Mycoplasma Contaminations in the Cell Culture ... - Minerva Biolabs

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:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical l 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
• Colloquial: l Mycoplasma or PPLO (pleuropneumonia-like lik 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)

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 to consume cholesterol, l amino acids, fatty acids, vitamins i and other
catabolites
• Typically arginine, glucose or urea metabolism
• Lacks cell wall, but has simple plasma membrane
– extremely flexible in relation to environment, however sensitive in relation to
chemical influences
– resistant to penicillin, contains no peptido p glycan gy wall
– osmotically unstable
• Occurring extra cellularly, only in rare cases intracellularly

Mycoplasma (continued)
• Parasites for humans, animals, plants, insects, etc.
• Effects latent infections of human beings
respiratory tract : M. pneumoniae
genital tract: M. genitalium, Ureaplasma urealyticum, M. hominis
joints:
M. fementans, M. arthritidis
central nerve system: M. pneumoniae
heart:
M. pneumoniae
• Effects on plants:
blossom greening (clover, strawberry)
yellowing, stall (vine, 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 the development of the cell culture
in order to find their actual biological 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 => p H shift
• arginine depletion => inhibition of protein biosynthesis, cell division and growth
• Influence of immunological reactions
(macrophage activation, inhibition of antigen presentation, ti induction of signal transduction)
ti Mühlradt et al. (1996) Biochemistry 35:7781
• Influence of virus proliferation and the infection rate
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 ii 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 profiles
bioinformatics.picr.man.ac.uk/experiments/mycoplasma/
• Mycoplasma can constitute t up to 50% of the total t protein and 15- 30% of
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)
20,2 %
25,3 %
A. laidlawii
others
9%
18% M. argininii
17%
M. hyorhinis
20%
M. salivarium
5%
36,9 %
M. orale
23%
M. hominis
5%
M. fermentans
3%

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, 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
• droplet transfer
• lacking disinfection
• careless technical work
From:Toni 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 10 7 to 10 8 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
!! 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 267 2.6.7, Suppl. 58 5.8
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)
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 proteins
• Seminars and experience required
• Eur. Ph.-listed evidence

Culture method
• Strict requirements for the culture
medium and growth conditions
(aerobic/anaerobic), generally
requires non-standard adjustments
for the individual id species
• Extremely long testing times of 1 to
4 weeks
• Difficult analysis
• Broth and disk possible
• Advantage: only living mycoplasma
is detected
t d
• Sensitivity: 1 CFU corresponds to
average 30 GU
M. argininii
Source: Mycoplasma Experience Ltd.
bar = 1 mm

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

Alternative ti Mycoplasma Diagnostic Methods
Method
Necessary Devices and Evaluation
Biochemical Verification Methods
combined with luminescence detection
Biochemical Verification Methods
Adenosinphosphorylase Test
(6-MPDR-Test)
Enzyme Immuno Verification
requires luminescence reader, low sensitivity
of approx. 10 5 CFU per test, high demands
for sample quality
easy usable
none / requires indicator cell line, low
sensitivity, easily performed
ELISA-Reader / specific for mycoplasma
species, intermediate sensitivity (10 6 /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
• Specific for > 25 mycoplasma species
• Detection limit 1,5 copies/µL, LOD 95% = 4,5 copies/µl
• Clear yes/no-result after 3 hours
• Package sizes: 25, 50, 100 und 250 tests,
• User friendly aliquotes á25tests
tests.
• Aliquots of Master-Mix can be stored frozen
including hot-start Taq possible.
• Also available for real-time PCR

Analysis using Venor ® GeM
100 bp DNA ladder
negative control / internal control amplification
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
• Upon modification of the cell characteristics
• 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 samples
• 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, 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
liquid extraction
• Autoclave

Effective Elimination with Mynox ® Gold
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
• Never use contained cultures; reject immediately or treatedt • Disinfect working surfaces and hands with alcoholic spray before and after
working 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