muhmina micro. .pdf

Cell disruption

1. CELL
DISRUPTION
(Physico-
mechanical and
Chemical
method)
Submitted by: Muhmina sherin
2nd semester M.Sc Food science and technology

2. CELL
DESTRUCTION
Cell destruction / cell disruption is the
process of breaking open ( otherwise
known as lysing) cells in order to obtain
the intercellular fluid ( commonly referred
to as lysate).
There are a variety of intercellular
components that may be desired to obtain
via cell disruption, including proteins and
viral vectors that are not expressed extra
cellularly .
Cell disintegration methods can be
physical, chemical, or biological.

4. •A “physico-mechanical” method of cell destruction
refers to a technique that uses physical forces, like
pressure, shearing, or impact to disrupt and break
open cells, essentially achieving cell lysis without
the use of chemical agents; common examples
include
1.Bead milling
2.High pressure homogenisation
3.French press
4.Rotor-stator homogenizers
5.Sonication

5. BEAD MILLING:
•Cells are mixed with small beads in a chamber and agitated vigorously, causing the
beads to collide with and disrupt the cells
•Bead milling, also known as bead beating, disrupts cells by vigorously shaking a
suspension of cells mixed with small, hard beads within a closed container, causing the
beads to collide with and physically break the cell walls, effectively releasing the
intracellular components.
Key points about bead milling cell disruptions:
•Bead material: The beads used are typically made of glass, ceramic, or metal, and their
size is crucial for the disruption efficiency, with smaller beads generally being better
for more delicate cells.
•Agitation process: The cell suspension with beads is vigorously shaken or agitated in a
specialized machine, causing the beads to collide with the cells, creating shear forces
that disrupt the cell membrane.

6. Factors affecting bead milling efficiency:
•Bead size: Selecting the appropriate bead size based on the cell type, with larger
beads being better for tough cells and smaller beads for fragile cells.
•Bead loading: The ratio of beads to cell suspension can influence disruption efficiency.
•Agitation speed and duration: Adjusting the shaking speed and time to optimize cell
disruption without causing excessive heat generation .
•Lysis buffer: The choice of lysis buffer can impact cell disruption and protect sensitive
intracellular components

7. HIGH PRESSURE HOMOGENISER:
•Cell suspension is forced through a small orifice under high pressure, sudden pressure
drop and cell disruption.
•High –pressure homogenisation (HPH) is a mechanical process that uses high pressure to
disrupt cell walls. It’s a common method for disrupting microbial cells in large –scale bio
process.
•HPH works by forcing a fluid through a small opening at high pressure, which creates
turbulence, shear stress and cavitation. This process break down particles into smaller,
more consistent sizes.
Working:
1.A high pressure pump forces a pressurized fluid through a small valve or orifice.
2.The fluid is exposed to high shear stress, cavitation and impact forces.
3.The fluid is homogenised and its temperature increases.
4.The particles are broken down into smaller and more consistent sizes.

8. •Advantages of HPH cell disruption:
High disruption efficiency: HPH can
effectively disrupt a wide range of cell
types, including bacteria, yeast, and
microalgae with tough cell walls due to the
intense shear forces generated under
high pressure.
Controllable parameters:Pressureand
number of passes through the
homogenizer can be adjusted to optimize
cell disruption based on the sample type.

9. French press:
•Similar to high pressure homogeniser, but often used on a smaller scale.
•A French press is a device that a destruct cell walls and membrane by forcing a liquid
sample through a small valve at high pressure.
•Can disrupt cell walls while leaving the cell nucleus intact.

10. Sonication:
•Ultrasonic waves creates cavitation bubble within cell suspension, causing cell
disruption when they implode.
•A vibrating probe is immersed in a liquid sample containing cells.
•The probe generates high frequency sound waves that agitate the samples
•The sound waves create microscopic bubble that implode, causing shock wave.
•The shock wave disrupts the cell membrane, releasing the cells content.

11. •Physical method of cell disruption use pressure, contact or denaturation to break down
cells.
•These methods are efficient and don’t create waste, but they do require energy.
•Physical method of cell disruption
1.Freeze-thaw
2.Microwave/ Thermolysis
3.Osmotic shock
4.Electric discharge.

12. Freeze-thaw:
•It is suitable when working with soft plant material and algae.
•Disruption is achieved via a series of freezing and thawing
cycle
•Freezing forms ice crystals, which expand upon thawing, and
this ultimately causes the cell wall to rupture.
Microwave/ Thermolysis:
•Microwave (along with autoclave and other high temperature
method) are used to disrupt the bonds within cell walls, and
also to denature protein.
•However, uncontrolled amount of heat can easily denature or
damage target protein and other substances.

13. Osmotic shock:
•Through the process of osmosis, water can be moved
into the cell causing its volume to increase to the point
that it burst.
•The method however can only work with animal cells and
protozoa since they do not have cell walls.
Electric discharge:
•It is also possible to achieve cell destruction why
electrical charges in mammalian and other cells that are
bounded by plasma membrane only.

14. •Cell disruption chemical method use chemicals to disrupt cell membrane or cell walls.
The chemicals used depends on the type of cell and its cell wall composition.
•Chemical methods:
Osmotic lysis:
•Cell swell and burst when suspended in a hypotonic solution, like dilute sucrose. This
method is often used for mammalian blood cells and RNA extraction
Detergent:
•Also called surfactant these compounds lower surface tension and disrupt cell
membranes. Detergents can be used to isolate membrane proteins.
Chaotropic agents:
•These agents disrupt the structure of water, making it less hydrophilic.
EDTA
•Ethylene diamine tetra acetic acid chelate cation, leaving Holes in cell walls.

15. •Acid treatment :
•High temperature acid treatment ( 58-160°C) can disrupt cell.
•Alkaline pH:
•Exposing cell to alkaline pH in the range of 11.5 –12.5 for 20 to30 min can cause cell
lysis.
DISADVANTAGES OF CHEMICAL METHODS OF CELL DISRUPTION
•A major drawback using chemical methods of cell disruption in manufacturing is the
cost. Using small amounts of chemicals and enzymes in the R & D laboratory is
acceptable; but the cost of using the large volume required for large scale production
is often not feasible.
•Harsh chemicals and detergents can often damage or destroy the content of the cell if
use incorrectly. Lastly using large volume of potentially hazardous chemicals created
significant health and safety risk.

16. THANK YOU