cell line development, animal cell culture

1. CELL LINE DEVELOPMENT
AND
IT'S CHARACTERIZATION

2. What is Animal Cell Technology ?
• Discipline of cell biology- aims to understand structures,
functions and behaviors of differentiated animal cells.
• Also to ascertain their abilities to be used in industrial
and medical purposes.
• Goal is the accomplishment
 Clonal expansion of differentiated cells with useful ability,
 Optimization of their culture conditions,
 Modulation of their ability to produce medically and
pharmaceutically important proteins
 The application of animal cells to gene therapy and
artificial organs.

3. History
• Ross Harrison (1907)- frog embryo nerve fiber outgrowth
in vitro.
• Carrel(1912)- explants of chick connective tissue, heart
muscle contractile for 2-3 months.
• Rous & Jones(1916)- trypsinization and subculture of
explants.
• Keilova(1948)- use of antibiotics in tissue culture.
• Gey et al. (1952)- First Human cell line HeLa
established.
• Eagle(1955)- development of defined media.

4. • Kleinsmith & Pierce (1964)- Pluripotency of embryonal
stem cells.
• Wiktor (1964)- Rabies, Rubella vaccines in WI-38 human
lung fibroblasts.
• Raham & Van der Eb(1973)- DNA transfer- calcium
phosphate.
• Kohler & Milstein (1975)- Hydridomas-monoclonal
antibodies.
• Ham & McKeehan (1978) -Serum free media.
• Freshney(2004)- Exploitation of tissue engineering.

5. CELL GROWTH KINETICS

6. GROWTH CURVE

7. FEW BASICS
ANIMAL
CELL
CULTURE
PRIMARY
CELL
CULTURE
CELL LINE
CELL
STRAIN

8. PRIMARY CELL CULTURE
• Cells taken directly from a tissue to a dish
• Can be passaged after this with a limited number of
times. After the limit, the cell will die.
 TYPES OF PRIMARY CELL CULTURE
• Mouse embryos
• Chick embryos
• Human biopsy materials
• Transplantable animal tumour
• Chick embryo organ rudiments (brain, heart, lungs, liver,
gizzard, kidney, spinal cord, skin, muscle)

9. ISOLATION OF TISSUES

10. • Mouse, mammals,
• Embryo
• Embryonated Eggs
• (best: for TC : embryo, young)
• because stage of differentiation)
cell culturing
organ
explant
Finely cut
Finely cut
tissue or explant
Enzymic digestion
Grow in media
-monolayer
-suspension cells

11.  Enzymatic disaggregation
• Warm trypsin, 37˚C for 30 mins, cell damaged if too long
exposure.
• Cold preexposure, soak at 4C overnight and 37C for less 30
mins. Advantage: higher yield of viable cells, preserve
more cell types
• Other enzyme
-collagenase benefit for connective tissues and muscle
(fibrous tissue)
- pronase, dipase, DNase, hyaluronidase
 Mechanical disaggregation (prevent proteolytic damage)
• Scrapping or spillage
• Sieving
• Syringes
• Trituration by pipette

12. DEVELOPING A CELL LINE

13. CELL STRAIN
• If a subpopulation of a cell line is positively
selected from the culture by cloning or
some other method, this cell line becomes
a cell strain.
• Acquires additional genetic changes

14. CELL LINE
CELL LINE
FINITE IMMORTAL
• After the first subculture, the primary culture
becomes known as a cell line or subclone.

15. • After the first subculture, primary culture may be called
secondary cultures, and thereafter, if continued passage
is possible, a cell line.
• An established or immortalised cell line has acquired the
ability to proliferate indefinitely either through random
mutation or deliberate modification, such as artificial
expression of the telomerase gene.

16. SERIAL SUBCULTURING

17. CRITERIAS
FOR
SUBCULTURING
1)Density of culture- confluency factor
2)Exhaustion of media- pH monitoring
3)Time since last subculture-seeding
density

18. MEASURING PARAMETERS OF
GROWTH
 increasing the number of cells
 increasing the size of the cells
 increasing the amount of intercellular substance.
• Cell counting
 Hemocytometer
 Electronic Particle Counter
• Cell viability assay
• Measurement of
 DNA amount
 RNA amount
 Protein amount

19. HEMOCYTOMETER
• tryphan blue dye stains
non viable cell and used
to calculate the viability %

20. ELECTRONIC PARTICLE COUNTER
cells counted by the
change in electrical
resistance produced by
them on passing them
between electrodes

21. CELL LINE IDENTIFICATION
• Karyotype
• Isozyme patterns
• Antibody labeling
• DNA fingerprinting

22. Subculturing
• Subculturing or "splitting cells," is required to
periodically provide fresh nutrients and growing
space for continuously growing cell lines.
• The frequency of subculture and the split ratio,
or density of cells plated depend on the
characteristics of each cell line being carried.
• Subculturing -
Adherent Cells
 Suspension culture.

23. SUBCULTURING
MONOLAYER SUSPENSION

24. COMPARISION

25. TYPES OF CELL LINES
CELL LINE
FINITE IMMORTAL

26. IMMORTAL CELL LINES?
• Transformed cell lines divide more rapidly and do not
require attachment to surface for growth, the loose
contact inhibition(tumors), It occurs spontaneously or
through interaction with viruses, oncogenes, radiation, or
drugs/chemicals.
• Characteristics
 Infinite life span
 High growth potential
 Low growth factor dependence
 Suspension growth
 Aneuploid

27. HOW DO THEY BECOME IMMORTAL?
• Mutagens
• Viruses
• Oncogenes
• Spontaneous

28. ROLE AND USES OF CELL LINE
Immortalized cell lines are widely used as a simple
model for more complex biological systems.
for example:
• The biochemistry and cell biology of mammalian
(including human) cells.
• Immortalized cell lines can also be cloned giving
rise to a colonal population(genetically identical
cells).
• The testing toxicity of compounds or drugs to
production of eukaryotic proteins.