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Cell Disruption Methods & Role of Liquid Nitrogen in Cell Disruption

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Cell Disruption Methods & Role of Liquid Nitrogen in Cell Disruption Cell Disruption Methods & Role of Liquid Nitrogen in Cell Disruption Cell is the basic unit for any living system & it contains all the necessary components that enable Cellular Metabolic processes. Cell Disruption (CD) is the process of Breaking the cell boundary (Cell Wall or Plasma Membrane) & obtaining the intracellular metabolic components with minimal damage. Depending on the type of cell and its cell wall composition the CD methods vary. Irrespective of the methods used the main aim of the CD: Method should not be too harsh & the product recovered remains in its active form. Cell Disruption methods broadly categorized in to two types. Those are: 1. Mechanical Methods & 2. Non- Mechanical Methods Mortar & Pestle Method: Also Called Homogenization. Mortar & Pestle are a set of two tools using since the Stone Age. Used for Grinding/Crushing the Substances in to a fine paste or powder in the Kitchen, Pharmacy and Laboratory Practices. Mortar is a bowl, typically made of hard wood, metal, ceramic or granite & the Pestle is a blunt, club shaped object. While disruption, the Biological material first need to be submerged in the Liquid Nitrogen (-196 ˚C) for Freezing Purpose. The Frozen Biological Material finely ground with Mortar & Pestle & Made into Powder. The Powdered Material then Dissolve in Suitable Solvent & Extract the Desired Component. Blending Method: It is a physical method for rupturing the mammalian and plant cells by shear force. The process is usually carried out in a typical domestic kitchen blender. In this method, the tissue is cut into small pieces and blended with buffer for about one minute to disrupt the cell. The process is usually carried out in a typical domestic kitchen blender. A blender with small beads can also be used for microorganism. Bead Beating: It is also called Grinding with Abrasives. It is a physical method to rupture cell in presence of abrasives like sand, alumina, glass beads, etc. It can be carried out in Mortar- Piston or Dynomill. Grinding in mortar and piston with sand or alumina in presence of buffer cause rupturing of the cell wall. This method is useful for disrupting bacterial or plant cells. However, this is applicable to the relatively small amount of sample. Dynomill is a large scale version of grinding in mortar and piston. It comprises a chamber containing glass beads and numbers of rotating impeller discs. It is an electrical process of rupturing cells. Cells get ruptured when they are caught between colliding beads. It can process up to 5kg cells per an hour. This method helps in the production of industrially important proteins and enzymes. Ultra Sonication: This method is suitable for a small volume of the suspension of cultured or microbial cells. A Sonicator probe generates high-frequency sound waves (<20 KHz) for 30-60 Sec. These sound waves cause disruption of cells by shear force and cavitations. Cavitation refers to an area of alternate compression and rarefaction which causes tension in a cell that causes rupturing of the cell. In Cavitation, gas bubbles are formed which explodes as they decompress after applying pressure that produces a local shock wave. The cells are disrupted by pressure change. Since a considerable amount of heat is generated during this process, the sample must be kept on ice during treatment. Chemical Disruption of Cells by Detergents: Detergents are cleaning agents that directly damage the cell wall or membrane, lead to release of intracellular content. One of the most commonly used anionic detergents is sodium dodecyl sulfate (SDS) which reorganizes the cell membrane by disturbing protein-protein interactions. Another commonly used compound for cell lyses is Triton X100, which is non-ionic detergent. Its mechanism of action is to solubilize membrane proteins. The disadvantage of using detergents for cell lysis is that many proteins will be denatured in lysis process and also disturb subsequent downstream processing steps. Enzymatic Disruption of Cells: Another strategy for cell lysis is to use digestive enzymes which decompose the cell wall. Different cell types possess different kind of cell walls and membranes, and thus the used enzyme depends For Eg. Lysozyme is commonly used enzyme to digest cell wall of gram positive bacteria. Lysozyme hydrolyzes β-1-4-glucosidic bonds in the Peptidoglycon. The cell wall of gram negative bacteria differs from the cell wall of gram positive bacteria so Lysozyme is not very efficient in the case of gram negative cell wall. The Cell wall of Yeast and Fungi differs and commonly used enzyme mixture for degradation of its cell wall Zymolyase. It has β-1, 3 glucanase and hydrolase activities. Microwave / Thermolysis: Thermolysis is potential in isolation of Periplasm proteins in G (-) bacteria which are released when the cells are heated up to 50ºC. Cytoplasmic proteins can be released from E. coli within 10min at 90 ºC. Freeze Thaw: This can cause the cells to burst due to the formation and melting of ice crystals. Gradual freezing, leading to the formation of larger crystals, can cause an extensive damage to the cell. By combining this method with cell grinding offers great results. Osmotic shock: This is a technology which can be utilized in biotechnical applications to cause cell lysis. In this technology, cells are first exposed to either high or low salt concentration. Then the conditions are quickly changed to opposite conditions which lead to osmotic pressure and cell lysis Electric Discharges: It is also possible to achieve cell disruption via electrical discharges in mammalian cells, which are cells that are bounded by plasma membranes and, unlike plant cells. This method allows researchers to examine secretion by exocytosis. Role of Liquid Nitrogen: Liquid nitrogen (LN2) is nitrogen in a liquid state at low temperature (−195.79 °C). It is produced industrially by fractional distillation of liquid air. It is a colorless, low viscosity liquid that is widely used as a coolant. Liquid nitrogen has many uses, mainly based on its cold temperature and low reactivity. Examples of common applications include: 1. The freezing and transporting of food products; 2. Use as a coolant for superconductors, vacuum pumps, and other materials and equipment; 3. The cryopreservation of biological samples, such as sperm, eggs, and animal genetic samples; 4. Use in cryotherapy to remove skin abnormalities; 5. The shielding of materials from oxygen exposure; 6. The cooling of materials for easier machining or fracturing; 7. Cell Disruption Methods to Stabilize the Components .
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