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Immobilized cell techniques(survey of recent advances)

GUNJA SRIVASTAVAM.PHARM (II SEM) PHARMACOGNOSY

PRESENTED TOPRESENTED BYMR.RAJIV GUPTAMR.ANURAG MISHRAMR.VINEET GUPTAMRS.DIPALI PANDEY

DATE-25.06.2011

CELL IMMOBILIZATION TECHNIQUE

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Cell immobilization is a technique to fix plant cells in a suitable matrix.

As a result of immobilization ,cells can be encouraged to grow together in a multicellular , partially organized conditions.

If the cells are maintained stationary the physical & chemical gradient established can provide an environment which may closely resemble the in vivo environment of the plant.

Plant cells grow much more slowly, they produce targeted compounds more slowly, they are more easily disrupted by physical stress and their behaviour (growth and synthesis) is influenced by chemical signals by neighbouring cells.

Immobilization is the newest culture technology for plant cells & perhaps it can be regarded as most natural in its approach

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Then by immobilization, the plant cells are protected from liquid shear forces.

It is the technique that facilitates the sequential manipulation of plant cells derived from the homogenous suspension culture & permits some structural & biochemical differentiation of those cells.

Freely suspended plant cells mostly accumulate their secondary metabolites in the stationary phase of their growth cycle, point of time when their growth stop.

Entrapment of plant cells is one the means to create non-growth condition under which the production of secondary metabolites may be improved.

Immobilization facilitates the importance of cellular cross talk, which can establish inter-cellular communication by the action of signalling the molecules.

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Rationale for plant cell

immobilization

Immobilization of the plant cells , protoplast & subcellular organelles is likely to be associated with one or more of the following properties

Sequential chemical treatment of the cells & harvest of metabolites from the medium.

Controlled aggregation of cells .

The facilitated establishment of directional gradients of physical &chemical factor.

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CONCEPT

The whole concept is based on the idea that regulation of metabolism is largely determined by the interaction between the genome & the cytoplasmic environment.

The cytoplasmic environment is in turn influenced by surrounding cells & their effect , biosynthesis of metabolic intermediates & their interference to the diffusion of physical & chemical factors supplied exogenously .

The growth rate of artificially aggregated(immobilized) cells is usually less than that of freely suspended cells ,there is always a correlation between reduced culture growth rate & enhanced production of secondary metabolites

Another assumption indicates that there is an antagonism between primary and secondary pathways .So if the rapid growth & division is encouraged precursors will tend to produce primary metabolites like protein but if the growth is limited then the same precursor will produce secondary metabolites such as phenolic or alkaloid.

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This therefore suggest that immobilized cells in which aggregation & organization are encouraged , might be particularly suited for higher production of secondary metabolites.

Some practical approaches that are helpful in the immobilized cell culture systems .

if cells are cultured on flat bed or in column reactor vessel and the medium is supplied from a separate reservoir by passing it over the cells than it is easy to

Supply precursor of secondary product in large quantity but in low concentration

Perform sequential chemical treatment on the cellsHarvest metabolite

from nutrient medium

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Gel entrapment by ionic network

formation

Gel entrapment by polymerisation

Entrapment in the preformed structures.

Gel entrapment by precipitation.

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GEL ENTRAPMENT BY

IONIC NETWORK

FORMATION

Entrapment by ionic network formation, especially in the form of alginate beads, is the most widely used method.

Alginate is a polysaccharide that forms a stable gel in the presence of cations, most frequently used with calcium

Beads of alginate-containing cells, are formed by dripping a cell suspension-sodium alginate solution mixture into a stirred calcium chloride solution. K-Carragenan can also be used in similar manner instead of alginate, using either calcium or potassium.

Advantage of this method is that the gel can be reversible by adding EDTA.

Cultivation of Morinda citrifolia, Catharanthus roseus and Digitalis lanata were successfully done by this method of immobilization, with significant increase of metabolite and the stability of metabolic capacity was also extended for long periods of time .

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After

cylindrical beads

Gel entrapment b

y

precipita

tion

Preparations of agar and agarose can be used to trap plant cells by precipitation. The polysaccharides forms gel, when a heated aqueous solution is cooled.

The gel can be dispersed into particles in the warm liquid state by mixing in a hydrophobic phase, e. g. olive oil.

When particles of the desired size are obtained the entire mixture is cooled and this results in solidification . I

Gel entrapment by polymerisation

Gel entrapment by polymerisation is most commonly carried out using polyacrylamide, but it affect the cell viability adversely.

The toxicity of the initiator and cross-linking agents used in the polymerisation has in some cases caused a loss of cell viability.

Catharanthus roseus cells entraped by polyacrylamide gel did not grow at all and hence respiration and plasmolysis were not detected.

On the other hand, by suspending plant cells in aqueous solution of prepolymerised linear polyacrylamide partially substituted with acrylhydrazide group , viability was maintained, with better growth.

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Gel Advantages/disadvantages Concentration

Agar Non toxic & freely permeable of inactivated cells

2%

Polyacrylamide Gelatin

•Adverse effect on cell viability

•Form cross linked gelatin foam with 25% glutaraldehyde

-

20%

K-Carrageenan Forms gel in the presence of K+

ions 3%

VARIOUS TYPES OF GELS USED FOR IMMOBILIZATION

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oEntrapment in preformed structures involves some form of open network through which nutrient medium may pass, but which entraps plant cells or cell aggregates.

oSuch structures can be facilitated by using cotton fibre, fibreglass mats, reticulate polyurethane foam, and in a cloth nonwoven polyester short fibres.

oThe polyurethane foam has some merit as a matrix, no reagent which might toxic to plant cells, and no complicated operation causing microbial contamination are required. Cells are immobilized by their invasion present in pre-formed polyurethane foam , cut into blocks.o Immobilized Capsicum frutescence cells in reticulate polyurethane foam, showed that the immobilized cells produced more capsaicin than the free cells.

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Bioreactors involved in Immobilization Technique.FLATBED

BIOREACTOR

The flat bed bioreactor is one of the simplest cell immobilization systems available

The cells obtained from callus cultured or cell suspension are loaded on substratum of polypropylene fabric within a glass vessel .

Liquid nutrient medium is supplied from a reservoir set above the culture vessel.

It is dipped onto the fabric & moved across it by capillary action, there by feeding the cells ,liquid medium is removed by using peristaltic pump & returned to the reservoir.

The technique provides cell to cell contact & sequential chemical treatments on stationary culture.

Column Bioreactor

Column reactor is the modification of the flatbed bioreactor in the vertical mode rather than horizontals one .

The culture vessel is a vertical glass column through which the nutrient medium is circulated before being pumped back to reservoir.

The column is supported with inert matrix along the length to prevent settling of the cells at the bottom of column.

column bioreactor system provides better control over the supply of nutrients ,saves the space.

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NUTRIENT MEDIUMRESERVOIR

CULTURE VESSEL

POLYPROPYLENE SUBSTRATUM

CELLS

PERISTALTIC PUMP

PERISTALTIC PUMP

COLUMN OF IMMOBILIZED CELLS

THE FLAT BED CULTURE SYSTEM THE COLUMN CULTURE SYSTEM

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• 1 Cell-Matrix Interaction. Initial interaction of cells with surface of matrix is necessary for proper

immobilization ,when using reticulated polyurethane foam, in order for any immobilized cells to grow well, the volumetric fraction of the foam has to be sufficient enough for all the cells to interact, and the reticulated pores of the foam should be large enough to contain the cells.

• 2. Aeration of Immobilized Cells Relationship between metabolism and dissolved oxygen concentration is complex, a conclusion can not be reached about the effect of reduced availability of oxygen in immobilized plant cell system on secondary metabolite production and growth. Alginate entrapped cells of Thalictrum minus were found to turn black

owing to the insufficient supply of oxygen and they failed to produce berberine.

On other hand, reduction in the dissolved oxygen concentration of the medium resulted in the production of capsaicin by Capsicum frutescens entrapped in polyurethane foam particles.

Factor Affecting Cell

Immobilization

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• LightMetabolism of cultures can be affected by periodic exposure to light, and the quality and intensity of the light are significant. Only the outer cell layers of the cultures in the immobilized matrix may receive some light, this may be advantageous in the case where some precursors are form in light and some in dark condition, such as Catharanthus alkaloids. In other cases supply of light to the interior of the immobilized cell matrix may be possible by the use of optical fibres.

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APPLICATION OF IMMOBILIZED CELL TECHNIQUES

1.The greatest potential of the technique is in their utility in the large –scale production of secondary metabolites either by replacing immobilized enzymes or catalyst in biotransformation reaction or in complex process.

2.Immobilization might increase the capacity for long term storage or transport of cells & protoplast , it can also be used in cell –cell recognization.

3.Immobilization can be valuable in studying the biochemical response of plant cells to infection.

4.Immobilization is also helpful in developmental studies of cells, protoplast & cell metabolism

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5.Cultivation of plant cells in aqueous two-phase polymer systems:Suspension cultures of Nicotiana tabacum have been successfully grown in aqueous, two-phase systems comprised of polyethylene glycol (PEG) and dextran in a modified LS medium. Aqueous two-phase systems may be advantageous for plant tissue cultivation since cells can be immobilized in one phase while secondary products are collected and withdrawn in the other phase, thus enhancing productivity.

6.Immobilisation has been used for several cell lines for either production of metabolites or their biocoversion. For Capsicum frutescens and Catharanthus roseus, immobilisation significantly affects the production of capsaicin and alkaloids, respectively.

7.Immobilization of plant cell may be used in bioconversion of Codeinone(-) to codeinine(-) ,digitoxin to digoxin.

8 .D.lanata by suspending them in sodium alignate solution , peletting & allowing the product to harden. The granules catalyzed the conversion of digitoxin to purpurea glycoside A & hydroxilated β-methyldigitoxin to give β-methyldigoxin

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9.Decarboxylation of L-tyrosine & L-DOPA has also been recorded by plant cell immobilization 20

SPECIES CELL TYPE IMMOBILIZED SUBSTRATUM

Catharanthus roseus Cells Calcium alegnate

Digitalis lanata Cells Calcium alegnate

Cannabis sativa Cells Calcium alegnate

Glycine max - Hollow fiberDhatura innoxia Cells Matting, Calcium alegnate

Capsicum. frutescens Cells Reticulate Polyurethane

Mucuna pruriens Cells Calcium alegnate

Papavar somniferum Cells Calcium alegnate

Spinacia oleracea Cell(Chloroplast ) Calcium alegnate

Persea americana Cell(mitochondria) -

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Recent Advancement

in Cell

immobilization technique

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Recent Advancement

Description References

1.Immobilization of Andrographis paniculata leaf cells for enhanced productionof andrographolide

Andrographolide is the major alkaloid found inAndrographis paniculata, has been used for the treatment of various diseases and disordersparticularly related with liver. Leaf cell homogenate was immobilized in calcium alginate andcarragennan and the effect of immobilization on cell growth, cell viability and cells biosynthetic activity was observed, calcium alginate was found to be the better option for higher andrographolide synthesis.

Der Pharmacia Lettre, 2010, 2(2): 198-201.

2.Immobilization of soybean (Glycine max) α-amylase onto Chitosan and Amberlite MB-150 beads

α-Amylase from soybeans was immobilized on two different matrices, Chitosan beads and Amberlite MB-150. Maximum immobilization of 62% and 70.4% was obtained with Chitosan and Amberlite MB-150, respectively. Amylase immobilized on Chitosan showed an apparent Km of 4 mg/mL, whereas Amberlite immobilized enzyme showed an apparent Km of 2.5 mg/mL

Journal of Molecular Catalysis B: Enzymatic, Volume 69, Issues 1-2, April 2011, Pages 8-14

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3)Performance improvement of araujiain (Araujia hortorum), a cystein phytoprotease, by immobilization within calcium alginate beads

The effect of immobilization within alginate beads on the performance of araujiain in aqueous and non-aqueous media was studied.

Immobilized araujiain retained 95% of their initial activity after 45 days of storage at 4 °C. Furthermore, it showed a good performance in non-aqueous medium, used for the peptide synthesis of interest for the food industry, with a yield 2.6-fold greater than using free enzyme.

The entrapped enzyme was more stable over a wide range of temperatures, pH, and storage time as compared to free enzyme.

Process Biochemistry, Volume 46, Issue 4, April 2011, Pages 1029-1034

4)Prolonging the lifetime and activity of silica immobilised Cyanidssium caldarium

The photosynthetic activity of an acidophilic thermotolerant algal cell, Cyanidium caldarium, has been preserved within silica gel, yielding living hybrid materials capable of CO2 bioconversion to carbohydrates.

Journal of Colloid and Interface ScienceVolume 356, Issue 1,April 2011, Pages 159-164

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5)Immobilization of apricot pectinesterase (Prunus armeniaca ) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzymeThe immobilized PE exhibited better thermostability than the free one. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.

Journal of Molecular Catalysis B: EnzymaticVolume 56, Issue 1, January 2009, Pages 13-19

6)Immobilization of microbial cells on inner epidermis of onion bulb scale for biosensor application

Inner epidermis of onion bulb scales was used as a natural support for immobilization of microbial cells for biosensor application. Whole cells of Sphingomonas sp. were immobilized on inner epidermis of onion bulb scale by adsorption followed by cross-linking methods.

Biosensors and BioelectronicsVolume 26, Issue 11, 15 July 2011, Pages 4399-4404

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7) Agricultural aspect Long-term tissue culturesand cell suspensions were derived from watermelon seedlings. Immobilization technique increased the watermelon crop.

Journal of Applied Sciences Research, 5(12): 2466-2476, 2009

8)Comparative study of bio-ethanol production from mahula (Madhuca latifolia ) flowers by Saccharomyces cerevisiae cells immobilized in agar agar and Ca-alginate matrices.

Batch fermentation of mahula (Madhuca latifolia ) flowers was carried out using immobilized cells (in agar agar and calcium alginate) and free cells of Saccharomyces cerevisiae. The ethanol yields were 151.2, 154.5 and 149.1 g kg−1 flowers using immobilized (in agar agar and calcium alginate) and free cells, respectively.Also cell entrapment in calcium alginate was found to be marginally superior to those in agar agar (2.2% more) as well as over free cell (3.5% more) regarding ethanol yield from mahula flowers .

Applied EnergyVolume 87, Issue 1, January 2010, Pages 96-100

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9)Immobilization of nitrifying bacterial consortia on wood(Ailantus altissima) particles for bioaugmenting nitrification in shrimp culture systems

An inexpensive and user-friendly technology using immobilized nitrifying bacterial consortia (NBC) as bioaugmentors has been developed and proposed for adoption in shrimp culture systems.

Indigenous NBC stored at 4 °C were activated at room temperature (28 °C) and cultured in a 2 L bench top fermentor.

The consortia, after enumeration by epifluorescence microscopy, were immobilized on delignified wood particles of a soft wood tree Ailantus altissima (300–1500 μm) having a surface area of 1.87 m2 g− 1.

The products of nitrification in all experiments were undetectable due to denitrifying potency, which made the NBC an ideal option for biological nitrogen removal

AquacultureVolume 294, Issues 1-2, 1 September 2009, Pages 65-75

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References 1.Narayanswami.S, “Plant cell & Tissue Culture”2005, Tata Mc Graw-Hill Publishing Company Limited ,New Delhi, page no -573,575.

2.Kalia A.N, “Text book of Industrial Pharmacognosy”,published by C.B.S Publisher page no-130.

3.Rangari Vinod, “Pharmacognosy and Phytochemistry, Part-II , Ist edition, published by Career publication,page no:444-452.

4.Brodelius, P., Deus, B., Mosbach, K., and Zenk, M. H., 1979, Immobilized plant cells for the production and transformation of natural products, FEBS Letters, page no- 93 – 97.

5. Evans W.C; “Trease and Evans Pharmacognosy”,14thedition,Saunders publisher ,Page no- 76,77. 28

11.Journal of Molecular Catalysis B: Enzymatic Volume 56, Issue 1, January 2009, Pages 13-19

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6.Der Pharmacia Lettre, 2010, 2(2): 198-201.

7.Journal of Molecular Catalysis B: Enzymatic, Volume 69, Issues 1-2, April 2011, Pages 8-14.

8.Process Biochemistry, Volume 46, Issue 4, April 2011, Pages 1029-1034

9.Applied Energy ,Volume 87, Issue 1, January 2010, Pages 96-100

10.Aquaculture Volume 294, Issues 1-2, September 2009,Pages 65-75

11. Plant Cell Reports ,Volume 8, 546-549.

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12.Biosensors and Bioelectronics, Volume 26, Issue 11, July 2011, Pages 4399-4404

13. Journal of Colloid and Interface Science,Volume 356, Issue 1, April 2011, Pages 159-164

14. http://www.oocities.org/tumbur/cellimm.pdf

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