CELL

CELL DISRUPTION
DISRUPTION
At the end of this topic, student will acquire
the ability:

1) To recognize two classes of cells and the

structure of cells that cause cell lysis.
2) To choose between chemical and
mechanical cell disruption methods for general
classes of applications.
 INTRODUCTION
Many biological products of interest are secreted
from cells into the medium (extracellularly).
However, a large number of undesired cell
constituents and products are either present inside
the cells or produced intracellularly.
Consequently, many downstream processes must
include a cell disruption step for recovery of the
desired product, including those listed in Table 1.
 Table 1: Product of which cell disruption is required
Product Type Examples
Vaccines Tetanus, meningitis

Enzymes L-asparginase, glucokinase,

glycerokinase, invertase, mandelate
dehydrogenase, phenylalanine
dehydrogenase, sarcosine dehydrogenase
Spore release Spore preparations
Spore breakage Spore enzymes
Toxins Enterotoxin A
Subcellular Mitochondria, chloroplasts
constituents
 Different types of cell need to be
disrupted in the bio-industry:
1. Gram positive bacterial cells
2. Gram negative bacterial cells
3. Yeast cells
4. Mould cells
5. Cultured mammalian cells
6. Cultured plant cells
 Some Elements of Cell Structure
1) Prokaryotic Cells
Gram ve cells has 3 layers:
a) Outer layer 8 nm thick, consists of protein and
lipopolysaccharide
b) Second thinner layer peptidoglycan
below the second layer is a gap, called
periplasmic space (8 nm thick). Proteins and enzymes are
often located in this gap.
c) Plasma membrane (inner membrane) consists
largely of phosphilipids and dispersed proteins and
ions.
Gram +ve: outer membranes and the peptidoglycan layer
provide mechanical strength.
The weaker plasma membrane controls the permeability of
the cells including transport of nutrients into the cells and
export of metabolites into the surrounding solution.
The membrane consists largely of zwitterionic phospholipids,
and also contains a significant amount of protein.
 Bacteria
Gram positive bacteria
Sub-micron to 2
microns in size
Have thick cell walls, 0.02-
0.04 microns,
peptidoglycan +
polysaccharide+ teichoic
acid
Phospholipid cell
membrane present
Susceptible to lysis by
antibacterial enzyme.
Gram negative bacteria
Sub-micron to 1 micron in
size
Cell capsule present
Peptidoglycan layer is thin
Periplasmic space present
Mechanically less robust
than gram +ve bacteria
Chemically more resistant
than gram +ve bacteria
 Yeast and mould
Yeast: 2-20 microns in size, spherical or ellipsoid
Moulds: Bigger and filamentous
Yeasts are unicellular while moulds are multicellular
Very thick cell walls are present in both
Cell wall is mainly composed of polysaccharides
such as glucans, mannans and chitins
Plasma membranes are mainly made up of
phospholipids
2) Eukaryotic Cells
Cells like these carry their DNA in a nucleus and have more
complex walls than those in prokaryotic cells
 Animal and plant cells
Animal cells do not have cell walls
Animal cells are very fragile
Cultured animal cells are several microns in size
Spherical or ellipsoid

Plant cells can be bigger in size

Plant cells have very thick and robust cell walls mainly
composed of cellulose, pectin and lignin
Plant cells are difficult to disrupt (2 cell wall)
Cellulose can undergo degradation by the enzyme
cellulase
 Shear sensitivity of cells

Bacteria (small, thick cell wall) 1 m

Ease of Yeasts (slightly larger, cell wall) 5-8 m
cell Fungi (moulds)(filamentous, cell wall)
breakup Plant cells (large, cell wall) 20-50 m
Animal cells (no cell wall) 10-50 m

Bigger cells, easier to disrupt, cell wall protects!

 CELL DISRUPTION

Mechanical Chemical

French press
Physical Enzymatic
Homogenizer - Heat shock
Bead mill - Osmotic shock

Chemical
Ultrasound
- Detergents
- Solvents
 Cell Disintegration Techniques
A) Chemical methods
Technique Principle Stress on Cost Examples
product
Osmotic Osmotic Gentle Cheap Rupture of
shock rupture of red blood
membrane cells
Enzyme Cell wall Gentle Expensive Micrococcus
digestion digested, lysodeikticus
providing treated with
disruption egg
lysozyme
Solubilization Detergents Gentle Moderate- Bile salts
solubilize cell expensive acting on E.
membrane coli

Alkali Saponificatio Harsh Cheap

treatment n of lipids
solubilizes
membrane
 B) Mechanical methods
Technique Principle Stress on Cost Examples
product
Homogenization Cells Moderate Moderate Animal tissue
(blade type) chopped in and cells
Waring
blender
Homogenization Cells Harsh Moderate Large scale
(orifice type) forced treatment of
through cell
small hole suspension,
are broken except of
by shear bacteria
Ultrasonication Cells Harsh Expensive Cell
broken suspension at
with least on a
ultrasonic small scale
cativation
Crushing in ball Cells Harsh Cheap Large scale
mill crushed treatment of
between cell
glass and suspension
steel balls and plant
cells
 Cell disruption using homogenizer

A common apparatus for high pressure homogenization of

cells is Manton- Gaulin homogenizer.
Shear forces generated in the treatment are sufficient to
completely disrupt many types of cell.
The high pressure pump incorporates an adjustable valve with
restricted orifice and the cells are forced at pressures up to 550
atm.
Applicable for cell disruption, although the valve can become
blocked when used with highly filamentous organisms.
 Cell disruption using bead mills

Disruption takes place due to the grinding action of the

rolling beads and the impact resulting from the cascading ones
Bead milling can generate enormous amounts of heat
Cryogenic bead milling: Liquid nitrogen or glycol cooled unit
Application: Yeast, animal and plant tissue
Small scale: Few kilograms of yeast cells per hour
Large scale: Hundreds of kilograms per hour.
 Cell disruption using French press

Application: Small-scale recovery of intracellular proteins

and DNA from bacterial and plant cells
Primary mechanism: High shear rates within the orifice
Secondary mechanism: Impingement of jet to cause further
cell disruption
Operating pressure: 10,000 to 50,000 psig
 Cell disruption using ultrasonic vibrations
Frequency greater
than 18 kHz.
Ultrasonic tip is
dipped in the cell
suspension.
Ultrasonic vibration
is emitted
continuously or in the
form of short pulses.

The duration of ultrasound depends on: cell type, sample

size, cell concentration.
High frequency (25 kHz) creates tiny bubbles, when reach
resonance size, they collapse releasing mechanical energy in
the form of shock waves that disrupt the cells in suspension.
For E. coli, 30-60 sec for small samples, yeast cells need 2-10
min.
 Cell disruption using detergents
Detergents disrupt the structure of cell membrane by
solubilizing the phospholipids.
Mainly used to rupture mammalian cells
For bacterial cells, detergents have to be used with lysozyme.
Categories of detergents:
a) cationic e.g.: tetraalkylammonium salts
b) anionic e.g.: sodium dodecylsulphate (SDS)
c) non-ionic preferred in bioprocessing; least amount of
damage to sensitive biological molecules
such as proteins and DNA.
e.g.: Triton-X series, Tween series
Needs further purification/polishing step in the process,
avoid where possible.
 Cell disruption using enzymes
Lysozymes (an egg based enzyme) lyses bacterial cell wall
(gram +ve type).
Lysozyme on its own cannot disrupt bacterial cells it does
not lyse the cell membrane.
The combination of lysozyme and a detergent is frequently
used.
Lysozyme is also used in combination with osmotic shock or
mechanical cell disruption methods.
Disadvantages:
a) Enzymes are expensive not practical for large scale
application
b) Lysozyme needs to be removed from the product increase
bioprocessing cost
c) Other enzymes such as proteases are also present in
lysozymes samples.
 Cell disruption using organic solvents

Acetone - mainly act on the cell membrane by solubilizing

its phospholipids and by denaturing its proteins.
Toluene - disrupt fungal cell wall.
Limitations:
a) The need to remove organic solvents from products
however a lot easier than detergents based on their
volatility
b) Denaturation of proteins
 Cell disruption using osmotic shock
Mainly used to lyse mammalian cells.
For bacterial and fungal cells, the cell wall need to be weakened
before cell being disrupted.
This method is also used to remove periplasmic substances
(especially proteins) from cells without physical cell disruption.
The process is carried out by simply dumping a given volume
of cells into pure water. The cells swell because they contain
solutes which cause an osmotic flow of water into the cells.
E.g.: if red blood cells are suddenly introduced into water, they
hemolyse, i.e.: disrupt and subsequently release hemoglobin.
Animal cells can be lysed, only after the tissue has been
mechanically homogenized.
Plant cells are much more difficult to lyse; cell walls often
contain strong woody material which is relatively impermeable
to osmotic flow.