Mycoplasma Contaminations in the Cell Culture - · PDF fileMycoplasma Contaminations in the...
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Mycoplasma Contaminations in the Cell Culture -Background Information, Detection, Treatment
Dirk Vollenbroich, Ph.D.
Minerva Biolabs GmbHwww.minerva-biolabs.com
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Cell Threats
• Contamination with other cell lines
• Yeast
• Fungi
• Viruses:especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrheaCSFV Vi f h l i l iCSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
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Mycoplasma
• Found in1898 classified as virus
• Bacteria, origin in gram-positive Bacillus/Lactobacillus/ Streptococcus line, but own class
C ll i l M l PPLO ( l i lik i )• Colloquial: Mycoplasma or PPLO (pleuropneumonia-like organism)
• Class of Mollicutes (soft skin) with the families:
Mycoplasmataceae: Mycoplasma (animal), Ureaplasma (animal)
Acholeplasmataceae: Acholeplasma (animal, plant)( )
Spiroplasmataceae: Spiroplasma (plant, anthropodes, rodent)
Anaeroplasma (ruminant)Anaeroplasma (ruminant)
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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
N d t h l t l i id f tt id it i d th• Need to consume cholesterol, amino acids, fatty acids, vitamins and other catabolites
• Typically arginine glucose or urea metabolismTypically arginine, glucose or urea metabolism
• Lacks cell wall, but has simple plasma membrane
t l fl ibl i l ti t i t h iti i l ti t– extremely flexible in relation to environment, however sensitive in relation to chemical influences
– resistant to penicillin, contains no peptido glycan wallp , p p g y
– osmotically unstable
• Occurring extra cellularly only in rare cases intracellularlyOccurring extra cellularly, only in rare cases intracellularly
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Mycoplasma (continued)
• Parasites for humans, animals, plants, insects, etc.
• Effects latent infections of human beingsrespiratory tract : M. pneumoniaegenital tract: M. genitalium, Ureaplasma urealyticum, M. hominisj i t M f t M th itidijoints: M. fementans, M. arthritidiscentral nerve system: M. pneumoniaeheart: M. pneumoniae
• Effects on plants:blossom greening (clover, strawberry)yellowing, stall (vine, pear, ster)y g ( p )midget growth (rice)sproud shooting (appel)transfer by cicada and leaf fleas
• Average cell culture contamination rate:5 % in industry, 47 % in academics
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Mycoplasma
it d th d l t f th ll ltawaited the development of the cell culture
in order to find their actual biological nichein order to find their actual biological niche.
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Mycoplasma in the Electron Microscope
Bovince cell line MDBK(a) without and (b) with Mycoplasma(a) without and (b) with Mycoplasma
Source: Lünsdorf & Rohde, GBF BraunschweigBG Chemistry BookletB 004
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Effects on Cell Culture
• Inhibition of cell proliferation up to 50% by nutrient withdrawal and secretion of harmful metabolic productsof harmful metabolic productsMcGarrity 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( h ti ti i hibiti f ti t ti i d ti f i l t d ti )(macrophage activation, inhibition of antigen presentation, induction of signal transduction)
Mühlradt et al. (1996) Biochemistry 35:7781
I fl f i lif ti d th i f ti t• Influence of virus proliferation and the infection rateNar-Paz et al.(1995) FEMS Microbiol. Lett. 128:63
• Cause chromosomal aberrations and multiple translocationsCause chromosomal aberrations and multiple translocationsMcGarrity et al. (1978) In: Mycoplasma infection of cell cultures. Plenum Press S. 213ff
• Disturbance of the hybridoma technique, contaminated cells become i i i HAT disensitive in HAT medium
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Effects on Cell Cultures (continued)
• Accumulation of mycoplasma at cells wall alters cell wall integrity
• Significant changes in micro array and gene expression profilesbioinformatics.picr.man.ac.uk/experiments/mycoplasma/
M l tit t t 50% f th t t l t i d 15 30% f• Mycoplasma can constitute up to 50% of the total protein and 15- 30% of the isolated DNA
• Decrease of the transfection rates by 5% through electroporation• 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
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Frequency and Source of Mycoplasma SpeciesFrequency and Source of Mycoplasma Species Occurring in Cell Cultures (Literature comparison)
others18%
A. laidlawii9%
25,3 %18% M. argininii
17%20,2 %
M. hominis5%
M. fermentans3%
5%M. hyorhinis
20%
M. orale23%
M. salivarium5%
36 9 %36,9 %
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Sources of Contamination
• Primary cultures from the original tissue (incidence approximately 4 %)(incidence approximately 4 %)
• Cross contamination • contaminated cultures• contaminated cultures
• new cultures from unknown sources, also partly from cell banks
• virus suspensions antibody- solutions or other additions of contaminated cell• virus suspensions, antibody- solutions or other additions of contaminated cell cultures
• Direct contamination
• serum (only treated serums, e.g. UV or γ-radiated are presumably mycoplasmafree)
• laboratory instruments, media and reagents, which came into contact with contaminated cultures
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Sources of Contamination (continued)
• The User
• direct entry while handling, usually from the oral flora
• droplet transferp
• lacking disinfection
• careless technical work
From:Toni Lindl Zell-und GewebekulturFrom:Toni Lindl, Zell-und Gewebekultur
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Importance of the Mycoplasma Tests
• Cell cultures offer ideal living conditions to parasitic mycoplasma: contamination is possible at any timecontamination 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 cellprojection referring to the contaminating mycoplasma species and the cell type
• Microscopically unrecognizable• Microscopically unrecognizable
• Standard antibiotics can allow contamination levels lower than detection levels Pen/Strep does not provide protection from contaminationlevels, Pen/Strep does not provide protection from contamination
!! only each 10th cell culture user regularly tests for mycoplasma contamination !!
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Instruction for Testing
• Regulations: FDA Points to Consider (May 1993), Regularien 21CFR610.30USDA federal code #9CFR113.28European Pharmacopoeia 2 6 7 Suppl 5 8European Pharmacopoeia 2.6.7, Suppl. 5.8ICH Guideline for biotechnological/biological products
• Obliged to test:Obliged to test:Master cell cultures, cell cultures, virus stocks, control cell culturesBioproducts 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
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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 cytadherent proteinsy p
• Seminars and experience required
• Eur Ph listed evidence• Eur. Ph.-listed evidence
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Culture method
• Strict requirements for the culture medium and growth conditionsmedium and growth conditions (aerobic/anaerobic), generally requires non-standard adjustments f h i di id l ifor the individual species
• Extremely long testing times of 1 to 4 weeks4 weeks
• Difficult analysis
• Broth and disk possible
• Advantage: only living mycoplasmai d t t dis detected
• Sensitivity: 1 CFU corresponds to average 30 GU
M. argininiiSource: Mycoplasma Experience Ltd.b 1average 30 GU bar = 1 mm
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NAT method
• Nucleic acid amplication test with primers andcommercial kits free of choicecommercial kits free of choice
• Eur. Ph. 2.6.7, v 5.8, valid since 01. July 2007
• Validation must show equality to established• Validation must show equality to establishedmethods according sensitivity, specificity, androbustness
• Can replace indicator methods if sensitivity below100 cfu/ml
• Can replace culture method if sensitivity is below• Can replace culture method if sensitivity is below10 CFU/ml
• Can replace both methods if results are requiredquickly
• Cell culture enrichement possible to increasesensitivitysensitivity
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Alt ti M l Di ti M th dAlternative Mycoplasma Diagnostic Methods
M th d N D i d E l tiMethod Necessary Devices and Evaluation
Biochemical Verification Methods combined with luminescence detection
requires luminescence reader, low sensitivityof approx. 105 CFU per test, high demandspp p , gfor sample qualityeasy usable
Biochemical Verification Methods none / requires indicator cell line lowBiochemical Verification MethodsAdenosinphosphorylase Test (6-MPDR-Test)
none / requires indicator cell line, low sensitivity, easily performed
Enzyme Immuno Verification ELISA-Reader / specific for mycoplasmaspecies, intermediate sensitivity (106/ml), time intensive
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Features of Venor®GeM
• Validated according to Eur. Ph. 2.6.7, 5.8
• Recommended by the WHO
• More than 100x cited in publicationsp
• 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• User friendly aliquotes á 25 tests.
• Aliquots of Master-Mix can be stored frozen including hot-start Taq possible.including hot start Taq possible.
• Also available for real-time PCR
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Analysis using Venor®GeM
negative control / internal control amplification
100 bp DNA ladder
g p
100.000 copies
10.000 copies
1000 copies1000 copies
100 copies
10 copies
1 copy1 copy
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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
• Upon modification of the cell characteristicsp
• In case of problems with result reproducibility
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Procedure for Contaminated Cell Cultures/ Virus
• Isolate the culture immediately (incubator and if possible autoclave)
• Immediately lock and test cryo- conserves
• Isolate all cryo-conserved samplesy p
• Inform all possible receipts
• Standard disinfection of the laboratory
• Initiate immediately treatment with irreplaceable cells
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Mycoplasma Elimination Methods
• Antibiotics
average activity: Ciprofloxacin (Ciprobay, Ciproxin), Doxycyclin
low activity: Plasmocin, Chloramphenicol, Clindamycin, Azithromycin,Clarithromycin, Tetracyclin, TiamulinClarithromycin, Tetracyclin, Tiamulin
no activity: Penicillin, Streptomycin, Polymyxin, Vancomyin, Erythromycin(only active against some species), Cephalosporine, Sulfametaxol, G418 (Geneticin Gentamycin-Analogon) BacitracinG418 (Geneticin, Gentamycin Analogon), Bacitracin
• Complement Fixation• Co-Cultivation with Macrophages• Physical and chemical methods
heat inactivation at 40-42 °Cphoto inactivation with Hoechst 33258/5 Bromuracilphoto inactivation with Hoechst 33258/5-Bromuracilliquid extraction
• Autoclave
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Effective Elimination with Mynox®Goldy
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
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Effect of Mynox® on Mycoplasma
Electron micrographs of mink lung cells (ML cells), contaminated withMycoplasma hyorhinisMycoplasma hyorhinis
Source: M. Özel, Robert-Koch-Institut Berlin
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Recommendations for Improvement
• Regularly and sensitive testing (monthly); weekly testing in cases of laboratory contaminationlaboratory contamination
• Operate free of standards antibiotics
• Whenever possible: separate the work benches and incubators for handling contaminated and mycoplasma free materials
N t i d lt j t i di t l t t d• Never use contained cultures; reject immediately or treated
• Disinfect working surfaces and hands with alcoholic spray before and after working procedures or with the change of the working materialworking procedures, or with the change of the working material
• Quarantine new cells of any origin and integrate into the laboratory only after testing negativeafter 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