In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain

A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h.     

The Effects of Mycoplasma Contamination upon the Ability to Form Bioengineered 3D Kidney Cysts

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

Detection of mycoplasma contamination in tissue cultures by fluorescence microscopy

Summary The in situ staining method of Chen (1977) for the detection of mycoplasma contaminants in tissue cultures was tested in cultures of human skin fibroblasts after controlled contamination with Mycoplasma arginini. It is concluded that this method is reliable only at infection rates of 100% or higher, i.e., at one mycoplasma or more per tissue-culture cell.     

Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations

Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.     

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.     

一种快速判定细胞培养中支原体污染的方法——瓜氨酸测定法

目的：开发一种简便、快速、能及时发现细胞培养中支原体污染的方法。方法：用HPLC检测细胞培养中瓜氨酸是否存在及其量的大小。结果：当细胞培养被支原体污染时,培养基中精氨酸量明显下降,同时有瓜氨酸出现;当支原体被消除后,瓜氨酸即消失。结论：在细胞培养中瓜氨酸的出现与支原体污染的关系是特异的,用HPLC在2h内即可检出,表明该方法可靠、简便、快速,可作为细胞培养过程中支原体污染的常规监测手段。     

Detection of mycoplasma contaminations in cell cultures by PCR analysis

Mycoplasma contamination is still one of the main problems in using cell cultures in biological and medical research and in the production of bioactive substances, because mycoplasma can alter nearly all parameters and products of the cell. They can persist undetected in the culture if no special detection methods are applied. In recent years, the PCR technology has become a commonly used method to analyze genomic DNA and the expression of genes, with both high specificity and sensitivity. This technique can be effectively employed for the detection and even the identification of mycoplasma contaminations in cell cultures applying primers complementary to the 16S rDNA region. Although this technique, once established, is characterized by simplicity and speed, PCR is still a complex process and its sensitivity and specificity can be influenced by a number of different parameters, e.g. inhibiting compounds originating from the preparation process of the DNA, RNA or cDNA, contamination of the solutions with PCR products, and the selection of a primer pair which does not cover all the mycoplasma species occurring in cell cultures. Thus, adequate controls have to be included to obtain reliable results. The present review examines the use of different primers of the 16S rDNA region including their specificity, the sensitivity applying various DNA or RNA preparation procedures, and the methods to detect finally the amplicons. In conclusion, basic nucleic acid preparation and PCR product detection methods offer a simple, fast and reliable technique for the examination of mycoplasma contaminations in cell cultures, provided that the indispensable control assays are implemented.     

细胞培养过程中支原体污染的检测和去除研究

细胞培养过程中的支原体污染相当普遍。如何快速、简便地检测支原体,并且采取有效措施去除支原体一直是细胞培养中急待解决的难题。本文就近年来有关支原体检测及去除方面的工作加以综述。     

Elimination of mycoplasmas from cell cultures utilizing hyperimmune sera

Eighteen cell lines contaminated with various mycoplasmas have been treated with hyperimmune sera and mycoplasmas have been eradicated from all. After treatment the cell lines have been observed for a least one year and they are still free from mycoplasma contamination as ascertained by four independent mycoplasma detection assays. The hyperimmune sera used were of high titer, type-specific and growth-inhibiting. These sera were produced by immunization of rabbits with purified membranes from Mycoplasma orale, M. arginini, M. hominis, M. fermentans, M. hyorhinis and Acholeplasma laidlawii. In addition to elimination of mycoplasmas from cell cultures we have successfully used these sera for detection and typing of mycoplasma contamination in cell cultures.     

Effects of the PKC inhibitor PD 406976 on cell cycle progression, proliferation, PKC isozymes and apoptosis in glioma and SVG-transformed glial cells

It is well established that protein kinase C (PKC) isozymes are involved in the proliferation of glioma cells. However, reports differ on which PKC isozymes are responsible for glioma proliferation. As a means to further elucidate this, the objectives of our research were to determine how inhibition of PKC-alpha, PKC-beta and PKCmu with PD 406976 regulates the cell cycle, cell proliferation and PKC during glioma growth and development. To establish the cell cycle effects of PD 406976 on brain cells (SVG, U-138MG and U-373MG glioma cells), specimens were treated with either dimethylsulfoxide (DMSO; control) or PD 406976 (2 microm). Results from flow cytometry demonstrated that PD 406976 delayed the entry DNA synthesis phase in SVG cells and delayed the number of cells entering and exiting the DNA synthesis phase in both U-138MG and U-373MG cells, indicating that PD 406976 may inhibit G(1)/S and S phase progression. Assessment of cell viability demonstrated a cytostatic effect of PD 406976 on SVG, U-138MG and U-373MG glioma cell proliferation. The PD 406976-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified and demonstrated that exposure of SVG and U-373MG glioma cells to PD 406976 suppressed PKC activity. Western blotting demonstrated reduced PKC-beta1, PKC-gamma and PKC-tau protein content in cells treated with PD 406976. We determined that the growth inhibitory effect of PD 406976 was not as a result of apoptosis.     

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥10⁴ colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 10⁴ CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

细胞培养中支原体污染的PCR检测

根据支原体16s rDNA序列,选择RemyTeyssou设计的三条寡核苷酸链,组成两套引物:P_(1-2a)能检测出细胞培养中常见的各种支原体,P_(1-2b)能检出无胆甾原体。反应可检出体系中10CFV的菌体。此法先用于对实验室人为污染支原体Vero细胞的检测,后与DNA 染色法和培养法比较,检测了49份生物样品,其中24份传代细胞,PCR检测的阳性率为58％,DNA染色法为42％,培养法为33％;三者的灵敏性比较,PCR可检出10~(-3)稀释度的阳性样品,高于其他两种方法。此PCR方法快速、灵敏、特异,适用于细胞培养中支原体污染的检测。     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Cross comparison of rapid mycoplasma detection platforms

The use of animal and plant derived raw materials in mammalian cell culture processes may provide a possible route of entry for adventitious contaminants such as mycoplasma. Mycoplasma contaminations of cell culture represent a serious challenge to the production of biotechnology derived therapeutics. The slow growing nature of mycoplasma can disguise their infection of cultures since cells may continue to proliferate, though at reduced levels and with lesser output of engineered protein. Rapid identification of mycoplasma contaminated cell cultures and materials enables a faster response time to prevent the spread of the contamination. We describe here the comparison of different mycoplasma detection methods: two nucleic acid-based technologies, the standard mycoplasma culture procedure, and a hybrid culture-quantitative PCR assay. In this study, a cell line infected with two species of mycoplasma was used to compare the different detection methods. Our data demonstrates that the two nucleic acid-based techniques are robust methods for detection of mycoplasma and have similar detection capability. In contrast, no mycoplasma was detected in the standard culture assay or in a hybrid culture-quantitative PCR assay. This shows a potential limitation of the culture assay that relies on the ability of mycoplasma to grow in broth media.