THE CELL Mr. King. Animal Cell Cell Membrane Animal Cell Cell Membrane Vacuole.
Nur Istianah,ST,MT,M.Eng Cell...
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Cell Disruption Nur Istianah,ST,MT,M.Eng
© THP UB 2017
Definition
Cell Disruption is the method or process for
disrupting or lysing the cell in order to
release the contents out of the cell.
Mechanism
1
Fermentation (rx)
(intracellular product still exist inside of cell)
2
Homogenised cell
Cell separation
3
Cell disruption
Cell
1. Gram positive bacterial
2. Gram negative bacterial
3. Yeast cell
4. Mould cells
5. Cultured mammalian
6. Cultured plant cells
7. Ground tissue
Cell wall
• Cell wall wherever present is the main
barrier which needs to be disrupted to
recover intracellular products.
• A range of mechanical methods can be
used to disrupt the cell wall.
• Chemical methods when used for cell
disruption are based on specific targeting
of key cell wall components
lysozyme is used
to degrades
peptidoglycan
which is a key cell
wall constituent.
the peptidoglycan
layer is less
susceptible to lysis
by lysozyme,
helped by osmotic
shock
Cell wall
• The plasma membrane can be easily
destabilized by detergents, acid, alkali and
organic solvents.
• The plasma membrane is also quite fragile
when compared to the cell wall and can
easily be disrupted using osmotic shock
i.e. by suddenly changing the osmotic
pressure across the membrane.
Methods
1. Disruption in bead mill
2. Disruption using a rotor-stator mill
3. Disruption using French press
4. Disruption using ultrasonic vibrations
Physical methods
1. Disruption using detergents
2. Disruption using enzymes e.g. lysozyme
3. Disruption using solvents
4. Disruption using osmotic shock
Chemical and
physicochemical
The physical methods: cell wall disruption, chemical and
physicochemical: destabilizing the cell membrane
Cell disruption using bead mill
• The cell disruption takes place due to the grinding
action of the rolling beads as well as the impact resulting
from the cascading beads.
Cell disruption using bead mill
• Bead milling can generate enormous
amounts of heat so can be carried out at
low temperatures, i.e. by adding a little
liquid nitrogen into the vessel. This is
referred to as cryogenic bead milling. An
alternative approach is to use glycol
cooled equipment.
Cell disruption using bead mill
• A bead mill can be operated in a batch
mode or in a continuous mode and is
commonly used for disrupting yeast cells
and for grinding animal tissue.
• Using a small scale unit operated in a
continuous mode, a few kilograms of yeast
cells can be disrupted per hour. Larger
unit can handle hundreds of kilograms
of cells per hour.
Cell disruption using bead mill
• Cell disruption primarily involves breaking
the barriers around the cells followed by
release of soluble and particulate sub-
cellular components into the external
liquid medium.
• Empirical models are therefore more often
used for cell disruption:
rotor-stator mill
10,000 to 50,000 rpm
rotor-stator mill
• These mills are more commonly used for
disruption of plant and animal tissues
based material and are operated in the
multi-pass mode, i.e. the disrupted
material is sent back into the device for
more complete disruption.
• The cell disruption caused within the rotor-
stator mill can be described using the
equations discussed for a bead mill.
French press
rotor-stator mill
• commonly used for small-scale recovery of
intracellular proteins and DNA from
bacterial and plant cells
• The cell disruption takes place primarily
due to the high shear rates influence by
the cells within the orifice.
• Typical operating pressure ranges from
10,000 to 50,000 psig.
Ultrasonic cell disraption
Ultrasonic cell disraption
• A frequency of 25 kHz is commonly used
for cell disruption. The duration of
ultrasound needed depends on the cell
type, the sample size and the cell
concentration.
• These high frequency vibrations cause
cavitations, i.e. the formation of tiny
bubbles within the liquid medium
Ultrasonic cell disraption
• When these bubbles reach resonance size,
they collapse releasing mechanical energy in the
form of shock waves equivalent to several
thousand atmospheres of pressure. The shock
waves disrupts cells present in suspension.
• For bacterial cells such as E. coli, 30 to 60
seconds may be sufficient for small samples. For
yeast cells, this duration could be anything from
2 to 10 minutes.
Equation
The time constant θ depends on the processing conditions,
equipment and the properties of the cells being disrupted
• In a multi-pass operation:
• A batch of yeast cells was disrupted using
ultrasonic vibrations to release an intracellular
product. The concentration of released product
in the solution was measured during the
process:
• If the ultrasonic cell disruption were carried out
for 240 seconds, predict the product
concentration.
Time (s) Concentration (mg/ml)
60
120
3.49
4.56
Solution
Cell disruption using detergents
• Detergents disrupt the structure of cell
membranes by solubilizing their
phospholipids. These chemicals are
mainly used to rupture mammalian cells.
• For disrupting bacterial cells, detergents
have to be used in conjunction with
lysozyme. With fungal cells (i.e. yeast and
mould) the cell walls have to be similarly
weakened before detergents can act.
Detergent
cationic anionic and non-
ionic
Cell disruption using detergents
• Non-ionic detergents are preferred in
bioprocessing since they cause the least
amount of damage to sensitive biological
molecules such as proteins and DNA.
Commonly used non-ionic detergents
include the Triton-X series and the Tween
series.
Cell disruption using detergents
• However, it must be noted that a large
number of proteins denature or
precipitate in presence of detergents.
• Also, the detergent needs to be
subsequently removed from the product
and this usually involves an additional
purification/polishing step in the process.
• Hence the use of detergents is avoided
where possible.
Cell disruption using enzymes
• Lysozyme (an egg based enzyme) lyses
bacterial cell walls, mainly those of the gram
positive type.
• Lysozyme on its own cannot disrupt bacterial
cells since it does not lyse the cell membrane.
The combination of lysozyme and a
detergent is frequently used since this takes
care of both the barriers. Lysozyme is also used
in combination with osmotic shock or
mechanical cell disruption methods.
• Main limitation: high cost
Cell disruption using organic solvents
• Organic solvents like acetone mainly act
on the cell membrane by solubilizing its
phospholipids and by denaturing its
proteins (toluene are to disrupt fungal cell)
• The limitations of using organic solvents
are similar to those with detergents,
• However, organic solvents on account of
their volatility are easier to remove than
detergents.
Cell disruption by osmotic shock
Concentration difference
Rapid influx of water into the cell (osmotic)
rapid expansion in cell volume
Cell rupture
Cell disruption by osmotic shock
• 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.
Exercise
• An intracellular antibiotic is being
recovered by ultrasonication from 5 litres
of bacterial cell suspension having a cell
concentration of 15 g/1. Past experiences
have shown that 50% of the antibiotic can
be recovered in 40 minutes. Predict the
time required for 90% recovery of the
antibiotic.
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