Identification and Elimination of Contaminations in Cell Culture and Polymerase Chain Reaction Labor

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Identification and Elimination of Contaminations
in Cell Culture and Polymerase Chain Reaction
Laboratories

• Yih-Horng Shiao, Ph.D.
• Laboratory of Comparative Carcinogenesis
• National Cancer Institute at Frederick
• Maryland, USA

June 25, 2005
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The consequences of contamination

• Hazardous to humans
• Inaccurate experimental results
• Loss of cells and samples
• Waste of time, money, and other resources.

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Cell Culture Contamination
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Chemical contaminants and sources

• Exogenous Metals (glassware), reagent residues
  (glassware), endotoxin (culture media, sera, and
  water), other water impurities, CO2 impurities,
  disinfectant residues, etc.
• Endogenous Free radicals (photo-activation of
  tryptophan, riboflavin, or HEPES buffer by
  fluorescent light)

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Microorganism contamination and detection
Fungus
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Microorganism contamination and detection
Cells
Mycoplasma
Mycoplasma
Scanning electron microscopy
Hoechst 33258 stain
PCR-based detections for mycoplasmas and
viruses
HIV
Transmission electron microscopy
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Frequent mycoplasma contamination (Studies in
1990s)

• United States 11-15 of cell cultures
• Netherlands 25
• Former Czechoslovakia 37 (100 of the cultures
  from labs without routine testing but only 2
  from labs having mycoplasma screening regularly)
• Argentina 65
• Japan 80

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Cell line cross-contamination(Surveys in 1970s
and 1980s)

• A study in 1967 showed that 20 commonly used
  human cell lines were contaminated with HeLa
  cell.
• A report in 1976 demonstrated that 14 of 246
  cell lines were wrong species and 25 were
  replaced completely by HeLa cell.
• In a 1981 survey, over 60 cell lines were
  actually HeLa cell, 16 were contaminated by
  non-HeLa cells, and 12 were interspecies
  contamination.

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Sources of biological contamination

• Humans
• Newly arrived cell line
• Glassware
• The neck and outside of culture flasks and dishes
• Sera, culture media, and other reagents
• Airborne particles and aerosols
• Laminar-flow hood and safety cabinet
• Water bath and incubator
• Work surface
• Tubing and container for waste collection

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Cell culture management (1) Aseptic technique
and procedure

• Exercise procedures with the highest ethical and
  moral standards.
• Wear protective equipments (lab coat, gloves,
  etc.).
• Swab work surface, biosafety cabinet, and reagent
  bottles with disinfectant before and after use.
• Disinfect spill, splash, and any suspected areas
  immediately.
• Use sterile disposable tubes,
• pipettes, and culture vessels.
• Avoid generation of airborne particulates and
  aerosols.

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Cell culture management (2) Aseptic environment

• Disinfect water bath, incubator, tubing and
  container for waste collection routinely.
• Replace HEPA filter on schedule.
• Keep laminar-flow hood on all the time.
• Minimize the number of entrance and frequency of
  entering and exiting the cell culture room.

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Cell culture management (3) Monitoring and
surveillance

• Quarantine and test all incoming cell lines for
  contamination, except those from reliable
  sources.
• Perform tests of microorganism contamination and
  cell-specific markers for all active cell lines
  and freeze aliquots of clean passages
  periodically.
• Monitor the performance of biosafety cabinets.
• Conduct annual safety training and refreshment
  courses (classroom or on-line) to all personnel.
• Record and document all monitoring and
  surveillance items.

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Cell culture management (4)Curing for
contaminated cells

• Discard and heat-destroy all contaminated cells
  because contamination alters cell behaviors and
  functions.
• If cells are irreplaceable, antibiotics can be
  used to eradicate some bacteria and mycoplasmas.
  However, the experimental results need to be
  interpreted cautiously.

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Types of disinfectants
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PCR contamination
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Air flow during PCR
Heated lid
Air flow
Heating/cooling block
PCR mixture
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Sources of PCR contamination

• Humans
• Carryover products, especially from PCR using the
  same primer set over and over.
• Vector DNA containing insert of a target gene and
  other positive controls
• Dusts and aerosols
• PCR reagents, pipetters, and tubes
• Work surface
• Instruments

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Detection of PCR contamination

• Negative controls
• ? Include no template controls throughout the
  entire RNA and DNA analyses, beginning
  from nucleic acid extraction.
• ? Set up more than two negative controls each
  time to detect random contamination.
• Sequence polymorphism
• ? Unique gene sequence can be used to detect
  contamination.
• Repetition
• ? If the sample cannot be repeatedly
  amplified, it may indicate contamination.

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Good practices in PCR laboratory

• Be vigilant to avoid carrying vectors, genomic
  DNA, and PCR products onto human body.
• Use different sets of reagents, equipments, and
  supplies for pre-PCR and post-PCR experiments.
  Never bring items in the post-PCR areas into
  pre-PCR room.
• Wipe work surface with 10 Chlorox or other
  DNA-destructing agents before and after use.
• Aliquot reagents.
• Change gloves often and prevent static build-up
  on the gloves. Keep the working areas free of
  dusts.
• Limit the PCR cycle number.

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PCR laboratory set-up

• Physically separate Pre-PCR from post-PCR room,
  and each room has independent heater, ventilation
  and air conditioner.
• Need a biological cabinet with UV lamps in the
  pre-PCR room to provide clean area for steps,
  such as DNA extraction and PCR preparation.
• It is optional to install a dead-air biological
  cabinet in post-PCR room for steps, such as
  opening of PCR tube, gel electrophoresis, and
  staining, to contain PCR products and to destroy
  the products with cabinet UV.

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PCR cabinets
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Special measures to cure PCR contamination

• Discard contaminated samples and reagents.
• Pre-PCR
• ? Enzymatic digestion (endonuclease, DNase I,
  and exonuclease)
• ? UV irradiation
• Post-PCR
• ? Isopsoralen followed by UV
• ? Incorporation of dUTP followed by Uracil
  DNA glycosylase and heat treatment

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Conclusion

• Be alert and conscious to all potential
  contaminants.
• Practice safety procedures with the highest
  ethical and moral standards.
• Follow the schedule for monitoring and
  surveillance.
• Take refreshment courses or training periodically.