Cell Disruption- Definition, Methods, Types, Significance
Cell disruption is the process of breaking open the cell membrane or cell wall to release the intracellular contents, such as proteins, DNA, RNA, organelles, enzymes, metabolites, etc. Cell disruption is an important step in many biotechnological applications, such as recombinant protein production, enzyme extraction, DNA isolation, metabolite analysis, and cell-based assays. Cell disruption can also be used for studying the structure and function of cellular components, such as membranes, cytoskeleton, and organelles.
There are different methods of cell disruption, depending on the type of cells, the desired product, the scale of operation, and the available equipment. Generally, cell disruption methods can be classified into two main categories: mechanical and non-mechanical. Mechanical methods use physical force to rupture the cell membrane or cell wall by grinding, shearing, or sonication. Non-mechanical methods use chemical agents, physical treatments, or enzymes to weaken or dissolve the cell membrane or cell wall.
Each method of cell disruption has its own advantages and disadvantages. Some factors that need to be considered when choosing a suitable method are:
- The efficiency and yield of cell disruption
- The quality and integrity of the desired product
- The cost and availability of the equipment and reagents
- The safety and environmental impact of the method
- The scalability and reproducibility of the method
In this article, we will discuss some of the most common methods of cell disruption in detail, along with their types, principles, applications, and significance. We will also compare and contrast the different methods and provide some tips and recommendations for choosing the best method for your specific needs.
Mechanical methods of cell disruption are those methods that require some sort of force to separate out intracellular proteins without adding chemicals or enzymes. These methods are suitable for large-scale and rapid cell disruption, but they may also cause heat generation, foaming, and denaturation of proteins. Some of the common mechanical methods of cell disruption are:
- Mortar and pestle: This method involves the grinding of the cells such that they are disrupted. This does not have to be in suspension and is often done with plant samples frozen in liquid nitrogen. When the material has been disrupted, metabolites can be extracted by adding solvents.
- Blenders: The use of blenders which employ high speed can be used to disrupt cell walls. It is the same process used by centrifugation, which separates or concentrates materials suspended in a liquid medium.
- Bead beating: Glass or ceramic beads are used to crack open cells. The kind of mechanical shear is gentle enough to keep organelles intact. It can be used with all kinds of cells, just add beads to an equal amount of cell suspension and vortex.
- Ultrasonication: Ultrasonic homogenizers work by inducing vibration in a titanium probe that is immersed in the cell solution. A process called cavitation occurs, in which tiny bubbles are formed and explode, producing a local shockwave and disrupting cell walls by pressure change. This method is very popular for disruption of plant and fungal cells.
- Homogenization: Liquid-based homogenization is the most widely used cell disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or tissue suspension through a narrow space. Homogenizers use shearing forces on the cell similar to the bead method. Homogenization can be performed by squeezing cells through a tube that is slightly smaller than beads beating.
These are some of the mechanical methods of cell disruption that can be used to release intracellular proteins and other biomolecules from different types of cells. Each method has its own advantages and disadvantages, depending on the cell type, volume, and desired product quality. Therefore, it is important to choose the appropriate method for each application.
- It is one of the oldest and simplest methods of cell disruption.
- It involves the grinding of the cells such that they are disrupted.
- This does not have to be in suspension and is often done with plant samples frozen in liquid nitrogen.
- When the material has been disrupted, metabolites can be extracted by adding solvents.
- It is suitable for small-scale and low-throughput applications.
- It is inexpensive and easy to use, but it can be labor-intensive and time-consuming.
- It can also cause heat generation and oxidation, which may affect the quality of the extracted products.
- Blenders are devices that use high-speed rotating blades to chop and mix cell suspensions.
- Blenders can disrupt cell walls by applying shear forces and turbulence to the cells.
- Blenders are suitable for disrupting large volumes of soft plant or animal tissues, such as liver, spleen, or leaf.
- Blenders are not effective for disrupting bacterial or fungal cells, which have rigid cell walls.
- Blenders can generate heat and foam, which may damage the target proteins or substances. Therefore, cooling and anti-foaming agents may be needed.
- Blenders can also cause contamination or cross-contamination of samples, so proper cleaning and sterilization are required.
- Bead beating is a method of cell disruption that uses glass or ceramic beads to crack open cells.
- The beads are mixed with an equal volume of cell suspension and agitated vigorously in a device called a bead mill or a bead beater.
- The beads collide with the cells and break them apart by mechanical shear, releasing the intracellular contents.
- Bead beating can be used with all kinds of cells, including bacteria, yeast, fungi, algae, plant and animal cells.
- The size and type of beads, the speed and duration of agitation, and the temperature and pH of the buffer can affect the efficiency and quality of cell disruption.
- Bead beating is a simple, fast and scalable method that can process multiple samples simultaneously.
- However, bead beating can also generate heat, foam and aerosols that may damage the target proteins or substances. Therefore, cooling systems, anti-foaming agents and biosafety precautions are recommended.
- Additionally, bead beating may not be suitable for fragile or delicate cells that require gentle disruption.
Ultrasonication is a method of cell disruption that uses high-frequency sound waves to create vibrations in a liquid medium. The vibrations cause the formation and collapse of microscopic bubbles, a phenomenon known as cavitation. The collapse of these bubbles generates intense local pressure and temperature changes, which can rupture the cell membranes and walls of the suspended cells. Ultrasonication can also shear DNA and proteins, and release intracellular contents.
Ultrasonication is a versatile and efficient method that can be used to disrupt various types of cells, such as bacteria, fungi, algae, plant and animal cells. It can also be applied to different scales, from laboratory to industrial. Ultrasonication has some advantages over other mechanical methods, such as:
- It does not require grinding media or beads, which can contaminate the sample or damage the target molecules.
- It can be performed in a closed system, which reduces the risk of contamination and aerosol formation.
- It can be easily controlled by adjusting the power, frequency, duration and amplitude of the sound waves.
- It can be combined with other methods, such as freeze-thaw cycles or chemical agents, to enhance the cell disruption efficiency.
However, ultrasonication also has some limitations and challenges, such as:
- It can generate heat and noise, which can affect the quality and stability of the sample and the operator`s safety.
- It can cause oxidation and denaturation of proteins and other biomolecules due to the high temperature and pressure changes.
- It can be affected by the physical and chemical properties of the medium, such as viscosity, density, pH and conductivity.
- It can be difficult to scale up for large volumes or high cell densities.
Therefore, ultrasonication should be optimized for each specific application and cell type, taking into account the desired product yield and quality. Some factors that should be considered are:
- The type and size of the sonicator probe or horn, which determines the intensity and distribution of the sound waves.
- The distance between the probe and the sample, which affects the cavitation efficiency and heat transfer.
- The volume and concentration of the sample, which influences the energy input and output.
- The temperature and cooling system of the sample, which affects the enzyme activity and protein stability.
- The pulse mode and duty cycle of the sonication, which allows intermittent cooling and mixing of the sample.
Ultrasonication is a powerful tool for cell disruption that can offer high efficiency and versatility. However, it also requires careful optimization and monitoring to ensure optimal results.
Homogenization is a liquid-based method of cell disruption that involves forcing the cell or tissue suspension through a narrow space. This creates high pressure and shear forces that break apart the cell membranes and walls. Homogenizers use a piston, a valve, or a high-speed blade to create the narrow space. The cell suspension is pumped through the space repeatedly until the desired level of disruption is achieved.
Homogenization is similar to bead beating in that it uses mechanical shear to lyse cells. However, homogenization does not require beads or other solid materials, which can reduce contamination and sample loss. Homogenization can also be performed at high throughput and with large volumes of cells. Homogenization is suitable for disrupting animal cells, bacteria, yeast, fungi, and some plant cells. However, homogenization may not be effective for cells with very tough cell walls, such as algae or filamentous fungi.
Some advantages of homogenization are:
- It is fast and efficient
- It can handle large volumes and high densities of cells
- It does not require beads or other additives
- It produces uniform and reproducible results
Some disadvantages of homogenization are:
- It may generate heat and noise
- It may damage or denature some proteins or other molecules
- It may require special equipment and maintenance
Non-mechanical methods of cell disruption are those methods that do not require physical force or shear stress to break open the cells. Instead, they rely on other factors such as temperature, osmotic pressure, electric field, or chemical agents to disrupt the cell membrane or wall. Non-mechanical methods can be classified into three main categories: physical methods, chemical methods, and enzymatic methods.
Some of the non-mechanical methods of cell disruption are based on physical principles that can alter the structure and integrity of the cell wall and membrane. These methods include:
Freeze-Thaw: This method is suitable when working with soft plant material and algae. Disruption is achieved via a series of freezing and thawing cycles. Freezing forms ice crystals, which expand upon thawing, and this ultimately causes the cell wall to rupture. However, this method can be time-consuming and may not be effective for cells with thick or rigid walls.
Microwave/Thermolysis: Microwave (along with autoclave and other high temperature methods) are used to disrupt the bonds within cell walls, and also to denature proteins. This method can be fast and efficient, but it requires careful control of the temperature and exposure time to avoid excessive damage to the target molecules.
Osmotic Shock: Through the process of osmosis, water can be moved into the cell causing its volume to increase to the point that it bursts. The method however, can only work with animal cells and protozoa, since they do not have cell walls. Moreover, this method may not be suitable for cells that have high osmotic pressure or that are resistant to osmotic changes.
Electric Discharges: It is also possible to achieve cell disruption via electrical discharges in mammalian and other cells that are bounded by plasma membranes only. This method involves applying a high voltage pulse across the cell suspension, which creates pores in the membrane and allows the release of intracellular contents. This method can be very effective and selective, but it requires specialized equipment and expertise.
Chemical methods of cell disruption involve the use of substances that can alter the structure or permeability of the cell membrane or cell wall. These substances can be organic solvents, detergents, chaotropic agents, chelating agents, or acids and bases. Chemical methods are often used in combination with mechanical or physical methods to enhance the efficiency of cell disruption. Some examples of chemical methods are:
- Organic solvents: These are compounds that can dissolve lipids and other hydrophobic molecules in the cell membrane or cell wall. Examples of organic solvents are toluene, ether, benzene, methanol, ethanol, acetone, chloroform, and hexane. Organic solvents can be used to disrupt plant cells, fungal cells, bacterial cells, and animal cells. However, organic solvents can also damage or denature proteins and other biomolecules in the cell, so they should be used with caution and in low concentrations.
- Detergents: These are amphipathic molecules that can interact with both hydrophobic and hydrophilic regions of the cell membrane or cell wall. Detergents can form micelles around lipid molecules and disrupt the integrity of the membrane or wall. Examples of detergents are sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, and cetyltrimethylammonium bromide (CTAB). Detergents can be used to disrupt animal cells, plant cells, fungal cells, and bacterial cells. However, detergents can also interfere with protein interactions and solubility, so they should be used with care and removed after cell disruption.
- Chaotropic agents: These are compounds that can disrupt the hydrogen bonding network of water and affect the stability of biomolecules. Chaotropic agents can denature proteins and nucleic acids and increase their solubility in aqueous solutions. Examples of chaotropic agents are urea, guanidine hydrochloride (GdnHCl), sodium thiocyanate (NaSCN), and potassium iodide (KI). Chaotropic agents can be used to disrupt animal cells, plant cells, fungal cells, and bacterial cells. However, chaotropic agents can also cause irreversible damage to biomolecules and affect their activity and function, so they should be used with caution and at optimal concentrations.
- Chelating agents: These are compounds that can bind to metal ions and remove them from their coordination sites in biomolecules. Chelating agents can weaken the structure of the cell wall or membrane by removing metal ions that stabilize them. Examples of chelating agents are ethylenediaminetetraacetic acid (EDTA), citric acid, oxalic acid, and ethylene glycol tetraacetic acid (EGTA). Chelating agents can be used to disrupt bacterial cells (especially gram-negative bacteria), plant cells, fungal cells, and animal cells. However, chelating agents can also affect the activity and function of metal-dependent enzymes and cofactors in the cell, so they should be used with care and at optimal concentrations.
- Acids and bases: These are compounds that can alter the pH of the cell environment and affect the ionization state of biomolecules. Acids and bases can disrupt the electrostatic interactions and hydrogen bonds that maintain the structure of the cell wall or membrane. Examples of acids and bases are hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), sodium hydroxide (NaOH), potassium hydroxide (KOH), and ammonium hydroxide (NH4OH). Acids and bases can be used to disrupt bacterial cells (especially gram-positive bacteria), plant cells, fungal cells, and animal cells. However, acids and bases can also denature proteins and nucleic acids and affect their activity and function, so they should be used with caution and at optimal pH values.
Chemical methods of cell disruption have some advantages over mechanical or physical methods. They are relatively simple, inexpensive, fast, scalable, and compatible with various types of cells. However, chemical methods also have some disadvantages. They can cause unwanted side effects on biomolecules such as denaturation, degradation, precipitation, aggregation, oxidation, or modification. They can also introduce contaminants or impurities into the cell extract that may interfere with downstream processes or analyses. Therefore, chemical methods should be carefully selected and optimized according to the type of cell and the desired product.
Another strategy to achieve cell disruption is to use digestive enzymes that will decompose the microbial cell wall. Different cell types and strains have different kinds of cell walls and membranes, and thus the choice of enzyme depends on the microbe. For example:
- Lysozyme is a commonly used enzyme to digest the cell wall of gram-positive bacteria. Lysozyme hydrolyzes β-1-4-glucosidic bonds in the peptidoglycan layer, which is the main component of the bacterial cell wall.
- Zymolyase is a mixture of enzymes that can degrade the cell wall of yeast and fungi. It has activities such as β-1,3 glucanase and β-1,3-glucan laminaripentao-hydrolase, which can break down the β-glucans that are present in the fungal cell wall.
- Cellulases, pectinases, xylanases and chitinases are other examples of enzymes that can disrupt the cell wall of plants, algae and fungi. These enzymes can cleave the polysaccharides that form the structural components of the cell wall, such as cellulose, pectin, xylan and chitin.
Enzymatic methods have some advantages over mechanical and physical methods, such as:
- They are more specific and gentle, and can preserve the integrity and activity of the intracellular components.
- They are less likely to cause contamination or damage to the equipment.
- They can be combined with other methods to enhance the efficiency of cell disruption.
However, enzymatic methods also have some limitations, such as:
- They are more expensive and require optimal conditions of pH, temperature and concentration to work effectively.
- They may have unwanted side effects on the target proteins or substances, such as degradation or modification.
- They may not be effective for some types of cells that have resistant or complex cell walls.
Cell disruption is an important step in biotechnology and the downstream processes related to the production of biological products. It is necessary for the extraction and recovery of the desired products, such as proteins, nucleic acids, metabolites, and other biomolecules, from the cells. Cell disruption significantly enhances the recovery of biological products by breaking down the cell walls and membranes that protect them from the external environment.
Different methods of cell disruption have different advantages and disadvantages depending on the type of cells, the product of interest, and the scale of operation. Some factors that influence the choice of cell disruption method are:
- The efficiency and yield of product recovery
- The quality and purity of the product
- The cost and time of the process
- The safety and environmental impact of the process
Some examples of how cell disruption methods are applied in biotechnology are:
- Mechanical methods such as bead beating, ultrasonication, and homogenization are widely used for disrupting bacterial, fungal, plant, and animal cells. They are effective for large-scale operations and can release a wide range of products. However, they may also cause heat generation, foaming, and shear damage to the product.
- Non-mechanical methods such as freeze-thaw, microwave/thermolysis, osmotic shock, and electric discharges are suitable for disrupting soft or fragile cells such as algae, protozoa, and mammalian cells. They are relatively simple and inexpensive methods that can preserve the product quality. However, they may also cause denaturation, oxidation, or contamination of the product.
- Chemical methods such as organic solvents, surfactants, and EDTA are often used for permeating or dissolving cell walls of plant and bacterial cells. They can enhance the solubility and extraction of hydrophobic or lipophilic products. However, they may also affect the product stability, activity, or functionality.
- Enzymatic methods such as lysozyme, zymolyase, cellulase, pectinase, xylanase, and chitinase are specific for degrading certain types of cell walls or membranes of bacteria, yeast, fungi, and plants. They can release high-quality products with minimal damage or loss. However, they may also require optimal conditions such as pH, temperature, and enzyme concentration.
Cell disruption is a crucial step in biotechnology that enables the access and utilization of valuable biological products from various types of cells. It requires careful selection and optimization of the appropriate method to achieve the desired outcome.
We are Compiling this Section. Thanks for your understanding.