Cell Culture Conquests: Finding and Defeating the Invisible Enemy
Jtranslational research requires extensive preclinical development. During these early stages, scientists make extensive use of cell cultures to test and validate a wide range of research questions before moving on to animal and human models. As such, in vitro models are essential tools for advancing basic and applied science. Although culture protocols for different cell types vary widely, all cell cultures have one key consideration in common: the need to mitigate contamination.
Scientists use a variety of approaches to guard against contaminants, including using aseptic techniques, adding antimicrobial agents to cell cultures, filtering reagents, and routinely screening cultures. However, despite best efforts, cell cultures become infected, affecting cell viability and a range of other biochemical and biomolecular pathways. When this happens, scientists often have to discard the cells and start over, wasting valuable resources and time.
Mycoplasma – a submicron-sized bacterium with no cell wall – is a particularly formidable enemy because it is ubiquitous, resistant to commonly used antibiotics, small enough to pass through filters and notoriously difficult to identify in cell culture. How then do scientists combat mycoplasma contamination when the enemy is moving stealthily under the radar?
Several methods for detecting mycoplasmas in cell cultures are available, which together provide researchers with a toolbox of options to effectively identify mycoplasmas and take the necessary steps to eliminate them. For example, direct cultivation of culture samples on specialized mycoplasma plates provides scientists with a direct assay. However, the combined culture and assay time, which can exceed one month, often makes this approach impractical because it eliminates the possibility of rapidly isolating and treating contaminated cultures. Alternative approaches that stain bacterial DNA using fluorescent nuclear markers, such as DAPI or Hoechst, are rapid but do not provide definitive evidence on their own.
To overcome these speed and specificity limitations, scientists use highly sensitive polymerase chain reaction (PCR) technology to rapidly amplify even the smallest amounts of mycoplasma DNA from contaminated cultures. Widely regarded as the gold standard for mycoplasma detection, the accuracy of PCR depends on the use of appropriate reagents, such as primers, nucleotides and polymerase, and the removal of contaminating DNA non-mycoplasmas from the PCR environment.
The Complete MilliporeSigma Suite of mycoplasma detection and removal reagents, kits and techniques provide a robust end-to-end solution for detecting and removing contamination from mycoplasma cell cultures. Their Lookout® One-Step PCR Mycoplasma Detection Kit simplifies preparation and selection of appropriate primers, nucleotides, polymerase and internal controls. The freeze-dried reaction mix streamlines PCR workflows by providing an easy-to-use one-step solution. In addition, Lookout® DNA Eraser Wipes providing researchers with a reliable option to eliminate nucleic acid contamination in the PCR environment, thereby ensuring accurate PCR detection of mycoplasma. Once detected, scientists can use the Lookout® Mycoplasma Removal Kit to reliably process cell cultures. This two-step kit combines various biological reagents that are highly effective in completely eliminating mycoplasma with minimal cytotoxicity and maintaining normal cellular attributes. For a more comprehensive suite of options, MilliporeSigma also provides reagents for Mycoplasma culture detection and DNA staining.
Taken together, these kits and reagents help scientists identify and neutralize an otherwise formidable enemy, save time and resources, and ensure the accuracy and reliability of research data obtained from cell cultures. in vitro.