Cell immobilization technique.

how to increase production of secondary metabolite by using cell immobilization technique.

1. PLANT CELL IMMOBILIZATION Presented by MR. SURAJ B. MANATKAR B- Pharm SEMESTER VII TH Under guidance of PROF. SHANKUL KUMAR m- pHARM Dept. Of PharmaCOGNOSY S. G. S. P. S. Institute of Pharmacy, Akola 1

2. CONTENT : Introduction. Need of Immobilization. Immobilization Technique. Viability testing. Bioreactor. Advantages. Disadvantages . 2

3. 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. 3

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. 4

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. Adhesion 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. 5

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. 6

7. METHOD ENTRAPMENT : 1. Entrapment by polymerization. 2. Entrapment by ionic network formation. 3. Entrapment by precipitation. 7

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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. 9

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. 10

11. 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. 11

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. 12

13. Example : Cells mixed in aqueous polymeric solution Dispersed Oil phase stirred Droplet formation cool Solidification of polymer centrifugation Microencapsulated beads collect. 13

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. 14

15. Viability testing of immobilized cells. 1 . Viability staining a. Fluroscein diacetate ( FDA) – green b. Phenosafranin - Red 2. Plasmolysis : Determine Integrity of Plasma membrane by adding plasmolysing agent e.g – glycerol & sorbitol . 3. Cell Growth. 15

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 Law 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. 16

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. 17

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. 18

19. a. Flat plate membrane reactor 19

20. b. Multimembrane reactor 20

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. 21

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. 22

23. Reference : Medicinal Plant Biotechnology by Ciddi Veeresham, first edition 2008 published by CBS Publisher and Distributors. Plant Tissue Culture by S. S. Bhojawani and M. K. Razdan , First edition 2013 , published by Elsevier Pvt. Ltd. 23

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