Cell disruption
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Cell disruption is a method or process for releasing biological molecules from inside a cell.
Methods
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.
Major factors
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.
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.
Lysis
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.
Laboratory-scale methods
Enzymatic method
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.
Disadvantages include:
- 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.
Bead method
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. It has the advantage of being able to disrupt very small sample sizes and when processing many samples, to eliminate cross-contamination concerns.
At the simplest level of the method, an equal volume of beads are added to the cell or tissue suspension in a test tube and the sample is mixed on a common laboratory vortex mixer. While processing times are 3-10 times longer than that in specialty machines, it works for easily disrupted cells and is inexpensive.
At the more sophisticated level, beadbeating is done in closed vials, centrifuge tubes, or sealed microtiter plates. The sample and 0.1 to 3 mm diameter beads are agitated at about 2000 oscillations per minute in a specially designed vial shaker driven by a high energy electric motor. Cell disruption is complete in 1–3 minutes of shaking. In some machines hundreds of samples can be processed simultaneously using deep well microplates.
In fact, one of the most recent beadbeater innovations, "Simultaneous Multidirectional Bead-Beating" (SMBB)--whereby the precise combination of vertical and horizontal motion induces omnidirectional bead motion, and thus omnidirectional cell membrane impact--has allowed even the most impossible tissues and cells, whether endospores or chitin or compact bone, to often be lysed in under 40 seconds.
These SMBB machines have also drastically improved functional protein/RNA yields, due to the decreased amount of "Purgatory Time," the time during which a protein or an mRNA molecule remains inside of a punctured/lysed cell awaiting diffusion into the extracellular buffer. During this Purgatory Period, the protein (or RNA) of interest is trapped inside a punctured cell, a cell in which previously compartmentalized enzymes pervade the cytoplasm (along with the previously compartmentalized low-pH lysosomal fluids), awaiting to enter the neutralizing buffer, which can also have protease inhibitors (e.g., NaF) and RNase inhibitors (e.g. DEPC). Often this can lead to specific activities and yields upwards of 50% higher than non-SMBB machines.
To prevent degradation of RNA and proteins, some form of cooling is often required because samples warm about 10 degrees per minute of operation due to collisions of the beads. Sample warming can be controlled by placing vials in pre-chilled aluminum vial holders or by beadbeating for several small time intervals with cooling on ice between each run. A different configuration, suitable for larger sample volumes, uses a rotor inside a sealed 15, 50 or 200 ml chamber to agitate the beads. The chambers can be surrounded by a cooling jacket. Using this later configuration, commercial machines are available to process in a continuous manner many liters of cell suspension. These machines, however, are limited to processing monocellular organisms such as yeast and bacteria.
Disadvantages include:
- Occasional problems with foaming and sample heating, especially for larger samples. Can be ameliorated with a dry-ice adapter.
- 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. Not so much a problem with SMBB machines.
Sonication
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.
Disadvantages include:
- Heat generated by the ultrasound process must be dissipated.
- High noise levels (most systems require hearing protection and sonic enclosures)
- Yield variability
- Free radicals are generated that can react with other molecules.
Detergent methods
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, 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.
Solvent Use
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.[1]
Simple and rapid method for disruption of bacteria for protein studies.
S Bhaduri and P H Demchick
Disadvantages include:
- Proteins are denatured
'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.
Disadvantages include:
- 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 high risk of personal hazard if not handled carefully.
High-shear mechanical methods.
High-shear mechanical methods for cell disruption fall into four major classes: rotor-stator disruptors, valve-type processors, fixed-geometry processors and fixed orifice and constant pressure 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.
Rotor-stator Processors and blenders
The rotor/stator homogenizer is commonly used for small volumes of tissue suspended in 3 to 10 times its volume of homogenizing media (1-100 ml total). Larger volumes of tissue in homogenization media (100-2000 ml total) are processed either by larger rotor/stator machines or by blade blenders (often called a drink blenders). Both of these homogenizers rely on rotory cutting and/or chopping action using compact blades or paddles turning at speeds of 10,000 to 30,000 rpm.
Disadvantages include:
- Does not work with microorganisms like bacteria, yeast and fungi and most monocellular tissue cultures.
- Often variable in product yield.
Valve-type processors
![](http://upload.wikimedia.org/wikipedia/commons/thumb/7/76/Homogenizing_valve.svg/400px-Homogenizing_valve.svg.png)
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. Three major implementations of the technology exist: the French pressure cell press, Constant Cell Disruption Systems 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:
- 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.
Disadvantages include:
- 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.
Fixed Orifice and Constant Pressure
Constant Cell Disruption Systems by Constant Systems part of Score Group plc - these systems are fully contained and operate using a finely controlled hydraulic system powered by electricity only. The sample is taken in and instantly pressurised up to a maximum of 40,000 PSI before being passed through a very small and fixed orifice and then returned to atmospheric pressure. As the sample is being processed this type of cell disruptor ensures that the pressure is maintained throughout the process, ensuring repeatability throughout the sample run.
Both fluid and non fluid samples can be processed through this type of cell disruptor, plant leaves and skin samples being a good example of non fluid samples. Having a maximum process pressure achievable of 40,000 PSI enables this type of unit to process more difficult sample types with fewer repeat passes. A built-in cooling jacket ensures temperature control of the sample (Water Bath or Chiller Unit is required)
See also
External links
- Cell Disruption by GEA Niro Soavi
- Cell Disrupters: A Review
- Microfluidics - Cell Disruption using a Microfluidizer processor
- Constant Systems - How the sample is processed
- Physical methods of Cell Disruption
- Homogenizer types for tissue and cell disruption