Cell disruption physical methods

Physical methods such as osmotic shock, in which the cells are exposed to high salt concentrations to generate an osmotic pressure difference across the membrane, can lead to cell-wall disruption. Similar disruption can be obtained by subjecting the cells to freeze/thaw cycles, or by pressuriziug the cells with an inert gas (e.g., nitrogen) followed by a rapid depressurization. These methods are not typically used for large-scale operations. 

In general, the physical structure of the tissue must be broken down mechanically followed by an extraction procedure, before the sample can be analyzed. Homogenization using blenders, probe homogenizers, cell disrupters, sonicators, or pestle grinders is particularly useful for muscle, liver, and kidney samples. Regardless of the method used for tissue disruption, the pulse, volume of extraction solvent added, and temperature should be validated and standardized in order to ensure reproducible analytical results. During cell disruption, care should be taken to avoid heat build-up in the sample, because the analyte may be heat labile. 

After cell disruption, gross fractionation of the properly stabilized, crude cell homogenate may be achieved by physical methods, specifically centrifugation. Figure 7.11, Chapter 7, outlines the stepwise procedure commonly used to separate subcellular organelles such as nuclei, mitochondria, lysosomes, and microsomes. 

Physical Methods Physical methods include mechanical disruption by milling, homogenization, or ultrasonication. Typical high-speed bead mills are composed of a grinding chamber filled with glass or steel beads which are agitated with disks or impellers mounted on a motor-driven shaft. The efficiency of cell disruption in a bead mill depends on the concentration of the cells, the amount and size of beads, and the type and rotation speed of the agitator. The optimum wet solid content for the cell suspension for a bead mill is typically somewhere between 30 percent to 60 percent by volume. The amount of beads in the chamber is 70 percent to 90 percent by... 

There are a range of physical and chemical methods available at laboratory scale for cell disruption which involve the use of reagents or temperature and pressure changes to break the cell wall to release the desired products. However, at an industrial scale it is more common to use a mechanical disruption technique, and a number of companies have developed efficient... 

In addition to these various chemical treatment methods, a number of physical methods of cell disruption can be used in a chip-based system. These physical methods include osmotic shock, which occurs when cells are suspended in a hypotonic solution shearing and fracturing of cells walls and membranes using... 

One of the most efficient techniques for physical cell disruption is the grinding of the cells in a ball mill (solid shear). These mills consist of either a vertical or a horizontal cylindrical chamber with a motor-driven central shaft supporting a collection of off-centred discs or other agitating elements. In this process, cells are agitated in suspension with small abrasive particles. Cells break because of shear forces, grinding between beads and collisions with beads. The beads disrupt the cells to release biomolecules but do not break the target molecules. The kinetics of biomolecule release by this method is a first-order process. 

Physical or mechanical methods of cell disruption are the most widely researched in terms of containment. The underlying principle is either by breakage of the cell wall by mechanical contact, the application of liquid or hydrodynamic shear forces, or the application of solid shear forces. Cell disruption by non-physical methods generally involve simple operations which may be carried out in large tanks or vessels, which may or may not require agitation. 

If non-physical methods of cell disruption can be developed to the stage where they are cost-effective at large-scale, the requirement for physical methods will decrease. There is no doubt that high pressures and high speed rotating shafts increase the risk of a breach of containment and subsequent production of hazardous aerosols. It is predictable that the trend of research will be towards non-physical methods of cell disruption and their application at large-scale. 

If non-physical methods of cell disruption are developed to a stage where they are cost effective then the requirement for physical methods of disruption will decrease. Disruption which can be carried out in a bioreactor or simple stirred tanks that can be adequately sealed to comply with legislation will reduce the need for high pressures and highspeed rotating shafts. 

The detailed mechanism of action of cationic peptides is described in Section IVC. The most prominent effect on cells is the formation of channels m or disruptions of the cytoplasmic membrane. Thus, these molecules appear to kill by a physical method that takes advantage of the specific composition of bacterial membranes. In contrast, most... 

Physical Methods. Although there are many methods (eg reciprocating pump, ball mill) to physically disrupt microbial cells, there have been few processes which are based on these principles proposed for the large-scale recovery of PHAs. A process in which a biomass suspension was heated to 220° C... 

Physical and mechanical methods are more suitable for cell disruption in general, because these methods cost less and do not affect intracellular biopolymer chemical integrity. Depending on the chemical method, temperature and material, it may affect chemical integrity if no judicious choice of solvents and operating conditions is made. Physical methods can be ultrasound, hydrocyclone, mill balls, Hughes press or osmotic pressure. 

A variety of cell disruption methods are available. Physical, chemical, and enzymatic methods have all been used. The proper method should be carefully selected to ensure maximum cell disruption with minimum enzyme damage. This depends on the enzyme source, nature and stability. 

Once the cellular materials are separated, those with intracellular proteins need to be ruptured to release their products. Disruption of cellular materials is usually difficult because of the strength of the cell walls and the high osmotic pressure inside. The cell rupture techniques have to be very powerful, but they must be mild enough so that desired components are not damaged. Cells can be ruptured by physical, chemical, or biological methods. 

Figure 5.2 Methods used in the disruption of tissues or organs, such as liver and skin. The initial step is usually some physical technique dicing with scissors is illustrated. The fragments are then treated with an enzyme such as trypsin or collagenase to disrupt the fragments further to obtain single cells.

The steps involved in purification include harvesting of the bioreactor, followed by inactivation of cells and concentration of the starting material, which is the cells, or the medium when the product is secreted. Concentration is achieved by using centrifugation or membrane filter systems by ultrafiltration. Cells are disrupted using the physical, chemical or enzymatic method that is best suited to the particular situation (Hopkins, 1991 Papoutsakis, 1991). 

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