Animal Biotechnology PDF

Serum-free Media: Using serum in a medium has a number of disadvantages: the physiological variability the shelf life and consistency the quality control, the specificity, the availability, the downstream processing, the possibility of con...

Serum-free Media: Using serum in a medium has a number of disadvantages: the physiological variability the shelf life and consistency the quality control, the specificity, the availability, the downstream processing, the possibility of contamination, the growth inhibitors, the standardization and the costs Using serum-free media and defined media supplements (Nutridoma-CS, Nutridoma- SP, Transferrin) offers three main advantages: The ability to make a medium selective for a particular cell type. The possibility of switching from growth-enhancing medium for propagation to a differentiation-inducing medium. The possibility of bioassays (e.g., protein production) free from interference with serum proteins (easier downstream processing). But serum-free media are not without disadvantages: It increases the number of media. It can lead to the selection of a sublineage that is not typical of the whole population. Cell proliferation is often slower. Tissue culture Flasks: Alexis Carrel developed the first glass flasks in the 1920s. Harry Earle developed the more traditional straight neck rectangular (also hexagonal) glass T-flasks in the 1940s. Today, plastic flasks are available with a range of growing areas, a variety of shapes, with several different neck designs. Choice of design depends on the cell culture techniques used as well as personal preference. The more common sizes are listed below:  Cell culture dishes: Cell culture dishes offer the best economy and access to the growth surface. This makes them the vessels of choice for cloning or other manipulations such as scraping that require direct access to the cell monolayer. They must be used with incubators that control CO2 and humidity. Most manufacturers offer dishes in four diameters: 35 mm, 60 mm, 100 mm, and 150 mm. These are nominal diameters and may not be the actual diameter of the growth surface. Cell culture dishes are available with either specially treated surfaces for growing anchorage-dependent cells, or untreated (native) surfaces for growing suspension cultures where attachment is not desired.  Multiwell plates: These widely used vessels were originally designed for virus titration, but have since become popular in many other applications, especially hybridoma production, high-throughput screening, and toxicity testing. Multiwell plates offer significant savings in space, media, and reagents when compared to an equal number of dishes. They are more convenient to handle, especially if the pipettors, plate washers, readers, and other equipment for processing these plates are used. They must be used with incubators that control humidity and CO2 levels. (continued): Characteristics of a ‘normal’ cell:  Defined by Hayflick and Moorhead following their work with human embryonic cells.  Main characteristics: a diploid chromosome number (e.g.: 46 chromosomes for human cells). This indicates that no gross chromosomal damage has occurred; anchorage dependence. The cells require a solid substratum for attachment and growth. Growth continues until a confluent monolayer of cells is formed on the substratum; a finite lifespan. This is a reflection of the intrinsic growth potential of the cells; nonmalignant. The cells are not cancerous. This can be shown by the inability of the cells to form a tumor.  Anchorage-dependence: Anchorage-dependence is the requirement of cells for a solid substratum for attachment before growth can occur. At the laboratory scale this substratum can be provided by the solid surface of Petri dishes, T-flasks, or Roux bottles which are made of specially treated glass or plastic. The interaction between the cell membrane and the growth surface is critical and involves a combination of electrostatic attraction and van der Waal’s forces. Cell adhesion occurs by divalent cations (usually Ca2+) and basic proteins forming a layer between the solid substratum and the cell surface. In most cases the cell-surface interaction is provided by a range of nonspecific proteins which form a 2.5 nm-thick layer on the substratum prior to cell attachment. The adhesion of anchorage-dependent cells to a solid substratum. Figure outlines the process of cell attraction to the substratum and the involvement of various proteins in cell-surface bonding.  Anchorage-dependence (continued): Serum-derived glycoproteins (e.g. fibronectin) can provide a surface coating conducive to cell attachment. Conditioning factors are released by cells into the medium and help in forming a bond between cell surface glycoproteins and the substratum. The density of the electrostatic charge on the solid substratum is also critical in maximizing cell attachment. A negative charge is provided on glass surface containers by alkali treatment. Tissue culture-grade plasticware consists of sulfonated polystyrene with a surface charge of 2–5 negatively charged groups per nm2.