Structural Biochemistry/Proteins/Purification/Cell Disruption

=Cell Disruption

Cell disruption is the process of obtaining intracellular fluid via methods that open the cell wall. The overall goal in cell disruption is to obtain the intracellular fluid without disrupting any of its components. Though many cell disruption methods exist, certain factors must be considered in order to obtain viable cellular products.

Factors of Cell Disruption

Sample Size

In most cases, sample size limits the ability to obtain pure forms of the intracellular fluid. It is necessary to use precise and accurate procedures when handling samples sizes on the order of micro liters or less. Large sample sizes pose problems in reproducibility of pure product.

Ability to disrupt the cell and the necessary conditions

The ability to disrupt cells is dependent on the different components of the cell itself. The harder it is to disrupt the cell, more time and power are required to obtain the intracellular fluid.

Efficiency of disruption

Disruption efficiency must be sacrificed for experiments that require pure and intact forms of the product under scrutiny. Not only that, the cost (explicitly and implicit) of processing cells is a major factor in laboratory experiments as well.

Stability of the component needed to be isolated

It is important to combine materials in cell disruption with the conditions required to keep the component intact and pure. Different cell disruption methods have been created and enhanced to ensure the safety of the component under investigation.

Problems with Cell Disruption methods

Though cell disruption is necessary for obtaining intracellular fluid, the process of doing so could pose problems in purification of certain biomolecules. Some adverse effects of cell disruption include, but not limited to:

Heat generation
Release of proteases
Contamination (Nucleic acids, heavy metal, etc.)
Foaming

These effects must be considered before beginning any cell disruption procedure to ensure the viability of the cellular product under scrutiny

Cell Disruption Methods

Cell disruption can be carried out via mechanical or non-mechanical methods. Within the non-mechanical method, there are three subcategories: physical, chemical, and enzymatic methods. The method if choice is dependent on the factors discussed above. However, these methods are not the only pathways to the end product. A combination of these methods can lead to a higher release of the product and a more effective result. The figure below shows the various methods that can be used depending on the cell and its wall structure.
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Lab Scale Methods of Cell Disruption

Enzymatic Method

The use of enzymes to breakdown cell walls is a novel idea in obtaining a certain cellular components without the risk of high contamination. It outbeats any mechanical method because of its high specificity and capabilities. Enzymes used in this process include, but not limited to: lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase. Although there are advantages in this method, some disadvantages include its incapability to reproduce the same non-membrane cell and to produce on a large scale sample.

Bead Method

The beach method uses glass, ceramic, zirconium, or steel beads as an abrasive to break down cell walls. This process is similar to sonication in that the sample and beads are placed into a vial and the vial is placed on a vortex to induce mechanical abrasion. It is important to cool the sample. Most procedures include a lysis solution to begin cell degradation.

Detergent Method

The use of a detergent is ideal for breaking up the cell wall because it increases the solubility of the cell wall. Saponins in the detergent bind to the lipid bilayer of the cell wall, which dissolve the cell wall and leave the intracellular fluid intact. It is important though to understand the chemical composition of the detergent at use; Ionic detergents such as SDS (sodium dodecyl sulfate) denature proteins. Common factors to be considered include temperature, pH, and buffer.

Cell Bomb Method

Pure pressure force can be used to “explode” the cell wall with rapid decompression. The idea is similar to narcosis in which a high pressure is placed on the sample (25,000 psi) and then the pressure is dropped rapidly to release dissolved gases in the intracellular fluid. This released gas creates a high positive pressure on the inside lining of the cell wall until the wall breaks.

Sonication

Sonication uses high frequency (20–50 kHz) of ultrasound to propagate the liquid with pressure waves that expand and contract. The expansion and contraction creates cavities and as these cavities collapse, the pressure that is generated can disrupt the cell membranes. This method is useful, but there is a large variable yield due to the nature of random vibrations. The best method is to do short bursts and cooling of the sample to prevent over heating.

An instrument called the SonicMan was developed in order to carry out this particular method. It contains interchangeable 96, 384, and 1536 format disposable pin lids to transfer sonic energy to each individual well. These disposable and specific pin lids prevent cross contamination between the wells. This instrument also has variable power settings between 1 and 1150 watts, and variable sonication time intervals from 0.1 to 20 seconds. The touch screen panel can control the environment and settings in order to effectively carry about the method to obtain the desired sample.

File:SonicMan.jpg

High Shear Mechanical Methods

Rotor-stator Processors

These processors, also known as homogenizers, use pressure (turbulance and cavitation) and shear force to break up cell walls. The apparatus is closed loop so a sample may be processed many times to ensure that most of the cell walls have been ruptured. A rotor pulls the sample into a chamber (stator) where the sample is pushed through a very fine grid that breaks up the cell wall. The use of this apparatus has many factors that must be considered before using.

Valve-type Processors

These processors use a method in which the sample is placed into a narrow valve under high pressure (20,000-30,000 psi). A liquid is passed over the sample, shearing off the cell wall and leaving the intracellular fluid. This process creates a lot of heat and constant cooling is required. Different types of methods include the French press, pump fluid, and Constant Cell Disruption Systems.

Gentle vs. Vigorous Lysis Methods

Gentle lysis methods are used when the cells, such as tissue culture cells, blood cells, and microorganisms, are easily disrupted and one particular cellular portion is analyzed. These methods include osmotic, freeze-thaw, detergent, and enzymatic lysis.
File:Gentle lysis.png

Vigorous lysis methods can be used when the cells are more difficult to disrupt. However, this method requires more attention because heating and foaming can occur, which will complicate the process and result in damaging or altering the desired cell. These cell disruption methods include sonication, French pressure, grinding, mechanical homogenization, and glass bead homogenization.
File:Vigorous lysis.png