Mycoplasma Uncover the Hidden Enemy in Cell Culture

Transcription

1 Pharma&Biotech BioResearch Mycoplasma Uncover the Hidden Enemy in Cell Culture Webinar 8 March 2016 Session 1: Mr. Joseph Camp Session 2: Dr. Nazim El-Andaloussi Lonza Andrea Cologne Toell / Lonza GmbH, Cologne GmbH, / 2016 Cologne Lonza / 6 January 2016 Lonza

2 Agenda An Introduction to the Hidden Enemy Mycoplasma Detection Mycoplasma Prevention and Elimination Summary 2

3 What Contaminations can Occur in Cell Cultures? Bacteria Small black specks ph change Often mistaken as cell debris Definite movement Fungi Filamentous strands Web-like mesh Yeast Oval/round in shape Smaller than cells Reflect light ( bright beads ) Formed branched chains Mycoplasma Invisible Only visible via electron microscopy 3

4 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 4

5 Mycoplasma So What? 5

6 Mycoplasma Impact Gene Expression Miller CJ et al (2003) Mycoplasma infection significantly alters microarray gene expression profiles. BioTechniques 35: 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 6

7 Mycoplasma Impair Transfection Efficiency HeLa cells uninfected HeLa cells infected with Mycoplasma fermantans Program I-013 no DNA no program + DNA Program I DNA 57.7% GFP % GFP + Preliminary data kindly provided by customer 7

8 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 8

9 Mycoplasma Prevalence and Sources Prevalence % of continuous cell lines Source Organisms/ cm 2 5% of early passage cell cultures Scalp % primary cell cultures Humans Forehead 10 5 Sneeze Sources Cross-contamination from infected cultures Laboratory personnel Culture reagents (e.g. bovine serum) Original isolate tissue (<1%) Sterile clothing Saliva 10 7 After 6 hours 1-6 Air Indoor Table adapted from: Crueger, W. Sterile Techniques in Biotechnology. In Biotechnology Focus 2, (Hanser Publishers, Munich, 1990) p

10 Mycoplasma Testing Best Practice Mycoplasma testing of new cells is essential Routine monitoring of running cultures to detect crosscontamination 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 10

11 Agenda An Introduction to the Hidden Enemy Mycoplasma Detection Mycoplasma Prevention and Elimination Summary 11

12 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 s 12

13 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: Mycoplasma Mycoplasma enzymes + specific mollicute substrate ATP Luciferase + Luciferin + O 2 LIGHT + Oxyluciferin + AMP + PPi + CO 2 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 13

14 MycoAlert Protocol MycoAlert Substrate Specific Substrate for Mollicutes MycoAlert Reagent Lysis, Luciferase, Luciferin 10 min 5 min Read B Read A ATP generated by mycoplasma enzymes is measured Background ATP generated by cells is measured while mycoplasma are lysed Sample 100 µl Ratio Negative Positive Borderline MycoAlert Assay < 0.9 > MycoAlert PLUS Assay < 1.0 >

15 RLUs MycoAlert Kit Assay Kinetics MycoAlert Reagent added Reading A + MycoAlert Substrate added Reading B Clean sample Infected sample* Time (minutes) *Infected sample = supernatant from K562 cell culture infected for 72 hours with M. hyorhinis Data generated with first generation MycoAlert Assay 15

16 MycoAlert Kit Species Testing Species Result Species Result Species Result Acholeplasma laidlawii Mycoplasma canadense Mycoplasma lipophilum Acholeplasma modicum Mycoplasma cloacale Mycoplasma muris Acholeplasma morum Mycoplasma conjunctivae Mycoplasma neurolyticum Mesoplasma entomophilum Mycoplasma crocodyli Mycoplasma opalescens Mesoplasma florum Mycoplasma equirhinis Mycoplasma orale Mycoplasma agussizii Mycoplasma faucium Mycoplasma pirum Mycoplasma alkalescens Mycoplasma fermentans Mycoplasma pneumoniae Mycoplasma alligatoris Mycoplasma gallinacium Mycoplasma primatum Mycoplasma arginini Mycoplasma gallisepticum Mycoplasma pulmonis (human) Mycoplasma arthritidis Mycoplasma genitalium Mycoplasma pulmonis (rat) Mycoplasma bovirhinis Mycoplasma hominis Mycoplasma salivarium Mycoplasma bovis Mycoplasma hyopneumoniae Mycoplasma spermatophilum Mycoplasma bovoculi Mycoplasma hyorhinis Mycoplasma synoviae Mycoplasma buccale Mycoplasma hyosynoviae Spiroplasma citri Mycoplasma californicum Mycoplasma iguanae 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 16

17 MycoAlert MycoAlert Ratio The Speed of An Infection h 22 h 40 h 46 h 64 h 70 h 136 h 0,1 Time after innoculation K562 control M. hyorhinis M. salivarium Data generated with first generation MycoAlert Assay 17

18 Ratio MycoAlert Assay Compared to a PCR Kit K562* U937* HL60 JURKAT CHO* BJAB COS7* MycoAlert Assay PCR * Positive cell lines (infected with M. hyorhinis) Data generated with first generation MycoAlert Assay 18

19 RLUs 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 ,2 0,02 0,002 0 ATP concentration (nm) MycoAlert Assay MycoAlert PLUS Assay MycoAlert PLUS generates a 25x higher light output at 2000 nm ATP 19

20 1:10 1:100 1:1000 1: :10 1:100 1:1000 1: :10 1:100 1:1000 1: Ratio B/A MycoAlert PLUS Assay vs. MycoAlert Assay MycoAlert PLUS Assay detects a 100x higher control dilution 1000,0 100,0 10,0 1,0 0,1 Tube luminometer Lucetta Luminometer (Lonza) MycoAlert Assay Plate luminometer Orion (Berthold Detection Systems) MycoAlert PLUS Assay Multifunctional Reader SpectraMax M5 (Molecular Devices) 20

21 1:10 1:100 1:1000 1: :10 1:100 1:1000 1: :10 1:100 1:1000 1: Ratio B/A MycoAlert PLUS Assay vs. MycoAlert Assay MycoAlert PLUS Assay detects a 100x higher control dilution 1000,0 100,0 10,0 1,0 0,1 Tube luminometer Lucetta Luminometer (Lonza) MycoAlert Assay Plate luminometer Orion (Berthold Detection Systems) MycoAlert PLUS Assay Multifunctional Reader SpectraMax M5 (Molecular Devices) 21

22 Ratio B (30 min) / A (20 min) 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 untreated spiked with M.orale 0 undil. 1:10 plus 10% FBS plus 10% FBS (1:10) undil. undil. 1:10 plus 10% FBS plus 10% FBS (1:10) undil. DMEM Neg C RPMI Neg C 22

23 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 1 st generation MycoAlert Assay Scintillation counters might be used in luminescence mode List of MycoAlert Assay compatible luminometers is available at: 23

24 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 24

25 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 25

26 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 26

27 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 27

28 Agenda An Introduction to the Hidden Enemy Mycoplasma Detection Mycoplasma Prevention and Elimination Summary 28

29 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) 29

30 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 30

31 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 2-6 days 2-6 days 2-6 days 2-6 days Test with MycoAlert Assay MycoZap Reagent 1 MycoZap Reagent 2 MycoZap Reagent 2 MycoZap Reagent 2 31

32 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 32

33 Agenda An Introduction to the Hidden Enemy Mycoplasma Detection Mycoplasma Prevention and Elimination Summary 33

34 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 34

35 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 35

36 Sources of Information Our Online Resources Background: Product Instructions and Luminometer List: FAQs: Cell Culture Experts Scientific Support Team EU: Scientific Support Team US: (toll free) 36

37 Interested in Learning More? Do not miss Lonza s 2016 webinars series! Check the schedule of upcoming webinars on our website Follow us on LinkedIn View or download our BioResearch Catalog 37

38 Thank You! All trademarks belong to Lonza or its affiliates or to their respective third party owners. 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 warranty is expressed or implied concerning the use of these products. 38