Lecture 2 animal cell biotechnology

Industrial Microbiology Dr. Butler 2011

1. Lecture 2 - Animal Cell Biotechnology Viral vaccines Monoclonal antibodies Recombinant glycoproteins Hormones, growth factors Enzymes Why study Animal Cell Biotechnology?

2. 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:

3. 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.

4. 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.

5. Lecture 2 - Animal Cell Biotechnology Applications for Animal Cell Cultures investigation of the normal physiology or biochemistry of cells (effect of substrates on metabolic pathways) biochemical toxicity - study the effects of compounds on specific cell types (mutagens, metabolites, growth hormones, etc) to produce artificial tissue by combining specific cell types in sequence – may be able to produce artificial skin for burn victims, etc. the synthesis of valuable biological products from large-scale cell cultures

6. 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 Cultures 1. 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

7. 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

8. Lecture 2 - Animal Cell Biotechnology Ross 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.

9. Lecture 2 - Animal Cell Biotechnology Alex 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.

10. Lecture 2 - Animal Cell Biotechnology Alex 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

11. Lecture 2 - Animal Cell Biotechnology Hayflick and Moorhead and the finite lifespan of isolated animal cells Hayflick 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

12. Lecture 2 - Animal Cell Biotechnology Hayflick 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.

13. Lecture 2 - Animal Cell Biotechnology Hayflick 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

14. Lecture 2 - Animal Cell Biotechnology Hayflick and Moorhead and the finite lifespan of isolated animal cells Immortal 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

15. Lecture 2 - Animal Cell Biotechnology Hayflick 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

16. Lecture 3 Animal Cell Biotechnology Characteristics 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:

17. Lecture 3 Animal Cell Biotechnology Characteristics 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

18. 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.

19. 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)

20. Telomere

21. 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