Cell disruption is an integral element of biotechnology, as are the subsequent processes involved in the creation and production of biologic products. Cell disruption is required to extract and retrieve of desired substances because cell disruption dramatically improves the process of recovering biological products. Cell disruption cannot be thought of as an isolated processbecause it alters its physical characteristics of slurry that is produced by cells, consequently influencing downstream processes. There are a variety of cell disruption techniques are available in the context of biological products, which can be intracellular, extracellular or the periplasmic.
Cell Disruption Definition / What is Cell Disruption?
Cell Disruption or Cell Lysis involves breaking the down cells’ wall and/or membranes and releasing intracellular fluids which contain particles or molecules that are of interest, for example, viruses or proteins. The principal objective for cell disruption is kill suspended cells to recover the highest yield possible of the particles or molecules of interest.
Methods of Cell Disruption
Different cells have different structure and therefore require different methods to disrupt. Cell walls are additional disruptors and yeast cells are especially difficult to destroy due to the fact that the cell’s wall restricts the solvent’s access to the products you want. Other kinds of cells that require disruption are bacterial cells moulds, plant cells mammalian cells, and ground tissue. Bacterial cells could use different methods of disruption, dependent on whether they’re either gram positive or negative, since the amount of peptidoglycan in the cell and the existence of an envelope impact the entire process. Mammalian cells can be the easiest ones to destroy because they do not have walls, as opposed to plants, which tend to be more challenging dislodge.
Drying cells can enhance the methods of disruption and could aid in reducing costs. In certain situations multiple disruption method might be required to ensure full recovery of the product. The factors that determine the choice of the disruption method are
- Size of the cell
- The toughness of the cell determines regardless of whether it’s bacteria or fungi, plants or animal cells.
- The effectiveness of the disruption method
- The stability of the item with respect to the procedure used.
- Simple extraction and purification
- The cell’s biohazard potential is a factor or not
- Time and cost
- Training or expertise required, or not.
Important note: before cell disruption occurs the cells need to be removed from the culture medium. Extracellular substances secreted by cells must be reduced and non-utilized media components should also be eliminated. It is ideal that the method for cell disruption is appropriate for the cells to be affected, uses a known mechanism, and is sterilisable, contained and verified, and has the possibility of automated. Other benefits include a consistent and compact technique that is affordable and effective.
A. Mechanical physical methods of Cell Disruption
The principle behind mechanical methods of disruption is that cells are exposed to extreme stress through pressure, abrasion and rapid agitation using beads or ultrasound. There are a variety of methods for disruption, including impingement, shearing, cavitation or a combination of these. The suspension must be cooled extensively after treatment is needed in order to get rid of the heat generated through the dissipation process of the mechanical energy. Certain high-pressure methods are only able to be utilized in laboratory scale, like French presses and Hughes press. For industrial applications bead mills, as well as homogenizer with high pressure are appropriate.
1. Bead mill
The basic principle of a bead mill
It’s a method of destroying various microbial cells since different designs have been created. The principle is based on an enclosed grinding chamber that has rotating shaft that runs in the center. Agitators are equipped with the shaft and supply an energy source to the tiny beads inside the chamber. This causes the beads to meet and collide. The dimensions and the weight of each is dependent on the nature of cell. The size of the beads can impact the effectiveness of cell disruption by influencing the location of the enzyme desired in the cell.
The greater number of beads-to-bead interactions. The increase in beads also impacts the power and heating consumption. The ideal beads is between 80 to 85 percent of the volume free. The discs operate at a speed between 1500 and 2500 rpm. Glass beads that have larger diameters than 0.5 millimeters are thought to be the ideal for yeast cells and a diameter less than 0.5 millimeters is the ideal size for bacteria. The key variables in the process are: the speed of the agitator, the percentage in the bead, the size of beads and concentration of the cell suspension as well as the flow rate of cell suspension and the design of the agitator disc.
In = (Rm/Rm-R) +kt
K is determined by the rate at which agitation occurs (1500-2250 rpm) and cell concentration (30-60 percent wet solids) and beads’ in diameter (0.2-1.0 millimeters) and the temperature. The R is the protein released (kg biomass/kg protein) Rm – maximum protein released K -rate constant, and is dependent on temperature.
Advantages
- It’s extremely useful for smaller sized materials, and doesn’t emit dangerous aerosols.
- Bead mills can be performed in batch or continuous manner.
- Commonly used to disrupt yeast cells and to grind animal tissues.
Limitation
The most important issues relating to bead mills include:
- the temperature increases as the bead volume increases,
- Poor scale-up
- Most important the most important thing is that there is a good likelihood of contamination.
Application
Bead mills were initially employed in the paint industry and were later adapted to disrupt cells in both large and small-scale production.
2. Ultrasonic disruption/Ultrasound
Principle
Ultrasonic disruption occurs due to ultrasonic vibrators which produce high-frequency sound with the density of around 20 kHz/s (figure 3). A transducer converts the sound waves into mechanical oscillations using an aluminum probe, which is submerged into suspended cell fluid.
Limitation
- Sonication is extremely effective when working on small-scale projects; however, scaling up is extremely ineffective.
- It is a high energy-demanding area,
- It poses risk of health and safety, because of the noise.
- It’s not continuous.
Uses
- This method is employed for fungal and bacterial cell destruction. Bacterial cells can be broken within 30 to 60 seconds and yeast can take between 2 and 10 minutes.
- This method is often employed in conjunction with a chemical process (mostly lysis)
3. French press and high pressure homogeniser
Principle
In the case of a French press, also known as high homogenization of pressure in a French press, the suspension of cells is pumped through a valve to an cylinder pump. It is then pushed under pressures of up to 1500 bar through an annular gap with a small gap, and discharge valves, at which point the pressure decreases to atmospheric pressure. Cells are disrupted by the abrupt reduction in pressure on the discharge, which causes the cells to explode.
When the press is operated in higher pressures it increases the amount of times the Slurry through the press is increased. it is possible to reduce it to achieve the desired level of disruption.
Limitation
- The deactivation of some heat-sensitive proteins could make it more difficult to complete the passages needed.
- The release of protein is influenced by many factors including temperature, the location inside the cell of enzymes, quantity of passages, as well as operating pressure.
- The process is influenced by the biomass concentration.
- A French press can be described as a small-scale process and the homogenizer may be used for a large-scale production. Homogenizers differ in their design and have large quantities of solids that can be up to 50 percent in the food. The heat generation can be extremely high – 1.5oC/1000 psi.
Uses
This is among the most well-known and employed techniques.
- It is most commonly used to treat yeast cells.
- It is an important component of the dairy production industry to homogenize milk.
4. Mortar and pestle
Grinding by hand is the most popular method to break up the plant cell. The tissue is then stored in nitrogen liquid, and later crushed using the use of a pestle and mortar. Because of the tensile force of cellulose and the other polysaccharides that make up the wall of cells.
Benefits
- It is effective with difficult tissues (e.g. plants)
- Reconstitution of buffers in the buffer of your the choice
Limitation
- Time-consuming
- The reproducibility of a product may differ
Use
- This is the most efficient and fastest method of gaining access to DNA and plant proteins.
5. Ultra sonicator
APV is a high pressure homogenizer utilized on a large-scale. Pressure is delivered by valve pressure. When the vessel’s pressure is at the atm pressure, cells explode during this process. No heating is generated as the inert gases are employed inside the vessels. This is a method that is gentle employed for the disruption of animal cells however, it is not applicable to the destruction of plant cells or the fungi.
6. Microfluidizer:
In this method , pressure is applied to cause the production of tiny particles. This technique is then applied to the lab scale. Shear force and pressure can causes cell damage.
Advantages
- Cost-intensive method
- Not suitable for large-scale production
7. Liquid homogenization
Cells are killed by pushing the suspension of tissue or cell through a small space, and thereby securing the cell membranes. Three types of homogenizers are commonly used.
- A Dounce homogenizer is an round glass pestle which is manually inserted into the glass tube.
- A Potter-Elvehjem homogenizer consists of a mechanically or manually driven pestle made of PTFE, which is designed to fit into a round or conical vessel. The amount of strokes performed and how fast strokes are administered affect the efficiency in Dounce and Potter-Elvehjem methods for homogenizing. Both homogenizers are available in various sizes that can accommodate a range of sizes.
- A French press is made up of a piston employed to apply high tension to the sample of 40 to 250 mL. It then pushes it through a tiny opening within the press. Two passes are all that is required to ensure lysis is efficient due to the pressures that are high in this procedure. The equipment is costly, however it is a good investment. French press is typically the most effective method to break down bacteria mechanically.
Benefits
- Simple to use even with smaller quantities
Limitation
- Low throughput
- Dounce: reproducibility can vary
Use
- Liquid-based homogenization is among the most commonly used cell disruption method for the small-sized volumes of cell lines that have been cultured.
8. Sonication
The technique uses pulsed high-frequency sound waves to stimulate and lyse cells, bacteria, spores and tissues that have been finely chopped. Sound waves are transmitted through an apparatus that has vibrating probes that are submerged in the cell suspension in liquid. The probe’s mechanical energy causes the formation of tiny bubbles of vapor that appear and then explode and cause shock waves to be released through the sample. To avoid excessive heating by ultrasonic treatment, it is applied in a series of small bursts to the sample that is immersed in an in an ice bath. Sonication is most effective for samples of less than 100 milliliters.
Benefits
- Independent of cell type
- Highlysing effectiveness
- Shears chromosomes and eliminates the necessity for nuclease treatment
Disadvantages
- Produces heat, which must be controlled to avoid injury to proteins sensitive to it.
- Produces cellular debris
- The process is loud and requires sound reduction
Use
- It is commonly employed to break open cells..
B. Non-mechanical physical methods
It is divided into three categories such as;
- Physical Method
- Chemical Method
- Biological Method
1. Physical Method
A. Thermolysis
Thermolysis is a process that has been shown to be capable of increasing its use in large-scale production. Periplasmic proteins found in G(–) bacteria release when cells are heated to 50oC. Cytoplasmic proteins are released from E.coli in just 10 minutes at 90oC. The increased release of proteins has been achieved after short high temperature shocks, but not when exposed to longer temperatures at lower levels. However these results are inconclusive, because the solubility of proteins changes with changes in temperature.
The freezing and thawing process of a cell slurry could cause cells to explode due the melting and formation of crystals of ice. The gradual freezing process, which leads to the growth of larger crystals can cause enormous damage to the cells. When this technique is combined with grinding of cells this method has yielded impressive results.
Limitations
- It’s very expensive.
- Limited to small-scale labs.
- A few reports also show an absence of enzyme activity.
b. Decompression
In the process of explosive decompression the suspension of cells is mixed with subcritical pressurized gas for a specific period of duration, based on the type of cell. The gas gets into the cell, and then expends upon release, which causes the cell to explode. Decompression has been utilized in smaller scale labs to aid in the destruction of E.coli. This technique has demonstrated promising results using yeasts.
Benefits
- Supercritical CO2 has the ability to remove off-flavours due to lipids.
- This method is showing to be very promising
- Gentle on cells
- This results in large amounts of debris that is easier to get rid of so that you can get the product you want.
Limitation
- Its low efficiency
- Its dependence on the release of pressure and time for contact between suspension of cells as well as the gas.
C. Osmotic shock
The correct functioning of cell’s processes generally requires strictly defined chemical conditions. This implies e.g. that the cell’s inner pH or salt concentrations must not differ significantly from ideal values. The ideal conditions and the ability to withstand conditions that are suboptimal are a matter of species. Cells are able to control internal environment, however drastic and sudden changes in the environment surrounding cells can trigger shock, which can lead to cell death and disturbance.
Osmotic Shock is a technique that can be used in biotechnological applications to trigger cell destruction. With this technique cells are exposed to moderate or high salt concentrations. Then, the conditions are switched to the opposite condition that result in the osmotic pressure to cause cell destruction. The reason is because water rapidly flows from low salt concentration environments to conditions that have a high salt concentration. So, when cells first are exposed to a the solution with a high salt concentration the cell will be flooded with water after exposure to a low salt concentration.
The result is that cell pressure increases and the cell explodes. In contrast, if cells are exposed to a high salt concentration (~1 milliliter solution) following exposure to a low concentration, the water flows out of cells, which results in cell destruction.
Limitation
- Osmotic shock isn’t a widely employed as a method of cell disruption due to its inefficiency.
- The most effective disruption could require enzymatic pre-treatment to reduce the cell’s resistance.
- Furthermore to this, this technology calls for the the addition of large amounts of salts. Water usage is very high.
- The product can also be dilute, which can result in higher processing costs downstream.
D. Freeze-thaw lysis
The freeze-thaw technique is widely employed to kill bacterial and mammalian cells. This method involves freezing a suspension of cells in a bath of dry ice and ethanol or freezer and then cooling the suspension at room temperature, or 37degC. This method of cell lysis causes cells to expand and then break off as crystals of ice develop during the freezing process , and they expand during melting. A series of cycles is required for effective lysis, and the process may take a long time. However, freeze/thaw was proven to be effective in releasing Recombinant proteins that are that are found in the cytoplasm bacteria, and is suggested to lyse mammalian cells in certain protocols.
Benefits
- Inexpensive technique
Limitation
- Not suitable for extraction of the cellular components that are that are sensitive to temperature
- Time-consuming process
E. Dessication
This method is where the suspension of cells is dried using either vacuum drying or air drying. Following dessication, cell shrinks. If the cell is flooded with water then the cell will explode. The process repeats for many times.
Advantages
- Process is slower and may require multiple repetitions.
- Always in conjunction with other processes.
F. Electrolysis (Electroporation):
If cell suspension is subjected to the electric field, with an intensity greater than a certain threshold, there will be the development of a nanoscale pores on the cell’s surface. This pore may be reversible, or not, based on the the applied field and the direction of the field. Through the pores, intracellular substance releases.
Advantages
- Heat generation
- Cost-intensive process
2. Biological methods or enzymatic methods of cell disruption
Cell lysis by enzyme is a managed operation that is low in energy and requires little capital investment. It may result in biological specificity, and occurs under moderate operating conditions. The physical stress of the stress on shear that results from mechanical breakdown can be prevented. The selection of the right enzyme as well as the determination of the specific reaction conditions to achieve efficient lysis is required.
Three different approaches are described:
- autolysis,
- the addition of foreign enzymes as well as
- Phage Lysis.
The application of enzymic lysis at the scale of a vast scale is limited by the availability of enzymes and costs.
A. Autolysis
The process of autolysis, which is used to make yeast extracts is not well comprehended, and consequently difficult to control. The production of lytic enzymes in yeast is usually triggered by a mild thermal or chemical shock, and hence its classification is not always clear.
B. Lysemic enzyme
Different enzymes like cellulase and lysozyme proteases, etc. are employed to lyse wells. This technique is employed on a small-scale.
C. Phage lysis
T4-phage, OX174, ssRNA phage, etc. are bacteria called bacteriophage. They enter and multiply within bacteria, and eventually cause cell lysis through the production of endolysin and hydrolase enzymes.
3. Chemical Methods
Microbial disruption through chemical methods depends on cell’s structure to be broken. Common agents used include pH extremes, particularly the alkali condition, as well as solvents cleaning agents, detergents reducers and chaotropic substances. In this section we will focus on the most commonly employed agents, which have general applicability are addressed.
a. Alkaline cell lysis
Alkaline cell lysis has already been studied in a variety of bacterial systems using pH 10.5-12.5 in a interval of 30 s up to 30 min. Examples of the tested systems include Erwinia carotovora E. coli, and C. necator. For use, a material stable at a high pH is needed. Additionally, the need for neutralization can affect the inventories of materials.
b. Solvents
Solvents can be utilized to extract lipids from cell membranes, which can trigger the release of intracellular components. Be cautious in the use of these solvents due to their potential for ignitability and the possibility to trigger protein denaturation. Solvents that are used to release of intracellular compounds comprise alcohols like ethanol isopropanol, butanol (at concentrations of 10 to 80 percent) dimethyl sulfoxide toluene (2 percent) and methyl ethylketone. The application of these compounds across a wide range of microorganisms, including E. coli, S. cerevisiae and Kluveromyces kinds has already been proven. While permeabilization is a natural process at room temperature, the increased release can result in elevated temperatures between 25 and 45 degrees Celsius.
Diadvantages of Solvents method
- The disadvantage of this method is that the chemical costs to neutralize alkali are very high.
- Furthermore it is possible that the product will not be stable under alkali conditions.
c. Detergent treatment
Detergent treatment is extensively used at the laboratory scale to permeate or lyse cells for the release of liquid components by altering protein-lipid interactions by interplay between the hydrophobic nonpolar tail as well as the polar hydrophilic head of a detergent molecule. Stability of the product within these systems is essential.
Detergents are classified based on what the head of hydrophilia is made up. It is classified as anionic, cationic, or nonionic.
- The anionic detergents (e.g. sodium dodecyl sulfate SDS) cause cell disorganization. membrane.
- Cationic detergents are believed to affect the lipopolysaccharide part in the cell’s envelope, as well as interaction with phospholipids. Cetyltrimethylammonium bromide (CTAB) has been used for permeabilization of both yeast and bacteria at a concentration in the range of 0.02-0.4%.
- Nonionic detergents like Triton X-100 and Pluronic F-68 cause partial solubilization of proteins within the membrane’s inner structure, which results in the permeabilization.
The component of the lipopolysaccharide layer on the membrane’s outer layer provides resistance to detergent in the event that the combination of chemical methods is utilized. Triton X-100, at concentrations that range between 0.1 percent and 4percent is known to facilitate the permeabilization to E. coli, S. cerevisiae, P. pastoris, Nocardia rhodocrous, and Yarrowia lipolytica.
The disadvantages of treatment with detergents
- The drawback of using detergents in cell lysis , is the fact that a lot of proteins will denature during the the lysis process.
- Detergents can also disrupt downstream processing processes. A second step of purification could be needed following cells have been lysed, and this may limit their application in large-scale processes.
- But, they are also used to kill cells in the laboratory, for instance when DNA, proteins or RNA are removed from cells.
d. Chaotropic agents mediate cell lysis
Chaotropic agents cause cell lysis by disrupting H-bonding and altering hydrophobic interaction, which reduces cross-linking within the wall of cells. Common agents include guanidine chloride and the urea. The impact of both chaotropic agents as well as detergents can be enhanced when they are used in conjunction with a chelator, like EDTA. It chelates divalent ions dissociating membranes, and destabilizing the layers of lipopolysaccharides.
e. Chelating agent
EDTA is an excellent example of a chelating agent. It is a chelating agent that binds Cations (bivalent) and makes them unusable for cells, which causes rupture of the cell membrane. (Mg2+, Ca2+).
f. Peroxide and hypochloride
H2O2 and HClO oxidize the cells’ structure. HClO causes damage to the lipid bilayer and also inhibits the -SH protein. The oxidation of the cell membranes causes cells to release cell constituents.
g. Antibiotics
Polymyxin, Azoles, Nystatin have been identified as cell membrane inhibitors and can destroy cell membrane inhibitors and cause cell membrane destruction which results in the release of cell content.
Benefits and Disadvantages of Physical disruption methods
Benefits
- A reliable method of lysing the vast majority of cells
- Control of the buffers used, and eliminating any potential containments that might hinder downstream applications
- Highlysing effectiveness
Limitation
- Requires equipment
- Reproducibility could be different
- Mechanical methods are typically not compatible with small amounts
- Denaturation of proteins and aggregation may be caused by localized heat
- Cells break down at various time points, and subcellular components can be subjected to constant disruption forces
Benefits and Disadvantages of Reagent-based methods
Benefits
- Quick, light, efficient and repeatable process
- It is able to separate total protein, organelles, subcellular fractions or subcellular fragments from different types of samples
- It is easily adaptable to small quantities or for higher throughput
- The cell can be utilized in conjunction with mechanical techniques to cause total disruption of the cell
Limitation
- Certain buffer components might need to be removed prior to further analysis
- The high concentrations of salts and detergents may not be compatible with certain protein assays or mass spectrum analysis
- It works well with cells that have been cultured however it may not be efficient for certain tissues.
References
- Harrison, S.T.L. (2011). Comprehensive Biotechnology || Cell Disruption. , (), 619–640. doi:10.1016/B978-0-08-088504-9.00127-6
- https://info.gbiosciences.com/blog/cell-lysis-5-common-cell-disruption-methods-g-biosciences
- https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.html
- https://www.microfluidics-mpt.com/blog/what-is-cell-disruption
- https://en.wikipedia.org/wiki/Cell_disruption
- https://analytik.co.uk/what-is-cell-disruption/
- https://www.slideshare.net/AishwaryaBabu2/cell-disruption-methods
- https://www.bio-rad.com/en-in/applications-technologies/cell-disruption?ID=LUSP2D1FX
