Lecture 2 animal cell biotechnology
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Transcript of Lecture 2 animal cell biotechnology
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