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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