Mycoplasma Contaminations in the Cell Culture Background Information, Detection, Treatment Dirk Vollenbroich, Ph.D. Minerva Biolabs GmbH www.minerva-biolabs.com Cell Threats • Contamination with other cell lines • Yeast • Fungi • Viruses: • Bacteria • Mycoplasma especially bovine Pestiviruses BVDV – Virus of Bovine Virus diarrhea CSFV – Virus Vi off the h classical l i l swine i but also BDV (Borna Disease Virus) Mycoplasma • Found in1898 classified as virus • Bacteria, origin in gram-positive Bacillus/Lactobacillus/ Streptococcus line, but own class • C ll Colloquial: i l Mycoplasma M l or PPLO (pleuropneumonia-like ( l i lik organism) i ) • Class of Mollicutes (soft skin) with the families: ¾ Mycoplasmataceae: Mycoplasma (animal), Ureaplasma (animal) ¾ Acholeplasmataceae: Acholeplasma ((animal, plant)) ¾ Spiroplasmataceae: Spiroplasma (plant, anthropodes, rodent) ¾ Anaeroplasma (ruminant) Mycoplasma (continued) • Pass cellulose- and polyvinyl filter with 0,45 µm pore width • Smalles, self-replicating organisms (0,3 to 0,8 µm, 600 kb to 1700 kb (1/5 of E. coli-genome) with approx. 500 genes • Need N d tto consume cholesterol, h l t l amino i acids, id ffatty tt acids, id vitamins it i and d other th catabolites • Typically arginine, arginine glucose or urea metabolism • Lacks cell wall, but has simple plasma membrane – extremely t l fl flexible ibl in i relation l ti to t environment, i t however h sensitive iti iin relation l ti tto chemical influences – resistant to p penicillin,, contains no p peptido p glycan g y wall – osmotically unstable • Occurring extra cellularly, cellularly only in rare cases intracellularly Mycoplasma (continued) • Parasites for humans, animals, plants, insects, etc. • Effects latent infections of human beings • respiratory tract : genital tract: j i t joints: central nerve system: heart: M. pneumoniae M. genitalium, Ureaplasma urealyticum, M. hominis M fementans, M. f t M arthritidis M. th itidi M. pneumoniae M. pneumoniae Effects on plants: blossom greening (clover, strawberry) yellowing, y g stall ((vine, p pear, ster)) midget growth (rice) sproud shooting (appel) transfer by cicada and leaf fleas • Average cell culture contamination rate: 5 % in industry, 47 % in academics Mycoplasma awaited it d the th development d l t off the th cell ll culture lt in order to find their actual biological niche. niche Mycoplasma in the Electron Microscope Bovince cell line MDBK (a) without and (b) with Mycoplasma Source: Lünsdorf & Rohde, GBF Braunschweig BG Chemistry BookletB 004 Effects on Cell Culture • Inhibition of cell proliferation up to 50% by nutrient withdrawal and secretion of harmful metabolic products McGarrity et al. (1984) In Vitro Cell. Dev. Biol. 20:1 • • fast glucose reduction and formation of acids => pH shift • arginine depletion => inhibition of protein biosynthesis, cell division and growth Influence of immunological reactions ( (macrophage h activation, ti ti iinhibition hibiti off antigen ti presentation, t ti iinduction d ti off signal i l ttransduction) d ti ) Mühlradt et al. (1996) Biochemistry 35:7781 • I fl Influence off virus i proliferation lif ti and d th the iinfection f ti rate t Nar-Paz et al.(1995) FEMS Microbiol. Lett. 128:63 • Cause chromosomal aberrations and multiple translocations McGarrity et al. (1978) In: Mycoplasma infection of cell cultures. Plenum Press S. 213ff • Disturbance of the hybridoma technique, contaminated cells become sensitive i i iin HAT medium di Effects on Cell Cultures (continued) • Accumulation of mycoplasma at cells wall alters cell wall integrity • Significant changes in micro array and gene expression profiles bioinformatics.picr.man.ac.uk/experiments/mycoplasma/ • Mycoplasma can constitute M l tit t up to t 50% off the th total t t l protein t i and d 1515 30% off the isolated DNA • Decrease of the transfection rates by 5% through electroporation • Induction of leopard cells (condensation of the chromatins) Influence almost all functions of the host cell metabolism Frequency and Source of Mycoplasma Species Occurring in Cell Cultures (Literature comparison) A. laidlawii 9% others 18% 20,2 % 25,3 % M. argininii 17% M. hominis 5% M. hyorhinis 20% M. fermentans 3% M. salivarium 5% M. orale 23% 36 9 % 36,9 Sources of Contamination • Primary cultures from the original tissue (incidence approximately 4 %) • • Cross contamination • contaminated cultures • new cultures from unknown sources, also partly from cell banks • virus suspensions, suspensions antibody- solutions or other additions of contaminated cell cultures Direct contamination • serum (only treated serums, e.g. UV or γ-radiated are presumably mycoplasma free) • laboratory instruments, media and reagents, which came into contact with contaminated cultures Sources of Contamination (continued) • The User • direct entry while handling, usually from the oral flora • p transfer droplet • lacking disinfection • careless technical work From:Toni Lindl, Lindl Zell-und Gewebekultur Importance of the Mycoplasma Tests • Cell cultures offer ideal living conditions to parasitic mycoplasma: contamination is possible at any time • Despite titers from 107 to 108 mycoplasma/ml in cell cultures, no apparent projection referring to the contaminating mycoplasma species and the cell type • Microscopically unrecognizable • Standard antibiotics can allow contamination levels lower than detection levels Pen/Strep does not provide protection from contamination levels, !! only each 10th cell culture user regularly tests for mycoplasma contamination !! Instruction for Testing • Regulations: FDA Points to Consider (May 1993), Regularien 21CFR610.30 USDA federal code #9CFR113.28 European Pharmacopoeia 2.6.7, 2 6 7 Suppl. Suppl 5.8 58 ICH Guideline for biotechnological/biological products • Obliged to test: Master cell cultures, cell cultures, virus stocks, control cell cultures Bioproducts from cell cultures (antibodies, hormons, immune stimulators, blood products from cell cultures)) p Vaccines for humans and the veterinary field • Test necessary for: Editors who are aware of the significance of mycoplasma contamination Fluorescence method • Simple, direct indicator for vital mycoplasma • Little operative expense, but very time consuming • Poor indicator for mycoplasma species with tendencies of extra cellular cell absorption via y proteins p cytadherent • Seminars and experience required • Eur Ph Eur. Ph.-listed listed evidence Culture method • Strict requirements for the culture medium and growth conditions (aerobic/anaerobic), generally requires non-standard adjustments f the for h individual i di id l species i • Extremely long testing times of 1 to 4 weeks • Difficult analysis • Broth and disk possible • Advantage: only living mycoplasma i detected is d t t d • Sensitivity: 1 CFU corresponds to average 30 GU M. argininii Source: Mycoplasma Experience Ltd. b = 1 mm bar NAT method • Nucleic acid amplication test with primers and commercial kits free of choice • Eur. Ph. 2.6.7, v 5.8, valid since 01. July 2007 • Validation must show equality to established methods according sensitivity, specificity, and robustness • Can replace indicator methods if sensitivity below 100 cfu/ml • Can replace culture method if sensitivity is below 10 CFU/ml • Can replace both methods if results are required quickly • Cell culture enrichement possible to increase sensitivity Alt Alternative ti Mycoplasma M l Di Diagnostic ti Methods M th d M th d Method N Necessary D Devices i and dE Evaluation l ti Biochemical Verification Methods combined with luminescence detection requires luminescence reader, low sensitivity pp 105 CFU p per test,, high g demands of approx. for sample quality easy usable Biochemical Verification Methods Adenosinphosphorylase Test (6-MPDR-Test) none / requires indicator cell line line, low sensitivity, easily performed Enzyme Immuno Verification ELISA-Reader / specific for mycoplasma species, intermediate sensitivity (106/ml), time intensive Features of Venor®GeM • Validated according to Eur. Ph. 2.6.7, 5.8 • Recommended by the WHO • More than 100x cited in publications p • Specific for > 25 mycoplasma species • Detection limit 1,5 copies/µL, LOD95% = 4,5 copies/µl • Clear yes/no-result after 3 hours • Package sizes: 25, 50, 100 und 250 tests, • User friendly aliquotes á 25 tests. tests • Aliquots of Master-Mix can be stored frozen including hot hot-start start Taq possible. • Also available for real-time PCR Analysis using Venor®GeM 100 bp DNA ladder negative g control / internal control amplification p 100.000 copies 10.000 copies 1000 copies 100 copies 10 copies 1 copy Frequency of Testing • New cell and virus cultures • Each month for continuous cell lines; each week in cases of laboratory contamination • Before each liquid nitrogen storage • p modification of the cell characteristics Upon • In case of problems with result reproducibility Procedure for Contaminated Cell Cultures/ Virus • Isolate the culture immediately (incubator and if possible autoclave) • Immediately lock and test cryo- conserves • y samples p Isolate all cryo-conserved • Inform all possible receipts • Standard disinfection of the laboratory • Initiate immediately treatment with irreplaceable cells Mycoplasma Elimination Methods • Antibiotics average activity: Ciprofloxacin (Ciprobay, Ciproxin), Doxycyclin low activity: Plasmocin, Chloramphenicol, Clindamycin, Azithromycin, Clarithromycin, Tetracyclin, Tiamulin no activity: Penicillin, Streptomycin, Polymyxin, Vancomyin, Erythromycin (only active against some species), Cephalosporine, Sulfametaxol, G418 (Geneticin, (Geneticin Gentamycin Gentamycin-Analogon) Analogon), Bacitracin • Complement Fixation • Co-Cultivation with Macrophages • Physical and chemical methods heat inactivation at 40-42 °C photo inactivation with Hoechst 33258/5-Bromuracil 33258/5 Bromuracil liquid extraction • Autoclave ®Gold Effective Elimination with Mynox y Basically no cytotoxicity Highly effective: up to 100% permanent elimination with first treatment Universal for cells Universal for Mycoplasma Low resistance risk Convenient Format Interoperable with other antibiotics Effect of Mynox® on Mycoplasma Electron micrographs of mink lung cells (ML cells), contaminated with Mycoplasma hyorhinis Source: M. Özel, Robert-Koch-Institut Berlin Recommendations for Improvement • Regularly and sensitive testing (monthly); weekly testing in cases of laboratory contamination • Operate free of standards antibiotics • Whenever possible: separate the work benches and incubators for handling contaminated and mycoplasma free materials • N Never use contained t i d cultures; lt reject j t iimmediately di t l or treated t t d • Disinfect working surfaces and hands with alcoholic spray before and after working procedures procedures, or with the change of the working material • Quarantine new cells of any origin and integrate into the laboratory only after testing negative • For larger loads of serum, incubate on indicator cells 3T3 or Vero over 4 passages before integration and use, and the test supernatants by means of PCR