Detection of mycoplasma contamination in cell cultures and bovine sera

Dvořáková, Hana; Valíček, L.; Reichelova, M.

doi:10.17221/5622-vetmed

Cited by 4 publications

(6 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although there is not much information about the expected rate of contamination, some authors have reported 0-19% of FBS batches to be positive for mycoplasma. 1,2,6,9,22 Our pestiviral RT-rtPCR results indicated that a high proportion (84%) of the FBS batches contained viral RNA. Our data are in accordance with previous studies; multiple authors have reported pestiviral contamination rates of 22-100% in commercial FBS batches.…”

Section: Discussionmentioning

confidence: 80%

“…The most likely sources of cell culture contamination with mycoplasma appear to be laboratory personnel, FBS, and other infected cell cultures. 9,17 Microbiologic culture is the gold standard test to detect every kind of mycoplasma contamination in cell cultures without considering the origin and species of mycoplasma. However, culture is time-consuming and may lead to false-negative results.…”

Section: Discussionmentioning

confidence: 99%

“…Although there is not much information about the expected rate of contamination, some authors have reported 0–19% of FBS batches to be positive for mycoplasma. 1,2,6,9,22…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents

et al. 2020

View full text Add to dashboard Cite

Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them.

show abstract

Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents

et al. 2020

View full text Add to dashboard Cite

show abstract

“…The materials such as cell seed, virus seed, tissue culture medium, fetal bovine serum [13] were screened qualitatively for the mycoplasma contamination by polymerase chain reaction before initiating any activity. The detection of mycoplasma through PCR is sensitive, rapid and suitable method for the cell culture facility [14]. This primer pairs can able to detect the species of mycoplasma, (Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma arginini, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma hominis, Mycoplasma pulmonis, Mycoplasma arthritidis, Mycoplasma neurolyticum, Mycoplasma hyopneumoniae,Mycoplasma capricolum) and one species Ureaplasma (Ureaplasma urealyticum) with specific amplified product for each species.…”

Section: Mycoplasma Detectionmentioning

confidence: 99%

Standardization of microcarrier based bioreactor culture in parallel with roller bottle for rabies virus (PV-11) propagation in Vero cell using MEM eagle’s and RPMI 1640 medium

Sekar¹,

Premkumar²,

Mohan³

et al. 2022

World J. Bio. Pharm. Health Sci.

View full text Add to dashboard Cite

The Vero cell is the continuous cell line used as a cell substrate for many viral vaccines manufacturing including Rabies vaccine. The Pitman and PV-11 strains of rabies virus are commonly used for production of rabies vaccines. In our study the PV - 11 strain is used as seed virus for the production of Vero cell derived inactivated Anti Rabies Vaccines (Lyophilized). The working cell bank and working virus seed lot were prepared and tested for its sterility and mycoplasma contamination. In this study the roller bottle method and micro carrier (cytodex-1) based bioreactor culturing method were standardized with MEM Eagles and RPMI 1640 medium. The outcome of the two methods were compared ,to find out the medium and culturing method which can give higher concentration of Vero cells to get higher viral titre in mass production. The quantities of Vero cells obtained from passage 144 to 148 were assessed using a haemocytometer cell count procedure. The quantity of the Vero cell obtained per roller bottle ranged from 222.82x106 cells to 236.67x106 cells in MEM Eagles medium and 227.80x106 cells to 230.66x106 cells in RPMI 1640 medium. The average quantity of Vero cells in roller bottles were 1.12 x 106 cells per ml of culture. In the bioreactor culturing method the Vero cells obtained in the concentration of 1.69 to 1.75 million cells / ml of culture by MEM Eagles medium and 1.67 to 1.70 million cells / ml by RPMI 1640 medium. In the bioreactor the average yield of vero cells ranged 1.70 x106 cells per ml of culture. It is estimated that about 35% increase in Vero cell quantity in bioreactor system than the roller bottle culturing method. 0.3 MOI (Multiplicity of Infection) of rabies virus was kept as constant for infection of confluent monolayered Vero cells in both roller bottle and bioreactor culture. Four viral harvests were collected at three day intervals and replenished with fresh culturing medium each time. The viral titres in each viral harvest were quantified using in-house standardized RT-PCR method; the roller bottle method yielded viral titre log 6.044 to 7.106 and the bioreactor culture yielded log 6.559 to 7.216 rabies viral particles. It is observed that the bioreactor culture yielded more viral titre than the roller bottle culture using both the medium.

show abstract

“…The contamination can cause disastrous effects on eukaryotic cells as it tends to alter the cells at a molecular level and compromises the value of the contaminated cell lines in providing accurate data for life science research. It can induce alterations in cellular parameters (e.g., chromosome aberrations, changes in metabolism and cell growth) leading to unreliable experimental results and potentially unsafe biological products [ 2 , 3 ]. In cell culture laboratories, infection usually occurs with the same mycoplasma species, and this proves that mycoplasma infections are often spread from one culture to another [ 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy

2018

View full text Add to dashboard Cite

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells. Graphical abstractFTIR microspectroscopy is able to differentiate between mycoplasma infected cells (LC for low contamination and HC for high contamination) and control non-infected cells (CN).

show abstract

Detection of mycoplasma contamination in cell cultures and bovine sera

Cited by 4 publications

References 23 publications

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents

Standardization of microcarrier based bioreactor culture in parallel with roller bottle for rabies virus (PV-11) propagation in Vero cell using MEM eagle’s and RPMI 1640 medium

Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy

Contact Info

Product

Resources

About