scholarly journals Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Cord C. Uphoff ◽  
Sabine-A. Denkmann ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Detection Method ◽  
Mycoplasma Species ◽  
Mycoplasma Contamination ◽  
Cure Rate ◽  
Mycoplasma Detection ◽  
Additional Cell ◽  
Limited Period

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

scholarly journals Detection of mycoplasma contamination in cell cultures and bovine sera

2012 ◽  
Vol 50 (No. 6) ◽  
pp. 262-268 ◽  
Author(s):  
H. Dvorakova ◽  
L. Valicek ◽  
M. Reichelova
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Control Measures ◽  
Mycoplasma Species ◽  
Virus Propagation ◽  
Culture Methods ◽  
Animal Virus ◽  
Elisa Kit ◽  
Acholeplasma Laidlawii ◽  
Mycoplasma Detection

Contamination of cell cultures and sera used for animal virus propagation with mycoplasmas represents a serious problem, especially in virology. Therefore specific control measures must be used. To achieve this we introduced PCR for the detection of mycoplasma species in cell cultures and compared its results with ELISA and microbiological culture. Seven mycoplasma species which are the most common contaminants of cell lines (Mycoplasma arginini, M. fermentans, M. hyorhinis, M. bovis, M. orale, M. hominis, and Acholeplasma laidlawii) were used to verify the method. Then we assessed five selected cell lines and three bovine sera by the PCR, ELISA and culture methods and compared the results. PCR was positive for all of the mycoplasma species tested. ELISA kit used (Mycoplasma detection kit, Roche, Germany) allowed detection of only four species of contaminating mycoplasmas (Acholeplasma laidlawii, Mycoplasma arginini, M. hyorhinis, and M. orale). All the methods detected contamination of the VERO and RK13 cell lines. The agents of contamination were determined by the species-specific ELISA kit as Mycoplasma arginini and M. orale, respectively. Other cell lines and sera tested were not contaminated with mycoplasma. The results confirmed that the PCR method used in the present study is a sensitive, fast and specific detection method of mycoplasma contaminations and is suitable for routine mycoplasma detection in cell cultures and bovine sera.

Detection of Mycoplasma hominis and Mycoplasma orale in cell cultures by immunofluorescence

1978 ◽  
Vol 24 (6) ◽  
pp. 689-692 ◽  
Author(s):  
P. Payment ◽  
M. Corbeil ◽  
A. Chagnon
Keyword(s):  
Cell Cultures ◽  
Culture Medium ◽  
Mycoplasma Hominis ◽  
Mycoplasma Species ◽  
Detection And Identification ◽  
Human Cell Cultures ◽  
Hypotonic Treatment ◽  
Immunofluorescent Technique ◽  
Indirect Immunofluorescent ◽  
Mycoplasma Contaminants

Mycoplasma contaminants of animal and human cell cultures were rapidly detected and identified by an indirect immunofluorescent technique. Cells suspected of being contaminated by mycoplasmas were grown as monolayers on chamber slides in a culture medium selected to promote mycoplasmal growth. Before fixation by acetone, the monolayers were subjected to a hypotonic treatment to cause swelling of the mycoplasmas. Detection and identification were then performed by indirect immunofluorescence using rabbit antisera to various mycoplasma species. The correlation between results obtained by the standard isolation procedure and those obtained by this method was very close.

Effects of Deoxycytidine on Mycoplasma-Associated Inhibition of Thymidine Incorporation and Growth in Antifolate-Containing Media

1988 ◽  
Vol 1 (2) ◽  
pp. 123-137
Author(s):  
James W. Mier ◽  
John Przygoda ◽  
Mark Allegretta ◽  
Peeter A. Poldre ◽  
Ruth B. Kundsin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Cellular Proliferation ◽  
Thymidine Incorporation ◽  
Cytidine Deaminase ◽  
Leukemia Cell ◽  
Lymphoid Cells ◽  
Mycoplasma Species ◽  
Cell Counts ◽  
Selection Medium

Several mycoplasma species markedly inhibit lymphokine- and mitogen-induced 3H-thymidine incorporation in cultured lymphoid cells, but have negligible short-term effects on cellular DNA synthesis as assessed by cytofluorography or by cell counts. The deoxyribonucleotide precursor deoxycytidine (dC) reverses this inhibition, but has little effect on isotope incorporation in uninfected cultures. Human lymphoblastoid leukemia cell lines contaminated with mycoplasma and hypoxanthine guanosine phosphoribosyl transferase (HGPRT)-deficient subclones do not grow in conventional HAT medium, but the unselected parent lines proliferate when dC is included in the culture medium. The beneficial effect of dC on the growth of contaminated cultures in selection medium is amplified by the addition of the cytidine deaminase inhibitor tetrahydrouridine (THU). These observations and corroborating nucleotide pool analysis suggest that dC may exert its beneficial effects on cellular proliferation and isotope utilization by inhibiting a mycoplasma-associated enzyme, thymidine phosphorylase. The data also suggest that the conversion of dC to dU by the cellular enzyme cytidine deaminase reduces the ability of dC to salvage contaminated cultures in the presence of an antifolate. The addition of dC to the culture medium in various 3H-thymidine incorporation assays makes possible the detection of stimulatory lymphokines despite the presence of mycoplasma contamination of the indicator cells. The normalization of nucleotide pools and cellular growth of mycoplasma-infected HGPRT (+) human leukemic cell lines with the addition of dC to HAT selection medium has made possible the use of infected HGPRT-deficient subclones as fusion partners in the generation of T-T hybridomas. Our studies also suggest that the ability of cells to grow in HAT medium only when dC is included is presumptive evidence for mycoplasma infection.

scholarly journals Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture

2020 ◽  
Vol 21 (11) ◽  
pp. 3784 ◽  
Author(s):  
Quanyuan Wan ◽  
Xiaohui Liu ◽  
Zihua Zeng ◽  
Zhenghu Chen ◽  
Yanting Liu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Cultured Cells ◽  
Detection System ◽  
Mycoplasma Contamination ◽  
Detection Model ◽  
Mycoplasma Detection ◽  
Infected Cells ◽  
Microplate Reader ◽  
Multiple Species

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

scholarly journals PRESENT KNOWLEDGE AND EXPERIENCE ON THE STRATEGIES EMPLOYED BY MYCOPLASMA CONTAMINATION OF THE HUMAN CELL CULTURES

2016 ◽  
Vol 4 ◽  
pp. 763-766
Author(s):  
Nevenka Velickova ◽  
Misko Milev ◽  
Gorgi Sumanov ◽  
Biljana Petrova
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
External Surface ◽  
Micronucleus Assay ◽  
Mycoplasma Species ◽  
Mycoplasma Contamination ◽  
Growth Morphology ◽  
Aerosol Droplet ◽  
Human Cell Cultures ◽  
A Cell

Introduction: Mycoplasma species often contaminate cell cultures and other cell-derived biological substances, leading to detrimental effects on the host that include changes in growth, morphology, metabolism and protein synthesis. In cell cultures, mycoplasma are extracellular parasites, usually attached to the external surface of a cell membrane. Many researchers use a mixture of penicillin and streptomycin in the cell culture to prevent contamination. Material and methods: We prepared cell cultures of lymphocytes from peripheral blood of 12 subjects and used micronucleus, assay which is the standard method, for detection of micronuclei in binuclear lymphocytes.Results: Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack cell wall. Streptomycin inhibits about half the mycoplasma strains but is ineffective against others. In fact, mycoplasma is generally resistant to most antibiotic mixtures commonly used in cell culture. We didn’t find any mycoplasma contamination in the cell culture where penicillin-streptomycin mixture was absent, but confirmed infection in the culture containing mixture of antibiotics.Conclusion: Antibiotics and mixture of antibiotics like penicillin-streptomycin mixture does not protect the cell culture against mycoplasma contamination. Hence, contamination can spread rapidly to other cell lines through aerosol droplet dispersion.

scholarly journals Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

2008 ◽  
Vol 74 (17) ◽  
pp. 5383-5391 ◽  
Author(s):  
Dmitriy V. Volokhov ◽  
Hyesuk Kong ◽  
Joseph George ◽  
Christine Anderson ◽  
Vladimir E. Chizhikov
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Mdck Cell ◽  
Insect Cell ◽  
Incubation Temperature ◽  
Insect Cell Line ◽  
Mycoplasma Species ◽  
Mycoplasma Detection ◽  
Mammalian Cell Lines

ABSTRACT In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.

scholarly journals Methyl Jasmonate Induces Enhanced Podophyllotoxin Production in Cell Cultures of Thracian Flax (Linum thracicum ssp. thracicum)

2015 ◽  
Vol 10 (7) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Pavlina Sasheva ◽  
Iliana Ionkova ◽  
Nadezhda Stoilova
Keyword(s):  
Mass Spectrometry ◽  
Methyl Jasmonate ◽  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Sustainable Production ◽  
Chemotherapeutic Drugs ◽  
Full Strength ◽  
Half Strength ◽  
Strength Medium

The Linum thracicum ssp. thracicum cell lines developed in this study are a feasible source for the sustainable production of podophyllotoxin, a lignan with an aryltetralin skeleton that is used for the manufacture of the chemotherapeutic drugs etopophos and teniposide. We used mass spectrometry to confirm the presence of the aryltetralin lignan in the thracian flax cell cultures. Next, we explored how changes in the culture medium influenced the podophyllotoxin content. Out of six developed cell lines, four were selected for further experiments and challenged with elicitors. The selected cell lines clustered into two groups: developed in full strength medium (Li) vs developed in half strength medium (HS). While podophyllotoxin production in the Li cell lines was boosted by 80% upon administration of the elicitor methyl jasmonate, the HS lines produced high amounts of the target metabolite triggered by reduced concentration of nutrients and were only slightly influenced by the elicitor.

scholarly journals Practical aspects on identification, cultivation and characteristics of varicella-zoster virus isolates

2020 ◽  
Vol 10 (2) ◽  
pp. 387-396
Author(s):  
F. G. Nagieva ◽  
E. P. Barkova ◽  
A. N. Lisakov ◽  
A. V. Sidorov ◽  
V. V. Zverev ◽  
...  
Keyword(s):  
Herpes Zoster ◽  
Varicella Zoster Virus ◽  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Target Cells ◽  
Varicella Zoster ◽  
Extracellular Virus

Until now, it has been considered that infectivity of the varicella-zoster virus (VZV) is closely related to target cell, and newly formed virus is not released into the culture medium. It is also known that it is hard to grow VZV in cell cultures, due to its slow replication rate and a limited range of sensitive cell cultures. In addition, VZV isolation depends on type of cell culture used, nature of clinical material, presence of viable virus and transport time. Objectives. To study production of infectious extracellular VZV in various cell cultures. Materials and methods. Eight cell cultures were used, including human embryonic diploid lung cells and human embryonic dermomuscular tissue (KM-27), as well as continuous human and monkey cell lines. Crusts detached from vesicular lesions were used as clinical isolates, which were placed into cryo-vials added with transport medium and transferred in liquid nitrogen. VZV infectivity was assessed in cell cultures by using hemo-adsorption assay with erythrocyte suspension isolated from guinea pig or human zero group blood and confirmed by indirect immunofluorescence with polyclonal sera from varicella or herpes zoster convalescents. Results. There were examined 27 clinical samples consisting of crusts from vesicular lesions isolated from patients with chickenpox, as well as one sample from 63-year old patient with exacerbated recurrent herpes zoster. Primary infection with clinical isolates was performed on diploid human lung embryo cells (HLEC) at low temperature. It was found that clinical samples collected within day 1–18 inclusive after the onset of skin eruption were able to induce cytopathic effects in HLEC cell monolayer such as cytolysis around dermal crusts. Specificity of cytopathic effect was confirmed by using real-time polymerase chain reaction (RT-PCR). Viral antigens were prepared on 7 cell lines infected with the laboratory strain Ellen VZV (USA) to assess the immune sera. A high anti-VZV specificity of mouse sera was detected by ELISA while all the lysates of infected cell lines were used as the solid-phase sorbent. In experiments on VZV reproduction demonstrated that extracellular virus was released into the culture medium starting from day 1 after infection of target cells, and infectivity of the virus-containing fluid ascends during further cultivation.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

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