Lecture 5 - Animal Cell Biotechnology

Cell growth

Lecture 5 Animal Cell BiotechnologyThe Phases of Cell Growth

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P50.

Lecture 5 Animal Cell BiotechnologyThe 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

Lecture 5 Animal Cell BiotechnologyThe Lag Phase

energy charge (EC) gives an indication of viability of a cell population

for healthy cells, EC = 0.8, 0.9

][][][][2/1][

chargeenergy AMPADPATP

ADPATP

Energy Charge

Lecture 5 Animal Cell BiotechnologyThe 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

Lecture 5 Animal Cell BiotechnologyThe Lag Phase

not all inoculum cells are viable → use trypan blue dye test → viable cells exclude trypan blue

100xcellsnumberoftotal

cellsstainednoncellsviable%

clones may require a feeder layer of cells – irradiated cells that can’t grow but release growth factors into the medium

Lecture 5 Animal Cell BiotechnologyGrowth/Exponential Phase

cells undergo mitosis and divide

mammalian cells double 18-24 hours

follows exponential growth

Lecture 5 Animal Cell BiotechnologyGrowth/Exponential Phase

N = final cell concentrationNo = initial cell concentrationX = number of generations of

cell growth

2x.logNologNlog

No.2N

101010

x

Equation only works for exponential growth

Lecture 5 Animal Cell BiotechnologyGrowth/Exponential Phase

T = total elapsed time (h)

X = number of generations

(h) XT )(t time doubling D

Lecture 5 Animal Cell BiotechnologyGrowth/Exponential Phase

Specific growth rate (μ) = measure of the rate of increase of cell number or biomass

TNoN

hN1

dTdN

lnln

)1(

or

Dt0.6931

Dt2ln

μ

Lecture 5 Animal Cell BiotechnologyGrowth/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

Lecture 5 Animal Cell BiotechnologyGrowth/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 contentButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P80.

►► ►

Flow cytometer and cell sorter

Fig. 5.12

Lecture 5 Animal Cell BiotechnologyGrowth/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.

Lecture 5 Animal Cell BiotechnologyStationary 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

Lecture 5 Animal Cell BiotechnologyStationary 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

Lecture 5 Animal Cell BiotechnologyThe Decline Phase - Necrosis

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

Two different death mechanisms:

Lecture 5 Animal Cell BiotechnologyThe 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

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.

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)

Lecture 5 Animal Cell BiotechnologyThe Decline Phase - Apoptosis

Smith and Wood, Eds. 1996. Cell Biology 2nd Ed. London:Chapman and Hall. P 507.

Lane 1: Mr DNA markersLanes 2-4: from mouse thymocytesshowing DNA laddering

Lecture 5 Animal Cell BiotechnologyThe Decline Phase - Apoptosis

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P51.

Lecture 5 Animal Cell BiotechnologyThe 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

Lecture 5 Animal Cell BiotechnologyNecrosis vs. Apoptosis – a comparison

http://www.niaaa.nih.gov/publications/arh25-31/images0.1.gif - accessed Jan 12/05

Measurement of specific productivityFig. 11.2

• 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).

The final yield of the product will depend on :-

Cell specific productivity

= P.

=

P.

1

Nt

0

dt

Qs

=

P.

=

P.

Time (hour)

0 20 40 60 80 100 120 140

IgG

con

cent

ratio

n (u

g/m

l)

0

20

40

60

80

Cel

l den

sity

(x1

06

cells

/ml)

0

2

4

6

8

10

12

14Mab from TB/C3.bcl2

Mab from TB/C3.pEF

Growth of TB/C3.bcl2

Growth of TB/C3.pEF

Determination of specific rate of productivity

t

0 X.dt

18.3 pg/cell per day

28.5 pg/cell per day

(105 cell-hours/ml)

Viability index

0 20 40 60 80 100

IgG

(m

g/L

)

-20

0

20

40

60

80

TBC3.bcl-2

TBC3.pEF

Wurm,F (2004) Nature Biotech 22: 1393

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.