Industrial Microbiology Dr. Butler 2011

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

2. Lecture 2 - Animal Cell Biotechnology
Cell Culture:
 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

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
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

6. Lecture 2 - Animal Cell Biotechnology
Advantages of using Cell Cultures
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

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

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
„Normal‟ mammalian cells have the following properties:
 a diploid chromosome number (46 chromosomes for
human cells)
 anchorage dependence
 a finite lifespan
 nonmalignant (non-cancerous)
 density inhibition

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