Animal Cell Culture Technology

Chapter 2(d)
What is animal cell culture?
• Cell culture can be defined as the process of
cultivating cells and tissues outside the body of an
organism(invitro) in an artificial environment, which
stimulates the invivo conditions such as temperature,
nutrition and protection from microorganisms.

• Animal cell culture technology is a new scientific

discipline closely associated with the further
development of biotechnological industrial field.
Animal cell culture technology
Major development:
- The use of antibiotics which inhibits the growth of
contaminants.
- The use of trypsin to remove adherent cells to subculture
further from the culture vessel.
- The use of chemically defined culture medium.
 Primary Culture
Definition
The growth of the attached cells whereby they are surgically or
enzymatically removed from an organism and placed in suitable
culture environment.

Techniques employed for the culture:

- Mechanical Disaggregation
- Enzymatic Disaggregation
Techniques
 Mechanical Disaggregation
Def: Chopping or slicing of tissue into pieces and collection of
spill out cells.

Ways to collect:
- Press the tissue pieces through a series of sieves with gradual
reduction in mesh size.
- Force the tissue fragments through a syringe and needle.
 Enzymatic disaggregation

Cold trypsinization

- It’s a process of trypsinization with cold pre-exposure.

- After chopping and washing, the tissue pieces are kept in a
vial (on ice) and soaked with cold trypsin for about 6-24 hours.
- However, the tissue pieces contain residual trypsin.
- These tissue pieces in a medium are incubated at 37°C for 20-
30 minutes.
- The cells get dispersed by repeated pi-pettings.
- The dissociated cells can be counted, appropriately diluted
and then used.
 Enzymatic disaggregation

Warm trypsinization

- The chopped tissue is washed with dissection basal salt

solution (DBSS), and then transferred to a flask containing
warm trypsin (37° C).
- The contents are stirred, and at an interval of every thirty
minutes, the supernatant containing the dissociated cells can
be collected.
- After removal of trypsin, the cells are dispersed in a suitable
medium and preserved (by keeping the vial on ice).
Figure A : Conceptual diagram for various types of tissue culture
Advantages of Tissue Culture
Control of the environment
Characterization and homogenity of sample
Economy, scale and mechanization
In-vitro modelling of In-vivo Conditions
Limitations
 To grow cells outside their normal environment,
three major controls are involved:
 Observing scrict asepsis
 Providing the right kind of physic-chemical environment
 Nutrients in its simplest absorbable form

 Culturing technique needs a great deal of expertise

 Tissue samples consists of a mixture of heterogenous
cell populations
 Continuously growing cells often show genetic
instability
 Differences in the behavior or cells in cultured and in
its natural form
 Should include proper balance of the hormones
Applications of Cell Culture
 Excellent model systems for studying:
– The normal physiology, cell biology and biochemistry of
cells
– The effects of drugs, radiation and toxic compounds on the
cells
– Study mutagenesis and carcinogenesis

 Used for gene transfer studies

– Large scale manufacturing of biological compounds
(vaccines, insulin, interferon, other therapeutic protein)
Micro-propagation

Chapter 2(e)
Plant Tissue Culture
A collection of techniques to maintain or grow plant cells,
tissues, organs, seeds or other Plant part in a sterile
environment on a nutrient culture medium of known
composition.

Micropropagation
Rapid clonal in vitro propagation of plants from cells, tissues, or
organs cultured aseptically on defined media contained in
culture vessels maintained under controlled conditions of light
and temperature.
Explants
An excised piece of tissue or organ taken
from the plant to initiate a culture.

They can be :
• shoot meristem, tip, bud
• leaf or stem (internode)
• root
• anther / microspore
• ovule
• embryo associated seed parts
Factors Affecting Tissue Culture
 Growth Media– Minerals, Growth factors, Carbon
source, Hormones
 Environmental Factors– Light, Temperature, Photoperiod.
 Explant Source– Usually, the younger, less differentiated
the explant, the better for tissue culture
 Genetics– Different species show differences in
amenability to tissue culture. In many cases, different
genotypes within a species will have variable responses to
tissue culture.
Steps of micro-propagation
 Stage 0- Selection and preparation of the mother
plant(Sterilization of the explant tissues)
 Stage I- Initiation of culture ( Explant placed into growth
media)
 Stage II- Multiplication ( Explant transferred to shoot
media and shoot can be constantly divided)
 Stage III- Rooting ( Explant transferred to rooting media)
 Stage IV- Transfer to soil ( Hardening off )
Significance of Micropropagation
 A single explant can be multiplied into several thousand
plants in less than a year.
 Continuous supply of young plants throughout the year could
be made possible
 Taking an explant does not usually destroy the mother
plant, so rare and endangered plants are not harmed
 Clones through micro-propagation are ‘true to type’ as
compared with seedlings, which show greater variability
 This allows fast selection for crop improvement - explants
are chosen from superior plants, then cloned.
 Disease and virus free plants can be produced through
this technique.
Applications of micro-propagation
 Somaclonal Variation
 Germplasm Conservation
 Mutation Breeding
 Inducing mutation
 Embryo culture
 Haploid and Dihaploid production
 In Vitro Hybridization- protoplast fusion
 Production of Disease free plants
 Molecular farming
 Genetic engineering
 Production of secondary metabolites
Applications..
Somaclonal variation

Somaclonal variation is a general phenomenon of all

plant regeneration systems that involve a callus phase.

There are two types of Somaclonal variation:

Heritable genetic variation

Non- Heritable genetic variation
Applications..
Advantages of somaclonal variation

 It helps in crop improvement

 Creates additional genetic variants
 Plants with resistant and tolerant to toxins,
herbicides, high salt and even mineral toxicity
 Suitable for breeding purposes
 Increased and improved production of secondary
metabolites
Applications..
Germplasmconservation

 Micropropgation is utilized in conserving genetic

resources.
 Depending upon the crop species and method of
preservation, tissue culture can help in the
preservation of genetic resources from 1 to 15 years.
 Cyopreservation – the preservation of germplasm
in a dormant state at ultra-low temperatures, usually
in liquid nitrogen (-196 °C) – is a type of tissue culture
which can be used to preserve seeds.
Applications..
Mutation Breeding
 1927: Muller produced mutations in fruit flies using
xrays
 1928: Stadler produced mutations in barley
 Basically, there are three groups of breeders
1) Mutation breeding is useless, we can accomplish the
same thing with conventional methods
2) Mutation breeding will produce a breakthrough
given enough effort
3) Mutation breeding is a tool, useful to meet specific
objectives
Applications..
Types of mutation:

Spontaneous (natural mutation)

 Some type of spontaneous mutation have
played an outstanding role in development of
valuable crop cultivars and hybrids.
 But limitation of this is that, it can not form the
basis of modern plant breeding

Induced mutation
 Any mutation found in nature can be induced
by mutation breeding.
Applications..
 Inducing Mutations

 Physical mutagens (Irradiation)

These mutagens helps in the chromosome
aberrations and point mutations.
Neutrons, Alpha rays
Gamma, Beta, X-rays
 Chemical mutagens
By using different carcinogenic chemicals which are
highly toxic in nature and eventually result in point
mutations.
Applications..
Embryo Culture
 Embryo culture is usually done from the need to
rescue embryo from wide crosses where fertilization
occurred, but not the embryo development.

 Objectives:
Rescue F1 Hybrid from wide crosses
Overcome seed dormancy by addition of hormone to
media. For ex. GA
To overcome immaturity in seed
To rescue valuable genotype from dead or dying plant
To speed up the generations in a breeding program
Applications..
Embryo culture as a source of genetic variation

 Hybridization
Can introduce new genetic combinations
through inter-specific crosses
Can transfer mutant alleles between species

 Polyploidy
It combines embryo culture with chromosome
doubling to create new polyploid species
Applications..
Haploid and dihaploid production

 Plants produced through the anther culture are the

haploids.
 Doubling the chromosomes without going into series
of backcrossing produce homozygous plants.
 This technique shortens the time of breeding by half.
Applications..
In Vitro Hybridization( Protoplast fusion)
 Created by degrading the cell wall using enzymes.
 Very fragile in nature.
 Protoplasts can be induced to fuse with one another.

Methods:
 Electrofusion
 Poly Ethylene Glycol(PEG)
 Addition of calcium ions at high PH Values
Applications..
Production of Disease free plant

Heat treatment
Plants grow faster than viruses at higher temperature.

Meristemming
Viruses are transported from cell to cell through
plasmodesmata and vascular tissue. Apical meristem are virus free
in nature. So, micropropagation of these cells gives virus free
plantlets.

Even, not all the cells in the plant are infected. For
example, adventitious shoots formed from single cells
can give virus free shoots.
Applications..
Molecular farming
 Where plants are treated as bioreactors for the production
of specific compounds.
 Range from simple peptides to a thermoplastic.

Two types of products:

1)High value compounds with small scale production
requirements such as pharmaceutical products.
For ex. Malaria epitope.
2)Compounds needed on a bulk scale with low production
costs (plant biotechnology has greatest potential in this
area)
For ex. α-Amylase (food + detergent industries)-starch
manipulation
Applications..
Genetic Engineering

 Genetic engineering would not be possible without the

development of plant tissue culture.
 Genetic engineering requires the regeneration of
whole plants from single cells.
 Efficient regeneration systems are required for
commercial success of genetically engineered
products.
 Protoplast fusion between male sterile cabbage and
normal cabbage was done and cybrids were selected
that contained the radish mitochondria and cabbage
chloroplast.
Applications..
Production of secondary metabolites
 Secondary metabolites are those cell constituents
which are not essential for survival.
 For example, alkaloids, glycosides, terpenoids,latex,tannins etc.
 In vitro production of secondary metabolites is much
higher from differentiated tissues compared to nondifferentiated
tissues.
 Azadirachtin from Azadirachta indica Insecticidal
 Berberine from Coptis japonica Antibacterial, anti
inflammatory
 Capsaicin from Capsicum annum Cures Rheumatic pain
Challenges of Micropropagation
 Expensive laboratory equipment and service
 No possibility of using mechanization
 Plants are not autotrophic
 Poor Acclimatization to the field is a common
problem (hyperhydricity)
 Risk of genetic changes if 'de novo' regeneration is used
 Mass propagation cannot be done with all crops to date. In
cereals much less success is achieved
 Regeneration is often not possible, especially with
adult woody plant material.
In vitro Fertilization

Chapter 2(f)
 Introduction
• Definition of In vitro Fertilization (IVF):
– A process by which an egg is fertilized with a sperm
outside the body, in laboratory glass containers –
‘in vitro’
• ‘In vitro’ – means ‘in glass’ in Latin. Fertilization
carried out externally in glass containers such
as petri dishes and test tubes.
• Babies conceived from IVF  ‘Test tube babies’
 Necessity of IVF
• IVF  a major treatment protocol for infertility
resulted from the following causes:
Steps in IVF
 Steps in IVF
• Initial Evaluation
Preliminary
• Suppression of natural Steps
hormonal cycle (NHM)
• Ovarian simulation
• Collection of oocytes (eggs)
• Collection of sperms Main Steps
• In vitro fertilization
• Embryo transfer
 (1) Initial Evaluation
• Blood Tests
• Seminal Fluid Examination
• Hysterosalpingogram
– a radiologic procedure to investigate the shape
shape and patency of the fallopian tubes.
• Trans vaginal ultrasound
 (2) Suppression of NHM
• To prevent spontaneous ovulation – if eggs
leave the ovary naturally, doctors are unable to
retrieve them
• Drugs:
– Oral Contraceptive Pills
– Lupron/Leuprolide Acetate
– Nafarelin
– Ganirelix Acetate Injection
 (3) Ovarian Stimulation
• Ovarian stimulation is used to produce multiple
matured egg-producing follicles in one go,
instead of single egg per month
• Produces many good follicles to be fertilized
• Only some eggs will be fertilized successfully and
develops normally – hence many follicles are
required
• Generally, 14 days of ovarian stimulation needed
(3) Ovarian Stimulation
 (3) Ovarian Stimulation

• Ovarian follicles, stimulated by ovulation medications, visible

on ultrasound. The dark, circular areas are the follicles
 (3) Ovarian Stimulation
• Main side effect: Ovarian Hyperstimulation
Syndrome (OHSS)
• Symptoms: Enlargement of ovaries and leakage
of fluid into abdomens and rarely, lungs (fatal)
• Mild OHSS can be treated conservatively
• Moderate to severe OHSS  IVF should be
postponed
 (4) Oocyte Collection
• After multiple follicles are formed, Human
Chorionic Gonadotropin (hCG) is injected 
induce maturation of oocytes
• Oocyte retrieval  34-36 hrs after hCG injection
• Procedure done under general anaesthesia, with
a period of 20-30 mins
 (4) Oocyte Collection

• The eggs are aspirated (removed) from the follicles

through the needle connected to a suction device.
 (4) Oocyte Collection
• Usually 10-15 oocytes are aspirated
• The oocytes are stripped of surrounding cells and
examined in lab for maturity and quality
• Mature oocytes placed in IVF culture medium 
transferred to an incubator to await fertilization
by the sperms
 (5) Sperm Collection
• Performed after oocyte collection
• Sexual abstinence of 3-4 days should be practiced
• Collected 1-1.5 hours before fertilization
• Liquefied ,centrifuged, suspended in culture
medium, and incubated for 30-60 mins at 37⁰ C
• Most active sperms are located in the surface of
the medium
 (5) Sperm Collection
• Sperm may be obtained from the testicle,
epididymis, or vas deferens if there is absence of
sperm in semen either due to an obstruction
 (6) In vitro fertilization
• Fertilization  adding 10,000-50,000 motile
sperms to about 100 μl to 1 ml culture medium in
which the oocytes are being incubated
• Intra-cytoplasmic sperm injection (ICSI) is
performed when semen does not contain sperm
– During ICSI, an embryologist isolates a sperm cell,
draws it up into a microscopic needle and injects it
inside an oocyte using a high power microscope.
(6) In vitro fertilization
 (6) In vitro fertilization
• Fertilisation check  approx. 18 hrs after sperm
injection/insemination.
• Typically, 65%-75% of matured oocytes will be
fertilized
• If the couple has genetic disease history and the
problematic gene is identified, a pre-
implantation genetic diagnosis may be done
 (6) In vitro fertilization
• Development of embryo:
 (7) Embryo Transfer
• Embryo transfer may be performed on day 2/3/4,
post-fertilization
• One or more embryos are drawn into a transfer
catheter - a long, thin sterile tube with a syringe
on one end
• The physician gently guides the tip of the transfer
catheter through the cervix and places the fluid
containing the embryos into the uterine cavity
(7) Embryo Transfer
Percentages of Success?
 Variations of IVF
• Gamete Intrafallopian Transfer (GIFT) 
Gametes are transferred to fallopian tubes
instead of uterus, and fertilization occurs in tubes
rather than in lab
• Zygote Intrafallopian Transfer (ZIFT) 
Fertilization occurs in lab, and the fertilized
embryo are transferred to fallopian tubes instead
of uterus
 Introduction: What it is about?
• Cloning describes a number of different
processes that can be used to produce
genetically identical copies of a
biological entity
• Clones are organisms that are exact
genetic copies. Every single bit of their
DNA is identical.
• Human Identical Twins are clones of each
other.
• Collectively refers to processes used to
create copies of DNA fragments.
• Animal cloning has been the subject of
scientific experiments for years, but
garnered little attention until the birth of
the first cloned mammal in 1996, a sheep
named Dolly.
• Since Dolly, several scientists have cloned
other animals, including cows and mice.

• No human cloning attempts have been

made because there have been many
disadvantages that involve the cloning of
both humans and animals.
History of Cloning
 Types of Cloning

CLONING

Artificial Natural
Embryo
Cloning
Twinning

Reproductive THERAPUETIC
cloning CLONING
 Natural cloning
• In nature, twins form very early in development when the
embryo splits in two. Twinning happens in the first days after
egg and sperm join, while the embryo is made of just a small
number of unspecialized cells. Each half of the embryo
continues dividing on its own, ultimately developing into
separate, complete individuals. Since they developed from
the same fertilized egg, the resulting individuals are
genetically identical.
 Artificial embryo twinning
• Artificial embryo twinning is a relatively low-tech way to make clones. As
the name suggests, this technique mimics the natural process that
creates identical twins.

• Artificial embryo twinning uses the same approach, but it is carried out
in a Petri dish instead of inside the mother. A very early embryo is
separated into individual cells, which are allowed to divide and develop
for a short time in the Petri dish. The embryos are then placed into a
surrogate mother, where they finish developing. Again, since all the
embryos came from the same fertilized egg, they are genetically identical.
Reproductive cloning
 Somatic cell nuclear transfer
• Somatic cell nuclear transfer (SCNT), also
called nuclear transfer, uses a different
approach than artificial embryo twinning, but
it produces the same result: an exact genetic
copy, or clone, of an individual. This was the
method used to create Dolly the Sheep.
Somatic cell
•A somatic cell is any cell in the body other than
sperm and egg, the two types of reproductive
cells. Reproductive cells are also called germ
cells. In mammals, every somatic cell has two
complete sets of chromosomes, whereas the
germ cells have only one complete set.
Nuclear
•The nucleus is a compartment that holds the cell's DNA. The DNA is divided into
packages called chromosomes, and it contains all the information needed to form an
organism. It's small differences in our DNA that make each of us unique.

Transfer
•Isolate a somatic cell from an adult female Next they remove the nucleus and all of
its DNA from an egg cell. Then we transfer the nucleus from the somatic cell to the
egg cell. After a couple of chemical tweaks, the egg cell, with its new nucleus, will
behave just like a freshly fertilized egg. It is developed into an embryo, which was
implanted into a surrogate mother and carried to term.
 THERAPUETIC CLONING
• you may have heard about researchers cloning, or
identifying, genes that are responsible for various medical
conditions or traits. What's the difference?

• When scientists clone an organism, they are making an exact

genetic copy of the whole organism.

• When scientists clone a gene, they isolate and make exact

copies of just one of an organism's genes. Cloning a gene
usually involves copying the DNA sequence of that gene into
a smaller, more easily manipulated piece of DNA, such as a
plasmid. This process makes it easier to study the function of
the individual gene in the laboratory.
 Advantages of Cloning
1. Solution to infertility
2. Provide organs for transplantation
3. Provide treatments for variety diseases
4. Genetic modification/ engineering
5. The healthiness of infants
6. Better understanding of genetic disease
7. Help improve lives
8. Protects endangered species
9. Improves food supply
 Disadvantages of cloning
1. Uncertainty of science and technology → cost
→ use in a wrong manner
2. Losing diversity in genes
3. The diseases/leading to distinction
4. Religious problems → unethical
5. Transgressing nature
6. Inhumane
 Potential benefits of human cloning
• JUST IMAGINE !!
• Human cloning technology could be used to reverse heart attacks.
Scientists believe that they may be able to treat heart attack
victims by cloning their healthy heart cells and injecting them
into the areas of the heart that have been damaged.
• Infertility - infertile couples can have children
• Down's syndrome - those women at high risk for Down's syndrome
can avoid that risk by cloning
• Leukemia - we should be able to clone the bone marrow for
children and adults suffering from leukemia. This is expected to
be one of the first benefits to come from cloning technology.
• Cancer - we may learn how to switch cells on and off through
cloning and thus be able to cure cancer
 Cloning vector
• The molecular analysis of DNA has been made possible by the
cloning of DNA. The two molecules that are required for
cloning are the DNA to be cloned and a cloning vector.
• A cloning vector is a small piece of DNA taken from a virus,
a plasmid or the cell of a higher organism, that can be stably
maintained in an organism and into which a foreign DNA
fragment can be inserted for cloning purposes.
• Most vectors are genetically engineered.

• The cloning vector is chosen according to the size and type of

DNA to be cloned.
• The vector therefore contains features that allow for the
convenient insertion or removal of DNA fragment in or out of
the vector, for example by treating the vector and the foreign
DNA with a restriction enzyme and then ligating the
fragments together.

• After a DNA fragment has been cloned into a cloning vector, it

may be further subcloned into another vector designed for
more specific use.
 Why cloning Vector
• Cloning vector is used as a vehicle to artificially carry
foreign genetic material into another cell, where it can be
replicated and expressed.

• It is used to amplify a single molecule of DNA into many

copes.

• Cloning vectors are DNA molecules that are used to

"transport" cloned sequences between biological hosts and
the test tube.

• Without Cloning Vector, Molecular Gene Cloning is totally

impossible
 Types of cloning vectors
• Plasmid
• Bacteriophage
• Cosmid
• Yeast Artificial Chromosome (YAC)
 Plasmids or Viruses
• Plasmids are molecules of DNA that are found in bacteria
separate from the bacterial chromosome.
• They:
are small (a few thousand base pairs)
usually carry only one or a few genes
are circular
have a single origin of replication
• Plasmids can replicate independently of the bacterial
chromosome content.
• Plasmids are similar to viruses, but lack a protein coat and
cannot move from cell to cell in the same fashion as a virus.
• Viruses can be used to place DNA into bacterial cells, too.
 Bacteriophage
• A bacteriophage is a virus which infects bacteria.
• The bacteriophage exemplifies the life cycle of viruses.
• It exists as an inactive virion until one of its extended
'legs' comes into contact with the surface of an bacteria.
• Sensors on the ends of its 'legs' recognize binding sites
on the surface of the host's cell, and this triggers the
bacteriophage into action.
• The bacteriophage binds to the surface of the host, punctures the cell with
its injection tube, and then injects its own genetic blueprint.
• This genetic information subverts the host cell's normal operation and sets
the cell's biosynthetic machinery to work creating replicas of the virus.
 Cosmid
• Cosmid are predominantly plasmids with
a bacterial oriV,
an antibiotic selection marker
and a cloning site,
but they carry one, or more recently
two cos sites derived from bacteriophage lambda.

• The loading capacity of cosmids varies depending on the size of the vector
itself but usually lies around 40-45 kb.
• The cloning procedure involves the generation of two vector arms which
are then joined to the foreign DNA
Yeast Artificial Chromosomes (YACs)
• Are useful for eukaryotic molecular studies
• Has following components
a) a centromere-distribute the chromosome to the daughter
cells
b) a telomere- to protect against degradation
c) an autonomously replicating sequences – enable the
molecule to replicate
• Cloning large DNA fragments
 Plant Cloning Vector
• DNA is being cloned into plants
• E.g. tobacco mosaic virus or TMV, Ti plasmid or the soil bacterium
• The "Ti" plasmid, or tumor-inducing plasmid is found in cells of the
bacterium known as Agrobacterium tumefaciens, which normally lives in
soil.
• The bacterium has the ability to infect plants and cause a crown gall, or
tumorous lump, to form at the site of infection.
• The tumor-inducing capacity of this bacterium results from the presence of
the Ti plasmid.
• The Ti plasmid itself, a large, circular, double-stranded DNA molecule, can
replicate independently of the A. tumefaciens genome.
• When these bacteria infect a plant cell, a 30,000 base-pair segment of the Ti
plasmid - called T DNA - separates from the plasmid and incorporates into
the host cell genome.
• This aspect of Ti plasmid function has made it useful as a plant cloning
vector.
 Mammalian Cell Vector
• Are used when a gene encoding a protein product contains
introns or the protein requires processing after synthesis.
• Coz these cells have the machinery to process DNA and
protein.
• Produce pharmaceutical compounds for therapeutic use. e.g.
Factor VIII used by hemophiliacs to aid blood clot formation,
growth hormone.
 Questions to ponder?
• Do you approve of human cloning ?
• Do you approve cloning of other animals ?
• Do you think it’s right to harvest organs
and tissue from clones ?
• If you had the option, would you want
to clone yourself ? Why ?
• Do you think the benefits outweigh the
risks with cloning humans ?