CELL LINE DEVELOPMENT AND

IT'S CHARACTERIZATION

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.

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.

• 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.

CELL GROWTH KINETICS

GROWTH CURVE

FEW BASICS

ANIMAL CELL

CULTURE

PRIMARY CELL

CULTURECELL LINE

CELL STRAIN

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)

ISOLATION OF TISSUES

• 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

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

DEVELOPING A CELL LINE

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

CELL LINE

CELL LINE

FINITE IMMORTAL

• After the first subculture, the primary culture becomes known as a cell line or subclone.

• 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.

SERIAL SUBCULTURING

CRITERIAS FOR

SUBCULTURING

1)Density of culture- confluency factor

2)Exhaustion of media- pH monitoring

3)Time since last subculture-seeding density

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

HEMOCYTOMETER

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

ELECTRONIC PARTICLE COUNTER

cells counted by the change in electrical resistance produced by them on passing them between electrodes

CELL LINE IDENTIFICATION

• Karyotype• Isozyme patterns• Antibody labeling• DNA fingerprinting

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.

SUBCULTURING

MONOLAYER SUSPENSION

COMPARISION

TYPES OF CELL LINES

CELL LINE

FINITE IMMORTAL

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

HOW DO THEY BECOME IMMORTAL?

• Mutagens• Viruses• Oncogenes• Spontaneous

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.