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1、第三章 细胞培养专用微载体第三章 细胞培养专用微载体 Chapter Three Microcarrier Specifically for Cell Culture ? Many cell lines are said to be “anchorage dependent,” which means that in order to grow and divide they must attach themselves to a suitable surface. At the research scale they are typically grown in small plastic
2、flasks or plates. However, large-scale production of anchorage- dependent cells or cell derived products has had to rely on methods other than a simple linear expansion of this process. ?Traditionally, roller bottles have been employed for this task. The rolling action ensures that the cells are alt
3、ernately exposed to growth medium and oxygen. The roller bottle method however, is very cumbersome and expensive for the production of large quantities of cells for a number of reasons: the roller bottles are an inefficient use of space, they require extensive handling, labor and medium, and the pro
4、cess is difficult to monitor. ?Over the years a number of schemes for growing large quantities of anchorage- dependent cells have been examined. These include spiral film, plastic bags, packed or fluidized beds, hollow fiber beds, and microcarrier suspension culture. In our experience, microcarrier
5、suspension culture has been the most successful of the different approaches. ?Microcarrier Suspension Culture ?The microcarrier technique was first developed at the Laboratory for Inactivated Virus vaccines (Bilthoven, The Netherlands) under the direction of Dr. Van Wezel. It involves allowing the c
6、ells to attach to microscopic polymer beads, or microcarriers. Later, a group was able to improve the process by modifying the surface charge of the beads which allowed cells on microcarriers to grow to densities equal to or greater than those achieved by cells adapted to growth in suspension. ? ?A
7、microcarrieris a support matrix allowing for the growth of adherent cells in bioreactors. ?In 1967, microcarrierdevelopment began when van Wezelfound that microcarriers could support the growth of anchorage-dependent cells. ?Microcarriersare typically 125-250 micrometre spheres and their density all
8、ows them to be maintained in suspension with gentle stirring. Microcarriers can be made from a number of different materials including DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen, and these microcarrier materials, along with different surface chemistries, can influence cellula
9、r behavior, including morphology and proliferation. ?Surface chemistries can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. ?Microcarriers are regularly used to grow protein-producing or virus-generating adherent cell population
10、s in the large-scale commercial production of biologics (proteins) and vaccines. ?Microcarrier cell culture is typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrier- based cultures. Th
11、e advantages of microcarrier technology in the vaccine industry include (a) ease of scale-up, (b) ability to precisely control cell growth conditions in sophisticated, computer- controlled bioreactors, (c) an overall reduction in the floor space and incubator volume required for a given-sized manufa
12、cturing operation, and (d) a drastic reduction in technician labor. ? Several types of microcarriersare available commercially including dextran- based (Cytodex, GE Healthcare), collagen- based (Cultispher, Percell), and polystyrene- based (SoloHill Engineering) microcarriers. They differ in their p
13、orosity, specific gravity, optical properties, presence of animal components, and surface chemistries. Requirements for an optimum microcarrier In order for a microcarrier to be suitable for animal cell culture at all scales, it must fulfil certain basic criteria. 1. Surface properties must be such
14、that the cells can adhere with a degree of spreading which permits proliferation. For homogeneous growth of cells the surface of the microcarriers must have an even continuous contour. The surfaces of all microcarriers in the culture should have consistent properties. 2. Density of the microcarriers
15、 should be slightly greater than that of the surrounding medium, thus facilitating easy separation of cells and the medium. The density should also be sufficiently low to allow complete suspension of the microcarriers with only gentle stirring. Under standard culture conditions the optimum density f
16、or microcarriers is 1.030-0.045 g/ml. ?3. Size distribution should be narrow so that even suspension of all microcarriers is achieved and that confluence is reached at approximately the same stage on each microcarrier. Best growth of cells occurs when microcarriers have a size distribution which lies within the limits of diameter in culture of 100-230m. ?4. Optical properties should be such that routine observation of cells on microcarriers can be achieved using standard microscopy techniques.
