CELL CULTURES and SEKONDARY METABOLITES

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CELL CULTURESand SEKONDARY METABOLITES
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ESTABLISHMENT OF CELL CULTURES
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• When callus pieces are agitated in a liquid
  medium, they tend to break up.
• Suspensions are much easier to bulk up than
  callus since there is no manual transfer or solid
  support.
• Large scale (50,000l) commercial fermentations
  for Shikonin and Berberine.

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Introduction of callus into suspension

• Friable callus goes easily into suspension.
• 2,4-D
• Low cytokinin
• semi-solid medium
• enzymic digestion with pectinase
• blending

• Removal of large cell aggregates by sieving.
• Plating of single cells and small cell aggregates
  - only viable cells will grow and can be
  re-introduced into suspension.

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Introduction into suspension
Sieve out lumps 1 2
Initial high density

Subculture and sieving
Pick off growing high producers
Plate out
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Small cells and agregate
Long cell
Actively dividing cells
Cell types
Giant cell (G),
Giant cell (G)
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Growth kinetics

• 1. Initial lag dependent on dilution
• 2. Exponential phase (dt 1-30 d)
• 3. Linear/deceleration phase (declining
  nutrients)
• 4. Stationary (nutrients exhausted)

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4
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Characteristics of plant cells

• Large (10-100mM long)
• Tend to occur in aggregates
• Shear-sensitive
• Slow growing
• Easily contaminated
• Low oxygen demand (kla of 5-20)

• Will not tolerate anaerobic conditions
• Can grow to high cell densities (gt300g/l fresh
  weight).
• Can form very viscous solutions

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Special reactors for plant cell suspension
cultures

• Modified stirred tank
• Air-lift
• Air loop
• Bubble column
• Rotating drum reactor

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Modified Stirred Tank
Glasses for shakers
Wing-Vane impeller
Standard Rushton turbine
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Airlift systems
Poor mixing
Bubble column
Airlift (draught tube)
Airloop (External Downtube)
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Ways to increase product formation

• Select
• Start off with a producing part
• Modify media for growth and product formation.
• Feed precursors or feed intermediates
  (bioconversion)

• Produce plant-like conditions (immobilisation)

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Shaker
Bioreactor
controoledchamber
Glasses for shakers
Shakers
Glasses
Glasses for shakers
Rotary shaker
Glasses for shakers
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• Cell growth and differentiation
• Somatic embryo production

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• Secondary metabolite production

Anthocyanine production in cell cultures
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Secondary metabolites from plant cell cultures

• Features of these compounds
• low molecular wt
• synthesis (in plant cell) is tightly regulated
• A few products are commercially produced
• examples shikonin, ginseng compounds, berberine
• more are not produced because of two main
  problems
• low yields in in vitro culture
• feedback inhibition from SMs stored
  intracellularly

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Secondary metabolites from plant cell cultures

• Transport mechanisms in cultivated plant cells
• cells that transport SMs to vacuolar space for
  storage
• final yield limited to storage capacity of medium
• cells that secrete SMs into the medium
• final yield limited to properties of the medium
• production is more easily manipulated, but
  separation of culture medium from cultures is
  required
• SMs originating from cells in the medium,
  secreted into the medium, taken up by other cells
  and stored

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Secondary metabolites from plant cell cultures

• Techniques for increasing yields
• usually attempt to redirect transport
• permeabilization
• objective is to permeabilize tonoplast and/or
  cell membrane to drive secretion of SM into the
  culture medium
• chemical agents such as DMSO or Triton X-100
• problem poor viability after treatment
• two-phase systems

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Secondary metabolites from plant cell cultures

• Other systems
• immobilized cell culture (2 approaches)
• entrapment in alginate beads
• adherence of cells to a porous membrane
• in both cases, the objectives
• control of growth (high nutrient levels first,
  then elicitation or starvation)
• release of SM into the medium where it can be
  harvested

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Secondary metabolites from plant cell cultures

• Other systems
• hairy root culture culture of fast-growing
  roots by
• inoculation with Agrobacterium rhizogenes (esp.
  useful where differentiated cells are required
  for SM production)
• transfer of T-DNA causing hairy roots, then
  elimination of Agrobacterium by antibiotic
• (transformed) hairy roots are first subcultured
  to solid, then liquid medium

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Secondary metabolites from plant cell cultures

• Other systems
• hairy root culture (contin)
• SM is released into the medium, then harvested
• primary advantage a simple system w/o problems
  inherent with cell suspensions
• limited to spp. susceptible to A. rhizogenes
• biotransformation

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Secondary metabolites from plant cell cultures

• Other systems
• biotransformation
• chemical conversion of an exogenously supplied
  substance by living cell cultures
• example addition of hydroquinone (inexpensive
  precursor) into liquid culture medium containing
  vinca cells results in efficient production of
  arbutin (a skin depigmentation agent)

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Secondary metabolites from plant cell cultures

• Outlook for SM production in plant cell cultures
  (where plant cell systems may be preferred over
  whole plants)
• rare and endangered plants of pharmaceutical
  interest
• plants that grow too slowly to match a sudden
  demand (e.g., Taxol present only in the bark of
  Taxus brefolia)
• some cell cultures can be made to produce a
  single compound, e.g., sanguinarine from poppy
  cell culture

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diosgenin, kodein, morfin, atropin, hiyosiyamin,
skopolamin, digoksin, digitoksin, kuinin,
reserpin, artemisinin and taxol.
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• Lithospermum erythrorhizon, red pigment
  (shikonin),

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Biotransformation