Cell immobilization is the process of fixing intact cells onto specific regions in a device or material without losing their biological function. Cells can be immobilized through physical adsorption, encapsulation, entrapment, and self-aggregation.

1. PRESENTED BY
Swapnil S. Tirmanwar
Pharmacognosy
(M. Pharm 1st year)
PRIYADARSHINI J. L. COLLEGE OF
PHARMACY,
Electronic zone, MIDC, Hingna road
NAGPUR-440016
2022-2023
PLANT CELL IMMOBILIZATION

2. CONTENT
• Introduction
• Need of Immobilization
• Immobilization Technique
• Viability testing
• Bioreactor
• Advantages
• Disadvantages.
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3. INTRODUCTION
• Definition :
• Immobilization is technique, which confines to a catalytically active
enzyme or to cell within reactor system and prevents its entry into the
mobile phase, which carries substrate and product.
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4. Need of Immobilization
• Plant cell culture has been use for production of secondary metabolite.
• Characteristics of plant cell culture such as slow growth, large size, sensitive to
shear and oxygen and need of cell to cell contact for metabolite production, the
compound produced should be of high value and low volume.
• The use of high biomass level for extended period would be one method of
increasing productivity and hence reducing the costs this can be achieved by the
immobilization of plant cell.
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5. Different types of immobilization technique
• 1. Physical retention within the framework of different pore size and
permeability.
a. Entrapment.
b. Micro-encapsulation
2. Direct intracellular binding due to natural affinity.
a. Adsorption.
b. Adhesionc.
c. Agglutination
3. Intracellular connection via bi or poly functional reagent
a. Cross-linking
4. Mixing with suitable material changing their consistency with temperature.
a. Embedding
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6. Selection of immobilization system
• The polymer material used for immobilization must be available in
large quantities, inert, non-toxic, cheap.
• Able to carry large quantities of biomass and its fixing potential must
be high
• The immobilization process must not diminish enzymatic activity of
biological catalyst.
• Manipulation of biological catalyst must be as simple as possible.
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7. METHOD
• ENTRAPMENT:
1. Entrapment by polymerization.
2. Entrapment by ionic network formation.
3. Entrapment by precipitation
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9. 1. Gel entrapment by polymerization
• A monomer or mixture of monomers is polymerized in the presence of a cell
suspension, which is entrapped cell inside the lattice of the polymer.
• The method is based on the free radical polymerization of acrylamide in an
aqueous solution.
• The free radical polymerization of acrylamide is conducted in an aqueous solution
containing the cell and the cross linking agent.
• Initiator - N, N,N',N'- tetramethy/ethylene
• An initiator & cross-linking agent are toxic to the cells & therefore
viability can be lost.
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10. 2.Gel entrapment by ionic network formation
• In this method the polymerization of poly electrolyte is achieved by addition of
multivalent ions the most common method is the entrapment in calcium alginate.
• This is non-toxic process in which sodium alginate solution is dropped into
mixture or counter ion solution such as calcium chloride.
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11. 3. Immobilization by Embedding:
• The temperature dependent solubility of macromolecules like agarose, agar,
carrageenam.
• Insoluble are formed under cold condition (agar) or in aqueous solution of CaCl2.
• Their structure in non-uniform, with different pore size at the surface and
in deeper layer.
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12. • Encapsulation:
Encapsulation is the process of forming a continuous membrane around
cells to be immobilized that denote the core of the system in which the
inner matrix is protected by means of the outer membrane. Liquid form
of active substance is the core material and polymeric wall is
the outer membrane.
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13. • Example:
Cells mixed in aqueous polymeric solution
Dispersed
Oil phase
Stirred
Droplet formation
Cool
Solidification of polymer
Centrifugation
Microencapsulated beads collect
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14. • Adsorption/Surface Immobilization:
• The adhesion of cells on the surface of support matrix is initiated by the attraction
of cells on the matrix followed by adsorption.
• The interaction between the cells and matrix is provided by vander Waals,
electrostatic, hydration, and hydrophobic forces.
• For the immobilization of viable cells adsorption process is well suited when
compared with entrapment technique. This type of immobilization is considered as
one of the easiest technique.
• Fiberglass mats, unwoven short fibrepolyster.
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15. • Viability testing of immobilized cells
1. Viability staininga.
a.Fluroscein diacetate (FDA) – green
b. Phenosafranin - Red2.
2.Plasmolysis:
Determine Integrity of Plasma membrane by adding
plasmolysing agente.g-glycerol & sorbitol.
3. Cell Growth.
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16. • Bioreactor for Cell Immobilization.
• 1. Packed bed reactors :-
In this reactor, cells can be
immobilizes either on surface or throughout the support.
When the cell are immobilized through the support the
placed bed can accommodate large no. of cells per reactor
volume.
Disadvantage
Low degree of mixing
Large incompressible support particle are needed.
The packed bed reactor are having filter, when the support
particle fragment during operation they will block the
pathways for fluid flow.
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v
EXIT
INLET
Packed bed reactor

17. • 2. Fluidized Bed Reactors
It utilize energy of the flowing fluid to suspend the
particle.
Small immobilized particle are employed
Fluid flow rate should be sufficient to suspend particle.
Large gas volume can be used to suspend the
immobilized cell while maintaining low fluid flow rates.
These condition leads to large degree of fluid mixing.
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Gas

18. •Membrane Reactors
In these, the cells are separated from growth medium by membrane are suitable for
fragile cells which can be entrapped more readily on membrane.
The environment in membrane reactor is more homogeneous.
a. Flat plate membrane reactor
1.One side flow.
2. Two side flow.
b. Multimembrane reactor.
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19. a.Flat membrane reactor
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Nutrient flow
Nutrient flow
Nutrient flow
Top plate
cell
membrane
s
membrane
A
B

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Sweep gas O2 CO2
Hydrophobic
membrane
Hydrophobic
membrane
cells
substrate product
Hydrophobic
membrane
product
b.Multimembrane reactor

21. • Advantages of Cell Immobilization
1. It may enable prolonged use of biomass.
2. The entrapped cells are protected against shear, reduce problem of aggregate.
3. High biomass level (compared to cell suspension culture).
4. Separates cell from medium and therefore if the product is extracellular. It can
simplify downstream processing.
5. It allows a continuous process, which increase volumetric productivity and allow
removal of metabolic inhibitors.
6.It uncouples growth and product formation which allows product optimization
without affecting growth.
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22. • Disadvantages of Cell Immobilization
1. The efficiency of the production process depends on the rate of release of
product rather than actual rate of biosynthesis.
2. The immobilization process may reduce biosynthesis capacity.
3. Product must be released from cell into medium
4. Secretion of secondary metabolite requires cellular transport or artificial altered
membrane permeability.
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23. Reference:
Medicinal Plant Biotechnology by Ciddi Veeresham, first edition 2008 published by
CBS Publisher and Distributors.
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