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.

Effects of Permeabilization on the Biotransformation of Phenylalanine by Immobilized Tobacco Cell Cultures

1989 ◽  
Vol 44 (3-4) ◽  
pp. 249-254 ◽  
Author(s):  
Jochen Berlin ◽  
Bernd Martin ◽  
Jerzy Nowak ◽  
Ludger Witte ◽  
Victor Wray ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Small Proportion ◽  
Culture Medium ◽  
Tobacco Cell ◽  
Permeabilized Cells ◽  
Oxidative Reactions ◽  
A Cell ◽  
Biotransformation Products ◽  
Severe Growth

Abstract Im mobilized tobacco cells excreted only a small proportion of their main secondary metabolite caffeoylputrescine into the culture medium . Experiments designed to release more of the com ­ pound by permeabilization caused the loss of caffeoylputrescine, probably by oxidative reactions. Moreover, rather mild treatments with permeabilizing agents (e.g. n-propanol) resulted in severe growth inhibition. The ability of permeabilized cells to form caffeoylputrescine and other hy-droxycinnam oyl conjugates from phenylalanine decreased considerably even when such cells were still able to metabolize phenylalanine into various ethyl acetate extractable com pounds (e.g. hydroxycinnamic acids and acetophenones). The formation of new biotransformation products suggests that permeabilized cells could be used as a tool for testing the enzymatic capabilities of a cell culture.

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.

scholarly journals IMMUNOFLUORESCENT STUDIES OF THE POTENTIATION OF AN ADENOVIRUS-ASSOCIATED VIRUS BY ADENOVIRUS 7

1967 ◽  
Vol 125 (5) ◽  
pp. 755-765 ◽  
Author(s):  
Neil R. Blacklow ◽  
M. David Hoggan ◽  
Wallace P. Rowe
Keyword(s):  
Cell Culture ◽  
Latent Period ◽  
Cell Cultures ◽  
Dose Response ◽  
Viral Antigen ◽  
Adenovirus Infection ◽  
Culture Systems ◽  
Cell Culture Systems ◽  
Sequential Inoculation ◽  
A Cell

A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA.

scholarly journals Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

2019 ◽  
Author(s):  
Zain Baaity ◽  
Sven Breunig ◽  
Kamil Önder ◽  
Ferenc Somoyvári
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
U937 Cell ◽  
Sample Volume ◽  
Dna Purification ◽  
Suspension Cell Culture ◽  
Mycoplasma Contamination ◽  
Monitoring Task ◽  
Cell Culture Sample ◽  
Dna Template

Abstract Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.

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.

The Use of Hydrogels as Biomimetic Materials for 3D Cell Cultures

2017 ◽  
Vol 70 (1) ◽  
pp. 1 ◽  
Author(s):  
Eric Y. Du ◽  
Adam D. Martin ◽  
Celine Heu ◽  
Pall Thordarson
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Three Dimensional ◽  
3D Culture ◽  
Biomimetic Materials ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
3D Cell Cultures ◽  
Recent Developments ◽  
A Cell

With the recent developments in cell cultures and biomimetic materials, there is growing evidence indicating that long-established two-dimensional (2D) cell culture techniques are slowly being phased out and replaced with three-dimensional (3D) cell cultures. This is due to the 3D cell cultures better mimicking the natural extracellular matrix (ECM) where cells are found. The emergence of self-assembled hydrogels as an ECM mimic has revolutionised the field owing to their ability to closely simulate the fibrous nature of the ECM. Here, we review recent progress in using hydrogels as biomimetic materials in 3D cell cultures, particularly supramolecular peptide hydrogels. With greater comprehension of the behaviour of cells in these hydrogels, a cell culture system that can be used in a wide array of 3D culture-based applications can be developed.

scholarly journals Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

2019 ◽  
Author(s):  
Zain Baaity ◽  
Sven Breunig ◽  
Kamil Önder ◽  
Ferenc Somoyvári
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
U937 Cell ◽  
Sample Volume ◽  
Dna Purification ◽  
Suspension Cell Culture ◽  
Mycoplasma Contamination ◽  
Monitoring Task ◽  
Cell Culture Sample ◽  
Dna Template

Abstract Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.

scholarly journals Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

2019 ◽  
Author(s):  
Zain Baaity ◽  
Sven Breunig ◽  
Kamil Önder ◽  
Ferenc Somoyvári
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
U937 Cell ◽  
Sample Volume ◽  
Dna Purification ◽  
Suspension Cell Culture ◽  
Mycoplasma Contamination ◽  
Monitoring Task ◽  
Cell Culture Sample ◽  
Dna Template

Abstract Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.

scholarly journals SAT-LB130 Tamoxifen Affects MiRNA Expression in Uterus and Breast Tamoxifen Affects MiRNA Expression in Uterus and Breast Tamoxifen Affects MiRNA Expression in Uterus and Breast

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Lyudmila Gulyaeva ◽  
Vladislav Kononchuk ◽  
Tatyana Kalinina ◽  
Andrey Yarushkin ◽  
Ekaterina Babayanz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Mammary Gland ◽  
Cell Cultures ◽  
Human Cell ◽  
Mirna Expression ◽  
Endometrial Hyperplasia ◽  
Primary Cell Culture ◽  
Target Genes ◽  
Primary Cell ◽  
Human Cell Cultures

Abstract In addition to genetic factors, environmental factors and lifestyle can play a significant role in the development of hormone-dependent tumors, such as endometrial cancer (EC) and breast cancer (BC). The discovery of microRNAs (miRs) involved in the post-transcriptional regulation of many genes, including those of hormonal carcinogenesis, namely, steroid receptors and their target genes, strengthened the epigenetic direction in the study of carcinogenesis mechanisms. A critical event in the development of hormone-dependent human tumors is violation in the metabolism of steroid hormones, primarily estradiol. An interesting aspect of the problem of ERα inhibition is the use of tamoxifen (TAM) in clinical practice in the treatment of hormone-dependent BC. A well-known side effect of TAM is increased proliferation in the endometrium and an elevated risk of EC. One of the mechanisms explaining such differences in the effects of TAM is formation of DNA adducts in endometrial cells, but this mechanism has not yet been substantiated. Therefore, the problem of carcinogenesis of the uterus with this drug remains unresolved and requires further research. The aim of our study was to evaluate the expression of miRs and target genes for hormonal carcinogenesis in the uterus and mammary gland under the exposure with TAM. As an object of study, we used female rats, primary human cell cultures and tissues of TAM-induced human endometrial hyperplasia. The results showed that estradiol enhances the expression of oncogenic microRNAs miR-21-, 221, -222 by three-ten times, both in the rat mammary gland and endometrium, which confirms its oncogenic properties. In the rat endometrium, TAM, to a greater extent than estradiol, increased the expression of oncogenic miRs, especially miR-419, -23a, 24-2,- 27, and significantly reduced the expression of their target genes. In addition, TAM caused a multiple (8-fold) increase in the expression of cyclin D in uterus compared with mammary gland. In most cases, TAM reduced expression of oncogenic miR-21,-221,-222 by 50% in BC primary cell culture whereas in EC primary cell culture expression of oncogenic 190a was increased. We also investigated the activity of estrogen-metabolizing enzymes in tamoxifen-induced human endometrial hyperplasia. A significant difference was found in the expression of estrogen-metabolizing genes (CYP1A,1B, CYP19, SULT1A1, SULT1E1, GSTP1,2, COMT, STS) in TAM-induced endometrial hyperplasia, which may be due to the difference in miRNA expression. Thus, both for the animal model and human cell cultures, it was shown that TAM causes other changes in the expression of microRNAs in the endometrium compared with the breast. Further studies with the identification of target miRNA genes will help identify molecular targets of TAM-induced endometrial hyperplasia. This work was supported by Russian Science Foundation, grant # 19-15-00319.

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