Yih-Horng Shiao, Ph.D. Laboratory of Comparative Carcinogenesis

1. 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

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

3. Cell Culture Contamination

4. 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)

5. Microorganism contamination and detection Fungus

6. Microorganism contamination and detection Cells Mycoplasma Mycoplasma Scanning electron microscopy Hoechst 33258 stain PCR-based detections for mycoplasmas and viruses HIV Transmission electron microscopy

7. 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%

8. 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.

9. 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

10. 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.

11. 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.

12. 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.

13. 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.

14. Types of disinfectants

16. PCR contamination

17. Air flow during PCR Heated lid Air flow Heating/cooling block PCR mixture

18. 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

19. 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.

20. 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.

21. 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.

24. PCR cabinets

25. 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

26. 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.