- Cell disruption
Cell disruption is a method or process for releasing biological molecules from inside a cell.
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 fractionationstudies 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.
Is the sample being subjected to the method biohazardous?
Preparation of cell-free extracts of pathogens presents unique difficulties. Mechanical disruption techniques are not always applicable owing to potential biohazard problems associated with contamination of equipment and generation of aerosols.
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 surfactantand 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,
hypotonicshock 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, mannaseetc.
*Not always reproducible.
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.
*Not usually applicable to large scale.
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.
Another common laboratory-scale mechanical method for cell disruption uses small glass, ceramic, or steel beads and a high level of agitation by stirring or shaking of the mix. The method, often referred to as "beadbeating", works well for all types of cellular material - from spores to animal and plant tissues.
At the lowest levels of the technology, beads are added to the cell or tissue suspension in a testtube and the sample is mixed on a common laboratory vortex mixer. While processing time is 3-10 times longer than that in specially machines (see below), it works for easily disrupted cells and is inexpensive.
At the more sophisticated level, beadbeating is done in closed vials. The sample and the beads are vigorously agitated at about 2000 oscillations per minute in a specially designed shaker driven by a high energy electric motor. [http://www.spexcsp.com/pdfs/applicationnotesp017-gg_basil_dna_extraction.pdf] In some machines hundreds of samples can be processed simultaneously. When samples larger that 2 ml are processed, some form of cooling is required because samples heat due to collisions of the beads. Another configuration suitable for larger sample volumes uses a rotor inside a sealed 15, 50 or 200 ml chamber to agitate the beads. The chamber can be surrounded by a cooling jacket. Using this same configuation, commercial machines capable of processing many liters of cell suspension are available.
*Occasional problems with foaming and sample heating, especially for larger samples.
*Tough tissue samples such as skin or seeds are difficult to disrupt unless the sample is very small or has been pre-chopped into small pieces.
Another 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 low 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.
*Heat generated by the ultrasound process must be dissipated.
*High noise levels (most systems require hearing protection and sonic enclosures)
*Free radicals are generated that can react with other molecules.
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
lipidbarrier 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, an 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.
A method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with sodium dodecyl sulfate followed by extraction of cellular proteins with acetone. This method is simple, rapid and particularly well suited when the material is biohazardous. [cite journal | author=Demchick PH and Koch AL | title=The permeability of the wall fabric of Escherichia coli and Bacillus subtilis | journal=Appl Environ Microbiol| year=1983 | pages=941–3| volume=46 | issue=4 [http://aem.asm.org/cgi/reprint/46/4/941] ]
Simple and rapid method for disruption of bacteria for protein studies. S Bhaduri and P H Demchick Disadvantages include:
*Proteins are denatured
The 'cell bomb'
Another 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.
*Only easily disrupted cells can be effectively disrupted (stationary phase E. coli, yeast, fungi, and spores do not disrupt well by this method).
*Large scale processing is not practical.
*High gas pressures have a small risk of personal hazard if not handled carefully.
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
homogenizationand 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.
*Do not work well with difficult-to-lyse cells like yeast and fungi
*Often variable in product yield.
*Poorly suited for culture use.
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 pressand 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:
*Not well suited to larger volume processing.
*Awkward to manipulate and clean due to the weight of the assembly (about 30 lb or 14 kg).
"Mechanically pumped-fluid processors" function by forcing the sample at a constant volume flow past a spring-loaded valve.
*Requires 10 mL or more of media.
*Prone to valve-clogging events.
*Due to variations in the valve setting and seating, less reproducible than fixed-geometry fluid processors.
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:
*Processing of more difficult samples
*Fewer repeat passes to ensure optimum sample processing
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.
Cell disruption by nitrogen decompression
* [http://www.microfluidicscorp.com/index.php?option=com_content&task=view&id=17&Itemid=36 Microfluidics - Cell Disruption using a Microfluidizer® processor]
* [http://www.desmech.com/?p=28 Physical methods of Cell Disruption]
* [http://www.biospec.com/Lab%20Cell%20Disrupters%20Review.htm Laboratory Cell Disrupters - A review covering apparatus and techniques of cell disruption]
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