Industrial Microbiology Dr. Butler 2011

1. Lecture 5 - Animal Cell Biotechnology
Cell growth

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

7. Lecture 5 Animal Cell Biotechnology
Growth/Exponential Phase
 cells undergo mitosis and divide
 mammalian cells double 18-24 hours
 follows exponential growth

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.

13. Fig. 5.12
Flow cytometer and cell sorter

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).

27. Cell specific productivity
Q s= P. t
1
N dt
= 0
P.

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)

30. Wurm,F (2004) Nature Biotech 22: 1393

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.