cell disruption technologies for bioprocess

Intact cell
Totally disrupted cell
Permeabilized cell
Cell disruption and permeabilization

Methods for Cell Disruption
Heat shock Detergents
Sequestrants

Physical methods- decompression
It also known as explosive decompression or cell bomb method.
In this process, cells are placed under high pressure (usually nitrogen or
other inert gas up to about 25,000 (psi) and the pressure is rapidly released.
The rapid pressure drop causes the
dissolved gas to be released as
bubbles that ultimately lyses the cell
Large quantities of nitrogen are first
dissolved in the cell under high pressure
within a suitable pressure vessel.
Then, when the gas pressure is suddenly
released, the nitrogen comes out of
the solution as expanding bubbles
that stretch the membranes of each
cell until they rupture and release
the contents of the cell.
Cell Disruption Bombs (Parr)

It can be conducted at low temperature by pre-chilling or by operating the
bomb in an ice bath.
There is no oxidation:
Means it offers protection against oxidation of cell components.
Although other gases: carbon dioxide, nitrous oxide, carbon monoxide and
compressed air have been used in this technique, nitrogen is preferred
because of its non-reactive nature and because it does not alter the pH of the
suspending medium.
In addition, nitrogen is preferred because of it’s low cost.
It is a gentle method:
Claimed to be more protective for enzymes and organelles than ultrasonic
and mechanical homogenizing methods

Any suspending medium can be used.
The suspending medium can be chosen for its comparability with the end
use of the homogenate and without regard for its adaptability to the
disruptive process.
This offers great flexibility in the preparation of cell suspensions and
produces a clean homogenate that will not require intermediate treatment to
remove contaminates which might be introduced when using other
disruption methods.
Each cell is exposed once:
Here substances are not exposed to continued attrition that might denature
the sample or produce unwanted damage.

Use any sample size.
Cell disruption by this method is independent of sample size or
concentration
Easy to control.
Can be easily controlled by adjusting the nitrogen pressure
The product is uniform:
Since nitrogen bubbles are generated within each cell, the same disruptive
force is applied uniformly throughout the sample, thus ensuring unusual
uniformity in the product
There is no heat damage:
Unlike other method nitrogen decompression procedure is accompanied
by an adiabatic expansion that cools the sample instead of heating it.

.
The technique is used to:
Homogenize cells and tissues
Release intact organelles and to prepare cell membranes
Release labile biochemicals
Produce uniform and repeatable homogenates
Well suited for treating mammalian and other membrane bound cells.
Used successfully for treating plant cells, for releasing virus from fertilized
eggs and for treating fragile bacteria.
Not recommended for untreated bacterial cells. Yeast, fungus, spores and
other materials with tough cell walls do not respond well to this method

Disadvantages includes:
Only easily disrupted cells can be effectively disrupted (stationary phase E.
coli, yeast, fungi, and spores do not disrupt well by this method).
Large scale processing is not practical.
High gas pressures have a small risk of personal hazard if not handled
carefully

Physical methods- Ultrasonic cell disruption
A common laboratory-scale method for cell disruption applies ultrasound
(typically 20-50 KHz) to the sample (sonication).
The treatment of microbial cells in suspension with inaudible ultrasound
(greater than about 18 kHz) results in their inactivation and disruption.
Ultrasonication utilises the rapid sinusoidal movement of a probe within the
liquid.
Ultrasound is characterised by:
high frequency (18 kHz - 1 MHz),
Small displacements (less than
about 50 µm),
moderate velocities (a few m s-1),
steep transverse velocity
gradients (up to 4,000 s-1)
and very high acceleration
(up to about 80,000 g).
Cell suspension
Ultrasound tip
Ultrasound
generator

Ultrasonication produces cavitation phenomena when acoustic power inputs
are sufficiently high to allow the multiple production of microbubbles at
nucleation sites in the fluid.
The bubbles grow during the rarefying phase of the sound
wave, then are collapsed during the compression phase.
On collapse, a violent shock wave passes through the medium.
The whole process of gas bubble nucleation, growth and
collapse due to the action of intense sound waves is called
cavitation.
The collapse of the bubbles converts sonic energy into
mechanical energy in the form of shock waves equivalent to
several thousand atmospheres (300 MPa) pressure.

Reasons for this may be the conformational liability of some (perhaps most)
enzymes to sonication and the damage that they may realise though oxidation
by the free radicals, singlet oxygen and hydrogen peroxide that may be
concomitantly produced.
Damage by oxidative free radicals can be minimized by flushing the solution
with nitrogen and/or including scavengers like
- cysteine, dithiothreitol or other -SH compounds in the media.
Use of radical scavenger N2O, have been shown to reduce this inactivation.
As with most cell breakage methods, very fine cell debris particles may be
produced which can hinder further processing.
Sonication remains, however, a popular, useful and simple small-scale
method for cell disruption

Disadvantages include:
Heat generated by the ultrasound process must be dissipated.
High noise levels (most systems require hearing protection and sonic
enclosures)
Yield variability
Free radicals are generated that can react with other molecules.
For bacterial cells, such as E. Coli, 30 to 60 seconds may be sufficient for
small samples
For yeast cells, the duration is 2 to 10 min.
Ultrasonic vibration is frequently used in conjunction with chemical cell
disruption methods – reduced energy required for cell disruption

Physical methods- Osmotic shock
Cytolysis, or osmotic lysis, occurs when a cell bursts due to an osmotic
imbalance that has caused excess water to move into the cell.
It occurs in a hypotonic environment, where water diffuses into the cell and
causes its volume to increase. If the volume of water exceeds the cell
membrane's capacity then the cell will burst

Cytolysis does not occur in plant cells because plant cells have a strong cell
wall that contains the osmotic pressure, or turgor pressure,
Contrary to organisms without a cell wall, plant cells must be in a hypotonic
environment in order to have this turgor pressure, which provides the cells
more structural support, preventing the plant from wilting.
In a hypertonic environment, plasmolysis occurs, which is nearly the
complete opposite of cytolysis: Instead of expanding, the cytoplasm of the
plant cell retracts from the cell wall, causing the plant to wilt
Plasmolysis is the term which describes plant cells when the cytoplasm
shrinks from the cell wall in a hypertonic environment.
Crenation is the contraction or formation of
abnormal notchings around the edges of a cell
after exposure to a hypertonic solution, due
to the loss of water through osmosis

Osmotic shock is usually used to lyse mammalian cells
With bacterial or fungal cells, the cell wall needs to be weakened before
application of osmotic shock
Osmotic shock is used to remove periplasmic products (mainly proteins)
from cells without cell disruption
In case of recombinant as well as non-recombinant gram negative bacteria,
proteins secreted into periplasmic space, such cells transferred to hypotonic
buffer, cell imbibe water through osmosis and volume confined in cell
membrane increases significantly
The cell wall, which is rigid not expand like cell membrane and material
present in periplasmic space is expelled into liquid medium
Cytorrhysis is the complete collapse of a plant cell's cell wall within plants
due to the loss of water through osmosis. This usually follows plasmolysis

Other Physical methods
Freeze and thaw
Repeated freezing and thawing of bacterial cells disrupts them because of the
repeated formation of sharp ice crystals - many beginning to grow in the
inside of the cells.
“Sort of like burst in balloon by using needles from the inside”
e.g. chilling a 37°c culture to 4°c by adding ice and back to 37°c

Mechanical Methods – Homogenization
Bead mill homogenizer
Cascading
beads
Cells being
disrupted
Rolling
beads
It consists of tubular
vessel made of metal or thick
glass within which a cell
suspension is placed along
with small metal or glass beads (typical beads loading is about 80-90%)
The tubular vessel is rotated about its axis and as a result of this the beads
starts rolling away from the direction of vessel rotation
At higher speed, some beads move up along with the curved wall and then
cascade back on the mass of beads and cells below
The disruption takes placed due to – the grinding action of the rolling beads
as well as the impact resulting from cascading beads
Wear resistant beads – zirconium oxide/silicate, titanium carbide, glass,
alumina ceramic

Disruption occurs by the crushing action of the
glass beads as they collide with the cells.
Compared to high-pressure methods of cell
disruption wet bead milling is low in shearing
forces.
Membranes and intracellular organelles can often
be isolated intact.
It is the method of choice for
disruption for spores, yeast and fungi and works
successfully with tough-to-disrupt cells like cyanobacteria, mycobacteria,
spores and microalgae.
More recently, it has been applied to soil samples and to plant and animal
tissue. If PCR techniques are to be used, this homogenization method is one
of the few that totally avoids possible cross-contamination between samples
because both vials and beads are disposable.

Highlights:
•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.
With bead mill – cell disruption involves size reduction – similarly like
grinding

• First Order
• k is a function of
– Rate of agitation (1500-2250 rpm)
– Cell concentration (30-60% wet solids)
– Bead diameter (0.2 -1.0 mm)
– Temperature
)
exp
1
( )
( kt
m
r R
R 

 )
exp
1
( )
(
max kt
r
r C
C 


or
Where,
Rr – concentration of released product (Kg/m3)
Rm –maximum concentration of released product (Kg/m3)
t – time (s)
K – time constant (s)
The value of Rm and k can be determined experimentally
The value of K, strongly depends on-type of impeller, bead size, bead load,
speed, and temperature

For continuous mode, mean residence time, and the residence time
distribution or number of continuous CSTR in series should be taken into
account to predict release
Optimal conditions for release of intracellular product are
a) The micro-organism used
- Cell wall thickness and composition
- Size
b) Location of the product
- in cytoplasm
- in cell organelles
- in periplasmic space
c) Type of bead mill
- bead diameter and type of impeller
- bead loading
- tip speed of impeller
d) Residence time of cell suspension
e) Cell concentration
f) Temperature (rise)
Bead mill performance

Scale up Bead mill
Removal of energy dissipated in the broth – problem in scale up
A power input is dissipated in heat and is needs to be removed by cylinder
wall. So the ratio of heat transfer area to the bead mill volume is:
T
L
T
TL
V
volume
Mill
A
area
surface
L
4
4
)
(
,
)
(
,
2





Where
T – Cylinder diameter (m) and L – length of bead mill (m)
On scaling up, the cylinder diameter will increase and the ratio 4/T will
decrease
5
3
,
, D
cpN
p
INPUT
POWER 
Where
C – dimensionless constant
ρ – suspension density (Kg/m3)
N – rotational speed of impeller (S-1)
D – impeller diameter (m)
C depends on type of flow (turbulent or laminar) and type of impeller

Homogenization is the widely used method for large scale operations as well
as lab scale.
This method employs equipment called Homogenizer or Cell disruptor
adapted from dairy industry which operates at extremely high pressures
(upto 400-2500 bars).
Cell disruptors and homogenizers are both positive displacement pumps
each differs in the way that they create pressure on the sample and transfer
it from pressurized chamber to another chamber which is at lower pressure.
Homogenizers pressurize the sample in a chamber which is then released
into a chamber of lower pressure through a homogenizing valve.
Cell disruptors use a hydraulic force to accelerate the sample to high
pressure and forcing them through a minute orifice to hit on a disruption
head which is at a lower pressure.
Homogenization

Pressure can be controlled by adjusting the force imparted on the valve,
which is controlled either pneumatically or hydraulically.
As the cell suspension is pumped through a minute orifice at high pressure
it causes a shear on the cell membranes. This is followed by the sudden
release of the suspension with instant expansion.
Disruption of the cell is accomplished at three stages causing the explosion
thereby releasing its contents.
1. Impingement on the homogenizing valve
2. High turbulence and shear combined with compression produced in the
minute gap
3. Sudden pressure drop upon release

The main disruptive factor in this process is the pressure applied on the
sample and consequent pressure drop across the valve.
This causes the impact and shear stress on the cells making them to break
which are proportional to the operating pressure.
The operating parameters which affect the
cell breaking efficiency of high-pressure
homogenizers are as follows:
-Operating Pressure
-Process Temperature
-Number of passes
-Valve/Orifice design
-Flow rate of the sample

There are certain variables to be considered while designing a
homogenizer/cell disruptor. They are:
-type of homogenizing
valve/orifice
-operating pressure
-stages of disruption
-viscosity of the sample
-temperature
-type of the surfactant
In the commonly-used operating range with pressures below about 75 MPa,
the release constant (k) has been found to be proportional to the pressure
raised to an exponent dependent on the organism and its growth history
- (e.g. k=k'P2.9 in Saccharomyces cerevesiae and k=k'P2.2 in
Escherichia coli, where P represents the operating pressure and k'
is a rate constant).
The higher the operating pressure, the more efficient is the disruption
process

The protein release rate constant (k) is temperature dependent, disruption
being more rapid at higher temperatures.
In practice, this advantage cannot be used since the temperature rise due to
adiabatic compression is very significant so samples must be pre-cooled
and cooled again between multiple passes.
At an operating pressure of 50 MPa, the temperature rise each pass is about
12 deg. C.
In addition to the fragility of the cells, enzymes/proteins are released at
various rates depending on their cellular location.
Proteins located in the periplasm are released faster whereas the proteins
located within the cellular components are released at a slower rate.
Unbound intracellular proteins may be released in a single pass whereas
membrane bound enzymes or proteins may require several passes for
reasonable yields to be obtained

Multiple passes are undesirable because, of course, they decrease the
throughput productivity rate and because the further passage of already
broken cells results in fine debris which is excessively difficult to remove
further downstream.
Consequently, homogenisers will be used at the highest pressures
compatible with the reliability and safety of the equipment and the
temperature stability of the enzyme(s) released.
High pressure homogenisers are acceptably good for the disruption of
unicellular organisms provided the
- enzymes needed are not heat labile and
- the shear forces produced are not capable of damaging enzymes
free in solution.
The valve unit is prone to erosion and must be precision made and well
maintained.

ROTOR-STATOR HOMOGENIZERS (also called colloid mills or Willems
homogenizers)
Cell
suspension
Rotor
Stator
Disrupted
cells
These are well suited for plant and animal tissue
and outperform cutting-blade type Benders.
Compared to a blender, foaming, swirling
and aeration are minimized and
smaller sample volumes are accommodated.
Mechanism of cell disruption:
-High shear and turbulence
The cellular material is drawn into the apparatus
by a rotor sited within a static tube or stator.
The material is then centrifugally thrown outward to exit through slots or
holes on the tip of the stator.
Because the rotor is turning at very high speed, the tissue is rapidly reduced
in size by a combination of turbulence and scissor-like mechanical shearing
occurring within the gap of the rotor and stator.
The process is quite fast and, depending upon the toughness of the tissue
sample, desired results are usually be obtained in 10-60 seconds.

For the recovery of intracellular organelles or receptor site complexes,
shorter times and/or reduced rotor speeds are used.
Samples often must be pre-chopped or - fragmented with a scissors, single-
edge razor blade or cryopulverizer (a device that quickly powders tissue at
liquid nitrogen temperatures ).
Unlike many other types of cell disrupters, rotor-stators homogenizers
generate negligible heat during operation.
Most laboratory rotor-stator homogenizers are top driven with a compact,
high speed electric motor which turns at 8,000 to 60,000 rpm and function
properly with viscosity range of <10,000 cps
the size of the rotor-stator probe can vary from the diameter for 0.5-50 mL
sample volumes to much larger units handling 10 liters or more.
Foaming and aerosols are the problems with rotor-stator homogenizers
Bottom-driven laboratory rotor-stator homogenizers
SINGLE OR MULTIPASS OPERATION











t
C
C
exp
1
max
Single pass
Multi pass
N
t
C
C


















exp
1
max
Where,
N – is the number of passes
ɵ - time constsant

FRENCH PRESS
Plunger
Cylinder
Cell
suspension
Impact
plate
Jet
Orifice
•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
•Operating pressure: 10,000 to 50,000 psig

FREEZE-FRACTURING
Both microbial pastes and plant and animal tissue can be frozen in liquid
nitrogen and then ground with a mortar and pestle at low temperature.
Presumably the hard frozen cells are fractured under the mortar because of
their brittle nature. Also, ice crystals at these low temperatures may act as
an abrasive
A freeze-fracturing device called the Bessman tissue pulverizer is useful for
fragmenting 10 mg to 10 g quantities of fibrous tissue such as skin or
cartilage to the size of grains of salt. (material is then easily homogenized by
other methods)
Looking somewhat like a tablet press, the pulverizer consists of a hole
machined into a stainless steel base into which fits a piston. The base and
piston are pre-cooled to liquid nitrogen temperatures.
Ten mg to ten grams of hard frozen animal or plant tissue is placed in the
hole. The piston is placed in the hole and given a sharp blow with a
hammer.
The resulting frozen, powder-like material can be further processed by Pestle
and Tube, Bead Mill or Rotor-stator homogenizers

Disadvantages include:
Not well suited for larger volume processing.
Awkward to manipulate and clean due to the weight of the
assembly (about 30 lbs/14 Kg).

Fixed-Geometry Fluid Processors
The patented technology of Microfluidics' fixed-geometry fluid processors
are marketed under the name of Microfluidizer® processors.
The processors disrupt cells by forcing the media with the cells at high
pressure (typically 20,000-30,000 psi) through a proprietary interaction
chamber containing a narrow channel that generates the highest shear rates
The ultra-high shear rates allow for:
Processing of more difficult samples
Fewer repeat passes to ensure optimum sample processing
Microfluidizer® systems provide a highly reproducible, convenient, and
efficient method for cell lysis.
The systems permit controlled cell breakage without the need to add
detergent or to alter the ionic strength of the media.

The fixed geometry of the interaction chamber ensures:
- Day-to-day reproducibility
- Machine-to-machine reproducibility
- Direct scalability from laboratory scale (20 ml to several liters) to
production scale (10s of liters per minute)
Disadvantages included:
In many circumstances, especially when samples are processed multiple
times, the Microfluidizer® processors do require sample cooling

Optimal conditions for release of intracellular product depends on
a) The micro-organism used
Cell wall thickness and composition
- Size
-b) Location of the product
- in cytoplasm
- in cell organelles
- in periplasmic space
c) Type of homogenizer
- pressure
- type of valve and seat
- temperature (rise)
- number of passages (N)
d) Residence time of cell suspension
e) Cell concentration
Homogenizer

Homogenizer –kinetics of release
N
p
f
K
C
C
C
h
r
r
r
)
(
ln max
max











Where,
f – is function of pressure difference, (Δp)
N – number of passages
)
exp
1
( )
)
(
(
max N
p
f
k
r
r
h
C
C 



For many cases f (Δp) = Δpβ
The exponent is of order 1.5 to 3. For bakers yeast exponent is 2.9,
therefore
)
exp
1
( )
(
max 9
.
2
N
p
k
r
r
h
C
C 




Homogenizer –kinetics of release
Scale up of homogenizer involves installing bigger plunger pump and
discharge valve
The power input is proportional to homogenization pressure, Δp and the
volumetric flow processed



 


 p
v
v c
p
p
Where,
ϕv – volumetric flow rate (m3/s)
ρ – density (Kg/m3)
Cp – specific heat (J Kg-1K-1)
Δɵ - Temperature difference (K)

Sonic Agitation Liquid
Pressing
Freeze pressing
Animal cells 7 7 7 7
Gram –ve bacilli &
cocci
6 5 6 6
Gram +ve bacilli 5 4 5 4
Yeast 3.5 3 4 2.5
Gram +ve cocci 3.5 2 3 2.5
Spores 2 1 2 1
Mycelia 1 6 1 5
Sensitivity of cells to disruption

Heat
All mechanical methods require a large input of energy, generating
heat. Cooling is essential for most enzymes. The presence of
substrates, substrate analogues or polyols may also help stabilise the
enzyme.
Shear
Shear forces are needed to disrupt cells and may damage enzymes,
particularly in the presence of heavy metal ions and/or an air
interface.]
Hazards likely to damage enzymes during cell disruption
Proteases
Disruption of cells will inevitably release degradative enzymes which
may cause serious loss of enzyme activity. Such action may be
minimised by increased speed of processing with as much cooling as
possible. This may be improved by the presence of an excess of
alternative substrates (e.g. inexpensive protein) or inhibitors in the
extraction medium.

cell disruption technologies for bioprocess

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