Cell culture and Secondary Metabolite Production

 What is Cell Culture?

Cell culture is the process of production of cells or growing out cells out of their normal environment (Plant/Animal bodies). It can also be defined as In-Vitro culture of single cells or group of cells.

It is also called as Tissue culture.

Depending on the part of the plant that is cultured, it is called as desired cell culture (gametic cells, cell suspension, and protoplast culture).Each type of culture is used for different basic and biotechnological application

Types Of Cell Culture: There are mainly two types of Cell Cultures

1. Cell Suspension Culture: Plant tissue culture on a liquid is said to be Cell Suspension Culture. For this the main condition is cell must be separated from each other, Mainly uses protoplast (cells without cell wall), microspores and macrospores.

2. Callus Culture: Plant tissue culture on a solid medium is said to be Callus Culture.

The plants still contribute significantly to the bulk of the market products, such as production of secondary metabolites. 

How cells grow?

The cell suspensions are grown in the closed (or batch) type where the cells are grown in fixed volume of liquid medium and which are routinely maintained through the transfer of a portion (ca 10%) of a fully-grown culture to fresh medium at regular intervals. The growth curve of a cell suspension culture has a characteristic shape consisting of four essential stages:

(1)  Lag phase, (2) Exponential phase, (3) Linear phase, (4) Stationary phase.

Secondary Metabolite production using Cell Cultures

Meaning of Secondary Metabolites: Plant products can be classified into two types: primary plant metabolites and secondary metabolites.

Primary plant metabolites are essential for the survival of the plant. It includes- sugars, amino acids and nucleotides synthesized by plants and are used to produce essential polymers. They are found in all and are produced using the same metabolic pathway. Primary metabolites provide the basis for normal growth and reproduction.

Secondary metabolites are the chemicals, which are not directly involved in the normal growth and development, or reproduction of an organism. Secondary metabolites are not indispensable for the plants but play an important role in plant defense mechanisms.

Major Advantages of Secondary metabolite production using cell culture

Ø  Compounds can be produced under controlled conditions as per market demands.

Ø  Culture systems are independent of environmental factors, seasonal variations, pest and microbial diseases and geographical constraints.

Ø  Cell growth can be controlled to facilitate improved product formation.

Ø  The quality of the product will be consistent as it is produced by a specific cell line.

Ø  Recovery of the product will be easy.

Ø  Plant cultures are particularly useful in case of plants which are difficult or expensive to be grown in the fields.

Ø  Mutant cell lines can be developed for the production of novel compounds of commercial importance, which are not normally found in plants.

The process of in vitro culture of cells for the large scale production of secondary metabolites is complex, and involves the following:

1. Selection of cell lines for high yield of secondary metabolites.

2. Large scale cultivation of plant cells.

3. Medium composition and effect of nutrients.

4. Elicitor-induced production of secondary metabolites.

5. Effect of environmental factors.

6. Biotransformation using plant cell cultures.

7. Secondary metabolite release and analysis.

1.Selection of Cell Lines for High Yield of Secondary Metabolites:

The very purpose of tissue culture is to produce high amounts of secondary metabolites.

However, in general, majority of callus and suspension cultures produce less quantities of secondary metabolites.

This is mainly due to the lack of fully differentiated cells in the cultures.

Some special techniques have been devised to select cell lines that can produce higher amounts of desired metabolites.

These methods are ultimately useful for the separation of producer cells from the non-producer cells.

The techniques commonly employed for cell line selection are cell cloning, visual or chemical analysis and selection for resistance.

Cell Cloning:

This is a simple procedure and involves the growth of single cells (taken from a suspension cultures) in a suitable medium.

Each cell population is then screened for the secondary metabolite formation.

And only those cells with high-yielding ability are selected and maintained by sub-cloning.

Single cell cloning:

There are certain practical difficulties in the isolation and culture of single cells.

Cell aggregate cloning:

Compared to single cell cloning, cell aggregate cloning is much easier, hence preferred by many workers.

A high yielding plant of the desired metabolite is selected and its explants are first cultured on a solid medium.

After establishing the callus cultures, high metabolite producing calluses are identified, and they are grown in suspension cultures.

Visual or Chemical Analysis:

A direct measurement of some of the secondary metabolites produced by cell lines can be done either by visual or chemical analysis.

Visual identification of cell lines producing coloured secondary metabolites (pigments e.g., β-carotene, shikonin) will help in the selection of high-yielding cells.

This method is quite simple and non-destructive.

The major limitation is that the desired metabolite should be coloured.

Certain secondary metabolites emit fluorescence under UV light, and the corresponding clones can be identified.

Some workers use simple, sensitive and inexpensive chemical analytical methods for quantitative estimation of desired metabolites.

Analysis is carried out in some colonies derived from single cell cultures.

Radioimmunoassay is the most commonly used analytical method.

Micro spectrophotometry and fluorescent antibody techniques are also in use.

2. Large Scale (Mass) Cultivation of Plant Cells:

In order to achieve industrial production of the desired metabolite, large scale cultivation of plant cells is required.

Plant cells (20-150 µm in diameter) are generally 10-100 times larger than bacterial or fungal cell.

When cultured, plant cells exhibit changes in volumes and thus variable shapes and sizes.

Further, cultured cells have low growth rate and genetic instability. All these aspects have to be considered for mass cultivation of cells.

The following four different culture systems are widely used:

1. Free-cell suspension culture

2. Immobilized cell culture

3. Two-phase system culture

4. Hairy root culture.

1. Free-cell Suspension Culture:

Mass cultivation of plant cells is most frequently carried out by cell suspension cultures. Care should be taken to achieve good growth rate of cells and efficient formation of the desired secondary metabolite.

Many specially designed bioreactors are in use for free-cell suspension cultures.

Some of these are listed below:

i. Batch bioreactors

ii. Continuous bioreactors

iii. Multistage bioreactors

2. IMMOBILIZED CELL CULTURE

Plant cells can be made immobile or immovable and used in culture systems.

The cells are physically immobilized by entrapment.

Besides individual cells, it is also possible to immobilize aggregate cells or even calluses.

Homogenous suspensions of cells are most suitable for immobilization.

Surface immobilized plant cell (SIPC) technique efficiently retains the cells and allows them to grow at a higher rate.

Further, through immobilization, there is better cell-to-cell contact, and the cells are protected from high liquid shear stresses.

All this helps in the maximal production the secondary metabolite.

The common methods adopted for entrapment of cells are briefly described:

1. Entrapment of cells in gels:

The cells or the protoplasts can be entrapped in several gels e.g., alginate, agar, agarose, carrageenin.

The gels may be used either individually or in combination.

The techniques employed for the immobilization of plant cells are comparable to those used for immobilization of microorganisms or other cells.

2. Entrapment of cells in nets or foams:

Polyurethane foams or nets with various pore sizes are used.

The actively growing plant cells in suspension can be immobilized on these foams.

The cells divide within the compartments of foam and form aggregates.

3. Entrapment of cells in hollow-fibre membranes:

Tubular hollow fibres composed of cellulose acetate siliconenpolycarbonate and organized into parallel bundles are used for immobilization of cells.

It is possible to entrap cells within and between the fibres.

Membrane entrapment is mechanically stable.

However, it is more expensive than gel or foam immobilization.

3. Medium composition and effect of nutrients.

The in vitro growth of the plant cells occurs in a suitable medium containing all the requisite elements.

The ingredients of the medium effect the growth and metabolism of cells.

For optimal production of secondary metabolites, a two-medium approach is desirable.

The first medium is required for good growth of cells (biomass growth) while the second medium, referred to as production medium promotes secondary metabolite formation.

The effect of nutrients (carbon and nitrogen sources,phosphate, growth regulators, precursors, vitamins, metal ions) on different species in relation to metabolite formation are variable, some of them are briefly described.

Effect of Plant Growth Regulators: Plant growth regulators (auxins, cytokinins) influence growth,

metabolism and differentiation of cultured cells. There are a large number of reports on the influence of growth regulators for the production of secondary metabolites in cultured cells.

4. Elicitor-induced production of secondary metabolites.

Elicitors are the compounds which induce the production and accumulation of secondary metabolites in plant cells. Elicitors produced within the plant cells include cell wall derived polysaccharides, like pectin, pectic acid, cellulose etc. Product accumulation also occurs under stress conditions caused by physical or chemical agents like UV, low or high temperature, antibiotics, salts of heavy metals, high salt concentrations which are grouped under abiotic elicitors. Addition of these elicitors to the medium in low concentration enhances the production of secondary metabolites.

The production of secondary metabolites in plant cultures is generally low and does not meet the commercial demands.

There are continuous efforts to understand the mechanism of product formation at the molecular level, and exploit for increased production.

The synthesis of majority of secondary metabolites involves multistep reactions and many enzymes.

It is possible to stimulate any step to increase product formation.

Elicitors are the compounds of biological origin which stimulate the production of secondary metabolites, and the phenomenon of such stimulation is referred to as elicitation.

Elicitors produced within the plant cells are endogenous elicitors e.g., pectin, pectic acid, cellulose, other polysaccharides.

When the elicitors are produced by the microorganisms, they are referred to as exogenous elicitors e.g., chitin, chitosan, glucans.

All the elicitors of biological origin are biotic elicitors.

5.Bioreactors for Use of Immobilized Cells:

Fluidized bed or fixed bed bioreactors are employed to use immobilized cells for large scale cultivation.

In the fluidized-bed reactors, the immobilized cells are agitated by a flow of air or by pumping the medium.

In contrast, in the fixed-bed bioreactor, the immobilized cells aremheld stationary (not agitated) and perfused at a slow rate with an aerated culture medium.

Biochemicals produced by using immobilized cells:

A selected list of the immobilized cells from selected plants and their utility to produce important

bio-chemicals is given in Table

3. Two-phase System Culture:

Plant cells can be cultivated in an aqueous two phase system for the production of secondary metabolites.

In this technique, the cells are kept apart from the product by separation in the bioreactor.

This is advantageous since the product can be removed continuously.

Certain polymers (e.g., dextran and polyethylene glycol for the separation of phenolic compounds) are used for the separation of phases.

4. Hairy Root Culture:

Hairy root cultures are used for the production of root-associated metabolites.In general, these cultures have high growth rate and genetic stability. For the production of hairy root cultures, the explant material (plant tissue) is inoculated with the cells of the pathogenic bacterium, Agrobacterium rhizogenes.

This organism contains root-inducing (Ri) plasmid that causes genetic transformation of plant tissues, which finally results in hairy root cultures. Hairy roots produced by plant tissues have metabolite features similar to that of normal roots. Hairy root cultures are most recent organ culture systems and are successfully used for the commercial production of secondary metabolites.