MODULE 1-

CELL
DISRUPTION
 Biological products synthesized by fermentation or cell culture are either intracellular or extracellular.
 Intracellular products either occur in a soluble form in the cytoplasm or are produced as inclusion
bodies (fine particles deposited within the cells).
Eg: recombinant insulin and recombinant growth factors.
 In order to obtain intracellular products the cells first have to be disrupted to release these into a liquid
medium before further separation can be carried out.
 Certain biological products have to be extracted from tissues, in order to obtain such a tissue-derived
substance, the source tissue first needs to be homogenized or ground into a cellular suspension and the
cells are then subjected to cell disruption to release the product into a solution.
 In the manufacturing process for intracellular products, the cells are usually first separated from the
culture liquid medium.
 This is done in order to reduce the amount of impurity: particularly secreted extracellular substances
and unutilized media components.
 In many cases the cell suspensions are thickened or concentrated by microfiltration or centrifugation
in order to reduce the process volume.
 The various methods of cell disruption may be classified broadly into three types:

(1) physical methods,

(2) chemical and enzymatic methods
(3) mechanical methods.

Physical Chemical & Enzymatic Mechanical

heat shock or thermolysis alkali treatment Ball mill

osmotic shock detergent solubilization High pressure

homogenizer
ultrasonication. lipid solubilization by
organic solvents
Enzymatic digestion
 Physical Methods
1. THERMOLYSIS.
 This is relatively an easy and economical method

 But can be used only if the products are stable to heat shock.

 Basically it inactivates the organisms by disrupting the cell wall without affecting the products.

 The effect of heat shock depends on parameters such as pH, ionic strength, presence of

chelating or sequestering agents such as ethylene diamine tetra acetic acid (EDTA) which
binds magnesium (thereby destabilizing the cell wall) and presence of proteolytic and other
hydrolytic enzymes.
Osmotic shock

 Osmotic shock of the cells is provided by simply dumping a given volume of cells into pure

water of about twice the volume of cells.

 The cells swell due to osmotic flow of water ultimately bursting, thereby releasing the products

into the surrounding medium.

 The osmotic pressure, , of the cytoplasmic solution inside the cell, which causes the osmotic

flow, is proportional to the concentration of the solutes and temperature, as given by van’t Hoff
equation. = RTC

 where refers to the difference in pressure inside the cell, P (in) being greater than atmospheric

pressure, P (out). R is the gas constant, T is absolute temperature and C (moles per litre) is the
total concentration of all the solutes in the cell.
 The susceptibility of the cells to undergo disruption by osmotic shock depends on their type.

 Red blood cells are easily disrupted.

 Animal cells can be disrupted only after mincing or homogenizing the tissues.

 Plant cells are more resistant to disruption by this method.

 Osmotic shock effect is often minimal on bacterial cells.

 Osmotic shock is used to remove periplasmic substances (mainly proteins) from cells without

physical cell disruption.

 In a large number of recombinant as well as non-recombinant gram negative bacteria, target proteins

are secreted into the periplasmic space.

 If such cells are transferred to hypotonic buffers, the cells imbibe water through osmosis and

the volume confined by the cell membrane increase significantly.

 The cell wall or capsule which is relatively rigid does not expand like the cell membrane and

hence the material present in the periplasmic space is expelled out into the liquid medium.
Ultrasonication

 This is essentially a laboratory method as it is expensive.

 Ultrasound waves of frequencies greater than 20 kHz rupture the cell walls by a phenomenon

known as cavitation.

 The passage of ultrasound waves in a liquid medium creates alternating areas of compression and

rarefaction which change rapidly.

 The cavities formed in the areas of rarefaction rapidly collapse as the area changes to one of

compression.

 The bubbles produced in the cavities are compressed to several thousand atmospheres.

 The collapse of bubbles creates shock waves which disrupt the cell walls in the surrounding region.
 The efficiency of the method depends on various factors such as the biological condition of the cells
(age and maturity), pH, temperature, ionic strength and time of exposure.
 Ultrasonication leads to a rapid increase in the temperature and to avoid heat denaturation of the
product it is necessary to cool the medium and also limit the time of exposure.
 CHEMICAL AND ENZYMATIC METHODS

Alkali treatment

 This is a cheap and effective method but very harsh.

 Alkali acts on the cell wall in a number of ways including saponification of the lipids.

 Alkali treatment is carried out at pH 11-12 for about 20-30 minutes.

Detergent solubilization

 This method involves the addition of a concentrated solution of detergent to about half the

solution’s volume of cells to disrupt the cell wall.

 The process depends on pH and temperature.

 Detergents are amphipathic capable of interacting with both water and lipid.

 The key mechanism involves the action of detergents in solubilizing the lipids in the cell wall to

form micelle.

 In dilute solutions, the detergents do not dissolve but at higher concentrations lipid solubilization

begins suddenly and thereafter increases linearly with detergent concentration.

 The surface tension of the medium shows abrupt change at the same concentration.

 The range of detergent concentration at which the abrupt changes in lipid solubility and

surface tension of the medium occur is called critical micelle concentration and corresponds to
the formation of micelle.

 Examples of detergents used in cell wall disruption include

 anionic detergents - sodium dodecyl sulphate (SDS), sodium sulphonate and sodium

taurocholate
 cationic detergents - cetyltrimethyl ammonium bromide (CTAB)

 non-ionic detergents - Triton X-100.

 In the case of mammalian cells, a non-ionic detergent such as saponin or steroid glycoside

digitonin (or digitin), which binds -hydroxysterols and is capable of complexing membrane
cholesterol has been used to release intracellular proteins by permeabilizing the plasma membrane
alone without affecting the organalle membranes.