Download PLANT AND ANIMAL CELL CULTURE 2015A

BIOCONVERSION
TECHNOLOGIES OF ANIMAL
CELL AND PLANT CULTURE
SALEHA SHAMSUDIN
0132081261
Considerations in Using Animal Cell
Culture (Chapter 12)
a) Structure and biochemistry of animal
cells
b) Methods used for the cultivation of
animal cells
c) Products of animal cell cultures
Eukaryotes: Animal cell
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
Animal cells are eukaryotic cells, or cells with a
membrane-bound nucleus that makes up many
tissues in animals..
contain other membrane-bound organelles, or tiny
cellular structures, that carry out specific functions
necessary for normal cellular operation.
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•
•
a)
b)
c)
d)
e)
f)
Size: between 10-30 μm.
Shape: (spherical or ellipsoidal).
Cell structure (typically Eukaryotes) :
Do not have cell wall.
Surrounded by thin and fragile plasma membrane
(composed of protein, lipid and carbohydrate) and has
significant shear sensitivity.
Some cells , a portion of plasma membrane is modified
to form a no. of projections called microvilli- to increase
surface area to provide more effective passage of
materials across the plasma membrane.
Composition of plasma is varies in different region.
Surface of the cell is negatively charged and cells tend
to grow on positively charge surface such as collagen
(for anchorage-dependent cells).
Posses specific cell surface receptors that adhere to
ligand on the surface.
Figure 1: structure of animal cell
Inside the cytoplasm
is an extensive
network of
membrane-bounded
channels called
endoplasmic
reticulum (ER)
Structure and function of organelles
Organelles
Function
Nucleus
• Regulate synthesis of proteins in cytoplasm through
mRNA
• Nucleolus- site of ribosome synthesis
• Chromosome: nuclear material (DNA) and some dark
granular structures called nucleoli
Plasma membrane
• selectively permeable to ions and organic molecules and
controls the movement of substances in and out of cells
Smooth Endoplasmic reticulum
(lumenal phase)
• Lipid synthesis
Rough endoplasmic reticulum
(cytosol)
(with ribosome)
• Critical in protein synthesis and posttranslational
processing
Mitochondria (Power house of cell)
• Site of respiration and production of ATP.
• Containing DNA
Golgi complex
• Completion of complex glycosylation
• Collecting and secreting extracellular proteins
• Directing intracellular protein traffic to other organelles.
Lysosomes
• Contain hydrolytic enzymes (proteases, nucleases
and esterases)
• Digestion of certain food particles ingested by the
cell
Peroxisomes and
glyoxysomes
Cytoskeleton
• Provide cell mechanical strength and control its shape
• critical in cell movement
• Transduction of mechanical forces into biological
responses
• Separation of chromosomes into two daughter cells
during cell division
Characteristic of animal cell
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Some animal cells are non-anchorage dependent
and grow in suspension culture.
Has cytoskeleton or system of protein filaments
(actin filaments, intermediate filaments and
microtubules)-provide cell mechanical strength,
control shape and guide cell movement.
Some animal cells contain cilia- used to transport
substrate across the cell surface.
Typical growth media culture

contains glucose, glutamine, non-essential and
essential amino acids, serum, mineral salts (e.g:
DME, Dulbecco’s Modified Eagle’s media)
Major metabolic pathways for
animal cell in culture (FIGURE 12.2)
• Glucose
is converted to pyruvate by glycolysis and
biomass synthesis through the pentose phosphate pathway
• Pyruvate is converted partly to CO2 and H2O by the TCA
cycle, partly to lactic acids and partly to fatty acids
•Part of glutamine deaminated to yield ammonium and
glutamate, which is converted to amino acids to biosynthesis
purposes
•Glutamine also enters into the TCA cycle to yield carbon
skeletons for other amino acids and to yield ATP, CO2 and
H2O
•The release of lactate and ammonia as waste products of
metabolisme (toxic to cells) is a problems to high-cell
density culture systems.
Methods Used for The
Cultivation of Animal Cells
Differ significantly from those used with bacteria, yeast
and fungi
Tissue excised from specific organs of animal such as
lung and kidney, under aseptic conditions are transfer
into a growth medium containing serum and small amount
of antibiotics in small T-flasks.
These cells form primary culture and do not normally
form aggregate
Grow in the form of monolayer on support surface such
as glass surface or flasks.
Using the proteolytic enzyme trypsin, individual cells in
a tissue can be separated to form single-cell cultures.
Steps in animal cells culture
1)Excised tissues are cut into small pieces (~
2mm3)
2)Placed in an agitated flask containing dilute
solution of trypsin (~0.25% w/v) in buffered
saline for 120 min at 37°C
3)The cell suspension is passed through a presterilized filter to clear the solution
4)Cell are washed in the centrifuged
5)Cells are re-suspended in growth medium
6)Placed in T-flasks or roller bottles
7)Cell attach to glass surface and form monolayer
Anchorage-dependent cells : Cells growing on support
surfaces
Nonanchorage-dependent cells : Cells grow in
suspension culture
Primary culture: The cells that directly derived from
excised tissues
Secondary culture: A cell line obtained from the
primary culture
Step for removing cell
1.
2.
3.
4.
5.
Removal solution for cells : EDTA, TRYPSIN,
COLLAGENASE OR PRONASE
The exposure time for cell removal : 5-30 min
(37°C)
After cells are removed from surfaces, serum is
added to the culture bottle
The serum-containing suspension is centrifuged,
washed with buffered isotonic saline solution and
used to inoculate secondary culture
Secondary lines can be adapted to grow in
suspension
Mamalian cells are divided by Normal (mortal) and immortal
(continuous/transformed)
Normal: Divide only for limited of generation (30generations)
Transformed: Can be propagated
Characteristic:
Contact inhibition: cell division is
inhibited when cell’s surface is in
contact with other cell
No contact inhibition: the cells do not
sense the presence of other cells and
keep dividing
The culture of Hybridoma Cell
Progeny are called hybridomas and can be
propagated indefinitely, can secrete highly specific
antibodies againts antigen.
Obtained by fusing lymphocytes (normal blood
cells that make antibodies) with myeloma (cancer)
cells
Lymphocytes producing antibodies grow slowly
and are mortal
After fusion with myeloma cells, hybridomas
become immortal, can reproduce and produce
antibodies.
Steps in formation of a hybridoma for making
(b) Lymphocytes in the
antibody
(a) Antigen is injected
into a mouse.
mouse are activated to
produce specific antibodies
to the antigen
(c) Lymphocytes are
collected from the
mouse
(d) Myeloma (cancer)
cells growing in tissue
culture are produced
(e) Myeloma are fused
with lymphocytes
(f) The hybrid cell grows
well in tissue culture and
makes a single
monoclonal antibody
Serum
A typical growth medium for mammalian cells
contains serum (5-20%), inorganic salts, carbon and
energy sources, vitamins, trace elements, growth
factor and buffer in water.
Serum is a cell-free liquid recovered from blood
(FBS-fetal bovine serum; CS-calf serum; HS-horse
serum)
Serum is known to contain amino acids, growth
factors, vitamins, certain protein, hormones, lipids and
minerals.
Serum’s function:
1. To stimulate cell growth and other cell
activities by hormone and growth
factors
2. To enhance cell attachment by certain
proteins such as collagen and
fibronectin
3. To provide transport proteins carrying
hormones, minerals and lipids
Kinetic Growth of Mammalian Cell Culture
No. Cells
1. Mammalian cells
2.
3.
Insect cells
Fish cells
Growth condition
37°C, pH ~7.3
Doubling time: 12 – 20 h
Need to be gently aerated
and agitated
Buffer used: Carbonate
buffer/CO2-enriched
air/HEPES
28°C, pH 6.2
25°C-35°C, pH 7 – 7.5
Similar to microbial growth
Stationary phase is relatively short
Concentration of viable cells drops sharply as a
result of toxic accumulation (lactate-from glucose
metabolisme and ammonium-from glutamine
metabolisme)
Reach peak value from 3 to 5 days
Product formation (monoclonal antibody
formation by hybridoma cell) can continue under
nongrowth conditions
Normally, most of mammalian cells cultures are
mixed-growth associated (during growth phase until
after growth ceases)
Products of Animal
Cell Cultures
Consists of high-molecular-weight proteins
with or without glycosidic groups
There are enzymes, hormons, vaccines,
immunobiologicals (monoclonal antibodies,
regulators-lymphokines, virus vaccines),
anticancer agents
Large molecules: 50-200 amino
acids
Produce by hormone-synthesizing
organ
May also produce by chemical
synthesis
Example: Erythropoetin
Hormones
Prophylactics
Virus is collected, inactivated
and used as vaccine
A weakened form will induce a
protective response but no
disease
Monoclonal
Antibodies
(Mab’s)
Products
Produced by hybridoma cell
Used for diagnostic assay systems
(determine drugs, toxins & vitamin);
theraopeutic purposes & biological
separations – chromatographic
separations to purify protein
molecules
Immunobiological
Regulators
Interferon – anticancer
glycoprotein (secreted
animal cell or recombinant
bacteria)
Virus
vaccines
Lymphokines
Interleukines (anticancer
agent)
Enzymes
Artificial organs and semi
synthetic bone and dental
structure
Whole
cells and
tissue
culture
Urokinase, rennin, asparaginase,
collaginase, pepsin, trypsin, etc..
Produced some insect
viruses that are highly
specific and safe to
environment
Products
Insecticides
Considerations in Using Plant Cell
Culture (Chapter 13)
a) The importance and advantages of plant
cell culture
b) Plant cells in culture compare to
microbes
c) Methods used for the cultivation of plant
cells Economics of plant cell tissue
culture
Many thousands of chemicals are
produced only in plant. The great
genetic potential of plants to
produced compounds has been little
exploited and the rapid destruction
of forest leading to the extinction
of many plants.
ADVANTAGES OF PLANT TISSUE CULTURE OVER
CONVENTIONAL AGRICULTURAL PRODUCTION
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In vitro grown plants is independent of geographical variations,
seasonal variations and also environmental factors, therefore it
offers a defined production system, continuous supply of products
with uniform quality and yield.
Novel compounds which are not generally found in the parent
plants can be produced in the in vitro grown plants through plant
tissue culture.
Stereo- and region- specific biotransformation of the plant cells
can be performed for the production of bioactive compounds
from economical precursors.
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Plant products can be classified into:
primary plant metabolites essential for the survival of
the plant. It consists of sugars, amino acids and
nucleotides synthesized by plants and are used to
produce essential polymers
secondary metabolites 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 a significant role in plant defense
mechanisms
Advantages of Plant Cell Culture for
Production of Secondary Metabolites
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Primary metabolites essentially provide the basis for normal
growth and reproduction, while secondary metabolites for
adaptation and interaction with the environment.
The economic importance of secondary metabolites lies in the
fact that they can be used as sources of industrially important
natural products like colours, insecticides, antimicrobials,
fragrances and therapeutics. Therefore, plant tissue culture is
being potentially used as an alternative for plant secondary
metabolite production. Majority of the plant secondary
metabolites of interest to humankind fit into categories which
categorize secondary metabolites based on their biosynthetic
origin. Secondary metabolism in plants is activated / created
only in particular stages of growth and development or during
periods of stress, limitation of nutrients or attack by microorganisms.
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Plants produce several bioactive compounds that are
of importance in the healthcare, food, flavor and
cosmetics industries. Many pharmaceuticals are
produced from the plant secondary metabolites.
Currently, many natural products are produced solely
from massive quantities of whole plant parts. The
source plants are cultured in tropical, subtropical,
geographically remote areas, which are subject to
drought, disease and changing land use patterns and
other environmental factors.
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- Plant cell cultures are maintained under controlled
environmental and nutritional conditions which ensure
the continuous yields of metabolites.
- Culture of cell may be more economical for those
plants which take long periods to achieve maturity
(E.g. Papaver bracteatum, the source of the banine ,
takes two to three seasons to reach maturity).
Steps involved in the production of secondary metabolites from
plant cell
Secondary Metabolites Produced in High Level by Plant Cell Cultures
YIELDS (% DRY WT)
COMPOUND
PLANT SPECIES
CULTURE
PLANT
CULTURE
TYPE*
Shikonin
Lithospermum
erythrorhizon
20
1.5
s
Ginsenoside
Panax ginseng
27
4.5
c
Anthraquinones
Morinda citrifolia
18
0.3
s
Ajmalicine
Catharanthus roseus
1.0
0.3
s
Rosmarinic acid
Coleus blumeii
15
3
s
Ubiquinone-10
Nicotiana tabacum
0.036
0.003
s
Diosgenin
Dioscorea deltoides
2
2
s
Benzylisoquinoline
Alkaloids
Coptis japonica
11
5 - 10
s
Berberine
Thalictrum minor
10
0.01
s
Berberine
Coptis japonica
10
2-4
s
Anthraquinones
Galium verum
5.4
1.2
s
Anthraquinones
Galium aparine
3.8
0.2
s
Plant Cell Culture
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Plasmodesmata (small pores that linked each cell together)whole plant are connected to one another by Plasmodesmata.
Important for cell-to-cell communication.
Allow interchange of lower molecular weight compounds from
cytoplasm of one cell to another by diffusion.
In large aggregate, there will be concentration gradients of
such metabolic products (ethylene) , nutrients(like O2, hormone).
As a result, cells in different position in aggregate may have
greatly different biochemical and morphological structure.
Cells in suspension can be made to undergo differentiation and
organization if correct environmental condition can be formed.
(so that adjustment levels of nutrients and hormone should be
made).
Embryos, shoots and roots can made from aggregates in
suspension.
Callus
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Can be formed from any part of plant containing
dividing cells.
Placed on solidified medium containing nutrients
and hormones that promote rapid cell
differentiation.
Form in large size but has no organized structure.
Suspension Culture
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Established from the friable(easily breaks into
small pieces) callus which is placed in liquid
medium in shake flask.
With gentle to moderate agitation , cells or small
aggregate of cells slough off at 27°C with pH 5.5
under dark condition, then replicate.
After 2 or 3 weeks the, suspended cells then
transferred to fresh medium, residual culture are
discarded.
Method 1 - Callus Culture

Callus is explants that cultured on the appropriate medium,
with auxin and a cytokinin, give rise to an unorganised,
growing and dividing mass of cells.
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Any plant tissue that have dividing cells can be used as an
explant.
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Exercised plant material is placed on solidified medium
containing nutrients and hormones that promote rapid cell
differentiation.

Callus forms can be quite large (>1cm) (Shuler and Kargi,
2002).
Method 2 - Suspension Culture

Callus culture is then transferred into liquid medium to
establish suspension culture, which is place on shaker to
supply the cells with sufficient oxygen.
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With moderate agitation, cells or small aggregates of cells
will slough off.
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A platform shaker is used to give a circular motion in a
viable speed control (30-150 rpm) (Shuler and Kargi, 2002).
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Callus culture is performed in the dark (27°C & pH 5.5) as
light can encourage differentiation of the callus (Shuler and
Kargi, 2002).
Compare Plant Cell Culture and
Microbes Culture.
Aspect
Degree of cellular
aggregation
Biochemical growth
Microbial Culture
Plant Cell Culture
Usually single cell
Usually aggregates
Typically, cell initially
adjust to new medium
(lag phase) till they start
dividing regularly (log
phase). When growth
limited, cells stop
dividing (stationary
phase), eventually show
loss of viability (death
phase).
•Plasticity (ability of
plant to endure extreme
by changing growth and
development of plant
organs).
•Totipotency (any part of
plant can give rise to
entire new plant to be
cloned and generated via
cell or tissues culture).
•Phytotoxic (compounds
that is toxic or inhibits
plant growth).
Types of culture media
Classified into several
categories depending on
composition or use:
•Chemically-defined
(synthetic) medium (exact
chemical composition
known).
Use: studying minimal
nutritional requirements of
microbes, enrichment
culture, wide variety of
physiological studies.
•Complex (undefined)
medium (exact chemical
constitution of medium is
unknown)
Use: cultivation of
bacterial pathogens and
other fastidious bacteria.
•For both callus and
suspension culture
•Use carbon or energy
source such as sucrose.
•Inorganic nutrients,
vitamins, plant hormones
(auxins, cytokinins,
giberellins).
Growth rate
Rapid; doubling time
of 0.5 to 1 hour.
Oxygen requirement High, thus high
respiration rate.
Slow; doubling time
of 2 to 5 days.
Low, thus low
respiration rate.
END