Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking

Abstract

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Keeping it clean: the cell culture quality control experience at the National Center for Advancing Translational Sciences

“…A month-by-month breakdown of historical data (Figure 1) shows a 33% positive return rate in the first month. These data are consistent with other reports on contamination rate 12,13 , and perhaps unsurprising given that routine testing had not been implemented previously. Subsequent to the first year of testing, 6-8% of cell lines tested positive (Table 1), with occasional 'spikes' (for example, see November 2018 and April 2019, Figure 1) that were associated with testing and re-testing of a number of cell lines received from external laboratories.…”

“…More recently, there has been an increasing awareness of the impact of mycoplasma contamination on reproducibility in the scientific literature. Screening of cell culture collections have produced estimates that 15-35% of cell lines are mycoplasma contaminated 12,13 (and higher numbers have been reported in individual collections). The recognition of the ease with which tissue culture can be contaminated and the implications for industries such as biotherapeutic production have stimulated the development of off-the-shelf mycoplasma detection kits to allow routine laboratory screening for mycoplasma contamination.…”

The human microglial HMC3 cell line: where do we stand? A systematic literature review

“…Thus, mollicutes can grow until extremely high titers without producing any turbidity in the supernatants. In addition, mycoplasma contaminations can significantly affect the biology of the cell culture, thus the quality of the experimental results [108]. Before freezing the cells, we excluded possible mycoplasma contaminations using the Venor®GeM Classic Mycoplasma detection kit, thus searching mycoplasma DNA in the incubation medium of almost confluent cultures at p3.…”

“…Interestingly, the production of ROS by HMC3 cells growth in either MEM or DMEMF12 was significantly lower, thus suggesting a less activated phenotype under basal conditions when kept in these media (not shown). In addition, we assessed the production of NO indirectly by measuring nitrite accumulation in the incubation media [108]. Nitrite levels were undetectable under basal conditions and remained so after a 24-h treatment with different pro-inflammatory stimuli, including LPS, LPS/IFNγ, and IL-1β, and TNFα used alone or in combination with IFNγ (data not shown).…”

The Use of Short Tandem Repeat Analysis for Cell Line Authentication

“…Однако в последнее десятилетие наиболее широкое распространение получили молекулярно-биологические методы определения подлинности КЛ. Самыми известными и эффективными являются методы, основанные на полимеразной цепной реакции (ПЦР) [7]. Данная группа методов является чрезвычайно чувствительной и селективной.…”