ERT211 BIOCHEMICAL ENGINEERING. Course Outcome Ability to describe the usage and methods for cultivating plant and animal cell culture Ability to discuss.

ERT211 BIOCHEMICAL ENGINEERING

Course Outcome Ability to describe the usage and

methods for cultivating plant and animal cell culture

Ability to discuss the technologies available in bioconversion

Chapter 5

Considerations in Using Plant and Animal Cell Cultures

Topic Outline Usage of plant cell cultures Potential product from plants cell

cultures Approaches to increase productivity Difference of plant cells from

microbes - implications for bioreactor design

What is plant cell culture?

Plant cell culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant.

The purpose of practicing plant and cell culture includes for regeneration/propagation, embryogenesis and secondary metabolites production.

In this technique, culture medium components, explant source and plant growth regulator is the important factors.

Procedures should be follows includes laboratory set up, medium preparation, sterilization, initiation and maintenance of cultures.

Plant cell culture techniques

Laboratory requirements for plant cell culture Laminar air flow cabinets Autoclave Oven for dry sterilization Water distillation apparatus Incubator Shakers Fermenters or bioreactors

Materials and methods

Plants – any part obtained from any plant species can be employed to induce callus tissue

Media - inorganic salt

- Carbon sources

- Vitamins

- phytohormones

- organic supplements Or ready made medium – Murashige and Skoog,

Gamborg, B5, Nitsch etc

Establishment of plant cell culture Callus and suspension cultures have been

established from hundreds of different plants A callus can be formed from any portion of the

whole plant containing dividing cells Callus – tissues arising by proliferation from

explants on agar medium/Mass of undifferentiated cells produced in tissue culture is called callus. The callus is highly vacuolated and unorganised cells.

Suspension cultures – cells or small cells aggregates growing dispersed in liquid medium

Basic protocols for suspension culture

Excised plant material is placed on solidified medium

Callus forms can be quite large (> 1 cm) Callus transfer to liquid medium (for propagation) Establishment of suspension culture from callus

is straightforward if callus is friable A piece of callus is placed in liquid medium in

shake flask. With moderate agitation, cells or small

aggregates of cells will slough off A platform shaker is used to give a

circular/orbital motion in a variable speed control (a range form 30-150 rpm).

Cond. 25°C, pH5.5 in the dark These suspended cell then replicate After 2/3 weeks, suspended cells are transferred

to fresh medium For large scale suspension cultures, bioreactors

system is being used for the purpose of producing high value plant products.

Implication for bioreactor design

Plant cell vs microbes

Characteristics Microorganism Plant Cell

Size

Shear stress

Water content

Duplication time

Aeration

Fermentation time

Product accumulation

Product phase

Mutation

Medium cost ($)

2Insensitive

75%

<1 hour

1-2 vvm

Days

Medium

Uncoupled

Possible

8-9/m3

>10Sensitive

>90%

Days

0.3 vvm

Weeks

Vacuole

Often growth linked

Required haploids

65-75/m3

Implication for bioreactor design

Many of the diff between plant cell and microbes have direct implication for the design and scale up of suspension culture

Differences Implication for bioreactor design

More shear sensitive May require operation under low-shear condition

Lower growth rate May need large bioreactor volume

Lower respiration rate Lower O2 transfer rates required

1. Shear Stress Plant cells are large compared to

microorganism When they are expose to high shear stress,

the cell will damage Reactors with high shear must be avoided Plant cells may require operation under

low-shear conditions.

2. Aeration In aerobic fermentation processes,

microorganisms require high aeration (have high metabolic actv) compare to plant cells

Require to change the bioreactor system that provide high aeration rate.

system can be equipped with mechanical devices used to agitate the liquid broth or by air compressor.

Eg airlift reactor

Airlift reactor•Mixing in airlift bioreactors usually accomplished without any mechanical agitation•The bioreactors are used for tissue culture because the tissues are shear sensitive and normal mixing (aeration plus mechanical agitation) is not possible

3. Size Plant cells are large compared to

microorganisms. Cell often grow as aggregates or clumps.

Cell often grow as aggregates or clumps and may have mass transfer limitations that limit the availability of nutrients to cells within the aggregate.

In some case, aggregates are necessary to achieve a mix of cell types essential for good secondary-metabolites formation

4. Duplication times Duplication time in microorganisms is within

days compare to plants within weeks. To contain the rapid multiplication of cells,

larger bioreactor volumes is needed and usually in the range of 10,000 – 100,000 L.

Products are produce within shorter period. Shorter period in the culture systems means

less contamination and less cost for the maintenance.

Advantages of using plant cell cultures for factory production of

chemicals

1. Control of supply of product independent of availability of the plant itself

2. Cultivation under controlled and optimized condition

3. Strain improvement with programs analogous to those used for microbial systems.

4. With the feeding of compounds analogous to natural substances, novel compounds not present in nature can be synthesized

1) Control of supply of product independent of availability of the plant

itself Many drugs contained active ingredients source from

plant parts. Paclitaxel (taxol) - anticancer agent - isolated from bark of Pacific

yew tree (Taxus brevifolia) - Three 100-year-old trees is

needed to supply enough paclitaxel for

one patient Supply problems greatly impeded the clinical

development of this drug

Production of paclitaxel in large-scale bioreactors provides a product with fewer contaminants, highly controllable and reproducible.

Recent, commercial production of paclitaxel in stirred tank vessel 30000 l

2) Cultivation under controlled and optimized condition

The accumulation substances extracted from plants affected by many factors etc. condition of the plant and environment

Production of saponin via tissue culture technique from Panax ginseng was affected by the concentration of hormones used.

2,4-D (mg/l)

5 10

Kinetin (mg/l)

1 1

Saponin (%)

3.62 8.78

Callus cultures of Oxalis reclinata are yellow in the light, but when they are transferred to the dark, they start producing red anthocyanin pigments.

3) Strain improvement with programs analogous to those used for microbial

systems

The physiological characteristics of individual plant cells are not always uniform

In tissue culture, selection of cell lines for specific compound production will increase in yield.

Selection using radioimmunoassay. Similar method to monocolony isolation of bacteria

radioimmunoassay (RIA), highly sensitive laboratory technique used to measure minute amounts of substances including antigens, hormones, and chemicals present in plant cells

A strain of Euphorbia milli was recognized to accumulate about 7 times higher amounts of anthocyanins than that of the parent strain after 24 selections.

4) With the feeding of compounds analogous to natural substances, novel compounds not present in the nature

can be synthesized

Addition to the culture media of appropriate precursors or related compounds sometimes stimulates secondary metabolite production

Precursors can be synthetic or natural substances

This approach is advantages if the precursors are inexpensive

The production of limonene is improved with the addition of mevalonic acid (primer precursor)

Acetyl CoA

3-hydroxyl-3-methylglutaryl CoA

Mevalonic acid (Primer Precursor)

+

dimethylallyl pyrophosphate

geranyl pyrophosphate Linalool (Immediate precursor)

Limonene

HOOCCH2CCH2CH2OH

CH3

OH

O PP PP O

O PP

OH

O PP

A diagram of limonene biosynthetic pathway.

Potential products from plants cultures

PharmaceuticalsFood additives

Morphinan alkaloids Codeine is an analgesic & cough-

suppressing drug Morphine from Papaver somniferum L.

(opium poppy) is a traditional commercial source of codein. Morphine can be converted to codeine

Thebaine from P.bracteatum also can be converted to codeine

Capsule of Papaver somniferum showing latex (opium) exuding from incision

Little success has been achieved from culture of undifferentiated cells of these plants for codiene production

After many attempts, researchers still failed to get direct codeine and morphine from cell culture

Later, Japanese researchers (Furuya et al), studied the biotransformation of codeinone to codeine using immobilized cells of P.somniferum

The conversion yield was 70.4% and about 88% of codeine converted was excreted into the medium

Shikonin compounds

Shikonin and its derivatives (e.g. acetyl shikonin & isobutyl shikonin) are reddish purple pigments accumulated in roots of Lithospermum erythrorhizon

They have been used in traditional dying and as a herbal medicine

Because of shortage of this plant, Mitsui Petrochemical company in Japan investigated mass cultivation of L. erythrorhizon cells to produce shikonin compounds.

Two-stage culture for shikonin production

Approaches to increase productivity

a) Optimization of cultural conditionb) Selection of high-producing strainsc) Addition of precursorsd) Biotransformatione) Elicitor treatment

In order to obtain products in high concentration, many effort have been made to stimulate or restore biosynthetic activities of cultured cells using various methods.

several typical approaches that may increase productivity of cultured plant cells :

a) Optimization of cultural conditionb) Selection of high-producing strainsc) Addition of precursorsd) Biotransformatione) Elicitor treatment

1) Optimization of cultural conditions

Medium -The most important is the medium that influence both the growth

of cells and yield of desirable products

-Various basal medium have been used and Murashige & Skoog (MS) is among the most widely applicable

-Sucrose and glucose are carbon source for plant tissue cultures and affects cell growth and yield of products

-Phytohormones such as auxins and cytokinin have shown the most remarkable effects on growth and productivity of plant metabolites.

Surrounding environment (temp,pH,light & O2)

- Generally, a temperature of 17-25°C is normally used for induction of callus tissues and growth of cultured cells

- The pH is usually adjusted between 5-6 before autoclave

- The use of light depends on the type of culture and the desire products

- Oxygen is not critical for plant cultures but still has an effect on the growth and production

Cell density- The use of high cell density cultures in a suitable

bioreactors found to increase yield in some cultures

TWO-STAGE CULTURE- Some plant cells needs different media for the cell

growth and secondary metabolite production. - This cultural condition is called two-stage culture. It means that 2 types of culture medium are used. both culture medium may differ in the

concentration or types of nutrient, hormone or vitamins used.

two types of medium were used because the medium used for promoting growth is not suitable for promoting metabolites production.

- In such system, the first medium is used to promote the growth of cells in the culture system.

- the second culture is to promote the production of metabolites in the culture.

- Cells of plant will be first cultivate in the first medium and after certain period, they will be transferred into the second growth until harvesting period.

Two-stage culture for shikonin production

2) Selection of high-producing strains

The physiological characteristics of individual plant cells are not always uniform

Therefore a rapid assay method is crucial in the selection of a high yielding cell line

The specific cell line is obtained from the selection a number of strains producing high level of desirable product

The strains then were subjected to further cell cloning to increase the level of secondary metabolites

3) Addition of precursors

Precursor – a compound that participate in the chemical reaction that produce another compound

Addition of precursors to the culture media sometimes stimulates secondary metabolite production

This approach is advantageous if the precursors are inexpensive

For exp : Phenylalanine is one of the biosynthetic precursors of rosmarinic acid. Addition of this amino acid to Salvia officialis suspension cultures stimulated the production of rosmarinic acid and shortened the production time as well.

4) Biotransformation A suitable substrate compound may be

biotransformed to a desired product using plant cell

Biotransformation has been extensively applied in the fermentation using microorganisms and their enzymes

For example, L-aspartic acid and L-malic acid can be biotransformed from fumaric acid using microorganisms

Using plant cells, for exp. the biotransformation of -methyldigitoxin to -methyldigoxin using Digitalis lanata has been investigated

- Digoxin has a large market as a cardiac glycoside

This approach beside precursors feeding are the most commercially realistic approaches because of economic reasons

5)Elicitor treatment Elicitor is an agent of microbial infections on

intact plants that cause the synthesis of specific secondary metabolite.

Some studies reviewed possible correlations between stress and secondary metabolism in cultured cells.

Some suggested that upon infection, plants shows their defense mechanism by secreting secondary metabolite.

Elicitors that have been used in plant cell cultures are yeast extract, chitosan, inorganic and organic molecules and many more

Plants grow under stress condition also show elicitation effects. Phosphate limitation in hairy root cultures of Hyoscyamus muticus had increased production of the sesquiterpene solavetivone

Examples of inorganic compounds used are sodium chloride, potassium chloride, sorbitol and abscisic acid

For economical use of the elicitors, they should be cheap and easy to obtain.