Animal Tissue Culture Facilities Lectures 11-15 PDF

Animal tissue culture facilities Infrastructural requirements Mechanical/plumbing/engineering: Dedicated or shared back-up generators; alarms and controlled access; CO2 and liquid N2 delivery to the laboratory and cryobank. A heating, ventilation and air conditioning (HVAC) system, for...

Animal tissue culture facilities Infrastructural requirements Mechanical/plumbing/engineering: Dedicated or shared back-up generators; alarms and controlled access; CO2 and liquid N2 delivery to the laboratory and cryobank. A heating, ventilation and air conditioning (HVAC) system, for both comfort in the laboratory and for the biological safety cabinets which play a major role in the construction of a tissue culture laboratory. Interior finishes: vinyl flooring; nonporous ceilings; washable, impermeable paint and coatings; impermeable bench-tops and furniture. Equipment: Biosafety cabinet  Laminar air flow cabinet A laminar airflow cabinet is an enclosed unit that creates a microbial free environment. This microbial free environment is created by high efficiency particulate air filters which are popularly known as HEPA filters. It is an acronym for "high efficiency particulate air [filter]" HEPA is a type of pleated mechanical air filter. This type of air filter can theoretically remove at least 99.97% of dust, pollen, mold, bacteria, and any airborne particles with a size of 0.3 microns (µm). Biosafety cabinet  Unlike the laminar airflow cabinets the biosafety cabinets not only give protection to the sample but they also protect the operator and the surrounding environment.  Different types basesd on level of protection provided: BSL Class1/2/3 CO2 incubator Inverted microscope Equipment Culture vessels Three common types: The choice of culture vessel depends on various factors such as: 1. Flasks Cell type (anchorage-dependent monolayer or 2. Multi-well plates. anchorage-independent suspension cells). 3. Petri dishes Total quantity of cells required. Cost and purpose for which cells are cultured. Sampling frequency Poly-D-lysine (PDL) and collagen I coated flasks are ideal for adherent cultures of primary cells that have difficulty attaching to the growth surface. The uniform coating present in these flasks builds a positive charge over the surface that ensures cell attachment, growth and differentiation. Serological pipette with dispenser Aseptic techniques Contamination sources Non-sterile supplies, media, and reagents Airborne particles laden with microorganisms, Unclean incubators Dirty work surfaces Aseptic technique Sterile work area Good personal hygiene Sterile reagents and media Sterile handling. The need for Artificial culture media  Initial attempts to culture cells were performed in natural media based on tissue extracts and body fluids, such as chick embryo extract, plasma, serum, and lymph.  With the propagation of cell lines (L929 cells, HeLa, etc.), the demand for larger amounts of a medium of more consistent quality led to the introduction of chemically defined media based on analyses of body fluids and nutritional biochemistry. Culture conditions for animal cells Controlled temperature, a substrate for cell attachment, an appropriate growth medium, and an incubator that maintains correct pH and osmolality are required for cell culture. Cell culture media comprise an appropriate source of energy and compounds which regulate cell cycle. Culture medium is composed of a complement of amino acids, vitamins, inorganic salts, glucose and serum as a source of growth factors, hormones and attachment factors. In addition to nutrients, the medium also helps maintain pH and osmolality. Since blood meets the nutritional requirements of mammalian cells, animal cell culture medium should be prepared in such a way that it contains all the components present in blood Broadly, culture media are divided into two classes: Natural and Artificial media. 1. Natural media were derived from different resources, such as plasma, serum, lymph, amniotic fluid, ascetic and pleural fluids, and aqueous humor from the eyes. 2. Natural media also include tissue extracts in the form of chick embryo extract and liver, bone marrow and spleen extracts. Artificial media A medium with a designed composition is called an artificial medium. The minimum requirements for artificial/synthetic media are: The medium must offer all the nutrients to the cell in a sustainable manner. The medium must be isotonic and sterilized before utilization. The medium has to offer optimum pH (around 7). In controlled conditions, a balanced salt solution is generally used to maintain biological pH and osmolality in a culture under in vitro conditions. To encourage growth, various essential and non-essential elements in the form of organic salts, growth factors (vitamins, amino acids, etc) and natural supplements are added. Supplementation of serum is required in most cases Physical and chemical properties of culture media 1. pH Desirable range is 7-7.4. pH of a culture can be stabilized by appropriate combinations of buffers. Phenol red is commonly used as an indicator. It is red at pH 7.4 and becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.5, more pink at pH 7.6, and purple at pH 7.8 Carbonate-Biocarbonate buffer A biocarbonate buffer system controls the pH of the culture medium. Sodium bicarbonate, when dissolved in water, results in the formation of sodium and bicarbonate ions. The bicarbonate ions reacts with hydrogen ions present in the solution to form carbonic acid, which ultimately forms carbon dioxide and water. The carbon dioxide present in the solution is in equilibrium with the carbon dioxide present in the gas phase. Thus, increasing the concentration of CO2 in gas phase enhances the concentration of carbon dioxide in the medium, and subsequently increases the carbonic acid concentration and decreases the pH. Conversely, if the carbon dioxide concentration in the gas phase is decreased, then the pH increases. 2. Oxygen Oxygen is vital for aerobic respiration. Mammalian cells are well adapted to O2 levels much lower than atmospheric conditions. The levels of O2 in the environment outside the cell must be properly maintained to meet the requirements of cells. This is applicable for both in vivo and in vitro environments. Generally, most animal tissue culture experiments are performed under higher oxygen concentrations, where the cells are exposed to high oxidative stress, which adversely affects the cells and tissues in culture. Supplementation of some radical scavengers or antioxidants, helps in relieving cells of oxidative stress, in particular in the case where serum is not supplemented to the medium, as serum contains vital antioxidants. 3. Balanced salt solutions (BSS) BSSs mainly contain inorganic salts and offer the required pH and osmolality. Occasionally, glucose, HEPES and sodium bicarbonate are also added to BSSs. During the late 18th century, Sydney Ringer formulated Ringer’s solution. This solution is called a balanced salt solution, as its composition is similar to that of body fluids. Several different BSS have been developed since then. The simple composition (inorganic salts, sometimes with glucose added as a nutrient) of BSS encourages its utilization. In addition, phenol red can be added to check pH variations BSSs can be utilized effectively to maintain tissues and cells under in vitro environments for short periods, usually up to a few days. Complete Culture Media:  In the early years, balanced salt solutions were supplemented with various nutrients (amino acids, vitamins, serum etc.) to promote proliferation of cells in culture. Eagle was a pioneer in media formulation.  He determined (during 1950-60) the nutrient requirements for mammalian cell cultures. Many developments in media preparation have occurred since then. There are more than a dozen media now available for different types of cultures. EMEM—Eagle’s minimal essential medium DMEM—Dulbecco’s modification of Eagle’s medium RPMI 1640—Media from Rosewell Park Memorial Institute. Compositi on of DMEM 5. Serum Serum is vitally important as a source of growth and adhesion factors, hormones, lipids, carrier proteins and minerals for the culture of cells in basal media. In addition, serum also regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell. The most commonly used serum is fetal bovine serum (FBS). Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can affect the growth of cells and it is therefore important to screen batches of serum for their ability to support the growth of cells. Serum composition Serum heat inactivation The practice of heat inactivating serum at 56º C was originally developed when only serum from adult animals was available for cell culture. Adult serum contains various immune factors, particularly serum complement, which may inhibit or destroy cells under certain conditions. For the media with serum, addition of hormones and growth factors is usually not required. They are frequently added to serum-free media Amino acids:  All the essential amino acids (which cannot be synthesized by the cells) have to be added to the medium.  In addition, even the non-essential amino acids (that can be synthesized by the cells) are also usually added to avoid any limitation of their cellular synthesis.  Among the non-essential amino acids, glutamine and/or glutamate are frequently added in good quantities to the media since these amino acids serve as good sources of energy and carbon. Vitamins: The quality and quantity of vitamins depends on the medium. For instance, Eagle’s MEM contains only water soluble vitamins (e.g. B- complex, choline, inositol). The other vitamins are obtained from the serum added. The medium M 199 contains all the fat soluble vitamins (A, D, E and K) also. In general, for the media without serum, more vitamins in higher concentrations are required. Advantages of serum in cell culture medium  It has basic nutrients present either in soluble or in protein-bound form.  Provides several hormones such as insulin and transferrin. Insulin is essential for the growth of cells in culture and transferrin acts as an iron binder.  It contains numerous growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-B), epidermal growth factor (EGF), and chondronectin. These factors stimulate cell growth and support specialized functions of cells.  It supplies protein, which helps in the attachment of cells to the culture surface (e.g., fibronectin).  It provides binding proteins such as albumin and transferrin, which helps transport molecules in cells.  It provides minerals such as Ca2+, Mg2+, Fe2+, K+ Na+, Zn2+, etc., which promote cell attachment. Disadvantages of serum in cell culture medium  Expensive: Fetal calf serum is expensive and difficult to obtain in large quantities.  Variation: Batch-to-batch variation occurs in serum, and there is no uniformity in composition of serum. This can affect growth and yields and can give inconsistent results.  Contamination: Serum medium carries a high risk of contamination with virus, fungi, and mycoplasma.  Cytotoxic and inhibiting factors: The serum itself may be cytotoxic and may contain inhibiting factors, which in turn may inhibit cultured cell growth and proliferation. The enzyme polyamine oxidase in serum reacts with polyamines such as spermine and spermidine to form cytotoxic polyamino-aldehyde. Advantages of Cell Culture  Role and effect of pH, temperature, O2/CO2 concentration, and osmotic pressure of the culture media can be altered to study their effects on the cell culture.  To study cell metabolism and investigate the physiology and biochemistry of cells.  Effect of various compounds or drugs on specific cell types such as liver cells can be studied.  These cultures help study the biology and origin of the cells.  Specific proteins can be synthesized in large quantities from genetically modified cells in large-scale cultures.  Reproducibility of the results that can be obtained by the use of a single type/clonal population.  Ethical, moral, and legal questions for utilizing animals in experiments can be avoided. Limitations of Cell Culture Expenditure and expertise: This is a specialized technique that requires aseptic conditions, trained personnel, and costly equipment. Dedifferentiation: Cell characteristics can change after a period of continuous growth of cells in cultures, leading to differentiated properties. Low amount of product: The miniscule amount of mAB and recombinant protein produced followed by downstream processing increases expenses tremendously. Contamination: Mycoplasma and viral infection are difficult to detect and are highly contagious. Instability: Aneuploidy chromosomal constitution in continuous cell lines leads to instability.