AMMA

CELL CULTURE TECHNIQUE AND

ITS APPLICATION
Source :
Microbiology by michael.j.pelczar.
Virology by murphy.
Veterinary microbiology by sharma.
Biotechnology4U by Dr. Zeba F. Alam,
General microbiology by quin and carter

INTRODUCTION
Now a days the culture of animal cells in artificial
media is an important aspect of any biotechnological
processes. These animal cells in culture are used in
recombinant DNA technology, genetic manipulations,
production of of transgenic animals and in a variety
of industrial processes. it has become possible to use
the cell and tissue culture in the areas of research
which have a potential for economic value and
commercialization. The animal cell cultures are
being extensively used in production of vaccines,
monoclonal antibodies, pharmaceutical drugs, cancer
research, genetic manipulations etc. it can also used
in vitro fertilization and transfer of embryos to
surrogate mothers.

WHAT IS CELL CULTURE?

When cells grow in controlled conditions this is called
cell culture.
The maintenance and growth of the cells of multicellular
organismsoutside the body in specially designed
containers and under precise conditions of temperature,
humidity, nutrition, and freedom from contamination. In
a broad sense, cells, tissues, and organs that are isolated
and maintained in the laboratory are considered the
objects of tissue culture , the term "cell culture" now
refers to the culturing of cells derived from multi-cellular
eukaryotes, especially animal cells. However, there are
also cultures of plants, fungi and microbes, including
viruses, bacteria and protists.

HISTORY
Sydney Ringer(19th-century) developed salt solutions containing the
chlorides of sodium, potassium, calcium and magnesium suitable for
maintaining the beating of an isolated animal heart outside of the
body.
Wilhelm Roux(1885) removed a portion of the medullary plate of an
embryonic chicken and maintained it in a warm saline solution for
several days, establishing the principle of tissue culture.
Ross Granville Harrison (1907 to 1910) establishing the methodology
of tissue .

Jonas Salk(1940s and 1950s) polio vaccine, The first product of cell
culture technique.
John Franklin Enders ,
Thomas Huckle Weller,
and
Frederick Chapman Robbins , who were awarded a Nobel Prize for
their discovery of a method of growing the virus in monkey kidney
cell cultures.

 CELL

Principle properties of a cell:

I.

Metabolic activity.
e.g. ETC, Glycolysis etc

2.

Replication, transcription & translation.

3.

Secretions.

4.

Phagocytosis & pinocytosis.

5.

Able to live IN-VITRO.

TYPES OF CELL CULTURE:

Primary cell culture

a) Anchorage Dependent /Adherent cells.

cell culture of kidney.
e.g. WRL-68, HepG2, HeLa etc.

b) Suspension Culture/Anchorage
Independent cells.
cell culture of blood cells.

WRL-68

Jurkat

e.g. Jurkat.

Secondary cell cultures

Subculturing

HeLa

HepG2

SECONDARY CELL LINES

H9c2

HeLa

Cell Line
a) Finite cell Lines. (usually 20-80 population doublings)
growth rate is 24-96 hours.
b) Continuous Cell Lines. (grow continuously)
growth rate is 12-24 hours.
i) Monolayer cultures
ii) Suspension cultures

Growth rate Number of multiplications per hour

.
Growth curve - A growth curve of a particular cell line
is established taking into consideration the population
doubling time,
Growth curve consist of:
1) Lag Phase:
2) Log Phase:
3) Plateau Phase:
4) Death phase:

SALIENT FEATURES OF CELL CULTURE WITH

EVOLUTION OF A CELL LINE

(i) Passage number- It is the number of times that the culture has
been subcultured.
(ii) Generation number- It refers to the number of doublings that a
cell population has undergone.

REQUIREMENTS OF CELL CULTURE LAB(EQUIPMENTS)

CO2-thermostats

Airflow
Solutions
Dishes
Freezers
Liquid nitrogen
Centrifuges

Autoclave

Vacuum ovens

Cryotubes

Microscopes

ELISA-readers

CELL CULTURE PROTOCOL

Isolation of cells
1) Purification,
e.g. blood sample,
2) Enzymatic digestion,
e.g. collagenase, trypsine.
3) Explant culture.
e.g. part of heart,

PLATING.
PLATING DENSITY:

Plating density is the number of cells per volume

of culture medium.
e.g.
Lower plating density
Granulosa cells exhibit
estrogen production,
Higher plating density
Progesterone.

MAINTAINING CELLS IN CULTURE

MEDIA
Temperature:
High
5%

37C (34 C, 41 C)

humidity.

CO2.

Biosafety

measures.

Antibiotics:
e.g. penicillin and streptomycin.
Antifungals:
e.g.amphotericin B.

CELL GROWTH MEDIA.

Growth media can vary in pH.
glucose concentration.
growth factors.
like serum such as fetal bovine serum, bovine
calf serum

AVOIDING CROSS-CONTAMINATION

Methods are used to identify cell lines:

1) Morphology,
2) Short tandem repeat(STR) analysis,
3) DNA fingerprinting,
4) Isoenzyme analysis,
5) Human lymphocyte antigen(HLA)
typing,
6) Chromosomal analysis,
7) Karyotyping,

MEDIA CHANGES

adherent cultures - aspiration,

non-adherent cultures - centrifuging and
resuspending.
Passaging cells
Passaging involves transferring a small
number of cells into a new vessel. adherent
cultures detachment by using trypsin-EDTA

TRANSFECTION AND TRANSDUCTION

Transfection
express a protein of interest.

Before transfection

After transfection

Transduction
DNA inserted into cells using viruses,

STEM CELL CULTURING

Stem cell :
Stem cells are the raw material from which all parts of
the bodys mature, different organs are made, They have
the potential to replace cell tissue that has been damaged
or destroyed by severe illnesses. Culturing of these cells
in cell culture media is called as stem cell culture.

There are two types of Stem Cells:

1) Embryonic/pluripotent stem cells
2) Adult stem cells

EMBRYONIC/PLURIPOTENT STEM
CELLS

Two Sources of Embryonic Stem Cells.

1)
2)

Excess fertilized eggs from IVF (in-vitro fertilization)

clinics .
Therapeutic Cloning (Somatic Cell Nuclear Transfer)

Adult stem cells

extracted from a 5-7 day old blastocyst, these are
less versatile and more difficult to identify, isolate,
and purify.

CULTURE OF NON-MAMMALIAN CELLS

Plant cell culture methods
plant growth hormones auxin and cytokinin.
Insect cell culture
Drosophila melanogaster, Spodoptera
frugiperda.
Bacterial and yeast culture methods
Viral culture methods

APPLICATIONS OF ANIMAL
CELL CULTURE
a) production of antiviral vaccines, which requires the standardization of cell
lines for the multiplication and assay of viruses.
b) Cancer research, which requires the study of uncontrolled cell division in cultures.
c) Cell fusion techniques.
d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
e) Production of monoclonal antibodies requires cell lines in culture.
f) Production of pharmaceutical drugs using cell lines.
g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell lines.
i) Use of artificial skin.
j) Study the function of the nerve cells.

SOME ANIMAL CELL LINES AND THE

PRODUCTS OBTAINED FROM THEM

HYBRIDOMA

Monoclonal antibodies

POLYCLONAL ANTIBODIES

DIAGNOSIS OF DISEASE:
BASED ON SPECIFIC CYTOPATHIC EFFECT.
E.G POX VIRUS

Production of Tissue Plasminogen activator

(t-PA) by recombinant DNA technology

PRODUCTION OF BLOOD CLOTTING FACTOR

VIII
Haemophilia is a sex-linked genetic disease in humans,
Blood clotting Factor VIII is produced by using Hamster kidney
cell lines.

Production of Vaccines
Vaccines for polio, measles, mumps, rubella, and chickenpox
H5N1, influenza vaccines .
Including recombinant DNA - based vaccines like adenovirus

PRODUCTION OF ERYTHROPOIETIN (EPO)

NORMALLY PRODUCED IN THE FOETAL
LIVER AND KIDNEYS OF THE ADULTS.
USED IN TREATMENT OF ANAEMIA, AIDS,
RENAL FAILURE ETC.

PRODUCTION OF CHIMERAS.

DETECTION OF BLADDER CANCER

The

probe was designed

to detect aneuploidy for
chromosomes 3, 7, 17
and loss of the 9p21
locus via fluorescence in
situ hybridization
(FISH) in urine
specimens from subjects
with transitional cell
carcinoma of the
bladder.

two copies of chromosome 3

(red), four copies of
chromosome 7 (green), five
copies of chromosome 17
(aqua) and one copy of p16
gene (gold)

Insulin production:
insulin secreted by the cultured cell line of islets of
langerhans.
Identification of species/karyotyping:
A live tissue can be classified and species of origin
can be identified based the cell lines on the culture
media & staining characters.

TRANSGENIC ANIMALS

Transgenic Mosquitoes

Aedes aegypti female mosquitoes transmits

Plasmodium falciparum cause malaria.

Dengue fever,

GLO FISH

 SPIDER

SILK PRODUCING GOAT

BRAINBOW

TRANSGENIC MICE

FEARLESS MICE

This goat contains a human gene that codes for a blood

clotting agent. The blood clotting agent can be
harvested in the goats milk.

PREPARED BY:
C.S.Darshan.
Ist M.V.sc