Author : Dr.

Saurav K Sarkar
Discipline : B. Tech (Biotech)
Module : BT 701
Topic : Growth Kinetics

Designation : Lecturer
Semester : VI

Phases of cell growth

Normal cells in culture typically show a sigmoidal pattern of proliferative activity that reflects adaptation to culture,
conditioning of the environment, and the availability of physical substrate and nutrient supply necessary to support the
production of new cells (Figure 2).
Growth curve for normal mammalian cells in culture.
Lag phase
Following seeding, cells exhibit a lag phase during which they do not
divide. The length of lag phase is dependent on at least two factors,
including the growth phase at the time of subculture and the seeding
density. Cultures seeded at lower density condition the medium more
slowly and have a longer lag phase. Cultures seeded from actively
growing stock have a shorter lag phase than cells from quiescent
stocks.
Logarithmic (log) growth phase
Period of active proliferation during which the number of cells
increases exponentially. In log phase the percentage of cells in the cell cycle may be as high as 90-100%, and at a given
time cells are randomly distributed throughout the phases of the cell cycle. During log phase the cell population as a
whole is generally considered to be at its 'healthiest', so it is common to use cells in log phase when assessing cell
function. Cell proliferation kinetics during log phase are characteristic of the cell line and it is during this phase that the
population doubling time is determined. Factors that influence the length of log phase include seeding density, rate of cell
growth, and the saturation density of the cell line.
Plateau (or stationary) phase
The rate of cell proliferation slows down and levels off as the cell population attains confluency. Many cell lines in
substrate-dependent culture still show some degree of mitotic activity at confluency. The cells may continue to divide and
pack tighter, or daughter cells may be released into the overlying medium. At plateau phase, cell division tends to be
balanced by cell death, and the percentage of cells in the cell cycle may drop to 0-10%. For some cell lines the plateau
phase can be extended if the medium is replenished. This is not a stable period for most cell lines and plateau phase
cells may be more susceptible to injury. Cells in suspension culture also reach plateau phase (saturation density)
dependent in part on availability of nutrients.
Decline phase
After the plateau phase is a period of decline in cell number (decline phase) due to uncompensated cell death. This is
not merely a function of nutrient supply.
Growth can be defined as Specific growth rate, -- the rate of growth per unit amount (weight / numbers of biomass):
where dx is the increase in cell mass. dt is the time interval and X is the cell mass. If the growth
rate is constant (e.g. during
logarithmic growth), then where X0 is
the biomass at time t0.
Calculation of in vitro age
Normal cell lines undergo in vitro age-related changes in structure and function and have a finite lifespan. Transformed
cell lines may also show changes with successive generations. Since cell lines do not remain stable throughout their
lifespan it is important to track the in vitro age of the line. Passage number documents the number of times a culture is
handled, but it is at best a crude estimate of the age of the cell line. A more useful method is to calculate the number of
cell generations the line has undergone, to determine the number of cumulative population doublings.
Population doubling level (generation number) : Cells in culture undergo sequential symmetric divisions such that the
population increases in number exponentially. At the end of n generations each cell of the original seeded inoculum
produce 2n cells. Thus, the total number of cells at a given time following inoculation is given by NH = N2n, where NH is
the number of cells harvested at the end of the growth period, and N1 is the number, of cells inoculated. The number of
generations is the number of population doublings, and can be expressed as:

Author : Dr. Saurav K Sarkar

Discipline : B. Tech (Biotech)
Module : BT 701
Topic : Growth Kinetics

Designation : Lecturer
Semester : VI

Example. 2.5 x 105 cells seeded

N H
NH
2
or n log 2 log
log N H log N1 N1 = 2.5 x 105
N1
N1
log 2.5 X 105 = 5.3979
Q log 2 0.301, 0.301n log N H log N1
6.0 X 106 cells harvested
n 3.32 log N h log N1
NH = 6.0 X 106
6
log 6.0 x 10 = 6.7782
n = 3.32 (6.7782 5.3979) = 4.58
Therefore, to yield 6.0 x 106 cells, 2.5 X 105 cells underwent 4.58 population doublings.
Add the number of generations for growth interval (time between seeding and harvest) to the previous PDL number to
give the cumulative PDL. Cumulative PDL is recorded on the flask, and similar calculations are performed at each
subsequent subculture.
PDL cannot be calculated without an accurate determination of cell number in the original inoculum and so is usually not
attempted with primary cultures. By convention the primary culture at confluency is designated PDL 1.
Multiplication rate (r) is the number of generations that occur per unit time and is usually expressed as population
doublings per 24 hours. Population doubling time (PDT) is the time in hours, taken for cell number to double, and is
the reciprocal of the multiplication rate.
PDT = total time elapsed/number of generations
Example. 2..5 x 10s cells were seeded at time zero (t1), and 6.0 X 106 cells harvested at 96 h (t2).
Multiplication rate (r)
= 3.32 (log NH - log NS) / (t2 t1)
= 3.32 (6.7782 - 6.3979)/96
= 0.048 generations per hour or 1.15 population doublings/24 hours PDT = 1/r PDT
= l/(1.15/24) h = 24/1.15 h = 20.9 h per doubling
Population doubling level, multiplication rate, and population doubling time describe the growth characteristics of the cell
population as a whole, and strictly speaking do not characterize the division cycle of individual cells. By definition, PDT
(commonly 15-25 h) is not the same as cell generation time (cell cycle time), which is the interval between successive
divisions (mitosis to mitosis) for an individual cell. In practice, however, PDT is used as an estimate of cell cycle time and
to determine the length of the phases of the cell cycle.
Cells must be subcultured at regular intervals and when the culture is in the log phase of growth.
Each subculture should be at the same seeding density, and culture conditions must be consistent, including the same
type of substrate, size of flask, volume of medium, and formulation of medium. When combining (e.g. averaging) data
from multiple cultures for the estimation of PDT, it is best to calculate the individual multiplication rates first, average
these, and finally calculate the PDT, otherwise a single slow-growing flask (giving a long PDT) can disproportionately
skew the result.
Haemocytometer counting : The simplest, most direct, and cheapest method of counting cells in suspension. It also
allows the percentage of viable (intact) cells in the preparation to be determined using the dye exclusion method. The
haemocytometer is a modified microscope slide that bears two polished surfaces each of which displays a precisely
ruled, subdivided grid. The grid consists of nine primary squares (area 1 mm) and limited by three closely spaced
lines. These triple lines are used to determine if cells lie within or outside the grid. Each of the primary squares is
further divided. The plane of the grid rests 0.1 mm below two ridges that support a coverslip. There is a bevelled
depression at the leading (outer) edge of each polished surface, where cell suspension is added to be drawn across the
grid by capillarity.
(a) Counts must be performed in the same way each time. Standardize the cell dissociation procedure for a given cell
line (or tissue for primary culture) and use the same counting protocol and conventions.
(b) When the haemocytometer is properly loaded, the volume of cell suspension occupying one primary square is
0.1 mm3 (1.0 mm2 X 0.1 mm) or 1 X 10ml.
(c) Mix 0.1 ml cell suspension with 0.1 ml isotonic Trypan blue solution. Load the haemocytometer with the help
of a pipette, avoiding overflowing. Diluent (like dye solution) must be isotonic. Dilution factor is then 2.
(c) Count the cells within ten primary squares (five primary squares per chamber), to give the number of cells within
10 x 0.1 mm or 1 x 10 ml. Total cell concentration in original suspension : Total count x 1000 cells/ml x dilution
factor.
n

Author : Dr. Saurav K Sarkar

Discipline : B. Tech (Biotech)
Module : BT 701
Topic : Growth Kinetics

Designation : Lecturer
Semester : VI

Determining the number of viable cells by dye exclusion : Dye exclusion involves mixing an aliquot of the cell
suspension in a volume of buffer or balanced saline containing a water-soluble (membrane lipid-insoluble) dye (e.g.
0.4% Erythrosin B, or Trypan Blue) that is visible when it leaks into cells that have damaged plasma
membranes. By counting total cells and stained cells (damaged cells) one can calculate the per cent viability.
Recently, a device has come on the market which automatically counts viable and dead cells as stained by Trypan Blue,
using the techniques of microscopy and irriage analysis. One should be cautious in interpreting cell 'viability' by dye
exclusion. Dye uptake marks cells that have grossly disrupted membranes and may not detect other forms of
injury that affect cell attachment or may progress to cell death. Also, this method does not account for cells that
have fully lysed (i.e. are no longer identifiable as cells). In addition, the choice of dye may influence results. Trypan Blue
has a greater binding affinity for protein in solution than for injured cells, and should only be used in protein-free solution.
Fluorescence in situ hybridization (FISH) involves the hybridization of appropriate DNA probes with the DNA in the host
cell chromosomes. The probe is labelled non-isotopically by incorporating nucleotides which have been modified with
molecules such as biotin or digoxigenin, which are then detected with a fluorochrome-labelled antibody against the biotin
or digoxigenin. The location and extent of amplification of both recombinant and non-recombinant genes can be detected
in metaphase spreads even when the sequences are only present in a single copy.
Chromosome painting (or spectral karyotyping, SKY) involves the use of a collection of probes which are chromosomespecific. These paints are available commercially from a number of suppliers, and are produced such that each
chromosome can be labelled with a different colour. This allows complex translocation events to be detected and
analysed even when only small fragments of a chromosome are rearranged.