ANIMAL AND PLANT CELL REACTOR TECHNOLOGY:

Growth of animal cells in culture is currently used for manufacture of

- Vaccines, proteolytic enzymes, Mabs, Interferons, etc.

These show substantial potential on production of lymphokines, other enzymes, growth
factors, clotting factors, hormones, etc.

Though r-DNA technology provides the opportunity of expressing foreign proteins in
microorganisms, animal cell cultivations also competes for the same.

Usually proteins synthesized in animal cells are often subjected to PTM, but this will not
happen in prokaryotes.

Molecules that required PTMs are better cultivated in eukaryotes.
Animal Cell Culture
Differences between procaryotes and eucaryotes
Eucaryotes Procaryotes
size 10-30 um 1-2 um
shape
spherical,
ellipsoidal
rods, ellipses,
etc.
locomotion no yes
border membrane wall
Cells are negatively charged.
attach to positively charges surfaces
some cells must attach to grow, others
not
examples of surfaces: sephadex,
collagen
positively charged vesicles will attach to cell
surfaces and be taken into the cell
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Animal Cell Culture Technique
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Historical events
- in the development of cell culture

1878: Claude Bernard proposed that physiological systems of an organism can be
maintained in a living system after the death of an organism.

1885: Roux maintained embryonic chick cells in a saline culture.

1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue
in serum and plasma.

1903: Jolly observed cell division of salamander leucocytes in vitro.

1907: Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging drop'
method and observed the growth of nerve fibers in vitro for several weeks. He was
considered by some as the father of cell culture.

1910: Burrows succeeded in long term cultivation of chicken embryo cell in plasma
clots. He made detailed observation of mitosis.


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Contd..
1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo
extract, salts and peptones. They observed limited monolayer growth.

1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long
periods.

1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of
adherent cells.

1923: Carrel and Baker developed 'Carrel' or T-flask as the first specifically designed cell
culture vessel. They employed microscopic evaluation of cells in culture.

1927: Carrel and Rivera produced the first viral vaccine - Vaccinia.

1933: Gey developed the roller tube technique
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Contd..
1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased
the problem of contamination in cell culture.

1948: Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer
developed a chemically defined medium, CMRL 1066.

1952: Gey established a continuous cell line from a human cervical carcinoma known as
HeLa (Helen Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent
monolayers of cultured cells.

1954: Abercrombie observed contact inhibition: motility of diploid cells in monolayer culture
ceases when contact is made with adjacent cells.

1955: Eagle studied the nutrient requirements of selected cells in culture and established
the first widely used chemically defined medium.

1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have
a finite lifespan in culture.

1964: Littlefield introduced the HAT medium for cell selection.

1965: Ham introduced the first serum-free medium which was able to support the growth of
some cells.

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Contd..

1965: Harris and Watkins were able to fuse human and mouse cells by the use of a
virus.

1975: Kohler and Milstein produced the first hybridoma capable of secreting a
monoclonal antibody.

1978: Sato established the basis for the development of serum-free media from
cocktails of hormones and growth factors.

1982: Human insulin became the first recombinant protein to be licensed as a
therapeutic agent.

1985: Human growth hormone produced from recombinant bacteria was accepted for
therapeutic use.

1986: Lymphoblastoid IFN licensed.

1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became
commercially available.

1989: Recombinant erythropoietin in trial.

1990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF,
EGF, mAbs, IL-2).

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Growth Medium
glucose, glutamine, amino acids,
serum: liquid extracted from blood of offspring removed from freshly-killed pregnant
cows.
proteins: cell attachment factors; metal binding proteins; protease inhibitors
peptides: various growth factors
hormones: stimulate growth and nutrient uptake
nutrients
metabolites
minerals
: Plasma
: Interstitial fluid
: Embryo extract
Growth factors
* cell require nutrients for use as substrate, catalysts or cofactor *
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Commercial cell culture media
: Minimum Essential Media (MEM)
: Dulbeccos Modified Eagle Media (DMEM)
: Opti-MEM
: RPMI-1630 (for suspension cell)
: RPMI-1640 (for mammalian cell)
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Metabolism
Animal cells can synthesize glucose from pyruvate via gluconeogenesis pathway
waste: lactate, ammonia
at high levels, these are toxic
challenge for high density cultures
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Cultivation of Animal Cells
1. Tissues are removed from animals and transferred to growth medium
2. Organs -> lung, kidney, etc. (cells grow attached)
3. These are primary cultures
4. Cells can be transferred to new flasks once they have grown into a monolayer
Remove cells with a protease trypsin, collagenase, pronase or EDTA
Wash cells with serum containing medium (centrifuge gently)
Resuspend in growth medium
Plate onto a fresh flask
Differentiated mammalian cells are mortal, however, cancer cell lines are immortal.
Animal cell lines include: mammal, insect, fish, crustaceans
Insect cells are easier to grow. They grow faster and you can use a baculovirus as a
vector for genetic engineering. Insect cells may not have post-translational
modifications like mammalian cells.
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Other Cells for Cultivation
Skin, epithelium cell, eye, kidney, liver, ovary
glands, bone, nerve
connective tissue, Skeletal muscle
tooth primmordia, bone marrow, lymphocyte
From young animals
Cell cloning, Separation, Hybrids,
Large scale production
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Commonly used cell lines
Chinese Hamster Ovary cells: CHO cells
HeLa cells
mouse kidney cells
Commonly used medium
nutrients + 5-20% serum ($100-$500 per liter)
Problems with serum
cost
virus safety issues
extra-cellular proteins
lot-to-lot variation
availability
foaming
Book: serum-free media contains insulin, transferrin, fibronectin, other protein
components
Serum-free media can also be protein-free
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Hybridoma Cells
antibody-producing lymphocytes fused with cancer cells myeloma
lymphocytes grow slowly and are mortal, hybridoma cells are immortal and
produce antibodies
Production of antibody fragments
by fungi and bacteria
See Nyyssonen and Eini;
Bio/Technology 1993 vol 11(5) p.
591.
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Tissue culture
Organ culture
: Maintain original structure and ability to differentiation
Primary explant culture
: 1-3 mm in size
Cell culture
: from single cell
: Lack cell-cell interaction and differentiation
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Growth Characteristics of Transformed cells
Transformed cells possess some common characteristics
but are not equivalent to cancer cells
Grown as Multilayer instead of Monolayer
Can grow in Suspension
Long life span
Less serum growth factor requirement
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Types of cell culture
Primary cell culture
Secondary cell culture
Diploid cell line
Continuos cell line (or Establish cell line)
*** Grown as Monolayer or in Suspension ***
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Monolayer culture
Requires substrate or solid surface
for attachment and growth
: Anchorage dependent of growth
Contact inhibition of movement: Monolayer
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Anchorage dependent of growth
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Suspension Culture
Mostly hemopoietic cells and a few others
Specialized culture medium may be required
Cells with high metabolic activities can be obtain
Cells that are not able to grow in suspension
: Those can be attached onto the surface
of carrier particles and grow
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Growth Requirement
Metabolites and intermediates from other cells
Population dependent growth requirement
: Diploid cells require a higher number to start with
To create a suitable condition, cells can be grown in
microenvironment in capillary tube
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Stationary cultivation
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Culture flask
Culture Plate
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Large scale cultivation
Roller Bottle
Culture dish Stack culture chamber
Culture bottle
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Medium Constituents
* Balance salt solution : Phosphate buffer, Mg
2+
, Ca
2+
* Inorganic ions and trace elements
: for membrane potential and osmotic pressure
: buffer
: Monovalent- and Divalent-cation
* Energy source : glucose, glutamine
* Amino acid : metabolism and biological synthesis
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Culture Medium Sterilization
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Role of Serum
Buffer, Chelator, Carrier proteins
Bind to toxin
Protease inhibitor
Promotes attachment of cell to substratum
Source of Intermediate metabolites,
hormone and growth factor
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culture vessel
* Glass or Plastics
* Polystyrene (gamma ray treated)
* Polyvinyl chloride
* Polycarbonate
*Polytetrafluoresthulene
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Microcarrier
* Polystyrene (gamma ray treated)
* Sephadex
* Polyacrylamide
Growth phase of Cells in culture
* Lag phase
: adapt to new environment; repair cell membrane damage
* Log phase
: exponential growth: 90-100% of cells are dividing
* Plateau or Stationary phase
: cell growth ~ 0-10%
: Contact inhibition of movement
: Density limitation of growth
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Advantages of using cell culture:
Can be observed microscopically
Genetic homogeneity
Environment
(pH, Temp., osmotic pressure, O
2
and CO
2
tension)
Rapid
Requires less amount of material
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Disadvantage of using cell culture:
Problem of contamination
May be expensive than using,
and may not represent the condition in, intact animal
Chromosome instability
Primary cell culture and Establishment of cell line
Preparation of cell suspension from intact tissue
1. Single cell preparation
: use mechanical, Chemical, and/or enzymatic method
2. Disaggregate or dissociate cell
: cutting, homogenizing, rotary shaker, vortex,
pipette, teasing
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** Enzymes used **
Trypsin (crude)
: from cattle and pigs pancrease
: contain Chymotrypsin, elastase, ribonuclease,
deoxyribonuclease and amylase
Collagenase
: for connective tissue
Pronase
: for fibroblast
Elastase
: for fibroblast protein
Deoxyribonuclease
: for DNA
** Enzymes used **
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** Chelating agents used **
Ethylene diamine tetraacetic acid (EDTA) or Versene
: bind permanently to Ca
2+
and Mg
2+

that maintain the cellular matrix
: prevent cell aggregation
: Its better to use in combination with Trypsin
Sodium citrate
Source of tissue
: Young animals
e.g. kidney (Monkey, Dog, Rabbit),
Chick embryo
: Old animal tissue contains a large amount
of connective tissue

Common Cell lines used for animal cell cultivation
BHK-21 : 1961
: from bay hamster kidney
: FMD and Rabies vaccine for animal use
CHO-K1: 1957
: from Chinese hamster ovary
: use in recombinant DNA technology
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HeLa : 1952
: from Henrietta Lach; cancer tissue
: harbors HPV type 18 genome
Vero : 1962
: from African green monkey kidney
: preparation of Poliovirus vaccine
Contamination sources of animal cells:
fungal contamination
Bacterial contamination
Mycoplasma contamination
Viral contamination
Other cell line
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Original tissue : primate virus, mycoplasma
Biological: Serum
Laboratory personnel : from body, aerosols
Laboratory environment
: culture vessel cap
: humidified Incubator
: Water bath
: Insect
Animal Cell Storage:
*** Prevent genetic drift ***
: Freezing Medium
* Serum (~ 20-90%)
* Culture medium
* Cryoprotective agent : ~ 5-10% (DMSO, Glycerol)
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: Temp. decline rate 1-10
o
C/min
* (-20
o
C) Freezer
* (-70
o
C) Freeze : 6M-2 yr
* liquid nitrogen: Years
: cell concentration ~ 5 x 10
6
-2 x 10
7
cells/ml
: % cell viability is decrease 2-3%/yr
** Slow Freeze : Quick Thaw **
Cell Thawing and culture
1. Quick thaw in 37
o
C water bath
2. Pipette to culture vessel
3. Slowly growth medium adding
4. Incubate for overnight
5. Refresh with new growth medium

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Subcultivation or Passage
Age of cells in culture can be determined by

* Number of subcultivation (Passage number)

* Number of population doublings
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Kinetics of growth are similar to bacterial culture
There is a difference between attached and suspended cultures
Disposable bioreactors
Cell growth measured by actual cell counts
Hemocytometer
Stain cells and drop on the slide count all the white ones
Cell growth is measured in days.
Production can continue in non-growth conditions hopefully!
Oxygen requirements: .06 - .2 x 10
-12
mol O
2/h/cell
OUR ~ 0.1-0.6 mmol O
2
/l/hr
Compare to bacteria at 10 200 mmol/l/hr!
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Animal Cells are shear sensitive cannot sparge reactors
cells respond to shear with apoptosis
Fritted metal fittings create very small bubbles
Chemical (e.g. Pluronic F-68) can be added to provide shear protection
Typical k
L
a of suspension cultures (10
6
cells/ml) 5 25 hr
-1

Bioreactor Considerations for Animal Cell Culture
Microcarriers: sephadex, etc: 70,000 cm
2
/liter: get ~ 10
7
cells/ml
cells grow in mono multi-layers on microcarriers
Hollow Fiber reactors
cells grow on the outside of the tubes, nutrients pass through the tubes
uncontrolled, unmixed environment
get high cell concentrations (eg hybridoma demonstrated at 5 50 mg/ml
antibody
Stirred-Tank reactors
use pitched blade or other impeller (10 30 RPM for stirrers)
Tank and bubble columns are used (especially with cells on multicarriers)
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Perfusion reactor
simultaneous cell cultivation and product concentration and byproduct removal
sonic separator
Products from Animal Cell Cultures
1. Immunobiologicals:
(i) monoclonal antibodies
(ii) immunobiological regulators
a) Used for diagnostic assay systems, therapeutics for biological
separation systems (affinity chromatography)
b) Interferon
2. Virus Vaccines
3. Hormones: glycosylated peptides (e.g. erythropoetin)
4. Enzymes: TPA, collagenase,factor VII, factor VIII, factor X
5. Insecticides
6. Whole cells and tissue culture

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Work in Safety cabinet Class-II
: 30 min UV
: 70% ethanol for decontamination
Culture medium and Reagent
1. (1x) PBS
2. Growth medium
: 5%FCS-DMEM
3. ( 0.1%) Trypsin-Versene
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PLANT CELL REACTOR
TECHNOLOGY
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Differences between plant cells and microbes and their implications for Bioreactor
design
Differences Implications for reactor design
Lower respiration rate Lower OTR required
More shear sensitive May require operation under low-shear
conditions
Cells often grow as aggregates or clumps Mass transfer limitations
Degree of aggregation Optimal aggregate size for product
Volatile compounds may be important for
cell metabolsim (ethylene)
May need to sparge gas mixtures
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Advantages and disadvantages of
Plant cell cultivation
Advantages
Can manipulate
environment
Can feed precursors
Possible to select in culture
Possible to get all cells in a
culture producing.
Can continuously extract.
Can retain biomass

Disadvantages
High cost
Contamination
Low intrinsic production
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Cost of production
Plant cells are slow growing.
Full of water (90% - 95%).
Easily contaminated.
Shear-sensitivity means specially modified
fermenters necessary
All this puts the cost of production of dry mass to
$25 per kilogram. Product only a fraction of this.
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Plant cell culture systems
Organised
Shoot cultures.
Hairy root cultures
Embryo fermentations.
Unorganised
Callus
Cell suspension culture
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Shoot cultures
Under conditions of high cytokinin, a culture
producing a mass of shoots may be produced by
adventitious shoot formation.
For light-associated products, may be much more
high yielding.
Sensitive to shear
Illumination a problem for scale up
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Hairy root cultures
Hairy roots are produced by infecting sterile plants
with a natural genetic engineer, Agrobacterium
rhizogenes.
Genes for auxin synthesis and sensitivity are
engineered into plant cells leading to gravity-
insensitive mass root production.
Very useful for products produced in roots.
Aggregration and shear sensitivity are a major
problem for scale-up
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Embryo Fermentations
Somatic Embryos may be produced profusely from
leaves or zygotic embryos.
For micropropagation, potentially phenomenally
productive.
Shear sensitivity is a problem.
Maturation in liquid is a problem.
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Shikonin production in culture
Shikonin production in the intact plant
Introduction into culture
Optimisation of production through medium
manipulations
Fermentation
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Callus
Equimolar amounts of auxin and cytokinin stimulate cell
division. Leads to a mass proliferation of an unorganised mass
of cells called a callus.
Requirement for support ensures that scale-up is limited
(Ginseng saponins successfully produced in this way).
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Cell suspension culture
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 (50,000 lit.) 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
+

Plate out

Sieve out lumps
1 2

Pick off
growing
high
producers
Initial high
density
Subculture
and sieving
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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)
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14 16 18 20 22
D
r
y

w
e
i
g
h
t

(
g
/
l
)
time (d)
Plant Cell Suspension typical
Growth curve
1
2
3
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 (k
L
a of
5-20)
Will not tolerate anaerobic
conditions
Can grow to high cell
densities (>300g/l fresh
weight).
Can form very viscous
solutions
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Shear and plant cells
Oxygen demand proportional to cell density.
Shear rate proportional to viscosity
shear rate proportional to **power of viscosity
3/9/2012 68
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
Standard Rushton turbine
Wing-Vane impeller
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Airlift systems
Bubble column Airlift (draught
tube)
Poor mixing
Airloop (External
Downtube)
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Rotating Drum reactor
Like a washing machine
Low shear
Easy to scale-up
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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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Selection
Select at the level of the intact plant
Select in culture
single cell is selection unit
possible to plate up to 1,000,000 cells on a Petri-
dish.
Progressive selection over a number of phases
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Selection Strategies
Positive
Negative
Visual
Analytical Screening
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EXAMPLES OF PLANT PRODUCTS OF POTENTIAL COMMERCIAL INTEREST
1. Pharmaceuticals
- Ajmalicine, atropine, berberine, codeine, digoxin, taxol,etc
2. Food colors & Dyes
- Anthocyamins, betacyanins, saffron, shikonin
3. Flavors
- Vanilla, strawberry, grape, onion, garlic
4. Fragrances
- Jasmine, lemon, mint, rose, sandalwood
5. Sweeteners
- Miraculin, monellin, thaumitin
6. Agriculture chemicals
- Alloepathic chemicals, rotenone, salannin, etc.