Lonza Webinar: Mycoplasma – Uncover the Hidden Enemy...
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Transcript of Lonza Webinar: Mycoplasma – Uncover the Hidden Enemy...
Pharma&Biotech
Andrea Toell / Lonza Cologne GmbH, Cologne / 6 January 2016 © Lonza
Mycoplasma –
Uncover the Hidden Enemy in Cell Culture Webinar – 8 March 2016
Session 1: Mr. Joseph Camp
Session 2: Dr. Nazim El-Andaloussi
BioResearch
Lonza Cologne GmbH, Cologne / 2016 ® Lonza
2
Mar-16
Agenda
An Introduction to the Hidden Enemy
Mycoplasma Detection
Mycoplasma Prevention and Elimination
Summary
3
Mar-16
What Contaminations can Occur
in Cell Cultures?
Bacteria
Small black specks
pH change
Often mistaken as cell debris
Definite movement
Yeast
Oval/round in shape
Smaller than cells
Reflect light (“bright beads”)
Formed branched chains
Fungi
Filamentous strands
Web-like mesh
Mycoplasma
Invisible
Only visible via electron
microscopy
4
Mar-16
What are Mycoplasma?
Smallest, simplest prokaryotes
Size ranges from 0.2 to 0.8 µm
Many species cannot be removed by standard
sterile filters
Cannot be visualized even at very high
concentrations
Lack of rigid cell wall
Not affected by traditional antibiotics used in cell
culture
Limited biosynthetic capabilities
Utilize nutrients from “hosts”
Parasites of humans, animals, plants, insects, etc.
Extracellularly, only in rare cases intracellularly
5
Mar-16
Mycoplasma – So What?
6
Mar-16
Mycoplasma Impact Gene Expression
Miller CJ et al (2003) Mycoplasma infection significantly alters
microarray gene expression profiles. BioTechniques 35:812-
814.
Mycoplasmas significantly altered gene expression profiles of
cultured cells by up- and down-regulation of e.g.
cytokines and growth factors
stress-response genes, receptors
transport proteins, ion channels
oxidases
tumor suppressors and oncogenes
7
Mar-16
Mycoplasma Impair
Transfection Efficiency
HeLa cells uninfected HeLa cells infected with
Mycoplasma fermantans
Program I-013
no DNA
no program
+ DNA
Program I-013
+ DNA
57.7%
GFP+ 23.0%
GFP+
Preliminary data kindly provided by customer
8
Mar-16
Mycoplasma – Types and Frequency
Mycoplasma orale (20-40%) - human
Mycoplasma argininii (20-30%) - bovine
Mycoplasma hyorhinis (10-40%) - pig
Mycoplasma hominis (10-20%) - human
Mycoplasma fermantans (10-20%) - human
Acholeplasma laidlawii (5-20%) - bovine
9
Mar-16
Mycoplasma
Prevalence and Sources
Prevalence
15-35 % of continuous cell lines
5% of early passage cell cultures
1% primary cell cultures
Sources
Cross-contamination from infected
cultures
Laboratory personnel
Culture reagents (e.g. bovine serum)
Original isolate tissue (<1%)
Source Organisms/
cm2
Humans
Scalp 106
Forehead 105
Sneeze 104-105
Saliva 107
Sterile
clothing
After 6
hours 1-6
Air Indoor 500-2000
Table adapted from:
Crueger, W. Sterile Techniques in Biotechnology. In Biotechnology
Focus 2, (Hanser Publishers, Munich, 1990) p. 393
10
Mar-16
Mycoplasma Testing
Best Practice
Mycoplasma testing of new cells is essential
Routine monitoring of running cultures to detect cross-
contamination from other cultures, laboratory equipment and
personnel
Recommendation: Ideally every 1- 2 weeks, minimum 1x per month
MycoAlert™ Assay can be easily included into passaging routine
11
Mar-16
Agenda
An Introduction to the Hidden Enemy
Mycoplasma Detection
Mycoplasma Prevention and Elimination
Summary
12
Mar-16
Mycoplasma – Classical Detection
Methods
Agar culture test (gold standard)
2 – 3 weeks
Often done externally
PCR methods
4-5 hours
Species detection depends on primer set
Also detects dead mycoplasma
Hoechst stain
Time-consuming, poor indicator
Experience required, risk of false positives
13
Mar-16
MycoAlert™ Mycoplasma Detection Kit
Unique 20 min bioluminescent assay for mycoplasma detection in cell cultures
Mechanism: The assay detects the activity of two enzymes found in
mycoplasma and other mollicutes
Enzymes are associated with energy generation pathways that result in ATP
synthesis:
The enzymes are found in all 6 of the main mycoplasma cell culture contaminants
and the majority of mollicute species
Being an enzyme assay, MycoAlert™ only detects viable mollicutes
The enzymes are not found in eukaryotic cells
ATP LIGHT + Oxyluciferin
+ AMP
+ PPi
+ CO2
Mycoplasma enzymes
+ specific mollicute substrate
Luciferase
+ Luciferin + O2
Mycoplasma
14
Mar-16
MycoAlert™ Protocol
Sample
100 µl
MycoAlert™
Reagent
MycoAlert™
Substrate
Lysis,
Luciferase,
Luciferin
Specific
Substrate for
Mollicutes
5 min
10 min
Read A
Read B
Background ATP generated by
cells is measured
while mycoplasma are lysed
ATP generated by mycoplasma
enzymes is measured
Ratio Negative Positive Borderline
MycoAlert™ Assay < 0.9 > 1.2 0.9 – 1.2
MycoAlert™ PLUS Assay < 1.0 > 1.2 1.0 – 1.2
15
Mar-16
MycoAlert™ Kit – Assay Kinetics
*Infected sample = supernatant from K562 cell culture infected for 72 hours with M. hyorhinis
Data generated with first generation MycoAlert™ Assay
Time (minutes)
1
10
100
1000
10000
0 2 4 6 8 10 12 14 16
RLU
s
Reading A
+
MycoAlert™ Substrate added
Reading B MycoAlert™ Reagent added
Clean sample
Infected sample*
16
Mar-16
MycoAlert™ Kit – Species Testing
Mollicute species obtained from the National Collection of Type Cultures UK: 44 of 118 species in the collection tested to date.
Data generated with first generation MycoAlert™ Assay. Bold: 6 main species
Species Result
Mycoplasma lipophilum positive
Mycoplasma muris positive
Mycoplasma neurolyticum positive
Mycoplasma opalescens positive
Mycoplasma orale positive
Mycoplasma pirum positive
Mycoplasma pneumoniae positive
Mycoplasma primatum positive
Mycoplasma pulmonis (human) positive
Mycoplasma pulmonis (rat) positive
Mycoplasma salivarium positive
Mycoplasma spermatophilum positive
Mycoplasma synoviae positive
Spiroplasma citri positive
Species Result
Mycoplasma canadense positive
Mycoplasma cloacale positive
Mycoplasma conjunctivae positive
Mycoplasma crocodyli positive
Mycoplasma equirhinis positive
Mycoplasma faucium positive
Mycoplasma fermentans positive
Mycoplasma gallinacium positive
Mycoplasma gallisepticum positive
Mycoplasma genitalium positive
Mycoplasma hominis positive
Mycoplasma hyopneumoniae positive
Mycoplasma hyorhinis positive
Mycoplasma hyosynoviae positive
Mycoplasma iguanae positive
Species Result
Acholeplasma laidlawii positive
Acholeplasma modicum positive
Acholeplasma morum positive
Mesoplasma entomophilum positive
Mesoplasma florum positive
Mycoplasma agussizii positive
Mycoplasma alkalescens positive
Mycoplasma alligatoris positive
Mycoplasma arginini positive
Mycoplasma arthritidis positive
Mycoplasma bovirhinis positive
Mycoplasma bovis positive
Mycoplasma bovoculi positive
Mycoplasma buccale positive
Mycoplasma californicum positive
17
Mar-16
The Speed of An Infection
0,1
1
10
100
1000
16 h 22 h 40 h 46 h 64 h 70 h 136 h
Myco
Ale
rt® R
atio
Time after innoculation
K562 control M. hyorhinis M. salivarium
Myco
Ale
rt™
Ra
tio
Data generated with first generation MycoAlert™ Assay
18
Mar-16
0.1
1
10
100
K562* U937* HL60 JURKAT CHO* BJAB COS7*
Ratio
MycoAlert™ Assay
Compared to a PCR Kit
* Positive cell lines (infected with M. hyorhinis)
Data generated with first generation MycoAlert™ Assay
MycoAlert™
Assay
39 32 0.4 0.4 23 0.8 25
PCR + + - - + - +
19
Mar-16
MycoAlert™ PLUS Assay
The Next Generation
Higher light output than 1st generation MycoAlert™ Assay
Better compatibility with less sensitive plate luminometers and
multifunctional readers
Suited for testing of unused media, media supplements or water
MycoAlert™ PLUS generates a 25x
higher light output at 2000 nM ATP
1
10
100
1000
10000
100000
1000000
2000 200 20 2 0,2 0,02 0,002 0
RL
Us
ATP concentration (nM)
MycoAlert™ Assay MycoAlert™ PLUS Assay
20
Mar-16
MycoAlert™ PLUS Assay
vs. MycoAlert™ Assay
MycoAlert™ PLUS Assay detects a 100x higher positive control dilution
0,1
1,0
10,0
100,0
1000,0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
Tube luminometer Lucetta™ Luminometer
(Lonza)
Plate luminometerOrion
(Berthold Detection Systems)
Multifunctional ReaderSpectraMax® M5
(Molecular Devices)
Ra
tio
B/A
MycoAlert™ Assay MycoAlert™ PLUS Assay
21
Mar-16
MycoAlert™ PLUS Assay
vs. MycoAlert™ Assay
MycoAlert™ PLUS Assay detects a 100x higher positive control dilution
0,1
1,0
10,0
100,0
1000,0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
1:1
0
1:1
00
1:1
000
1:1
000
0 0
Tube luminometer Lucetta™ Luminometer
(Lonza)
Plate luminometerOrion
(Berthold Detection Systems)
Multifunctional ReaderSpectraMax® M5
(Molecular Devices)
Ra
tio
B/A
MycoAlert™ Assay MycoAlert™ PLUS Assay
22
Mar-16
MycoAlert™ PLUS Assay –
Testing of Fresh Media
Testing of fresh media requires an alternative protocol that involves a
1:10 dilution step and prolonged incubation
0
1
10
100
1000
undil. 1:10 plus10%FBS
plus10%FBS
(1:10)
undil. undil. 1:10 plus10%FBS
plus10%FBS
(1:10)
undil.
DMEM Neg C RPMI Neg C
untreated
spiked with M.orale
Ratio B
(30
min
) / A
(20
min
)
23
Mar-16
Required Instrumentation
Cuvette/tube luminometers
Single sample throughput
E.g. Lucetta™ Luminometer with tailor-made MycoAlert™
Mode
Plate-reading luminometers
Up to 96 samples per plate
Can be semi-automated if fitted with reagent injectors for high
sample throughput
Some reader models are not sensitive enough for
measurements with 1st generation MycoAlert™ Assay
Scintillation counters might be used in luminescence
mode
List of MycoAlert™ Assay compatible luminometers is
available at: www.lonza.com/mycoalert
24
Mar-16
MycoAlert™ Tips and Tricks
Handling of Assay Components
Leave for 15 minutes at room temperature to ensure complete
rehydration
Ideally use freshly re-constituted components
If freezing is required:
Ideally store aliquots at -80°C
Avoid freeze-thaw cycles
Equilibrate to room temperature before use without the aid of artificial heat
25
Mar-16
MycoAlert™ Tips and Tricks
Sample Handling
Sample collection:
Suspension cells: Collect supernatant during passaging
Adherent cells: Collect supernatant prior to trypsinization
Leave cells at least 24 h under normal culture conditions before testing: Cells diluted
into fresh media after passaging result in a much lower signal
Supernatant from cells coming out of liquid nitrogen: Leave minimum of 1-2h under
normal culture conditions before testing
Remove remaining cells by centrifugation at 1500 rpm (200 x g) for 5 minutes: Cells
present in the sample will increase the background, resulting in loss of sensitivity
and possibly interfering with detection of low-level infections
Sample freshness:
For optimal assay performance, supernatant should be tested as soon as possible
after collection
If storage is required, please refer to respective product instructions
26
Mar-16
MycoAlert™ Tips and Tricks
Running The Assay
Optimal working temperature for all reagents is 22°C
Include Negative Control (assay buffer) and Positive Control to check assay
performance
Wear gloves: Skin has high levels of ATP on its surface that can contaminate
the reagents leading to falsely high readings
When using plate or multifunctional reader
Use white-walled plates with opaque bottom
Test background signal (empty wells)
Do not place MycoAlert™ Positive Control in close vicinity of samples, especially
with MycoAlert™ PLUS
Disable automatic background subtraction
27
Mar-16
MycoAlert™ Assay
Summary
Convenient & fast
No DNA extraction necessary
Simply add two reagents and perform two
luminescence readings
Easy interpretation of results
Universal
Confirmed to detect 44 species
Predicted to detect most common mollicute
contaminations (Mycoplasma, Acholeplasma,
Entomoplasma and Spiroplasma; except Ureaplasma)
Specific
No interference with bacteria (not lysed), fungi or yeast
Negligible interference with media components
28
Mar-16
Agenda
An Introduction to the Hidden Enemy
Mycoplasma Detection
Mycoplasma Prevention and Elimination
Summary
29
Mar-16
Elimination of Mycoplasma
Contamination
Mycoplasma cannot be fully removed by sterile filtration
Usual routine antibiotics (e.g. Penicillin) are ineffective against
mycoplasma due to the lack of cell wall
Some antibiotics are effective for preventing mycoplasma growth (e.g.
Neomycine, Tetracycline, Gentamycin) but
are restricted to specific mycoplasma species
only suppress mycoplasma growth (once treatment is stopped,
contamination will recur)
30
Mar-16
Antibiotic Resistance of Mycoplasma
From Infected Cell Cultures
Antibiotic Resistance
Chloramphenicol 30%
Chlortetracycline 11%
Ciprofloxacin 15%
Erythromycin 98%
Gentamycin 80%
Kanamycin 73%
Lincomycin 28%
Neomycin 86%
Spectinomycin 14%
Streptomycin 88%
Tetracycline 14%
Tylosin 21%
Table adapted from: Lundin DJ and Lincoln CK (1994)
Mycoplasmal Contamination of Cell Cultures within the Clinical
Diagnostic Laboratory. Amer. Clin. Lab. April (4):6
31
Mar-16
MycoZap™ Mycoplasma
Elimination Reagent
Universal:
Eradicates mollicutes (Mycoplasma, Acholeplasma, Spiroplasma,
Entomoplasma)
Total elimination of mycoplasma by using a biophysical treatment in
combination with an antibiotic agent
Effective but mild
Minimal toxic effects on eukaryotic cells
Suited for all cell cultures
MycoZap™
Reagent 1
MycoZap™
Reagent 2
MycoZap™
Reagent 2
MycoZap™
Reagent 2
2-6 days 2-6 days 2-6 days 2-6 days Test with
MycoAlert™ Assay
32
Mar-16
Prevention of Mycoplasma
Contamination
MycoZap™ Prophylactic
For prevention of mycoplasma contamination
in combination with your antibiotic formula of
choice (e.g. Pen/Strep) for preventing other
microbial contaminants
MycoZap™ Plus-CL and MycoZap™ Plus-
PR
For protection of cell lines (CL) or primary
cells (PR) against all common microbial
contaminants such as Gram(+) and Gram(-)
bacteria, fungi as well as mycoplasma
Complete solution, i.e. no further Pen/Step is
required
MycoZap™ Spray (DE and UK only)
For reliable disinfection of all laboratory
surfaces from mycoplasma contamination
33
Mar-16
Agenda
An Introduction to the Hidden Enemy
Mycoplasma Detection
Mycoplasma Prevention and Elimination
Summary
34
Mar-16
Summary – Mycoplasma
Mycoplasma are a real problem in cell culture
Changes in gene expression
Changes in cell function/cytotoxicity
and more
Mycoplasma contamination is often invisible
Routine monitoring detects cross-contamination from other cultures,
laboratory equipment and personnel
Mycoplasma testing of new cells is essential
35
Mar-16
Complete Product Portfolio for Managing
Mycoplasma Contaminations
Prevent
• Specific MycoZap™ Antibiotics
• MycoZap™ Spray (DE and UK only)
Detect
• MycoAlert™ PLUS Kit for basic research
• Lucetta™ Luminometer
Eliminate • MycoZap™ Elimination Kit
36
Mar-16
Sources of Information
Our Online Resources
Background: http://www.lonza.com/mycoplasma
Product Instructions and Luminometer List:
http://www.lonza.com/mycoalert
FAQs: http://www.lonza.com/faq
Cell Culture Experts
Scientific Support Team EU: +32 87 321 611
Scientific Support Team US: +1 800 521 0390 (toll free)
37
Mar-16
Interested in Learning More?
Do not miss Lonza’s 2016 webinars series! Check the schedule of
upcoming webinars on our website
www.lonza.com/researchwebinars
Follow us on LinkedIn®
www.linkedin.com/company/lonza-bioresearch-solutions
View or download our BioResearch Catalog
www.lonza.com/catalog
38
Mar-16
Thank You!
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The information contained herein is believed to be correct and corresponds to the latest state of scientific and technical knowledge. However, no
warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information and no
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