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TECHNOLOGY Topic: Methods of Cell Disruption Date of submission: 03-12-2009 Submitted to: Lect.Harsh Dept.Biotech (L.P.U) Submitted by:Jaskirat Singh 3040060128 B.Tech-M.tech(Biotechnology) L.P.U
Table of contents
1. Introduction 2. Cells 3. Cell disruption using bead mill 4. Cell disruption using rotor-stator mill 5. Cell disruption using French press 6. Cell disruption using ultrasonic vibrations 7. Cell disruption using detergents 8. Cell disruption using enzymes 9. Cell disruption using organic solvents 10. Cell disruption by osmotic shock
1. Introduction Biological products synthesized by fermentation or cell culture are either intracellular or extracellular. Intracellular products either occur in a soluble form in the cytoplasm or are produced as inclusion bodies (fine particles deposited within the cells). Examples of intracellular productsn include recombinant insulin and recombinant growth factors. A large number of recombinant products form inclusion bodies in order to accumulate in larger quantities within the cells. In order to obtain intracellular products the cells first have to be disrupted to release these into a liquid medium before further separation can be carried out. Certain biological products have to be extracted from tissues, an example being porcine insulin which is obtained from pig pancreas. In order to obtain such a tissue-derived substance, the source tissue first needs to be homogenized or ground into a cellular suspension and the cells are then subjected to cell disruption to release the product into a solution. In the manufacturing process for intracellular products, the cells are usually first separated from the culture liquid medium. This is done in order to reduce the amount of impurity: particularly secreted extracellular substances and unutilized media components. In many cases the cell suspensions are thickened or concentrated by microfiltration or centrifugation in order to reduce the process volume. 2. Cells Different types of cell need to be disrupted in the bio-industry: 1. Gram positive bacterial cells 2. Gram negative bacterial cells 3. Yeast cell 4. Mould cells Cultured mammalian cells 6. Cultured plant cells 7. Ground tissue
5.
Fig. 1 shows the barriers present in a gram positive bacteria. Themain barrier is the cell wall which is composed of peptidoglycan, teichoic acid and polysaccharides and is about 0.02 to 0.04 microns thick. The plasma or cell membrane which is made up of phospholipids and proteins is relatively fragile. In certain cases polysaccharide capsules may be present outside the cell wall. The cell wall of gram positive bacteria is particularly susceptible to lysis by the antibacterial enzyme lysozyme. Fig. 2 shows the barriers present in a gram negative bacteria. Unlike gram positive bacteria these do not have distinct cell walls but instead have multi-layered envelops. The peptidoglycan layer is bsignificantly thinner than in gram positive bacteria. An external layer composed of lipopolysaccharides and proteins is usually present. Another difference with gram positive bacteria is the presence of the periplasm layers which are two liquid filled gaps, one between the plasma membrane and the peptidoglycan layer and the other between the peptidoglycan layer and the external lipopolysaccharides. Periplasmic layers also exits in gram positive bacteria but these are significantly thinner than those in gram negative bacteria. The periplasm is important in bioprocessing since a large number of proteins, particularly recombinant proteins are secreted into it. An elegant way to recover the periplasmic proteins is by the use of osmotic shock.
Yeasts which are unicellular have thick cell walls, typically 0.1 to 0.2 microns in thickness. These are mainly composed of polysaccharides such as glucans, mannans and chitins. The plasma membrane in a yeast cell is composed of phospholipids and lipoproteins. Mould cells are largely similar to yeast cells in terms of cell wall and plasma membrane composition but are multicellular and filamentous. Mammalian cells do not possess the cell wall and are hence quite fragile i.e. easy to disrupt. Plant cells on the other hand have very thick cell walls mainly composed of cellulose and other polysaccharides. Cell wall wherever present is the main barrier which needs to be disrupted to recover intracellular products. A range of mechanical methods can be used to disrupt the cell wall. Chemical methods when used for cell disruption are based on specific targeting of key cell wall components. For instance, lysozyme is used to disrupt the cell wall of gram positive bacteria since it degrades peptidoglycan which is a key cell wall constituent. In gram negative bacteria, the peptidoglycan layer is less susceptible to lysis by lysozyme since it is shielded by a layer composed of lipopolysaccharide and protein.
Physical methods 1. Disruption in bead mill 2. Disruption using a rotor-stator mill 3. Disruption using French press 4. Disruption using ultrasonic vibrations Chemical and physicochemical methods 1. Disruption using detergents 2. Disruption using enzymes e.g. lysozyme 3. Disruption using solvents 4. Disruption using osmotic shock .(The physical methods are targeted more towards cell wall disruption while the chemical and physicochemical methods are mainly used for destabilizing the cell membrane.) 3. Cell disruption using bead mill Fig. 4.illustrates the principle of cell disruption using a bead mill. This equipment consists of a tubular vessel made of metal or thick glass within which the cell suspension is placed along with small metal or glass beads. The tubular vessel is then rotated about its axis and as a result of this the beads start rolling away from the direction of the vessel rotation. At higher rotation speeds, some the beads move up along with the curved wall of the vessel and then cascade back on the mass of beads and cells below. The cell disruption takes place due to the grinding action of the rolling beads as well as the impact resulting from the cascading beads. 1.Cascading beads 2.Cells being 3.Disrupted
Fig. 4 Cell disruption using bead mill Bead milling can generate enormous amounts of heat. While processing thermolabile material, the milling can be carried out at low temperatures, i.e. by adding a little liquid nitrogen into the vessel. This is referred to as cryogenic bead milling. An alternative approach is to use glycol cooled equipment. A bead mill can be operated in a batch mode or in a continuous mode and is commonly used for disrupting yeast cells and for grinding animal tissue. Using a small scale unit operated in a continuous mode, a few kilograms of yeast cells can be disrupted per hour. Larger unit can handle hundreds of kilograms of cells per hour.
Fig.5 shows an industrial scale bead mill. Cell disruption involves particle size reduction and has certain similarities with grinding. According to the Kick's law of grinding, the amount of energy required to reduce the size of material is proportional to the size reduction ratio: 1.Rolling 2.Beads
Fig. 7 shows a laboratory scale rotor-stator cell disruption mill. These mills are more commonly used for disruption of plant and animal tissues based material and are operated in the multi-pass mode, i.e. the disrupted material is sent back into the device for more complete disruption. The cell disruption caused within the rotor-stator mill can be described using the equations discussed for a bead mill. In a multi-pass operation:
REFERENCES
1.http://www.rpi.edu/dept/chem-eng/BiotechEnviron/DOWNSTREAM/disrupt.htm 2.http://en.wikipedia.org/wiki/Cell_disruption 3.http://www.scribd.com/doc/17976427/Lecture-6 4.http://www.scribd.com/doc/17976427/null