Lecture 5: Contamination in Mammalian Cell Cultures (PDF)

Week 1 Tue 10th Sept Lecture Module Introduction Week 2 Mon 16th Sept Lecture 1 Use of mammalian cells Tue 17th Sept Lecture 2 Cell Culture Laboratory Lab layout, Equipment and Materials Week 3 Mon 23rd Sept Lecture 3 Cont...

Week 1 Tue 10th Sept Lecture Module Introduction Week 2 Mon 16th Sept Lecture 1 Use of mammalian cells Tue 17th Sept Lecture 2 Cell Culture Laboratory Lab layout, Equipment and Materials Week 3 Mon 23rd Sept Lecture 3 Contamination control Tue 24th Sept Lecture 4 Contamination control Week 4 Mon 30th Sept Lecture 5 Contamination control Tue 01st Oct Lecture 2, 3, 4 and 5 recap and sample assessment questions Week 5 Mon 07th Oct Lecture 6 Nutrient uptake Tue 08th Oct Lecture 7 Nutrient uptake and sample assessment questions Week 6 Mon 14th Oct Lecture 8 Biology of Culture Cells Tue 15th Oct Lecture 9 Cell culture media Week 7 Mon 21st Oct Lecture 10 Cell culture media Tue 22nd Oct Lab 3 data analysis Reading Week Week 8 Mon 04thNov Lecture 11 Cell Culture Media Tue 05 Nov th Lecture 8, 9, 10 and 11 recap and sample assessment questions Week 9 Mon 11th Nov Lecture 12 Growing mammalian cells Tue 12th Nov Lecture 13 Monitoring growth Week 10 Mon 18th Nov Lecture 14 Cryopreservation of cells Tue 19th Nov Lecture 12, 13 and 14 recap and sample assessment questions Week 11 Mon 25th Nov Lecture 15 Innate immune response Tue 26th Nov Lecture 16 Adaptive immune response & Bioassays Lecture 15 and 16 recap and sample assessment questions Week 12 Mon 02nd Dec Revision Tue 03rd Dec BIOT6012 Mammalian Biotechnology Lecture 5 Slide 1  Contamination in Mammalian Cell Cultures Lecture Overview Introduction: Why discuss this topic Main discussion: Identify Most Common Contamination sources in the Cell Culture Laboratory Conclusion: Take home message BIOT6012 Mammalian Biotechnology Lecture 5 Slide 2  Contamination in Mammalian Cell Cultures Introduction Mammalian cell culture is the removal of animal cells and subsequent propagation and cultivation in vitro in an artificial environment that is suitable for growth An aseptic environment is created to reduce or eliminate incidences of contamination Contamination can be from biological or chemical sources This lecture will identify the most common cell culture contaminants BIOT6012 Mammalian Biotechnology Lecture 5 Slide 3  Contamination in Mammalian Cell Cultures Mammalian Cell Culture Biological Threats Any biological entity other than the desired mammalian cell is contamination Other cell lines (cross contamination) Yeast Molds Viruses Bacteria (particularly problematic Mycoplasma) BIOT6012 Mammalian Biotechnology Lecture 5 Slide 4  Contamination in Mammalian Cell Cultures Characteristics of Bacterial Contamination of Mammalian Cell Cultures pH - sudden change in pH is often a strong indicator of contamination, phenol red in the medium gives indication of pH Phenol red pH chart — Media containing phenol red will appear red with a pH of 7.4 and will range from yellow to purple as the pH changes. Contamination of a culture of relatively slowly dividing mammalian cells* can be quickly overgrown by microorganisms, resulting in the acidification of the medium, and the indicator turning yellow. Mammalian cell waste products themselves will slowly decrease the pH, gradually turning the solution orange indicating that the medium needs replacing, or the cells need to be sub-cultured. *Note: mammalian cell doubling time 12 – 48 hours bacteria e.g. E. coli 20 minutes BIOT6012 Mammalian Biotechnology Lecture 5 Slide 5  Contamination in Mammalian Cell Cultures Characteristics of Bacterial Contamination of Mammalian Cell Cultures Turbidity (adherent cells) – Media looks cloudy Microscopic evaluation – Can see individual contaminating microorganisms BIOT6012 Mammalian Biotechnology Lecture 5 Slide 6  Contamination in Mammalian Cell Cultures Characteristics of Bacterial Contamination of Mammalian Cell Cultures A B Both flask A and B were inoculated with BHK (adherent) cells and examined after 24 hours incubation at 37°C, 5% Co2. Which flask is contaminated and why? BIOT6012 Mammalian Biotechnology Lecture 5 Slide 7  Contamination in Mammalian Cell Cultures Characteristics of Yeast Contamination of Mammalian Cell Cultures Like bacterial contamination, cultures contaminated with yeasts become turbid, especially if the contamination is in an advanced stage. There is very little change in the pH of the culture contaminated by yeasts until the contamination becomes heavy, at which stage the pH usually increases. Under microscopy, yeast appear as individual ovoid or spherical particles, that may bud off smaller particles. Yeast contamination of cell culture BIOT6012 Mammalian Biotechnology Lecture 5 Slide 8  Contamination in Mammalian Cell Cultures Characteristics of Mold Contamination of Mammalian Cell Cultures pH of the culture remains stable in the initial stages of contamination, then rapidly increases as the culture become more heavily infected and becomes turbid. Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores. Mold contamination of cell culture BIOT6012 Mammalian Biotechnology Lecture 5 Slide 9  Contamination in Mammalian Cell Cultures Which type of microbial contamination easiest to observe? BIOT6012 Mammalian Biotechnology  Contamination in Mammalian Cell Cultures Virus Contamination of Mammalian Cell Cultures Viruses are microscopic infectious agents that take over the host cells machinery to reproduce Their extremely small size makes them very difficult to detect in culture, and to remove them from reagents used in cell culture laboratories Using virally infected cell cultures can present a serious health hazard to the laboratory personnel, especially if human or primate cells are cultured in the laboratory Some viruses cause morphological changes to infected cells (e.g. cytopathic effect) which are detectable by microscopy, however some viral contaminations causes no visual evidence of infection Viral infection of cell cultures can be detected by electron microscopy, ELISA assays, or PCR with appropriate viral primers BIOT6012 Mammalian Biotechnology Lecture 5 Slide 10  Contamination in Mammalian Cell Cultures Virus Contamination of Mammalian Cell Cultures The microscopic appearance of a monolayer of uninfected human fibroblasts grown in cell culture (A) and the same cells after infection with herpes simplex virus (B), demonstrating the cytopathic effect caused by viral replication and concomitant cell lysis. BIOT6012 Mammalian Biotechnology Lecture 5 Slide 11  Contamination in Mammalian Cell Cultures Cross-Contamination - with other cell lines Avoiding cross-contamination: - obtaining cell lines from reputable cell banks - periodically checking the characteristics of the cell lines - operating in an aseptic environment Detection: - DNA fingerprinting - Karyotype analysis - Isotype analysis BIOT6012 Mammalian Biotechnology Lecture 5 Slide 12  Contamination in Mammalian Cell Cultures Eradication of Contamination Microbial Contamination: Most reliable method of eliminating a microbial contamination is to discard the culture and the medium and reagents used with it Not recommended to treat a culture with antibiotic as this may lead to the development of an antibiotic-resistant microorganisms The general rule is to discard contaminated cultures and that decontamination is not attempted unless it is absolutely vital to retain the cell strain. Viral Contamination: There are no reliable methods for eliminating viruses from a culture at present; disposal is the only option BIOT6012 Mammalian Biotechnology Lecture 5 Slide 13  Contamination in Mammalian Cell Cultures Mycoplasma – common cell culture contaminant Major problems for basic research as well as for the manufacturing of biopharmaceuticals. Small bacteria cells (10% volume of E. coli) which lack a cell wall. Characteristic elasticity allowing them to form a variety of different shapes. Unaffected by many common antibiotics (that target cell wall synthesis). BIOT6012 Mammalian Biotechnology Lecture 5 Slide 14  Contamination in Mammalian Cell Cultures Structure of a typical mycoplasma cell Size comparison of Mycoplasma genitallium compared to other microscopic organisms BIOT6012 Mammalian Biotechnology Lecture 5 Slide 15  Contamination in Mammalian Cell Cultures Why is the small size of mycoplasma problematic for detecting contamination?  Contamination in Mammalian Cell Cultures Mycoplasma may induce cellular changes affecting metabolism and growth. Severe Mycoplasma infections may result in the death of a cell line. - inhibition of cell proliferation by up to 50%. - significant changes in gene expression profiles Mycoplasma can be retained by filtration with 0.1 µm filters. Need to routinely check cells for presence of Mycoplasma - immunoassays - PCR - DNA fluorescence staining - microbiological broth-agar colony assay (25 days before results available) BIOT6012 Mammalian Biotechnology Lecture 5 Slide 16  Contamination in Mammalian Cell Cultures Endotoxin Lipopolysaccharide (Lipid A) component of Gram negative cell walls. /upload.wikimedia.org/wikipedia/commons/thumb/8/82/LPS_en.svg/220px-LPS_en.svg.png Bound to the bacterium Small amounts shed during growth and large amounts shed following death (cell lysis) Can be toxic to specific hosts. Capable of producing systematic effects: - fever, shock, blood coagulation, intestinal haemorrhage. Detection: Limulus amoebocyte lysate (LAL) assay - aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. - LAL reacts with bacterial endotoxin – a gel clot forms. BIOT6012 Mammalian Biotechnology Lecture 5 Slide 17  Contamination in Mammalian Cell Cultures Endotoxin test Test sample (cell culture media, supernatant from cultured cells) is incubated with LAL reagent If endotoxin is present an enzyme cascade is triggered in the LAL reagent Formation of a clot is positive for endotoxin Variations in test methods available for quantitative measurement of endotoxin BIOT6012 Mammalian Biotechnology Lecture 5 Slide 18  Contamination in Mammalian Cell Cultures Source of Endotoxin in Cell Culture - water - serum - media and additives - glassware - plasticware Effects of Endotoxin on Cell Culture Vary greatly between cell lines May be direct or indirect - Membrane and morphological effects - Mitogenesis (endotoxin is a powerful mitogen) - Influence on adherence BIOT6012 Mammalian Biotechnology Lecture 5 Slide 19  Contamination in Mammalian Cell Cultures Conclusion Contamination can be biological or chemical Variety of biological contamination: bacteria, fungi, viruses, other cell lines Particularly problematic contaminants: - mycoplasma (small size) - endotoxin (affects cell growth) Key: routine testing for the presence of contamination BIOT6012 Mammalian Biotechnology Lecture 5 Slide 20