Mycoplasma Detection In Cell Culture – How We Do It In UMBI

Figure showing the presence of mycoplasma in two cell lines i.e. HepG2 and HTB112. The PCR product size of ~270 bp size correspond to the mycoplasma DNA sequence as amplified by PCR. The presence of the PCR product of ~160bp size only indicate no contamination.

Mycoplasma refers to a genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. Mycoplasma are the smallest living cells yet discovered, it can survive without oxygen and are typically about 0.1 µm in diameter. The exact source of the mycoplasma infections is not fully understood, because mycoplasmas are almost ubiquitously prevalent in or on most organisms.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory because these bacteria are common contaminants which persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment.

In UMBI, we always do a mycoplasma screening every 6 month in our stock of cell lines to make sure that the stock is clean from contamination. There are some basic procedures that are used for mycoplasma detection such as agar and broth culture, indirect staining and direct PCR (Young et al 2010). Polymerase chain reaction (PCR) detection is one of the widely accepted methodologies to detect mycoplasma in cell cultures and cell culture products. The advantages of the PCR assay are its sensitivity, specificity, speed, cost efficiency, and the potential to screen a large number of samples (Uphoff & Drexler 2004).

In UMBI, we are using the “e-Myco plus Mycoplasma PCR Detection Kit’ from Intron Biotechnology.  This kit involves a simple PCR method and using DNA primers sequence which code for highly conserved 16S rRNA. Internal controls of this product were constructed to identify false negative results in each reaction. This kit also can detect the mycoplasma using as low as 1 ng of sample. The kit is used for the detection of Mycoplasma species that a most commonly encountered in cell culture including M. arginini, M. fermentans, M. hyorhinis, M. orale and Acholeplasma laidlawii. Desura

To make sure that our cell lines are being protected from mycoplasma contamination, we are always improving our aseptic techniques and practices. To make this happen, all the new users of cell culture lab must undergo a comprehensive training program and competency assessment before doing the lab work. Besides that, if new cells arrive, the staff will grow and test the cells with mycoplasma detection kit before culturing it as a stock. Every 6 months, the cell lines stock will be screening for mycoplasma contamination and being recorded in our Mycoplasma Screening Form (LM-TC-003).  I am happy to say that with the preventive and monitoring schedule, the cell lines in UMBI are free from mycoplasma contamination.

Nurmi Nasir
Science Officer
UMBI