Lecture 2 - Animal Cell Biotechnology

1. Viral vaccines

2. Monoclonal antibodies

3. Recombinant glycoproteins

4. Hormones, growth factors

5. Enzymes

Why study Animal Cell Biotechnology?

Lecture 2 - Animal Cell Biotechnology

refers to the growth of cells as independent units. Once removed from animal tissue or whole animals, the cells will continue to grow if supplied with nutrients and growth factors.

cultures typically contain one type of cell which may be genetically identical (homogeneous population→clones) or show some genetic variation (heterogeneous population).

distinct from Organ culture, which requires maintenance of whole organs or fragments of tissues.

retains balanced relationship between associated cell types as in vivo

Cell Culture:

Lecture 2 - Animal Cell Biotechnology

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 12.

Lecture 2 - Animal Cell Biotechnology

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 13.

Lecture 2 - Animal Cell Biotechnology

Applications for Animal Cell Cultures

1. investigation of the normal physiology or biochemistry of cells (effect of substrates on metabolic pathways)

2. biochemical toxicity - study the effects of compounds on specific cell types (mutagens, metabolites, growth hormones, etc)

3. to produce artificial tissue by combining specific cell types in sequence – may be able to produce artificial skin for burn victims, etc.

4. the synthesis of valuable biological products from large-scale cell cultures

Lecture 2 - Animal Cell Biotechnology

1. consistency and reproducibility of results by using a batch of cells of a single type (clones)

2. allows for a greater understanding of the effects of a particular compound on a specific cell type during toxicological testing procedures; also less expensive than working with whole animals

3. during the production of biological products, can avoid the introduction of viral or protein contaminants using a well characterized cell culture

Disadvantage of using Cell Cultures1. after a period of time cell characteristics

can change and be different from those originally found in the donor animals

Advantages of using Cell Cultures

Lecture 2 - Animal Cell Biotechnology

Bacterial vs animal cell cultures

Advantages of bacteria

1. reliable, simpler system

2. cheap media

3. fast growing, high productivity

Disadvantages of bacteria

1. intracellular location of products

2. endotoxins produced, further purification steps required

3. lack of post-translational modification

Lecture 2 - Animal Cell BiotechnologyRoss Harrison and the Hanging Drop

Method

Harrison (1907) trapped small pieces of frog embryo in clotted lymph fluid and showed that:

1. cells require an anchor for support (coverslip and matrix of the lymph clot)

2. cells require nutrients (biological fluid contained in the clot)

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 4.

Lecture 2 - Animal Cell BiotechnologyAlex Carrel and the Carrel Flask

used aseptic technique to maintain long term cell cultures

used chick embryo extracts grown in egg extract medium mixed with blood plasma

developed carrel flask

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 4.

Lecture 2 - Animal Cell BiotechnologyAlex Carrel and the Carrel Flask

used surgical procedures for aseptic manipulation of cell cultures

claim to fame was the isolation of chick embryo fibroblasts and the maintenance of the cells from 1912-1946 (34 years!)

Carrel believed that he had isolated immortal cells

Lecture 2 - Animal Cell BiotechnologyHayflick and Moorhead and the finite lifespan

of isolated animal cellsHayflick and Moorhead (1961) studied the

growth potential of human embryonic cells.

cells could be grown continuously through repeated subculture for about 50 generations

pass through age-related changes until they reach the final stage when the cells are incapable of dividing further

the finite number of generations of growth is characteristic of the cell type, age and species of origin: referred to as the Hayflick Limit

Lecture 2 - Animal Cell BiotechnologyHayflick and Moorhead and the finite lifespan of

isolated animal cells

Phase 1. Cells are adapting to culture, relatively slow growth

Phase 2. Cells are growing @ doubling rate (~18-24 hours)

Crisis point. Cells recognize their own limited ability for cell division, growth slows

Phase 3. Growth slows further and eventually stops

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 5.

Lecture 2 - Animal Cell BiotechnologyHayflick and Moorhead and the finite lifespan

of isolated animal cells

Hayflick and Moorhead refuted Carrel’s conclusions about cellular immortality

Carrel’s use of plasma and homogenized tissue as growth medium reintroduced new cells into the culture from the egg extracts

therefore, cells in Carrel’s prolonged experiment were not derived from the original line

Lecture 2 - Animal Cell BiotechnologyHayflick and Moorhead and the finite lifespan

of isolated animal cellsImmortal Cells some cells acquire a capacity for infinite

growth (called ‘established’ or ‘continuous’ cell lines)

cells undergo a “transformation” which decreases cells’ sensitivity to the stimuli associated with growth control

requires a mutating agent such as: → mutagen (UV rays) → virus → spontaneous → oncogenes

Lecture 2 - Animal Cell BiotechnologyHayflick and Moorhead and the finite lifespan

of isolated animal cells

carcinogenesis in vivo analogous to transformation of cells in vitro, but not identical

transformed cells are not necessarily malignant

malignant transformation likely requires several mutations

non-malignant transformation requires a single mutation

Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture –

What’s Normal

a diploid chromosome number (46 chromosomes for human cells)

anchorage dependence

a finite lifespan

nonmalignant (non-cancerous)

density inhibition

‘Normal’ mammalian cells have the following properties:

Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture –

What’s Not

Transformed cell characteristics – a review

infinite growth potential

loss of anchorage-dependence

aneuploidy (chromosome fragmentation)

high capacity for growth in simple growth medium, without the need for growth factors

called an “established” or “continuous” cell line

Senescence: Evidence for a biological clock

Hayflick, Leonard (January 23, 1996). How and Why We Age, Reprint Edition, Ballantine Books. ISBN 0345401557.

Average human life-span is increasing

Maximum human life-span is not increasing ( 120 years). By calorie restriction ?

The maximum life span known for humans is 122.5 years, whereas the maximum lifespan of a mouse is about 4 years.

Lecture 2 Animal Cell Biotechnology Howard Cooke and the Biological Clock

Howard Cooke (1986) observed that the caps at the end of human germline chromosomes were longer than those found in somatic cells

caps consisted repeats of the nucleotide sequence TTAGGG/CCCTAA (15 kilobases)

shortened at each generation of growth (100 bases for human telomeres)

Telomere

Lecture 2 Animal Cell Biotechnology

hTRT+ clones = triangles; hTRT- clones = circles; closed symbols = senescent clones;half-filled symbols = near senescence (dividing less than once/ 2 weeks)

hTRT = human telomerase reverse transcriptase