Immobilized cell techniques(survey of recent advances) GUNJA SRIVASTAVAM.PHARM (II SEM) PHARMACOGNOSY
PRESENTED TOPRESENTED BYMR.RAJIV GUPTAMR.ANURAG MISHRAMR.VINEET GUPTAMRS.DIPALI PANDEY
Cell immobilization technique2
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 approach3
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.44
Rationale for plant cell immobilizationImmobilization 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. 5
conceptThe 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 metabolitesAnother 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.66m
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 toSupply precursor of secondary product in large quantity but in low concentration Perform sequential chemical treatment on the cellsHarvest metabolite from nutrient medium77
METHODS OF IMMOBILIZATIONGel entrapment by ionic network formationGel entrapment by polymerisation Entrapment in the preformed structures. Gel entrapment by precipitation. 8
Gel entrapment by ionic network formationEntrapment 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 .9 After
Gel entrapment by precipitationPreparations 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 10Gel 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.11
Gel Advantages/disadvantagesConcentrationAgarNon toxic & freely permeable of inactivated cells 2%Polyacrylamide GelatinAdverse effect on cell viability
Form cross linked gelatin foam with 25% glutaraldehyde -
20% K-CarrageenanForms gel in the presence of K+ ions 3%Various types of gels used for immobilization12
Entrapment in the preformed structuresEntrapment in preformed structures involves some form of open network through which nutrient medium may pass, but which entraps plant cells or cell aggregates.
Such structures can be facilitated by using cotton fibre, fibreglass mats, reticulate polyurethane foam, and in a cloth nonwoven polyester short fibres.
The 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. Immobilized Capsicum frutescence cells in reticulate polyurethane foam, showed that the immobilized cells produced more capsaicin than the free cells.
Bioreactors involved in Immobilization Technique.Flatbed bioreactorThe flat bed bioreactor is one of the simplest cell immobilization systems availableThe 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.14
Column BioreactorColumn 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. 15Nutrient MediumreservoirCulture vesselPolypropylene substratumcellsPeristaltic pumpPeristaltic pumpColumn Of immobilized cellsThe flat bed culture systemThe column culture system161 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
17 LightMetabolism of cult