Animal Cell and Tissue Culture Lecture 4 PDF

SIO2004 Animal Cell and Tissue Culture Lecture 4 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi Requirements for successful cell culture a) Culture surface b) Gas phase c) Temperature and humidity d) Media: amino acids, vitamins,...

SIO2004 Animal Cell and Tissue Culture Lecture 4 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi Requirements for successful cell culture a) Culture surface b) Gas phase c) Temperature and humidity d) Media: amino acids, vitamins, salts, energy source, etc. e) pH and buffering f) Osmotic balance g) Serum factors: growth factors, hormone, lipids, etc. h) Sterility Sterility Contaminating organisms (such as bacteria, yeast, etc.) grow much faster than animal cells in culture If culture is contaminated, the contaminants will overrun the culture and your cells’ growth will be affected and they will eventually die! Aseptic technique “Non-dirty techniques” To avoid/ minimize chances of contamination Antimicrobials additives to the media Antibiotics: protect against bacteria Antimycotics/anti-fungals: protect against fungi May interfere with some cell types or experiments Contamination Contaminating organisms Bacteria Fungi Most commonly encountered biological contaminants in cell culture, because of their Filamentous fungi (mold) ubiquity, size, and fast growth rates Yeast (unicellular fungi) Mycoplasma Viruses Other cell types Sources of contamination In the air: free organisms, dust particles or aerosols Surfaces or equipment The operator!  Phenol Red: pH indicator In many cell culture media, phenol red is added Its color exhibits a gradual transition from yellow to red over the pH range 6.8 to 8.2 Above pH 8.2, phenol red turns a bright pink (fuschia) color Below pH 6.8, it turns bright yellow At physiological pH (~7.4), it is bright red A convenient way to rapidly check on the health of tissue cultures In the event of problems, waste products produced by dying cells or overgrowth of contaminants will cause a change in pH, leading to a change in indicator color. Bacterial contamination Unicellular, typically a few micrometers in diameters, and can have a variety of shapes, ranging from spheres to rods and spirals. Detection (by eye and microscopy) Cloudy/turbid media, sometimes with a slight film or scum on the surface or spots on the growth surface that dissipate when the flask is moved Decrease in the pH of the media (orange or yellow in color) Under a 10X objective, spaces between cells will appear granular and may shimmer Under a 100X objective, possible to resolve individual bacteria and distinguish between rods and cocci. The shimmering that is visible in some infections will be seen to be caused moving bacteria. Some bacteria form clumps or associate with the cultured cells Phase contrast images of adherent 293 cells contaminated with E. coli (Image: Gibco Thermo Fisher) https://www.youtube.com/watch?v=z XPiZ2XBUzE VIDEO: Moving Bacteria in Cell Culture Yeast contamination Unicellular microfungi, ranging in size from a few micrometers (typically) up to 40 micrometers (rarely) Detection (by eye and microscopy) Very little change in pH unless the contamination is heavy, at which stage the pH usually decreases White powder-like particles floating in the medium Under microscopy (20X objective), yeast appear as individual ovoid or round particles, that may bud off smaller particles  In this particular incident of yeast contamination, all wells were contaminated but the contamination was heavy in 5 out of 6 wells, resulting in a decrease in the pH. The medium is not turbid, but white particles can be seen near the pieces of tissue. Phase contrast images of 293 cells in adherent culture that is contaminated with yeast. The contaminating yeast cells appear as ovoid particles, budding off (Image: Freshney et al.) smaller particles as they replicate (Image: Gibco Thermo Fisher) Mold (filamentous fungi) contamination Molds are eukaryotic microorganisms in the kingdom of Fungi that grow as multicellular filaments called hyphae. A connected network of these multicellular filaments contain genetically identical nuclei, and are referred to as a colony or mycelium. Detection (by eye and microscopy) Similar to yeast contamination, the pH of the culture remains stable in the initial stages of contamination, then may rapidly decrease as the culture become more heavily infected Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores (which may be blue or green) If fungal colony is big enough, can see with the naked eye! e.g. white fluffy balls Mycoplasma contamination Genus of bacteria that lack a cell wall around their cell membrane. Without a cell wall, they are unaffected by many common antibiotics! Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate until then, there are often no visible signs of infection Ways to detect: Fluorescent DNA staining (Hoechst 33258 dye) PCR Microbiological culture DBTRG cells; control versus contaminated cells after 24 h mycoplasma infection in T25 culture flasks (supernatant Myc+ corresponds to dilution 1:100 supernatant Myc+++) shown using an inverted microscope https://doi.org/10.1007/s00216-018-0987-9 Fluorescent staining of DNA by Hoechst 33258 is one of the easiest and most reliable methods and reveals mycoplasmal infections as a fine particulate or filamentous staining over the cytoplasm with a 50× or 100× objective. Media additives to prevent contamination Antibiotics (against bacteria) Combination of penicillin and streptomycin (“PenStrep”) Effective and not so toxic Antifungals e.g. Fungizone, nystatin Anti-mycoplasma gentamycin and kanamycin - increasingly used because more human tissues and cells are being used – main source of mycoplasma Detecting contamination In general, rapidly growing organisms are less problematic as they are often obvious and readily detected, after which the culture can be discarded. Difficulties arise when the contaminant is cryptic, either because it is too small to be seen on the microscope, such as mycoplasma, or slow growing such that the level is so low that it escapes detection. Use of antibiotics can be a common cause of cryptic contaminations remaining undetected! Cross-contamination by other cell types During the development of tissue culture, a number of cell strains have evolved with very short doubling times and high plating efficiencies. Although these properties make such cell lines valuable experimental material, they also make them potentially hazardous for cross-infecting other cell lines. The extensive cross-contamination of many cell lines with HeLa and other rapidly growing cell lines is now clearly established. Prevention Proper authentication of the cell lines used Do not share or reuse pipettes, reagents, etc. between different cell lines Do not mislabel tubes, flasks, etc. https://www.the- scientist.com/?arti cles.view/articleN o/50655/title/Pap ers-Based-on- Misidentified-Cell- Lines-Top-32-000/  So, what’s up with this flask? nope Aseptic Technique is the best prevention! Controlled environment Traffic, air flow Dedicated cell culture room Sterile media and reagents Avoid aerial contamination of solutions Avoid manual contamination of equipment Avoid repeated opening of bottles 70% ethanol swab UV irradiation of the cell culture hood before and after Only use disposable equipment once Hoods for Cell Culture Principle – to make available a space free of microorganisms and spores Types of hoods used for cell culture: Laminar Flow Hood Biological Safety Cabinet / Tissue Culture Hood Class II: most common Class III: provides maximum protection to the environment and the worker High Efficiency Particulate Air (HEPA) filter trap airborne pollutants including dust, allergens, and microorganisms  Sterilization UV radiation of cell culture hoods (and sometimes rooms!) Gamma radiation or using toxic gas at high pressure (e.g. ethylene dioxide) Sterilisation of utensils and vessels available commercially, esp. plastic Autoclaving of instruments and glassware used in cell culture Heating up to 121°C for a minimum of 15-20 minutes Filter sterilisation of media and solutions The main method for most solution components are heat sensitive and damaged by radiation. Use finely perforated filter 0.2 µm (to filtrate off particles size > 0.2 µm) Most bacteria and fungi > 0.2 µm Virus size (0.2 < virus=mikoplasma > 0.1 µm), therefore to filtrate virus, must use filter perforated with 0.1 µm Autoclave machines Filters Requirements for successful cell culture a) Culture surface b) Gas phase c) Temperature and humidity d) Media: amino acids, vitamins, salts, energy source, etc. e) pH and buffering f) Osmotic balance g) Serum factors: growth factors, hormone, lipids, etc. h) Sterility  Check the morphology of cells in culture! Regularly examining the morphology of the cells in culture (i.e., their shape and appearance) under the microscope is essential for successful cell culture experiments Confirm the healthy status of your cells! Signs of deterioration of cells include granularity around the nucleus, detachment of the cells from the substrate, and cytoplasmic vacuolation. Signs of deterioration may be caused by a variety of reasons, including contamination of the culture, senescence of the cell line, or the presence of toxic substances in the medium, or they may simply imply that the culture needs a medium change Allowing the deterioration to progress too far will make it irreversible. Media changes The purpose of media changes is to replenish nutrients and avoid the build up of potentially harmful metabolic by products and dead cells. In the case of adherent cultures the media can be removed directly by aspiration and replaced. In the case of suspension cultures, cells can be separated from the media by centrifugation and resuspended in fresh media.