Flashcards in Cell And Tisse Culture Deck (15):
What is cell and tissue culture?
The growth of cell, tissues or organs in artificial media in a laboratory. It can produce many identical clones.
What are the general culture requirements?
Some cells types can be grown in suspension using liquid medium. Other types can be grown on or within, a solid medium (substrate) such as agar jelly. To ensure rapid growth of the chosen cells, optimum conditions are provided in terms of nutrients, pH, and gases. Anaerobic organisms can be cultured in non-aerated suspension or within agar.
What are the aseptic requirements?
Contamination is the enemy of cell or tissue culture. Use of sterile materials and the treatment of sources tissue, are vital to prevent inoculation with unwanted cells or spores. Bacterial or fungal contamination will rapidly outcompete and spoil a culture of slower-growing plant in animals cells.
What are the principles of bacterial and fungal cell culture?
They are easily cultured in suspension in fermenters. In schools they are often grown in perti dishes on nutrient agar (agar with peptides and beef extracts added) or malt agar (agar with malt extract added). Plates are incubated at temperatures other than the human body temperature to reduce the chance of culturing pathogens. Bacteria and yeast are heterotrophs so they need to be provided with nutrients such as amino acids and an organic carbon source as well as minerals.
What are the specific culture requirements for mammalian cells?
Complex medium growth (salts, amino acids, vitamins and glucose). Growth factors provided by FBS - foetal bovine serum - many of the components appear to be essential for animal proliferation. FBS is a mixture containing growth factors, proteins, salts, vitamins and glucose. Antibiotics are also added to minimise the chances of spoilage by microoragnisms.
What happens in subculturing in mammalian cells?
Mammalian cells are usually culture in a flask. Cells for culture are detached from source tissue using proteolytic enzymes such as trypsin. When the cells are added to the flask the adhere to the surface of the agar (anchorage dependent), then flatten or spread out and then start to divide. The cells form a mono layer, a layer one cell thick. They stop dividing (contact inhibition) when they are confluent; that is, when they have 'flowed together' to form a complete covering of the surface. Cells soon use up the nutrients in the medium, so, to keep the cloned cell line alive, some must be subcultures into a fresh culture flask. During is process, cells have to be detached from the mono layer , again using proteolytic enzymes.
What happens in the selection of cell lines? Mammalian
Normal animal cells tend to die after am infinite number of divisions (about 60- hay flick limit). This reduces the length of time culture can be maintained. However, the limit does not exist in immortal cell lines, for example those derived from cancer cells of stem cells which can be subcultured indefinitely. Stem cells are much sought after for tissue culture as they can be pluripotent (able to differentiate into many cell types) or even totipotent (able to differentiate into all cell types).
What are the specific culture requirements for plant tissue?
To make a growth medium suitable for plant tissue culture, murashige and skoog (M&S) salts are often used. These salts contain an appropriate balance of macronutrients (for example N,P,K,Mg), micronutrients (such as Zn,Na,Cu), carbon sources (sugars) and vitamins. Growth regulators, such as cytokinins and auxins, can also be added to stimulate differentiation.
How is the selection of plant cells?
They are totipotent and so are able to divide into all the cell types required to form a whole new organism. The two methods of plant tissue culture use either explants of protoplasts.
What are explants?
Small pieces of plant tissue that are placed on a solid medium to either promote shoot growth or callus growth . Organ information is then stimulated by altering the ratio of cytokinins (to promote shoot growth) and auxins (to promote root growth).
What are protoplasts?
They are created by removing the cell walls with enzymes pectinase and cellulase. The returning cells can be grown in liquid medium as a cell suspension culture and treated with growth regulators to induce embryogenesis to generate whole new embryonic plants. Protoplasts can be encouraged to form a callus, as with explants. For protoplast fusion. The cell wall is removed to be fused prior to culturing.
What is the purpose of plant tissue?
Cell suspension cultures allow screening for beneficial traits (such as heat of salinity tolerance) or the selection of virus free cells for cloning. Micro propagation- the generation of many cloned plantlets from one source plant- allows rapid upscaling to field trials. In addition, cell suspension cultures are used to harvest useful plant cell secretion.
What is exponential growth?
Micro organisms are grown in a batch culture until a maximum density is achieved. The growth phase is called the log phase then it reaches stationary.
What does invitro mean?