Significance, Detection and Elimination of ... - Minerva Biolabs

Significance, Detection and Elimination of
Contaminations in the Cell Culture
Minerva Biolabs GmbH
www.minerva-biolabs.com
info@minerva-biolabs biolabs.com
Tel.: +49-30-2000-4370
Fax.: +49-30-2000-4379
-1-

The Usual Suspects
• Contamination with other cell lines
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-2-

Cross-contamination
The contamination of a cell line with a cells from another type
• Avoidance:
Only one cell line under the clean bench at a time
Separate medium bottles for each cell line
• Detection:
Difficult, especially with cell line of comparable morphology
Indications are changes in growth rates, morphology of prduct characterisation as well as
not reproducable results
Applicable detection techniques: Isoenzyme pattern analysis, DNA sequencing, Southern
blot bo analysis ayss
-3-

The Usual Suspects
• Contamination with other cell lines
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-4-

Yeast
unicellular fungi
size can vary greatly depending
on the species, typically
measuring 3–4 µm in diameter
smaller than eukaryotic cells
curable, but not recommended
antimycotics Amphotericin B and Nystatin
-5-

The Usual Suspects
• Contamination with other cell lines
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-6-

Fungi
Medium color turns yellow quickly
Under the microscope detectable as a
cotton-like fluff
Treatment with antimycotics possible at early stage
contamination
With fluffs strong spore formation expected:
Do not open ⇒ destroy immediately!
-7-

The Usual Suspects
• Contamination with other cell lines
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-8-

Contamination with Pestivirus
• BVDV typical bovine infection
i
• Contamination via sera: 20-30 % of raw sera, up to 49 % in
commercially available sera
• Testing via cell culture and CPE on indicator cell lines
• No statistics regarding the contamination of cell cultures available
-9-

Impact of Pestiviruses
Diagnosticsi
false-positive results with virus detection via plaque formation
Vaccine-production
Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of
such contamination for vaccine production.
Makoschey B, van Gelder PT, Keijsers V, Goovaerts D., Biologicals. 2003
Sep;31(3):203-8.
Fermentation/cell production
R&D
Diagnostic dilemma encountered when detecting bovine viral diarrhea virus in IVF
embryo production. Given MD, Riddell KP, Galik PK, Stringfellow DA, Brock KV,
Loskutoff NM. Theriogenology. 2002 Oct 15;58(7):1399-407.
-10-

The Usual Suspects
• Contaminations with other cells
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
but also BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-11-

Relevance of Bacteria Contaminations
• contamination i due to impure techniques and source materials
• typically recognized via organoleptic test
(turbidity, coloration of the media turns yellow)
• Approx. 6 % of the cultures are unrecognized contaminated (180
samples, 11 positive with Onar ® EUB)
Caulobacter sp.
Methylophilus sp.
Staphylococcus epidermis
Methylobacterium sp.
• Not recognized due to antibiotica in
the cell culture media
• No cure possible ⇒ Destroy!
-12-

Bacteria Contamination - Examples
• Blood bottles
• Organ culture
Incidence of bacterial and fungal contamination of donor corneas preserved by organ culture.Lagenah M, Bohnke
M, Engelmann K, Winter R. Cornea. 1995 Jul;14(4):423-6.
• Fermentation/cell culture
Safety of biological products prepared from mammalian cell culture. DNA. Dev Biol Stand. 1998;93:136-8.
• Tissue Engineering
Microbial contamination of cellular products for hematolymphoid transplantation therapy: assessment of the
problem and strategies to minimize the clinical impact. Lowder JN, Whelton P., Cytotherapy. 2003;5(5):377-90.
Endogenous microbial contamination of cultured autologous preparations in trials of cancer immunotherapy.Padley
DJ, Greiner CW, Heddlesten TL, Hopkins MK, Maas ML, Gastineau DA. Cytotherapy. 2003;5(2):147-52.
• R&D
Cell culture contamination by mycobacteria. Buehring GC, Valesco M, Pan CY. In Vitro Cell Dev Biol Anim. 1995
Nov;31(10):735-7.
-13-

Bacteria Detection with Onar ® EUB
One-step PCR with conventional gel evaluation
Needs EUB Polymerase or other DNA-free polymerases
Detection limit: 6 genomes/µl
Specific for more than 45 bacteria
Includes positiv control and internal amplification control.
yes/no-result after 3 hours
Package sizes: 25, 50, 100 und 250 tests, aliquotes á 25 tests.
Aliquots of Master-MixMix with hot-start Taq possible.
-14-

Analysis using Onar ® EUB
1 kb DNA ladder
positive control amplification
internal control amplification
positive sample / strong contamination
positive sample / moderate contamination
positive sample / weak contamination
negative sample with internal control
-15-

The Usual Suspects
• Contaminations with other cells
• Yeast
• Fungi
• Viruses:
especially bovine Pestiviruses
BVDV – Virus of Bovine Virus diarrhea
CSFV – Virus of the classical swine
• But also: BDV (Borna Disease Virus)
• Bacteria
• Mycoplasma
-16-

Mycoplasma
• Found in1898 classified as virus
• Bacteria, origin in gram-positive Bacillus/Lactobacillus/
Streptococcus line, but own class
• 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)
-17-

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, amino acids, fatty acids, vitamins and
other catabolites
• Typically arginine, i 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 glycan wall
– osmotically unstable
• occurring extra cellularly
-18-

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
zentral 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
-19-

Mycoplasma
awaited the development of the cell culture
in order to find their actual biological i l niche.
-20-

Mycoplasma in the Electron Microscope
Bovince cell line MDBK
(a) without and (b) with Mycoplasma
Source: Lünsdorf & Rohde,
GBF Braunschweig
BG Chemistry BookletB 004
-21-

Frequency of Mycoplasma in Cell Cultures
80%
70%
Ref.: S. Rottem, M. Wormser
60%
50%
5 % in industry
47 % in academics
40%
30%
20%
10%
0%
Deutschland USA Japan Israel Argentinien
-22-

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, induction of signal transduction)
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 in HAT medium
-23-

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 up to 50% of the total 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
-24-

Frequency and Source of Mycoplasma Species
Occurring in Cell Cultures (Literature comparison)
20,22 %
25,3 %
A. laidlawii
others
9%
18% M. argininii
17%
M. hominis
5%
M. hyorhinis
20% M. fermentans
3%
M. salivarium
5%
36,9 %
M. orale
23%
-25-

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
-26-

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
-27-

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 i 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 !!
-28-

Instruction for Testing
• Regulations:
FDA Points to Consider (May 1993), Regularien 21CFR610.30
USDA federal code #9CFR113.28
European Pharmacopoeia 2.6.7, Suppl. 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
-29-

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
-30-

Culture method
• Strict requirements for the culture
medium and growth conditions
(aerobic/anaerobic), generally requires
non-standard adjustments for the
individual species
• Extremely long testing times of 1 to 4
weeks
• Difficult analysis
• Broth and disk possible
• Advantage: only living mycoplasma is
detected
• Sensitivity: 1 CFU corresponds to
average 30 GU
M. argininii
Source: Mycoplasma Experience Ltd.
bar = 1 mm
-31-

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
-32-

Alternative Mycoplasma Diagnostic Methods
Method
Biochemical Verification Methods
combined with luminescence detection
Biochemical Verification Methods
Adenosinphosphorylase osp o Test
(6-MPDR-Test)
Enzyme Immuno Verification
Necessary Devices and Evaluation
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
-33-

Requirements of a Modern Mycoplasma Test
• Specificity for the “typical“ mycoplasma species
• Unique and sensitive result
• Applicable for diverse sample material
• Fast and time saving
• Samples directly testable
• Simple, without intensive study of the laboratory personnel
• Flexible, applicable for small and large sample numbers
• Applicable also for simple laboratory configurations
-34-

Performance
Sample
DNA-Extraction
PCR
Time: 120 - 180 min
Hands-on: approx. 30 min
Gel electrophoresis
-35-

Transport and Stability - Guidelines
• Non-treated at 4°C: max. 24 hours
• After heat treatment (5 min, 95 °C) or DNA extraction
at RT: approx. 3-4 days,
at + 2-8 °C: approx. 2 weeks
at< -18 °C: > 1 year
-36-

Preparation of the Venor ® GeM Kit Components
• Lyophylised components
• Primer/Nucleotides
• Positive Control
• Internal Control
• Rehydration procedure
1. Centrifugation
2. Addition of water
3. Incubation
4. Vortex
5. Centrifugation
• Storage
unopened kit at + 2-8 °C
after rehydration all components below - 18 °C
-37-

PCR Mastermix
• The correct order
1. Water
2. Buffer
3. Other components
• Important!
• Do not vortex Taq polymerase
• Mix your reaction samples
• Aliquot and then add your sample
• Mix and centrifuge
-38-

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, t
• User friendly aliquotes á 25 tests.
• Aliquots of Master-Mix can be stored frozen
including hot-start Taq possible.
• Also available for real-time PCR
-39-

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
-40-

Frequency of Testing
• New cell and virus cultures
• Each month for continuous cell lines; each week in cases of
laboratory contamination
ti
• Before each liquid nitrogen storage
• Upon modification of the cell characteristics
• In case of problems with result reproducibility
-41-

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
-42-

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), 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
-43-

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
-44-

Effect of Mynox ® on Mycoplasma
Electron micrographs of mink lung cells (ML cells), contaminated with
Mycoplasma hyorhinis
Source: M. Özel, Robert-Koch-Institut Berlin
-45-

Recommendations for the Improvement
of Mycoplasma Contamination
• 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 treated
• 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
-46-