2. Lecture 5 Animal Cell Biotechnology
The Phases of Cell Growth
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P50.
3. Lecture 5 Animal Cell Biotechnology
The Lag Phase
no apparent increase in growth
phase is associated with the synthesis of growth
factors that must reach a critical concentration before
growth starts
Length of lag phase is dependent on:
a) health of cells (metabolic status)
→ lag phase will be shorter if inoculum is taken from
a dense culture of highly active cells
4. Lecture 5 Animal Cell Biotechnology
The Lag Phase
Energy Charge
energy charge (EC) gives an indication of viability
of a cell population
for healthy cells, EC = 0.8, 0.9
[ ATP] 1/2 [ ADP]
energy charge
[ ATP] [ ADP] [ AMP]
5. Lecture 5 Animal Cell Biotechnology
The Lag Phase
b) need for metabolic adaptation
→ may need to adapt to different medium,
temperature, synthesize different enzymes, growth
factors
c) cell density of inoculum
→ should inoculate at 104-105 cells/mL
→ a high density of inoculum increases the ability of
cells to reach the initial critical concentration of
growth factors and enzymes more quickly
6. Lecture 5 Animal Cell Biotechnology
The Lag Phase
not all inoculum cells are viable
→ use trypan blue dye test
→ viable cells exclude trypan blue
non stainedcells
% viable cells x 100
total numberof cells
clones may require a feeder layer of cells –
irradiated cells that can’t grow but release growth
factors into the medium
8. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase
x
N No.2
log10 N log10 No x.log10 2
N = final cell concentration
No = initial cell concentration
X = number of generations of cell growth
Equation only works for exponential growth
9. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase
doublingtime(t D ) T (h)
X
T = total elapsed time (h)
X = number of generations
10. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase
Specific growth rate (μ) =
measure of the rate of dN 1 (h 1 )
increase of cell number or
biomass dT N
or
ln N ln No T
ln2 0.6931
μ
tD tD
11. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase – Cell Cycle
G1 – gap1 – uncharacterized
phase after mitosis
S – synthesis – period of DNA
synthesis
G2 – gap2 - uncharacterized
phase after synthesis
M – mitosis – cell division
12. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase – Cell Cycle Analysis
Stained cells forced
through a nozzle
Stream of cells exposed
to a laser ►
► ►
Fluorescence emission
detected by
photomultiplier
Fluorescence intensity
directly proportional to
the DNA content
Extrapolate distribution
of cells by DNA content
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P80.
14. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase – Cell Cycle Analysis
G1 – normal diploid
content (1x)
S – 1-2x diploid content
G2 – 2x diploid content
M – 2x diploid content
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P79.
15. Lecture 5 Animal Cell Biotechnology
Stationary Phase
stationary phase occurs when there is no further
increase in cell concentration
→ death rate = growth rate
Cell growth is limited by a number of conditions:
1) nutrients may have been depleted to a level that cannot
support cell growth
2) the accumulation of metabolic by-products to a level
that inhibits growth
→ build up of ammonia, lactic acid, etc
16. Lecture 5 Animal Cell Biotechnology
Stationary Phase
3) limitation of growth surface
→ cells have reached confluence (single monolayer of
cells covering the available substratum)
Cells may still be metabolically active in the absence of
growth
→ high cell density
→ many may still be viable
→ secrete product into media
17. Lecture 5 Animal Cell Biotechnology
The Decline Phase - Necrosis
Two different death mechanisms:
1) Necrosis
passive process that normally occurs when cells are
subjected to sudden severe cellular stress
leads to breakdown of the plasma membrane, leading
to cell swelling and eventual cell rupture
“extended” stationary phase
18. Lecture 5 Animal Cell Biotechnology
The Decline Phase - Apoptosis
2) Apoptosis (programmed cell death)
cell suicide mechanism that occurs in culture or in vivo
under normal physiological conditions
genetically programmed pattern of cellular events
abnormalities in process also lead to transformation
endogenous endonucleases are activated, cleave DNA
into fragments, forming a DNA ladder
19. Apoptosis
Definition: Cell death process which occurs during the
development and aging of animals
Also induced By: Cytotoxic lymphocytes, drugs, UV
irradiation, deprivation of survival factors and cytokines
called death factors.
20. Apoptosis
• -Cells Shrink
• -Microviolli disappear
• -Nucleus condensed and fragmented
• -Cells themselves fragmented with cellular
content inside.
• -Biochemical hallmark of apoptosis is the
fragmentation of chromosomal DNA into
nucleosomal size units (180bp)
21. Lecture 5 Animal Cell Biotechnology
The Decline Phase - Apoptosis
Lane 1: Mr DNA markers
Lanes 2-4: from mouse thymocytes
showing DNA laddering
Smith and Wood, Eds. 1996. Cell Biology 2nd Ed. London:Chapman and Hall. P 507.
22. Lecture 5 Animal Cell Biotechnology
The Decline Phase - Apoptosis
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P51.
23. Lecture 5 Animal Cell Biotechnology
The Decline Phase - Apoptosis
cell shrinks, the nucleus condenses, and the cell
fragments into apoptotic bodies, phagocytosed by
adjacent cells
have identified anti-apoptosis genes (gene products
inhibit apoptosis proteins)
→ inserted into cells to reduce/delay apoptosis
→ extends stationary phase, production period
24. Lecture 5 Animal Cell Biotechnology
Necrosis vs. Apoptosis – a comparison
http://www.niaaa.nih.gov/publications/arh25-31/images0.1.gif - accessed Jan 12/05
25. Fig. 11.2
Measurement of specific productivity
26. The final yield of the product will depend on :-
• the specific productivity of each viable cell
- expressed as μg of product formed per 106 cell-day.
• the viable cell density of the culture (x106 cells/ml).
28. 14 80
Mab from TB/C3.bcl2
Mab from TB/C3.pEF
Cell density (x106 cells/ml)
12 Growth of TB/C3.bcl2
IgG concentration (ug/ml)
Growth of TB/C3.pEF
60
10
8
40
6
4 20
2
0
0
0 20 40 60 80 100 120 140
Time (hour)
29. Determination of specific rate of productivity
80
28.5 pg/cell per day
TBC3.bcl-2
60
TBC3.pEF
40 18.3 pg/cell per day
IgG (mg/L)
20
0
-20
0 20 40 60 80 100
Viability index
t
0 X.dt (105 cell-hours/ml)
31. Problem demonstration 1
A bioreactor containing 20 liters of medium was
inoculated with a 1.5 L inoculum (3 x 106 cells/ml).
A lag phase was observed for the first 26 hours after
which cells grew exponentially until they reached a
maximum density of 2 x 106 cells/ml after 4 days
from the initial inoculation.
i) Determine the number of generations of cell growth.
ii) Determine the doubling time during cell growth.
iii) Determine the specific growth rate.