Vous êtes sur la page 1sur 25

CELL DISRUPTION

Cell disruption
-Key technique in DSP to release intracellular product.
Biological products:
1. Extracellular
2. Intracellular
3. Periplasmic
Cells
• Gram positive bacterial cells
• Gram negative bacterial cells
• Yeast cell
• Mould cells
• Cultured mammalian cells
• Cultured plant cells
• Tissue
What are we disrupting?
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
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


•Plant cells have thick and robust cell walls mainly composed
of cellulose
•Plant cells are difficult to disrupt
•Cultured plant cells are less robust than real plant cells
Cell disruption methods

Physical methods
• Disruption in bead mill
• Disruption using a colloid mill
• Disruption using French press
• Disruption using ultrasonic vibrations

Chemical and physicochemical methods


• Disruption using detergents
• Disruption using enzymes e.g. lysozyme
• Disruption using solvents
• Disruption using osmotic shock
Bead mill

Cascading
beads
Rolling
beads Cells being
disrupted

•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.
Colloid mill
Rotor
Disrupted
cells

Cell Stator
suspension

•Typical rotation speeds: 10,000 to 50,000 rpm


•Mechanism of cell disruption: High shear and turbulence
•Application: Tissue based material
•Single or multi-pass operation
French press

Plunger

Cell Cylinder
suspension

Jet
Orifice
Impact plate

•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
Ultrasonic vibrations
Ultrasound
generator

Ultrasound tip

Cell suspension

•Application: Bacterial and fungal cells


•Mechanism: Cavitation followed by shock waves
•Frequency: 25 kHz
•Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10 minutes
•Used in conjunction with chemical methods
Chemical Methods for Lysis

• Osmotic – drastic reduction in extracellular


concentration of solutes
• Enzymes and antibiotics – digestion of cell
wall or production of protoplasts
• Detergents – break plasma membrane
• Solvents – dissolve cell membrane and
excess fat, may aid in precipitation
van’t Hoff Law

• Estimates transmembrane osmotic pressure

π = RT (ci − co )
•R = gas constant
T = absolute temperature (K)
ci-co = difference between total solute molarity
inside and outside the cell
Chemical and physicochemical
methods

•Detergents
•Enzymes
•Organic solvents
•Osmotic shock
•Alkali treatment
Choice of Disruption Method
• The method selected for large scale cell
disruption will be different in every case,
but will depend on:
– Susceptibility of cells to disruption
– Product stability
– Ease of from cell debris
– Speed of method
– Cost of method
Adverse Factors during Disruption
• Heat
• Shear
• Proteases
• Particle size
• DNA, RNA
• Chemical
• Foaming
• Heavy-metal toxicity
Thank you