Disorder cell is a method or process for removing biological molecules from within a cell.
Video Cell disruption
Method
Biologically attractive molecular production using cloning and culture methods allows research and manufacture of relevant molecules. Except for excreted molecules, cells that produce attractive molecules should be disrupted. This page discusses various methods.
Maps Cell disruption
Bead Method
General laboratory-scale mechanical methods for cell disruption using small glass, ceramic or steel beads are mixed with suspended samples in aqueous media. First developed by Tim Hopkins in the late 1970s, a mixture of samples and beads underwent a high degree of agitation by stirring or shaking. Beads collide with cellular samples, cracks open the cell to release components between cells. Unlike some other methods, the mechanical shear is moderate during homogenization resulting in excellent membrane or subcellular preparation. The method, often called "beadbeating", works well for all types of cellular materials - from spores to animal and plant tissues. This is the most widely used lysis method of yeast, and can result in more than 50% damage. This has the advantage over other mechanical cell disruption methods to be able to interfere with very small sample sizes, process many samples at a time without the problem of cross-contamination, and not release potentially harmful aerosols in the process.
In the simplest method example, the same bead volume is added to the cell or tissue suspension in the test tube and the sample is strongly mixed on the general laboratory vortex mixer. While processing times are slow, take 3-10 times longer than on a special shaker machine, this works well for cells that are easily distracted and inexpensive. The processing time can be increased by using a bead dispenser to load the beads into the vial or plate. The LabTIE company produces a Bead dispenser that dispenses beads in a grid in just 8 seconds.
In most laboratories, beadbeating is done in closed plastic bottles, plastics, centrifuge tubes, or deep microtiter plates. Small samples and beads are agitated at about 2,000 oscillations per minute in special bottles designed by high power motors. Cell disorders complete within 1-3 minutes after vibrating. Machines are available that can process hundreds of samples simultaneously inside a good microplate.
The beadbeating success depends not only on the features of the shaking machine design (which takes into account the shaking oscillations per minute, shake throw or distance, shock orientation and bottle orientation), but also the correct selection of bead size (0.1-6 mm diameter), manic composition glass, ceramic, steel) and bead load in vials.
All high energy beadbeating machines warm up the sample by about 10 degrees/minute. This is due to collision friction of the beads during homogenization. Cooling of samples during or after beadbeating may be necessary to prevent damage to heat sensitive proteins such as enzymes. Example heating can be controlled by beadbeating for short time intervals by cooling in ice between each interval, by processing vials in a pre-cooled aluminum bottle holder or by gas cooling circulating through the machine during beadbeating.
Different beadbeater configurations, suitable for larger sample volumes, use fluorocarbon rotors in 15, 50 or 200 ml chambers to stir the beads. In this configuration, space can be surrounded by a static cooling jacket. Using the same rotor/cub configuration, large commercial machines are available to process many liters of cell suspension. Currently, these machines are limited to processing monocellular organisms such as yeast, algae, and bacteria.
Cryopulverization
Samples with hard extracellular matrix, such as animal connective tissue, multiple tumor biopsy samples, venous tissue, cartilage, seeds, etc., are reduced to fine powders by impact impact on liquid nitrogen temperatures. This technique, known as cryopulverization, is based on the fact that biological samples containing significant fractions of water become brittle at very cold temperatures. This technique was first described by Smucker and Pfister in 1975, which refers to techniques as cryo-impacting. The authors point out that cells are effectively broken with this method, which is confirmed by the phases and electron microscopes that damage cross cell walls and cytoplasmic membranes.
This technique can be done by using mortar and pitcher cooled to liquid nitrogen temperature, but the use of this classic tool is tiring and sample loss is often a concern. Special stainless steel pulverizers commonly known as Tissue Pulverizers are also available for this purpose. They require less manual effort, provide good recovery instances and are easy to clean among samples. The advantage of this technique is the higher yield of proteins and nucleic acids from small, hard tissue samples - especially when used as a first step for the above mentioned mechanical or chemical/solvent-disruptive methods.
Physical Cell Disorder
Since the 1940s high pressure has been used as a method of cell disorders, especially by French Pressure Cell Press, or French Press for the short term. This method was developed by Charles Stacy French and used high pressure to force cells through narrow apertures, causing melisis cells due to shear forces experienced throughout the pressure differential. While French Presses have become a staple in many microbiological laboratories, their production has largely been discontinued, leading to a revival in alternative technology applications alike.
Modern physical cell intruders usually operate either through pneumatic or hydraulic pressures. Although pneumatic machines typically have lower costs, their performance can be unreliable due to variations in processing pressure along the air pump stroke. It is generally assumed that hydraulic machines offer superior lysis capability, especially when processing harder to break down samples such as yeast or Gram-positive bacteria, due to their ability to maintain constant pressure along the piston stroke. As French Press, operated by hydraulic pressure, capable of more than 90% lysis of the most commonly used cell types, often taken as the gold standard in lysis performance and modern machines is often compared to not only in terms of lysis. efficiency but also in terms of security and ease of use. Some manufacturers also try to improve traditional design by altering properties within these machines in addition to the pressure that drives the sample through the hole. One example is Constant Systems, which recently showed that their Cell Disruptors not only match traditional French Press performance, but also that they are trying to achieve the same result with much lower forces.
Cycling Pressure Technology ("PCT"). PCT is a patented and enabling technology platform that utilizes alternating between ultra-high and ultra-high (up to 90,000 psi) environmental and hydrostatic cycles to safely, comfortably, and can be reproduced to control molecular actions in biological samples, for example, cell and tissue lysis from humans, animals, plants, and microbial sources, and pathogen inactivation. PCT-enhanced systems (instruments and consumables) address some of the challenging issues inherent in the preparation of biological samples. The advantages of PCT include: (a) extraction and recovery of more membrane proteins, (b) enhanced protein digestion, (c) differential lysis in mixed sample bases, (d) pathogen inactivation, (e) increased DNA detection, and (f) control the sample preparation process beautifully.
Microfluidizer Method
This method used for cell disorders greatly influences the physicochemical properties of the lised cell suspension, such as particle size, viscosity, protein yield and enzyme activity. In recent years the Microfluidizer method has gained popularity in cell disruption due to its ease of use and efficiency in disrupting various cell types. Microfluidizer technology was licensed by a company called Arthur D. Little and was first developed and used in the 1980s, originally started as a tool for the creation of liposomes. Since then it has been used in other applications such as cell interference nanoemulsion, and solid particle size reduction, among others.
By using microchannels with fixed geometry, and intensifier pumps, high shear rates are generated that break down the cell. This cell lysis method can result in damage of more than 90% of E. coli cells.
Many proteins are very sensitive to temperature, and in many cases can start to change the temperature to just 4 degrees Celsius. In microchannels, the temperature exceeds 4 degrees Celsius, but the engine is designed to cool rapidly so that the time of cells exposed to high temperatures is very short (residence time 25 ms-40 ms). Because of this effective temperature control, Microfluidizer produces higher levels of active proteins and enzymes than any other mechanical method when the protein is temperature sensitive.
Viscosity changes are also frequently observed when disrupting the cells. If the viscosity of the cell suspension is high, it can make downstream handling - like filtering and piping accurate - quite difficult. The viscosity change observed with the Microfluidizer is relatively low, and decreases with further additional passage through the machine.
In contrast to other mechanical distortion methods, Microfluidizer breaks down cell membranes efficiently but gently, resulting in relatively large cell wall fragments (450 nm), and thus makes it easier to separate cell contents. This can lead to shorter filtration times and better centrifugation separation.
The scale of Microfluidizer technology ranges from one milliliter to thousands of liters.
Nitrogen decompression
For nitrogen decompression, large amounts of nitrogen are first dissolved in a cell under high pressure in an appropriate pressure vessel. Then, when the gas pressure is suddenly released, nitrogen comes out of the solution as it dilates the bubbles that stretch the membranes of each cell until they break and release the cell contents.
Nitrogen decompression is more protective of enzymes and organelles than the method of ultrasonic and mechanical homogenization and better than the controlled interference actions obtained in PTFE and glass mortar and agitator. While other disturbing methods depend on friction or mechanical shear action that generate heat, the nitrogen decompression procedure is accompanied by adiabatic expansion that cools the sample rather than heats it up.
An inert nitrogen gas blanket that saturates cell suspensions and homogenates offers protection against cell component oxidation. Although other gases: carbon dioxide, nitrogen oxide, carbon monoxide and compressed air have been used in this technique, nitrogen is preferred because it is non-reactive and therefore does not alter the pH of the suspending medium. In addition, nitrogen is preferred as it is generally available at low cost and at appropriate pressure for this procedure.
Once released, subcellular substances are not subject to continued friction that may change the sample size or produce undesirable damage. No need to observe the peak between enzyme activity and percent disturbance. Because the nitrogen bubbles are generated in each cell, the same disturbance force is applied evenly throughout the sample, thus ensuring uniform uniformity in the product. Cell-free homogenates can be produced.
This technique is used to homogenize cells and tissues, release intact organelles, prepare cell membranes, release labile biochemistry, and produce uniform and repetitive homogenates without burdening the sample to extreme chemical or physical stress.
This method is particularly suitable for treating mammals and other membrane bound cells. It has also been successfully used to treat plant cells, to release viruses from fertilized eggs and to treat fragile bacteria. This is not recommended for untreated bacterial cells. Yeast, mold, spores and other materials with hard cell walls do not respond well to this method.
See also
- Homogenizer ultrasonic
- Sonication
- Homogenization (chemistry)
- Homogenizer
References
Source of the article : Wikipedia
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