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.eu@lonza.com

Scientific Support Team US: +1 800 521 0390 (toll free)

scientific.support@lonza.com

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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