Mycoplasma Contaminations in the Cell Culture -Background Information, Detection, Treatment

Dirk Vollenbroich, Ph.D.

Minerva Biolabs GmbHwww.minerva-biolabs.com

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

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)

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

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

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.

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

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

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

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 %

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

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

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 !!

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

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

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

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

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

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

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

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

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

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

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

Effect of Mynox® on Mycoplasma

Electron micrographs of mink lung cells (ML cells), contaminated withMycoplasma hyorhinisMycoplasma hyorhinis

Source: M. Özel, Robert-Koch-Institut Berlin

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