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)