Performance of PCR-based and Bioluminescent assays for mycoplasma detection

https://doi.org/10.1016/j.mimet.2015.08.010Get rights and content

Highlights

  • PCR and Bioluminescent assays to assess mycoplasma contamination were compared.

  • A controlled determination of the limit of detection of the PCR assay was performed.

  • Mycoplasma detection in frozen cell culture supernatants was also evaluated.

  • The assays presented similar performance.

  • Time of cell pre-culture without antibiotics was critical to obtain safe results.

Abstract

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed.

Introduction

Contamination of cell cultures by mycoplasma remains a significant problem in many laboratories. According to published reports, mycoplasma is present in about 5–35% of all cell cultures (Hay et al., 1989). However, the actual rates are probably higher in a significant number of laboratories that do not test for such phenomena. As reported by Rivera et al. (2009), the level of mycoplasma contamination of the cell cultures evaluated in Mexico was 88.7%.

The massive presence of mycoplasma is unfortunately a big issue, especially because an array of physiological and biochemical parameters are affected by the presence of mycoplasmas in cell culture. It is known that mycoplasma affects cell function, growth, metabolism, morphology, attachment, and membrane properties, contributes to virus propagation in the cell culture, and induces chromosomal abnormalities and DNA damage, as well as cytopathic effects including plaque formation (Lincoln and Gabridge, 1998). The use of contaminated eukaryotic cells may thus cause disastrous effects, since they can alter many cellular parameters, leading to unreliable experimental results and potentially unsafe biological products such as biopharmaceutical products used in cell therapy, tissue engineering and vaccine manufacturing (FDA, 2010). Moreover, mycoplasma contamination is a serious concern for both autologous (Gong et al., 2012) and heterologous (Albon et al., 2013) cell-based therapies and financially, represents considerable economic impact: a study estimated that approximately $350 million of NIH-funded research are potentially affected considering the contamination rate of 11% observed via a survey of NCBI RNA-Sequencing data for mycoplasma sequences (Olarerin-George and Hogenesch, 2014).

Mycoplasmas (class Mollicutes) are the smallest and simplest self-replicating organisms and are devoid of a cell wall. Despite the fact that over 180 mycoplasma species have been described, the vast majority of cell culture contaminants belong to only six species, primarily of human, bovine or porcine origin: Mycoplasma hyorhinis; Mycoplasma orale; Mycoplasma arginini; Mycoplasma fermentans; Acholeplasma laidlawii and Mycoplasma hominis. Due to their size, mycoplasmas are able to pass through 0.2 μm filters commonly used for the sterile filtration of media and media components (Young et al., 2010). Regarding their concentration, mycoplasmas can reach 108 cells per mL of tissue culture medium without causing obvious cloudiness and with no apparent effect on cell growth, unlike ordinary bacterial contamination. The absence of a rigid cell wall makes mycoplasmas unresponsive to antibiotics that target cell wall synthesis, commonly employed for the prevention of bacterial contamination in cell culture.

Detection of mycoplasmas may be difficult for routine cell culture and many cells support low levels of contamination that are only detectable using highly sensitive tests. Therefore, laboratories that do not test for mycoplasma, or use insensitive methods, have a high probability of being infected (UKCCCR, 2000). The source of contamination is usually traced to mycoplasma present in animal serum, or to human oral mycoplasma transferred by droplet infection during cell culture (UKCCCR, 2000); the problem of cross contamination when an infected cell line is obtained from an outside source may also be responsible for mycoplasma contamination.

The gold standard microbiological assay, currently recommended for the mycoplasma testing of biologics by the United States Pharmacopeia (USP), European Pharmacopeia (EP), Japanese Pharmacopeia (JP) and the US FDA, involves the culture of viable mycoplasmas in selective microbiological culture media. Although the procedure enables the specificity of mycoplasma detection in cell culture media ingredients and cell-derived products, the protocols are laborious and are usually very time consuming (up to 28 days for the test completion). The long-term culture required for these conventional assays does not allow their usage for timely “go/no-go” decisions during routine in-process testing (Volokhov et al., 2011).

Recently, PCR-based and other alternative methods for mycoplasma detection, such as Bioluminescent assays, have been proposed as potential approaches for routine cell culture screening. Nucleic acid testing methods employing PCR are widely used and it has been suggested that they have the potential superiority over conventional microbiological methods for mycoplasma testing in terms of analytical sensitivity, simplicity and turnaround time (Louie et al., 2000, Cenciarini-Borde et al., 2009, Molla Kazemiha et al., 2015). Bioluminescent assay, a biochemical method that detects mycoplasma carbamate kinase and/or acetate kinase, is claimed to be very sensitive and fast (30 min) (Mariotti et al., 2008, Molla Kazemiha et al., 2014).

Due to the importance of mycoplasma detection in cell cultures as aforementioned, the aim of this work is to compare the performance of two different commercial assays popularly used for routine cell culture screening, PCR-based and Bioluminescent assays, by assessing the mycoplasma contamination levels in incoming cell cultures at Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories that wanted to monitor the quality of their cell stocks. The results obtained by both assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants.

Section snippets

Cell cultures

Cell cultures used in the present study were from Rio de Janeiro Cell Bank (RJCB), a certified repository of cell lines and also from customers' laboratories that agreed to cooperate with this work and/or from customers' laboratories that intended to monitor the quality of their own cell stocks. All of them were located in Brazilian universities or hospitals. Eighty two (82) different cell cultures were analyzed, being 66 samples from RJCB, and 16 samples from other laboratories.

Sample preparation

Cell lines were

Results

A comparison between the results of the PCR-based and Bioluminescent analysis is presented in Table 1. The cell culture analysis by PCR (Fig. 1) showed a mycoplasma contamination in 20/82 samples (24.4%) and 23/82 samples (28.05%) were shown to be contaminated when the analysis was performed by the Bioluminescent assay. Overall, mycoplasmas were detected in 24/82 of the cell cultures (29.3%) by PCR and/or by Bioluminescent assays. The performance of both assays for detecting mycoplasma was not

Discussion

The purpose of the current study was to compare two different rapid commercial assays, PCR-based and Bioluminescent ones, used widespread for routine mycoplasma detection in laboratories. The present study has shown that these two different assays have a similar performance to assess mycoplasma contamination in cell culture. However, false negative results may be more inconvenient than false positive results, especially when cells or products obtained from cell cultures are used in humans. The

Acknowledgments

We would like to thank the CNPq (grant number 5663165/2010-3), Faperj (grant number E26/190.245/2012) and INMETRO (grant number 260413) for their financial support given to this project. P.F.L. is mainly supported by the CNPq/PROMETRO grant number 563165/2010-3. We would also like to thank the customers' laboratories that agreed to cooperate with this work.

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