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Cell disruption, sometimes referred to as digestion, is a method or process for releasing biological molecules from inside a cell.
In the simplest example of the method, an equal volume of beads are added to a cell or tissue suspension in a test tube and the sample is vigorously mixed on a common laboratory vortex mixer. While processing times are slow, taking 310 times longer than that in specialty shaking machines, it works well for easily disrupted cells and is inexpensive.
Successful bead beating is dependent not only on design features of the shaking machine (which take into consideration shaking oscillations frequency, shaking throw or distance, shaking orientation and vial orientation), but also the selection of correct bead size ( diameter), bead composition (glass, ceramic, steel) and bead load in the vial.
In most laboratories, bead beating is done in batch sizes of one to twenty-four sealed, plastic vials or . The sample and tiny beads are agitated at about 2000 oscillations per minute in specially designed reciprocating shakers driven by high power . Cell disruption is complete in 1–3 minutes of shaking. Significantly faster rates of cell disruption are achieved with a bead beater variation called SoniBeast. Differing from conventional machines, it agitates the beads using a vortex motion at 20,000 oscillations per minute. Larger bead beater machines that hold deep-well also shorten process times, as do Bead Dispensers designed to quickly load beads into multiple vials or microplates. Pre-loaded vials and microplates are also available.
All high energy bead beating machines warm the sample about 10 degrees per minute. This is due to frictional collisions of the beads during homogenization. Cooling of the sample during or after bead beating may be necessary to prevent damage to heat-sensitive proteins such as enzymes. Sample warming can be controlled by bead beating for short time intervals with cooling on ice between each interval, by processing vials in pre-chilled aluminum vial rack or by circulating gaseous coolant through the machine during bead beating.
A different bead beater configuration, suitable for larger sample volumes, uses a rotating fluorocarbon rotor inside a 15, 50 or 200 ml chamber to agitate the beads. In this configuration, the chamber can be surrounded by a static cooling jacket. Using this same rotor/chamber configuration, large commercial machines are available to process many liters of cell suspension. Currently, these machines are limited to processing unicellular organisms such as yeast, algae and bacteria.
The technique can be done by using a mortar and pestle cooled to liquid nitrogen temperatures, but use of this classic apparatus is laborious and sample loss is often a concern. Specialised stainless steel pulverizers generically known as Tissue Pulverizers are also available for this purpose. They require less manual effort, give good sample recovery and are easy to clean between samples. Advantages of this technique are higher yields of proteins and nucleic acids from small, hard tissue samples - especially when used as a preliminary step to mechanical or chemical/solvent cell disruption methods mentioned above.
Modern physical cell disruptors typically operate via either pneumatic or hydraulic pressure. Although pneumatic machines are typically lower cost, their performance can be unreliable due to variations in the processing pressure throughout the stroke of the air pump. It is generally considered that hydraulic machines offer superior lysing ability, especially when processing harder to break samples such as yeast or Gram-positive bacteria, due to their ability to maintain constant pressure throughout the piston stroke. As the French Press, which is operated by hydraulic pressure, is capable of over 90% lysis of most commonly used cell types it is often taken as the gold standard in lysis performance and modern machines are often compared against it not only in terms of lysis efficiency but also in terms of safety and ease of use. Some manufacturers are also trying to improve on the traditional design by altering properties within these machines other than the pressure driving the sample through the orifice. One such example is Constant Systems, who have recently shown that their Cell Disruptors not only match the performance of a traditional French Press, but also that they are striving towards attaining the same results at a much lower power.Under Pressure, M. Lougher, European Biopharmaceutical Review, July 2016; 12-16
Pressure Cycling Technology ("PCT"). PCT is a patented, enabling technology platform that uses alternating cycles of hydrostatic pressure between ambient and ultra-high levels (up to 90,000 psi) to safely, conveniently and reproducibly control the actions of molecules in biological samples, e.g., the rupture (lysis) of cells and tissues from human, animal, plant, and microbial sources, and the inactivation of pathogens. PCT-enhanced systems (instruments and consumables) address some challenging problems inherent in biological sample preparation. PCT advantages include: (a) extraction and recovery of more membrane proteins, (b) enhanced protein digestion, (c) differential lysis in a mixed sample base, (d) pathogen inactivation, (e) increased DNA detection, and (f) exquisite sample preparation process control.
The Microfluidizer method used for cell disruption strongly influences the physicochemical properties of the Lysis 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 at disrupting many different kinds of cells. The Microfluidizer technology was licensed from a company called Arthur D. Little and was first developed and utilized in the 1980s, initially starting as a tool for liposome creation. It has since been used in other applications such as cell disruption Emulsion, and solid particle size reduction, among others.
By using microchannels with fixed geometry, and an intensifier pump, high are generated that rupture the cells. This method of cell lysis can yield breakage of over 90% of E. coli cells.Evaluation of the Microfluidizer for Cell Disruption of Yeast and Chlorella by E. Uera-Santos, C.D. Copple, EA Davis and WG. Hagar.
Many proteins are extremely temperature-sensitive, and in many cases can start to denature at temperatures of only 4 degrees Celsius. Within the microchannels, temperatures exceed 4 degrees Celsius, but the machine is designed to cool quickly so that the time the cells are exposed to elevated temperatures is extremely short (residence time 25 ms-40 ms). Because of this effective temperature control, the Microfluidizer yields higher levels of active proteins and enzymes than other mechanical methods when the proteins are temperature-sensitive.agerkvist, Irene, and Sven-Olof Enfors.”Characterization Of E. Coli Cell Disintegrates from a Bead Bill and High Pressure Homogenizer.”Biotechnology and bioengineering Biotechnol.Bioeng.36.11 (1990):1083-089.Web.
Viscosity changes are also often observed when disrupting cells. If the cell suspension viscosity is high, it can make downstream handling—such as filtration and accurate pipetting—quite difficult. The viscosity changes observed with a Microfluidizer are relatively low, and decreases with further additional passes through the machine.agerkvist, Irene, and Sven-Olof Enfors.”Characterization Of E. Coli Cell Disintegrates from a Bead Bill and High Pressure Homogenizer.”Biotechnology and bioengineering Biotechnol.Bioeng.36.11 (1990):1083-089.Web.
In contrast to other mechanical disruption methods the Microfluidizer breaks the cell membranes efficiently but gently, resulting in relatively large cell wall fragments (450 nm), and thus making it easier to separate the cell contents. This can lead to shorter filtration times and better centrifugation separation.Evaluation of the Microfluidizer for Cell Disruption of Yeast and Chlorella by E. Uera-Santos, C.D. Copple, EA Davis and WG. Hagar.
Microfluidizer technology scales from one milliliter to thousands of liters.
Nitrogen decompression is more protective of and than ultrasound and mechanical homogenizing methods and compares favorably to the controlled disruptive action obtained in a PTFE and glass mortar and pestle homogenizer. While other disruptive methods depend upon friction or a mechanical shearing action that generate heat, the nitrogen decompression procedure is accompanied by an adiabatic expansion that cools the sample instead of heating it.
The blanket of inert nitrogen gas that saturates the cell suspension and the homogenate offers protection against oxidation of cell components. Although other gases: carbon dioxide, nitrous oxide, carbon monoxide and compressed air have been used in this technique, nitrogen is preferred because of its non-reactive nature and because it does not alter the pH of the suspending medium. In addition, nitrogen is preferred because it is generally available at low cost and at pressures suitable for this procedure.
Once released, subcellular substances are not exposed to continued attrition that might denature the sample or produce unwanted damage. There is no need to watch for a peak between enzyme activity and percent disruption. Since nitrogen bubbles are generated within each cell, the same disruptive force is applied uniformly throughout the sample, thus ensuring unusual uniformity in the product. Cell-free homogenates can be produced.
The technique is used to homogenize cells and tissues, release intact organelles, prepare , release labile , and produce uniform and repeatable homogenates without subjecting the sample to extreme chemical or physical stress.
The method is particularly well suited for treating mammalian and other membrane-bound cells. It has also been used successfully for treating , for releasing virus from fertilized eggs and for treating fragile bacterium. It is not recommended for untreated bacterial cells. Yeast, fungus, spores and other materials with tough cell walls do not respond well to this method.
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