finalized mycoalert presentation

MycoplasmaAn introduction to mycoplasma and its detection

What Contaminations Can You See Under A Microscope?

Bacteria small black specks pH change/ cloudy media mistaken for cell debris definite movement

Fungus Filamentous Strands web-like mesh

What Contaminations Can You See Under A Microscope?

Yeast oval/round in shape smaller than cells appear as bright beads reflect light form branched chains

Mycoplasma INVISIBLE!

Mycoplasma attaches to the cell surface

Mycoplasma

Smallest, simplest prokaryotes Size ranges from 0.2 to 0.8 Many species cannot be removed by

filtration Cannot be visualized, even at high

concentrations

Lacks Rigid Cell Wall Not affected by traditional antibiotics

used in cell culture

Limited Biosynthetic capabilities Utilize nutrients from “host”

Adverse Effects of Mycoplasma Contamination

DNA fragmentation associated with mycoplasmal nucleases Induction of Apoptosis Alters Gene Expression in contaminated cells Affects Cell Metabolism Changes in Signal Transduction

Mycoplasma - Effects

Inhibition of cell growth

Inhibition of cell metabolism

Disruption of nucleic acid synthesis

Chromosomal aberrations

Changes in cell membrane antigenicity

Alteration of DNA transfection efficiency

Increased sensitivity to inducers of apoptosis

DNA fragmentation due to Mycoplasmal nucleases NOT apoptosis

CELL DEATH

Production of viruses compromised

Most Common Species

Mycoplasma orale (human) 20-40%

Mycoplasma hyorhinis (swine) 10-40%

Mycoplasma arginini (bovine) 20-30%

Mycoplasma fermentans (human) 10-20%

Mycoplasma hominis (human) 10-20%

Acholeplasma laidlawii (bovine) 5-20%

Prevalence

15-35% of continuous cell lines 5% of early passage cell cultures 1% of Primary Cell Cultures

Sources

Cross contamination from infected cultures Laboratory Personnel Culture Reagents (i.e. bovine serum) Original isolate tissue (<1%)

Continuous Monitoring

Monitoring of U937 cells for the presence of mycoplasma

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Summary

Mycoplasma are a real problem in cell culture Changes in gene expression Changes in cell function\cytotoxicity

Mycoplasma contamination is often invisible Infection from Laboratory personnel and reagents requires

routine monitoring Mycoplasma testing of new cells is essential

Mycoplasma Detection Methods

Culture

PCR DetectionHoechst Stain

MycoAlert® for Mycoplasma detection

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 live mycoplasma

The enzymes are not found in eukaryotic cells

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Culture Method

Fluorescence Staining

PCR Amplification

MycoAlert® Assay

Time (hours)

2- 4 weeks

Mycoplasma Detection Methods

Culture Supernatant

MycoAlert® Reagent

MycoAlert® Substrate

Lysis, Luciferase, Luciferin

Specific Substrate for Mollicutes

5 min

10 min

ABSpecific

substrate formollicutes

Lysis,Lucerferase,Luciferin

If B/A < 1 = Negative

If B/A > 1.2 = Positive

If B/A 1- 1.2 = Borderline/retest

MycoAlert®- How it works

Assay Sensitivity

HepG2 cells were infected with mycoplasma at the concentration listed, the cells were then cultured for 4 hours before being tested with a range of techniques

A. Laidlawii Culture Fluorescence PCR MycoAlert®

0 Negative Negative Negative Negative

20 Positive Positive Negative Positive

200 Positive Positive Positive Positive



200000 Positive Positive Negative Positive

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Samples provided, enumerated and tested by the European Collection of Cell Cultures (ECACC) with the exception of the MycoAlert® testing which was performed by Lonza

Effect of Common Media Components

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RPMI

Penicillin/Streptomycin

10% DMSO

Trypsin/EDTA

Serum 20%

Non EssentialAmino Acids

Glutamate

Sodium Pyruvate

EMEM

Iscove’s

NegativeMycoplasma positive

M. faucium added to create the positive samples

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Conclusions

MycoAlert® technology enables: Simple methodology Rapid testing Routine monitoring Sensitive Generic mycoplasma detection

Bioluminescence gives: Speed Sensitivity Convenience

Best Practices

Receive cells

Quarantine & test for mycoplasma

NEGATIVE

Put cells in normal culturePrepare cryo samplesMonitor frequentlyUse in experimental protocols

POSITIVE

Destroy cellsInform source

Quarantine cellsTreat with MycoZap™Inform sourceMonitor daily for mycoplasmaKeep in quarantine until infection eradicated for 4 weeks

Best Practices

We strongly recommend to discard contaminated cultures It is not known if all cell functions return upon elimination In cases where fresh stocks cannot be obtained one should

consider treatment of the cells…

Treatments

Standard Antibiotics- most prominent Time-consuming (prolonged periods of >6 weeks needed) Usually the success rates are relatively low…

Low antibiotic concentration Heat-instability of the antibiotic Early termination of the treatment Formation of resistant mycoplasma Recontamination possible

MycoZap™ Reagent Fast (clear contamination in little as 4 days) Highly reliable and definite elimination of mycoplasma

MycoZap™ Reagent for Mycoplasma Elimination

Minimal toxic effect on host cellsOptimized reagent mix- combination of antibiotic and antimetabolic

agents Reagent 1 is a detergent

Integrates into the mycoplasma cell wall and disintegrates the mycoplasma particle

Pretreatment will reduce the mycoplasma titer drastically Reagent 2 is an antibiotic

Mycoplasma particles which survived this treatment by hiding in cell clusters or cell crinkles will be destroyed subsequently by the antibiotic

Results

MycoZap™ Summary

Easy to use Simply add Reagent to your culture

Universal One protocol to eliminate all mollicutes

Complete All required reagents are in one kit, one protocol

Effective clean up Combination of antibiotic and antimetabolic agents- disrupts

mycoplasma Minimum toxic effect on host cells

Allows you to rescue priceless or high value cell line from destruction and enables lab to continue using cell line and add value to their investment

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finalized mycoalert presentation

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