transposase accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq), which allow for the characterization of chromatin accessibility in an unprecedented manner. However, unlike RNA-seq samples that effectively mitigate the impact of mycoplasma contamination through poly(A) enrichment of RNA [7], ATAC-seq employs Tn5 transposase to detect chromatin accessibility on a genome-wide scale, which renders the sequencing results substantially subject to mycoplasma contamination [11]. Consequently, routine and frequent testing, rigorous cell culture techniques, accident prevention, maintaining a clean laboratory environment, and discarding infected cell culture are the most viable options to minimize contamination [12,13].
The objective of this protocol was to design and develop a reliable, rapid and standardized assay for preventing, detecting, and eradicating mycoplasma contamination in cell culture. Additionally, we compared the ATAC-seq results of samples before and after mycoplasma contamination to provide a valuable reference for studying epigenetic regulation.