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Cell disruption is a method or process for releasing biological molecules from inside a cell.
Additional recommended knowledge
Choice of disruption method
The production of biologically-interesting molecules using cloning and culturing methods allows the study and manufacture of relevant molecules.Except for excreted molecules, cells producing molecules of interest must be disrupted. This page discusses various methods.
Several factors must be considered.
Volume or sample size of cells to be disrupted
If only a few microliters of sample are available, care must be taken to minimize loss and to avoid cross-contamination.
Disruption of cells, when hundreds or even thousands of liters of material are being processed in a production environment, presents a different challenge. Throughput, efficiency, and reproducibility are key factors.
How many different samples need to be disrupted at one time?
Frequently when sample sizes are small, there are many samples. As sample sizes increase, fewer samples are usually processed. Issues are sample cross contamination, speed of processing, and equipment cleaning .
How easily are the cells disrupted?
As the difficulty of disruption increases (e.g. E. coli), more force is required to efficiently disrupt the cells. For even more difficult samples (e.g. yeast), there is a parallel increase in the processor power and cost. The most difficult samples (e.g. spores) require mechanical forces combined with chemical or enzymatic efforts, often with limited disruption efficiency.
What efficiency of disruption is required?
Over-disruption may impact the desired product. For example, if subcellular fractionation studies are undertaken, it is often more important to have intact subcellular components, while sacrificing disruption efficiency.
For production scale processes, the time to disrupt the cells and the reproducibility of the method become more important factors.
How stable is the molecule(s) or component that needs to be isolated?
In general, the cell disruption method is closely matched with the material that is desired from the cell studies. It is usually necessary to establish the minimum force of the disruption method that will yield the best product. Additionally, once the cells are disrupted, it is often essential to protect the desired product from normal biological processes (e.g. proteases) and from oxidation or other chemical events.
What purification methods will be used following cell disruption?
It is rare that a cell disruption process produces a directly usable material; in almost all cases, subsequent purification events are necessary. Thus, when the cells are disrupted, it is important to consider what components are present in the disruption media so that efficient purification is not impeded.
For easily disrupted cells such as insect and mammalian cells grown in culture media, a mild osmosis-based method for cell disruption (lysis) is commonly used. Quite frequently, simply lowering the ionic strength of the media will cause the cells to swell and burst. In some cases it is also desirable to add a mild surfactant and some mild mechanical agitation or sonication to completely disassociate the cellular components. Due to the cost and relative effort to grow these cells, there is often only a small quantity of cells to be processed, and preferred methods for cell disruption tend to be a manual mechanical homogenizer, nitrogen burst methods, or ultrasound with a small probe. Because these methods are performed under very mild conditions, they are often used for subcellular fractionation studies.
For cells that are more difficult to disrupt, such as bacteria, yeast, and algae, hypotonic shock alone generally is insufficient to open the cell and stronger methods must be used, due to the presence of cell walls that must be broken to allow access to intracellular components. These stronger methods are discussed below.
The use of enzymatic methods to remove cell walls is well-established for preparing cells for disruption, or for preparation of protoplasts (cells without cell walls) for other uses such as introducing cloned DNA or subcellular organelle isolation. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources (e.g. snail gut for yeast or lysozyme from hen egg white). The enzymes commonly used include lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase etc.
In addition to potential problems with the enzyme stability, the susceptibility of the cells to the enzyme can be dependent on the state of the cells. For example, yeast cells grown to maximum density (stationary phase) possess cell walls that are notoriously difficult to remove whereas midlog growth phase cells are much more susceptible to enzymatic removal of the cell wall.
Large scale applications of enzymatic methods tend to be costly and irreproducible.
The enzyme must be removed (or inactivated) to allow cell growth or permit isolation of the desired material.
A second common laboratory-scale mechanical method for cell disruption uses glass or ceramic beads and a high level of agitation. At the lowest levels of the technology, the beads are added to the cell suspension in a tube and the sample is mixed on a common laboratory vortex mixer. This process works for easily disrupted cells, is inexpensive, and multiple samples can be conveniently processed. The more sophisticated level, bead-based methods use a closed container holding the sample and the beads with an electric motor to provide vigorous agitation. When larger samples are processed, some form of cooling is provided (typically liquid CO2) as the sample heats significantly due to the extreme agitation. Another configuration uses an inert, rapidly rotating rotor in a small container containing the cells and beads.
A third common laboratory-scale method for cell disruption applies ultrasound (typically 20-50 kHz) to the sample (sonication). In principle, the high-frequency is generated electronically and the mechanical energy is transmitted to the sample via a metal probe that oscillates with high frequency. The probe is placed into the cell-containing sample and the high-frequency oscillation causes a localized high pressure region resulting in cavitation and impaction, ultimately breaking open the cells. Although the basic technology was developed over 50 years ago, newer systems permit cell disruption in smaller samples (including multiple samples under 200 µL in microplate wells) and with an increased ability to control ultrasonication parameters.
Detergent-based cell lysis is an alternative to physical disruption of cell membranes, although it is sometimes used in conjunction with homogenization and mechanical grinding. Detergents disrupt the lipid barrier surrounding cells by disrupting lipid:lipid, lipid:protein and protein:protein interactions. The ideal detergent for cell lysis depends on cell type and source and on the downstream applications following cell lysis. Animal cells, bacteria and yeast all have differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of the dense and complex nature of animal tissues, they require both detergent and mechanical lysis to effectively lyse cells.
In general, nonionic and zwitterionic detergents are milder, resulting in less protein denaturation upon cell lysis, than ionic detergents and are used to disrupt cells when it is critical to maintain protein function or interactions. CHAPS, a zwitterionic detergent, and the Triton X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, and ionic detergent that binds to and denatures proteins, is used extensively for studies assessing protein levels by gel electrophoresis and western blotting.
In addition to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, ionic strength and temperature.
The 'cell bomb'
A fifth laboratory-scale system for cell disruption is rapid decompression or the "cell bomb" method. In this process, cells in question are placed under high pressure (usually nitrogen or other inert gas up to about 25,000 psi) and the pressure is rapidly released. The rapid pressure drop causes the dissolved gas to be released as bubbles that ultimately lyse the cell.
High-shear mechanical methods.
High-shear mechanical methods for cell disruption fall into three major classes: rotor-stator disruptors, valve-type processors, and fixed-geometry processors. (These fluid processing systems also are used extensively for homogenization and deaggregation of a wide range of materials – uses that will not be discussed here.) These processors all work by placing the bulk aqueous media under shear forces that literally pull the cells apart. These systems are especially useful for larger scale laboratory experiments (over 20 mL) and offer the option for large-scale production.
Most commonly used as tissue disruptors.
Valve-type processors disrupt cells by forcing the media with the cells through a narrow valve under high pressure (20,000–30,000 psi or 140–210 MPa). As the fluid flows past the valve, high shear forces in the fluid pull the cells apart. By controlling the pressure and valve tension, the shear force can be regulated to optimize cell disruption. Due to the high energies involved, sample cooling is generally required, especially for samples requiring multiple passes through the system. Two major implementations of the technology exist: the French pressure cell press and pumped-fluid processors.
French press technology uses an external hydraulic pump to drive a piston within a larger cylinder that contains the sample. The pressurized solution is then squeezed past a needle valve. Once past the valve, the pressure drops to atmospheric pressure and generates shear forces that disrupt the cells. Disadvantages include:
Mechanically pumped-fluid processors function by forcing the sample at a constant volume flow past a spring-loaded valve.
Fixed-geometry fluid processors
Fixed-geometry fluid processors are marketed under the name of Microfluidizer® processors. The processors disrupt cells by forcing the media with the cells at high pressure (typically 20,000–30,000 psi or 140–210 MPa) through an interaction chamber containing a narrow channel. The ultra-high shear rates allow for:
The systems permit controlled cell breakage without the need to add detergent or to alter the ionic strength of the media. The fixed geometry of the interaction chamber ensures reproducibility. Especially when samples are processed multiple times, the processors require sample cooling.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Cell_disruption". A list of authors is available in Wikipedia.|