Cell Culture Techniques and Procedures PDF

Biotechniques (BMS 34010A) Fall semester 2024 -2025 Dr. Tania Tahtouh [email protected] Cell culture techniques  Learning outcomes  Describe tasks relevant to cell culture, including preparation and evaluation of media, maintaining cultures of animal cells and established cell line...

Biotechniques (BMS 34010A) Fall semester 2024 -2025 Dr. Tania Tahtouh [email protected] Cell culture techniques  Learning outcomes  Describe tasks relevant to cell culture, including preparation and evaluation of media, maintaining cultures of animal cells and established cell lines with good viability, minimal contamination, cryopreservation and recovery.  Distinguish between mammalian and bacterial cultures.  Recognize and troubleshoot problems common to routine cell culture.  Explore Gibco Cell culture Virtual Lab : https://www.thermofisher.com/ae/en/home/global/forms/cell-culture-basics/cell-culture- basics-virtual-lab.html  What is cell culture?  Cells, previously growing in a human or animal modified to grow in plastic or glass. ▪ Kept in an incubator to stay at body temperature. ▪ We use special media with nutrients so the cells can grow and divide. In the body = in vivo On plastic or glass = in vitro  Applications of cell line  Studying the activity of normal and pathological cells in isolation.  Production of valuable biological products: ▪ Viral vaccines. ▪ Antibodies. ▪ Therapeutic proteins. ▪ Recombinant proteins.  Testing drug metabolism and cytotoxicity.  Generation of artificial tissues (e.g., artificial skin)  Stem cell culture provides the possibility of treating diseases using cell-based therapy.  Study of gene function.  Genetic engineering.  Setting up a Cell Culture Laboratory  Sterile reagents, media and consumables.  Space allocated for the tissue culture should be dedicated to tissue culture ONLY.  Good aseptic techniques should be used at all times.  Minimize entry and exit, e.g. by having freezer for medium and supplements storage, centrifuge, etc.  Maintained and clean routinely/daily.  Surfaces should also be easy to clean and all waste generated should be disposed of immediately.  Equipment for Tissue Culture Laboratory  Basic equipment: ▪ Tissue culture hood where all the cell handling is carried out ▪ CO2 incubators to facilitate optimal cell growth under strictly maintained and regulated conditions ▪ Microscope for visualizing cells in culture ▪ Centrifuge to spin down cells ▪ Water bath for thawing frozen samples of cells and warming media to 37 °C before use ▪ Fridge and freezer for storage of media and other materials required for cell culture  Cell culture hood  Laminar flow hoods as they generate a smooth uninterrupted streamlined flow (laminar flow) of sterile air which has been filtered through a high efficiency particulate air (HEPA) filter.  Two types of laminar flow hoods: ▪ Vertical hoods provide a downward vertical flow. ▪ Horizontal hoods present airflow in a horizontal direction. Three classes of hood Three classes of hood  Cell culture hood rules  Must be maintained in a clutter-free and clean state at all times as too much clutter may affect air flow and contamination will introduce infections  Must be wiped clean with 70% ethanol before and during work, especially after any spillage.  Leave the hood running at all time, turning it off only when they will not be used for extended periods of time.  Turn the hood on for at least 10 min before starting work to allow the flow of air to stabilize.  HEPA (high efficiency particulate air) filtered air.  UV light to kill microorganisms also mutates human DNA, so it cannot be on when using the hood.  CO2 incubators  The purpose of the CO2 is to ensure that the culture medium is maintained at the required physiological pH (usually pH 7.2–7.4).  Should always be maintained at 37°C and supplied with 5% (or 10%) CO2.  Frequent cleaning of the incubator is a MUST.  2 types of incubators: ▪ Dry incubators (direct heat CO2 incubators) ▪ Humid CO2 incubators (water-jacketed CO2 incubators) Microscope  Inverted phase contrast microscopes are routinely used for visualizing cells in culture.  Light source is located above the slide and the objective lenses below the stage on which the cells are placed.  Visualization of cells by microscopy can provide useful information about the morphology and state of the cells. Inverted light microscope  Aseptic handling 1. Always wipe your hands and your work area with 70% ethanol. 2. Wipe the outside containers, flasks and dishes with 70% ethanol before placing them in the cell culture hood. Use only sterile glassware or other equipment. 3. Arrange your work area so that you have easy access to all items without having to reach over one to get at another. 4. Work within your range of vision. 5. Use sterile glass or disposable plastic pipettes to work with liquids, and use each pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be used. 6. Always keep caps on the bottles and flasks after use to prevent microorganisms from gaining entry. 7. Bottles of media and other reagents SHOULD NOT be shared with other people or used for different cell lines. 8. Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment. Return the cover as soon as you finished. 9. If you remove the cap or cover, and have to put it down on the work surface, place the cap with the opening facing down. 10. Wipe up any spillage immediately with 70% ethanol. 11. Be careful not to talk, sing or whistle when you are performing sterile procedure. 12. Perform your experiments as rapidly as possible to minimize contamination. 13. Remove everything once you’ve finished your work and wipe your work area with 70% ethanol. 14. Decontaminate all potentially infectious materials before disposal.  Aseptic techniques  Why is the Aseptic technique important? ▪ To prevent microbial contamination of cultures. ▪ To prevent cross-contamination of cell cultures as well. https://www.youtube.com/watch?v=nr1tV_LuqJk  Types of cell culture Types of tissue culture Primary Secondary Continuous Cells obtained cells obtained from an Immortal cell cultures. directly from an already established animal or plant tissue. primary culture. Finite Indefinite divide only a limited number of times ability to proliferate (20 to 100), before their growth rate indefinitely. declines and they eventually die.  Primary cultures  Derived directly from tissues following enzymatic dissociation.  Maintain the characteristics of donor tissue.  Disadvantages: ▪ Isolation can be labor-intensive and may produce a heterogeneous population of cells. ▪ Limited lifespan and can be used over only a limited period of time in culture.  Continuous cultures  Single cell type that has gained the ability for infinite growth.  Immortalized: ▪ Spontaneously (e.g.: spontaneous genetic mutation) ▪ By transformation vectors (e.g.: viruses &/or plasmids)  Disadvantage: ▪ Lose some of their original in vivo characteristics  Advantages: ▪ Require less serum for growth ▪ Have a shorter doubling time ▪ Can grow without necessarily needing to attach or adhere to the surface of the flask.  Systems for growing cells in culture  Adherent cells - Also referred to as anchorage-dependent cells, these are the type of cells that require attachment for growth. Adherent cells are immobile and grow by remaining attached in monolayers to a solid substrate.  Suspension cells - These are the type of cells that do not require attachment in order to grow. They are therefore also referred to as anchorage-independent cells and will float and grow suspended in the culture medium.  Examples of cell lines supplied by commercial sources  These cell lines can be obtained from European Collection of Animal Cell Cultures (ECACC) based in Salisbury, UK  Cell morphologies  Cell morphologies vary depending on cell type.  Cell culture media  Nutrients: ▪ Amino acids, carbohydrate (such as glucose), vitamins.  Inorganic salts: ▪ Magnesium, sodium, potassium, calcium, phosphate, chloride, sulphate and bicarbonate ions.  pH indicator: ▪ Phenol red.  Antibiotics and Antimycotics ▪ Penicillin, streptomycin, gentamicin, amphotericin B  Buffers (Bicarbonate and HEPES): ▪ Bicarbonate buffered media requires CO2 atmosphere ▪ HEPES Strong chemical buffer range pH 7.2 –7.6 (does not require CO2)  Supplements: ▪ Growth factors, hormones (e.g.: insulin), non-essential amino acids (NEAA), etc.  Foetal Calf/Bovine Serum (FCS & FBS)  Growth factors and hormones  Enhances cell attachment  Disadvantages: ▪ Infectious agents (prions) ▪ Variable composition ▪ Expensive How do we culture cells in the laboratory?  Growth curve  Lag phase: Enhanced cellular activity but no apparent increase in cell growth. The duration of this phase depends on several factors: ▪ Viability of the cells ▪ Density at which the cells are plated ▪ Media component  Log phase: Exponential increase in cell number with high metabolic activity.  Stationary phase: No further increase in growth. ▪ Depletion of nutrients in the medium ▪ Accumulation of toxic metabolic waste ▪ L imitation in available growth space  Decline phase: If left unattended, cells in the stationary phase will begin to die. Passaging cells  Why passage cells? ▪ To maintain cells in culture (i.e. don’t overgrow) ▪ To increase cell number for experiments/storage  Cell count  Manual cell count (haemocytometer): https://www.youtube.com/watch?v=WWS9sZbGj6A  Automated cell count:  Cryopreservation of cells Log phase of growth  Why cryopreserve cells? and >90% viability. ▪ Reduced risk of microbial contamination. ▪ Reduced risk of cross contamination with other cell lines. ▪ Reduced risk of genetic drift and morphological changes. ▪ Research conducted using cells at consistent low passage.  Resuscitation of frozen cells/Cell thawing Cryopreservant (DMSO) – precise mechanism unknown but prevents ice crystal formation.  Source of contamination  A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use.  Bacterial cell culture  Safety considerations for bacterial cell culture: ▪ Requires care and sterile techniques. Aseptic techniques and safety conditions described for animal cell culture should be adopted. ▪ Protecting the operator (from potentially harmful organisms). ▪ Instruments used during the culturing procedures should be sterilized before and after use by heating in a Bunsen burner flame. ▪ Areas of work must be decontaminated after use.  Culture procedures for bacterial cells: ▪ Liquid media are normally dispensed into flasks and inoculated with an aliquot of the organism to be grown. ▪ Solid medium is usually prepared by solidifying the selected medium with 1–2% of the seaweed extract agar. Plating techniques DNA transfer into cells  DNA cloning overview The inserted DNA is reproduced along with the vector DNA cloning overview  Extraction of plasmids  Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis in order to separate the two. ▪ Neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. ▪ Chromosomal DNA and proteins removed by centrifugation. Dirty minipreps References  Sanders ER. Aseptic laboratory techniques: plating methods. J Vis Exp. 2012;(63):e3064. Published 2012 May 11. doi:10.3791/3064