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Friday, June 22, 2012

Cell Disruption / Cell Lysis, Methods & Applications



Cell Disruption / Cell Lysis:

Cell Disruption is the method or process for disrupting or lysing the cell inorder to release the contents out of the cell.
Purpose

Cell disruption is done to release the cell contents. Cell Lysis or Disruption is done for isolating Nucleic acids (DNA / Plasmids), Proteins (Intracellular Proteins), Organelles, etc.

Methods

Cell-disruption / Cell Lysis methods include physical methods, Chemical methods and Enzymatic Methods.

Physical Methods

Manual Grinding:

Tissue is frozen and then crushed using a mortar and pestle. Minimum amount of bufer can be used while grinding. Because of the tensile strength of the cellulose and other polysaccharides constituting the cell wall, this method was the fastest and most efficient way to access plant proteins and DNA. Even for DNA isolation from liver tissue this method can be used.


Mortar & Pestle Used for manual grinding

Sonication:

Sonication is a physical method of cell disruption, in this method a sonicator probe is immersed into a solution containing cell suspension to disrupt, applying high frequency sound waves causes the cell to Lyse. Usually the solution will be kept in ice bath because lot of heat will be generated during the process.


A sonicator Device for cell Disruption

Liquid Homogenization:

Liquid-based homogenization is the most widely used cell-disruption technique for small volumes . Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer, Potter-Elvehjem homogenizer and French Press. A French press consists of a piston that is used to apply high pressure to a sample volume of 40 to 250 ml, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. The equipment is expensive,but the French press is often the method of choice for breaking bacterial cells mechanically.


Freeze Thaw Method:

The freeze/thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols.

Chemical Methods

Chemical agents can be used for lysing cell membranes, chemical agents acts on the cell wall of bacteria causing it to rupture the cells and release the product out. Urea and guanidium thiocyanate are widely used for cell lysis. Detergents are used in cell lysis buffers they help to solubilize membrane proteins and lipids there by causing the cell to lyse and release its contents outside.

Most widely used Detergents are:

Triton X – 100
Tween 20
Tween 80
Sodium Dodecyl Sulfate (SDS)
CHAPS

Enzymatic Methods:

Lysozyme, Chitinase, Pectinase

Enzymatic methods are easy and fast but cost for these methods are also high. Lysozymes are used for Bacterial cell lysis and Chitinase for Yeast cell lysis.Pectinases are used for the lysis of plant cell walls for isolating protoplast and other applications.

Lysis Buffer Composition used in Alkaline Lysis Method for Plasmid Extraction

(Tris pH 8.0, EDTA- Ethylene diamine tetra acetic acid and Glucose)
RNase
Lysozyme- optional
50mM glucose
25mM Tris-Cl (pH 8.0)
10mM EDTA (pH 8.0)

Applications of Cell-disruption
  1. Isolating intracellular Proteins.
  2. Downstream Processing.
  3. Isolating intracellular organelles.
  4. Nucleic acid isolation.
Disadvantages

  1. Whole cell contents are released out which makes it difficult to separate out product of interest from the mixture.
  2. Cell lysis increases viscosity of the solution making it difficult to process in the further steps.
  3. Product released into harsh environment causing the product to lose stability or activity.
  4. Might cause filtration membranes to foul during the filtration process.
  5. Enzymatic cell-disruption in large scale can be expensive.
Commercially Available Cell Disruptors / Cell Lysis Buffers / Enzymes

Qsonica Ultrasonicator

CelLyticTM MT - Mammalian Tissue Lysis/Extraction Reagent - Sigma Aldrich

Glo Lysis Buffer (GLB) - Promega

References

Tutorial on Cell Disruption - TELIOS T. TZANNIS - RPI 1991

Wikipedia

Thermo Scientific Technical Resource