Maintenance and

Characterization of cells
Maulik P. Suthar

© 2009, Maulik Suthar

 MEASURING PARAMETERS OF
GROWTH
(i) increasing the number of cells;
(ii) increasing the size of the cells; or
(iii) increasing the amount of intercellular substance.

Since the intercellular substance of a tissue is usually a secreted

product of the cell, for example collagen, it can be considered, so to
speak, as an extra-cellular extension of cytoplasm

© 2009, Maulik Suthar

 Cell counting

(i) Prepare the dishes in which the cells have been grown (this example is for 100-mm
dishes),
(ii) Prepare a trypsin solution, either 0.25 or 0.1 % (see note I below) in Hanks’ balanced
salt solution (containing no Ca2+ or Mg2+).
(iii) Pour Hanks’ solution (no Ca2+ or Mg2+) into 50-ml tubes,
(iv) Remove the medium from the dishes and set aside.
(v) Wash with 10 ml of Hanks’; remove.
(vi) Add 10ml of trypsin solution and leave for30sec-1.5 min (note i).
(vii) Remove the trypsin solution and let stand at room temperature for 2-3 min (note i).
(viii)Add the growth medium (which includes 5% calf serum), 10 ml. At this point the cells
will detach from the surface. In these days of very expensive serum we use the
conditioned medium obtained from step
(iv) to inhibit trypsin instead of fresh growth medium,
(ix) Mix well using a sterile pipette, drawing the cell suspension up and down thepipette 5-
10 times,
(x) Count in a haemocytometer (Figure 1), by depositing a few drops of cell suspension
under the coverslip. Use the four corners to count cells, divide by 4 and multiply by
1()4 to obtain cells/ml.
For instance, if you count 140 cells in the four corners:
• 140/4 = 35; 35 x 104 = 3.5 x 105 cells/ml

© 2009, Maulik Suthar

Cell counting

© 2009, Maulik Suthar

 Population Doublings

• The number of population doublings occurring in a culture in which

the cells are dividing mitotically can be determined by a geometric
progression expressed as y = x2n,
where
x = number of cells at the beginning of the growth cycle,
y = number of cells at the end of the growth cycle,
n = number of population doublings.

© 2009, Maulik Suthar

 Cell Viability

i. Dye exclusion
• The dye exclusion test is based on the concept that viable cells do
not take up certain dyes, whereas dead cells are permeable to
these dyes. Trypan blue (0.4%) is the most commonly used dye, but
has the disadvantage of staining soluble protein.
• In the presence of serum, therefore, erythrosine B (0.4%) is often
preferred . Cells are enumerated in the standard manner using a
haemocytometer.
• Some caution should be used when interpreting results as the
uptake of the dye is pH- and concentration-dependent, and there
are situations in which misleading results can be obtained.
• Two relevant examples are membrane leakiness caused by recent
trypsinization and freezing and thawing in the presence of
dimethylsulphoxide.
• A colorimetric assay using the MTT assay is being increasingly used
both to measure viability after release of cytoplasmic contents into
the medium from artificially lysed cells, and for microscopic
visualization withinthe attached cell
© 2009, Maulik Suthar
 DNA Amount
• The amount of DNA per dish can be determined, for instance, at 24-h
intervals after plating. It is also a very simple procedure; indeed, for
someone t/ained in biochemistry, it is easier than counting the number of
cells. Since the amount of DNA per cell is usually constant (in mammalian
cells, 6 x 10~12 g/ dipSoid cell in GI), the amount of DNA per dish is an
indirect measure of the number of cells.
• The G] amount of DNA in somatic cells is generally referred to as the In
amounT Cells in S-phase or in G2 have increased amounts of DNA with
respect to G, cells, but, if a population is truly growing, the increase in total
amount of DNA per dish will go way beyond the error caused by the
individual variations due to the distribution of cells throughout the cell cycle.
• DNA amount is probably the method of choice for sol id tissues. Using the
amount of DNA per cell given above, one can calculate (from the amount of
DNA per//g) the number of cells per ^g of tissue. A rough
• (very rough) estimate is that 1 /ug of tissue contains 5 x 1()8 cells, but this
estimate will vary greatly with the type of tissue.
• A possible source of error is polyploidy, that is an increase in the amount of
DNA per cell from 2n to 4n or even 8n, in which case one could have an
increase in the amount of DNA per dish without a concomitant increase in
the number of cells.
• This is not a frequent occurrence, but it can happen, for instance, in certain
pathological conditions, in response to certain drugs or when a large
proportion of cells is blocked in G2. There are several methods for
determining the amount of DNA in a culture dish or in a tissue. © 2009, Maulik Suthar
DNA Amount

© 2009, Maulik Suthar

DNA by the Diphenylamine Reaction

• The quantitative determination of DNA is made by analysis of the

deoxyribose content of the hot TCA extract in a colorimetric reaction
with diphenylamine. Since a cell contains about 5 pg of DNA, and
because microgram amounts are required to develop a detectable
color, 1 X 106 cells is the very minimum numberthat can be
analyzed.
• In the hot TCA extraction step, the DNA structure collapses;
deoxyribosides are released as both 5'- and 3'-phosphate bonds are
hydrolytically cleaved. Deoxy-ribose is jthen freed as glycosidic
linkages with purines are broken. Under theseconditions, the bond
between pyrimidines and deoxyribose is not cleaved, so only one
half the deoxyribose residues in DNA are available to react with the
reagent. The blue color develops in the reaction

© 2009, Maulik Suthar

DNA by the Diphenylamine Reaction

• Deoxyribose is hydrolyzed to levulinic acid, which forms a complex with

diphenylamine
• Diphenylamine purification. Diphenylamine can be recrystallized by
dissolving the reagent-grade compound in boiling hexane or in 70 percent
ethanol. Upon cooling, a white crystalline product is obtained
• Diphenylamine reagent. One gram of purified diphenylamine is dissolved
in 100 ml of reagent-grade glacial acetic acid and 2.75 nil of reagent-grade
concen-trated sulfuric acid. The reagent is prepared immediately before
use.’standard curve purified DNA between 10 and 200 fig is reacted with the
^(icnylamine reagent to obtain a standard curve. The intensity of the blue
colorI’-developed is directly related to concentration.
• A straight line is obtained when the concentration of DNA is plotted against
the absorbance.
• Deoxyribose in the range of 5 to SO ng can also be used to obtain a
standard curve. Since only deoxyribose linked to the purine reacts, a
conversion factor can be employed by considering that one molecule of 2-
deoxyribose is equivalent to a purine nucleotide plus apyrimidine nucleotide.
• Thus, the DNA content would be 4.87 times the measureddeoxyribose.

© 2009, Maulik Suthar

 RNA Amount

• For a given cell type, the amount of RNA ought to be constant. As with
DNA, G2 cells will have roughly twice the amount of RNA as G, cells. As a
measure of cell number, however, RNA amount is less accurate than DNA
amount because, in several conditions, cells can grow in size and increase
their RNA amount without cell division
• Indeed, RNA amount is a good indicator of cell size, rather than cell
number.
• Most of the cellular RNA that is measured by bulk chemical methods or
individual cell histochemical methods is ribosomal RNA (rRNA ~85% of total
cellular RNA).
• Since rRNA forms a part of the ribosome, on which protein synthesis is
carried out, it seems logical that RNA amounts ought to be a reasonable
indicator of cell size, a hypothesis that has been empirically confirmed.
• Classic methods for the determination of RNA amounts can be found
elsewhere. If one wishes to determine the amount of RNA in individual cells,
one needs expensive equipment that, in addition, require technical expertise
to operate.
• I prefer computerized microspectrophotometry to flow cytofluorimetry, but
both are complex and unless one is a devoted cell biologist who loves to
look at single cells, my advice is that when these instruments are needed
one should seek collaboration. Flow cytometry has already been discussed
in a previous book from this series . © 2009, Maulik Suthar
 RNA by the orcinol reaction
• The quantitative determination of RNA is made by reacting the ribose in the
macromolecule with orcinol. Since a single cell contains between 10 and 20
pg of RN A, 5 X 105 cells is the minimal number that can be used for the
assay.
• In the acid-extraction procedure, only ribose linked in a glycoside bond with
purine is freed and available to react with orcinol. A green-colored complex
formsbetween ribose and orcinol.
• Orcinol purification. Orcinol is purified by dissolving it in boiling benzene,
decolorizing with charcoal, and crystallizing after adding hexane.
• A white crystal-line product can be obtained in this manner.
• Orcinol reagent. One gram of purified orcinol is dissolved and 0.86 g of
Fe(NH4)2SO4-6H2O in 25.0 ml of double-distilled water.
• This is stored as a stock solution in the cold at 4°C. The working reagent is
prepared immediately before use by adding 5.0 ml of the orcinol solution to
85.0 ml of concentrated HC1; additional distilled water is added to bring the
volume to 100.0
• ml. Reaction.
• A 1-ml sample is mixed with 3 ml of orcinol reagent and heated in a boiling-
water bath for 20 minutes. After cooling, the intensity of the green color mat
develops is read at 670 nm. Standard curve. Purified RNA at a
concentration ranging from 10 to 100 jug is measured at required nm
© 2009, Maulik Suthar
 Protein Amount

• The same comments apply here as to RNA amount.

• The amount of cellular protein is a reasonable indicator of cell size,
but a poor indicator of cell number.

© 2009, Maulik Suthar

 Protein by the Lowry Reaction

• The quantitative determination of protein is made by reacting the

Folin-Ciocalteau reagent with the alkali-solubilized precipitate after
the extraction of DNA and RNA. The blue color that develops is a
result of (1) biuret reaction of proteins with copper ion in alkali, and
(3) reduction of tungstic reagent by the tyrosine and tryptophan
present in the treated protein.
• Since a cell contains between 300 and 600 pg of protein, the
reaction can be carried out using 0.5 to 1.0 X 10s cells.
Reagents
• Reagent A: dissolve 0.5 g of CuS04 -5H2O and 1 g of sodium or
potassium tar-trate in 100.0 ml of water.
• Reagent B: carbonate-copper solution. Mix 50.0 ml of 2 percent
Na2CO3 with 1.0 ml of reagent A. Discard after 1 day.
• Reagent C: alkaline copper solution. Mix 50.0 ml of 2 percent
Na2C03 in 0.1 A’ NaOIl with 1.0 ml of reagent A. Discard after 1
day.
• Reagent D: diluted Folin reagent. Dilute the Folin-Ciochalteau
reagent to make it 1 N in acid.
© 2009, Maulik Suthar
 Mitoses
• However, mitoses arefleeting; in most cells they last only 45 min and, unless
one looks at precisely the right moment, one may miss them. Furthermore,
the duration of mitosis can increase in certain cells, especially in
transformed cells.
• Everything else being equal, if the duration of mitosis in cell line A is twice
that of cell line B, the number of mitoses in A will also be twice that of B,
although the two cell lines may grow at the same rates. In tissues, the
number of mitoses per 1000 cells (the mitotic index) is a reasonable
measure of the proliferating activity of a cell population, but not of its growth.
• For instance, in the crypts of the lining epithelium of the small intestine,
there are many mitoses. Fortunately for us, the small intestine in the adult
individual does not grow, because, for
• every new cell produced in the crypts, one dies at the tips of the villi.
• So, in any given cell population, one must distinguish between cell division
and increase in cell number. There are also technical problems. To begin
with, if we wish to determine the mitotic index of cells in culture a 22-mm2
coverslip will have to be placed into the culture dish.
• Mitoses can then be counted directly on the coverslips after fixation and
staining .Staining and counting of mitoses directly on plastic surfaces is not
advisable. However, suppose that a wave of mitoses occurse 25-27 h after
stimulation of a quiescent cell population with growth factors. One may miss
it, unless samples are taken practically every hour.
© 2009, Maulik Suthar
 Mitoses

• More economic in terms of time and money is to add a drug that will
arrrest cells in mitosis. One can then count the percentage of cells
that accumulate in mitosis over a certain period of time.
• The three main drugs for this purpose are colcemid, colchicine and
nocodazole. Colcemid and colchicine are very similar but the latter
is more toxic.
• For mitotic arrest, the optimal concentration of colcemid is 0.16
,wg/ml for human cells or 0.04-0.08 ug/ml for rodent cells.
• I like to leave the drug in for 4 h, then fix the coverslips. By dividing
the time period into 4-h blocks (for instance, 16-20 h; 20-24 h; 24-28
h) after serum-stimulation, once should be able to get a pretty good
idea of the mitotic activity of a cell population.
• If cells are left in colcemid (and especially colchicine) for more than
4 h, cell damage occurs with loss of mitotic figures. Nocodazole
offers the advantage that it can be used for longer periods of time,
16-24 h.’We use it at concentrations of 0.04-0.2 ,Mg/ml, and
mitoses, clearly identifiable, continue to accumulate.
• Depending on the cell line, and up to 12-16 h, nocodazole arrest is
reversible (so is colcemid-arrest but only up to 4 h).
© 2009, Maulik Suthar
Cells Arrested in Mitosis by Nocadozole

(i) Coat the coverslips (four per 100-mm Petri dish) with poly-L-lysine
(Sigma 3000 mol. wt) at 1 ing/ml dissolved in Hanks’ (calcium,
magnesium freesolution). Leave for 24 h.
(ii) Remove the polylysine solution and allow the coverslips to dry.
(iii) Plate 5 x 10s cells per 100-mm dish in normal growth medium,
each dish containing two polylysine-coated coverslips.
(iv) After 18 h remove the medium and add fresh growth medium plus
0.1-0.2 ,wg/ml of nocodazole dissolved in dimethylsulphoxide
(DMSO).
(v) Leave for the desired period of time and then fix using the method
given above and stain with Giemsa/Sorenson’s buffer.

© 2009, Maulik Suthar

The Phases of a Culture

© 2009, Maulik Suthar

 The Growth Studies

• Populations of animal cells cultured in vitro increase in number as

the individual cell divides mitotically. Whether they are primary cells,
cell strains, or establishedcell lines, multiplication
• begins only after a period of adjustment and stops when anumber is
reached that is saturating for the system.
• If the logarithm of number of cells present at any time in the culture
is plotted as a function of time, a smooth growth curve of
characteristic of culture system i sobained
• The growth curve for mammalian cells can be divided into three
phases: the lag phase,the log phase or exponential phase, and the
plateau phase.

• The lag phase usually varies from 24 to 48hours. In this phase, the
cells do not divide but are in the process of adapting to the medium.
When the cells begin to divide, the population enters the
logarithmics of the culture cycle in which the cell number is
increasing at a constant rate is period, which lasts 2 to 8 days, the
population at any time is composed will points in the division cycle.
© 2009, Maulik Suthar
 The Growth Studies

• The population doubling time for cultured cells ranges from 12 to 48

hours. Since cultures are usually started with 50,000 to 200,000
cells, four or five population doublings occur during the culture
cycle.

• A population of cells will stop dividing when an essential nutrient is

depleted or an inhibitory substance is produced. At this point the
culture enters the plateau phase, which is characterized by the fact
that a constant cell number prevails over a period of time.

• The constancy of cell number may result because division ceases in

all cells or because some cells degenerate and die while others
continue to divide

© 2009, Maulik Suthar

 Mathematical Growth Curve

• The growth curve for a population of animal cells Analysis of Cell

Multiplication in Populations cultured in vitro can be subjected to a
mathematical analysis. Three useful parameters can be calculated
that define the cell population increases with time:
(1) the number of population doublings occurring throughout the growth
cycle,
(2) the population doubling time when the cells are dividing in the
exponential phase, and
(3) the rate of growth in the exponential phase. The formulas and
methods for calculating these parameters are presented in the
following discussion.

© 2009, Maulik Suthar

 The Lag Phase
• This is an early phase in which there is no apparent increase in cell
concentration. This phase is associated with the cellular synthesis of
growth factors I which may be required to reach a critical
concentration before growth takes place.
• The length of this phase is dependent upon the culture medium
formulation as well as the initial concentration and state of the cells.
The lag I phase tends to be longer at low inoculation densities or if
the viability of the inoculated cells is low.
• This may occur if subculture is delayed. If a high density of cells with
good viability is inoculated into the culture medium then the lag
phase may be eliminated altogether. Transformed cells have a I
lower requirement for growth factors and often show no lag phase
even I when inoculated at lower concentrations
• In some circumstances, there may be a requirement to inoculate at
a low I cell density, for example during cell cloning.
• Cloning is the establishment of a culture from a single cell. This
ensures that all cells in the culture are identical, i.e. a homogeneous
population. In this situation cell growth can be enhanced by adding a
feeder layer which consists of irradiated cells incapable of growth
but metabolically active and capable of releasing growth factors into
the medium. © 2009, Maulik Suthar
Exponential Phase

© 2009, Maulik Suthar

 The stationary Phase

• This phase occurs when there is no further increase in cell

concentration. During the stationary phase, death rate = growth rate.
• At this point cell growth is limited by one of a number of conditions
nutrients may have been depleted to a level that cannot support
further cell growth;
• the accumulation of metabolic by-products may have reached a
level which is inhibitory to cell growth; the cells may have formed a
complete cover over the growth surface.
• Growth may stop when a single monolayer of cells covers the
available substratum (called ‘confluence’).
• This phenomenon is associated with cell-cell interaction .
• During the stationary phase the cells may be metabolically active
even though growth is not occurring. For example, in some cases
high productivity of secreted proteins may occur during this phase.

© 2009, Maulik Suthar

 The Decline Phase

• This phase occurs as a result of cell death. Cell viability is lowest

during the decline phase of culture - shown by a large difference
between total and viable cell counts
• The measured viable cell concentration decreases as the cells lyse
and their intracellular metabolites are released into the growth
medium.
• There are two possible mechanisms of cell death in culture -
apoptosis or necrosis.

© 2009, Maulik Suthar

 In vitro
• In exponentially growing cell populations the growth fraction is close to
100% and gives very little information. Thus, if we take almost any cell
line that grows with a doubling time of 24 h or
• less during the exponential growth phase, and we label it with
[3H]thymidine for 24 h, the number of labelled cells would be close to
100%.
• The odd unlabelled cells can probably be considered as dying cells. The
fraction of labelled cells becomes important in experiments dealing with
the effect of growth factors on the state of quiescence of a cell
population.
• For instance, many cell lines stop proliferating when they reach
confluence. It is desirable to test the extent of quiescence in a cell
population in culture by exposing the cells to [3H]thymidine. The interval
usually taken, more for expediency than for any logical reason, is 24 h.
• Populations of cells that can be made quiescent should, at this point
have very low labelling indexes, that is not more than 1% of the cells
ought to be labelled by a 24-h exposure to [3H]thymidine .
• At this point if one wishes to determine the effect of growth factors on
the proliferation of this particular cell population, all one has to do is to
add the growth factors together with [3H]thymidine and let the
experiment run for 24 or 48 h. © 2009, Maulik Suthar
Flow of Cells Through the Cell Cycle
• At times it is desirable to determine whether there is a block or a
delay in a particular phase of the cell cycle. This can be caused by a
drug, by treatment with inhibitory factors or, for instance, in
temperature-sensitive mutants, by a defect in one of the gene
products that are necessary for cell cycle progression.
(i) A block in S-phase, due to direct inhibition of DNA synthesis, can be
detected by exposing the cells at various times after the treatment,
to a 30-min pulse of [3H]thymidine. An inhibition of DNA synthesis
will result in a quick decrease in the fraction of cells that can be
labelled by the pulse exposure, usually within 30 min of treatment.
(ii) If, instead, the decrease in the fraction of cells that are labelled by
pulse exposure to [3H]thymidine is delayed, then one should
suspect a block in Gj, or even later. For
• instance, suppose that a given drug acts at a point in Gt which is
roughly located 4 h before the S-phase. If that drug is given and the
cells are then pulsed with
• [:’H]thymidine at various intervals after treatment, what one will
observe is that the percentage of labelled cells will remain constant,
as in controls, for 4 h.
© 2009, Maulik Suthar
MAINTAINANCE OF CELL LINES

© 2009, Maulik Suthar

Common features of continuous cell lines

1. Smaller, more rounded, less adherent cells with a higher

nucleus/cytoplasm ratio.
2. Increased growth ratio; cell doubling time reduced to 12-36 hour
from 36- 48 hours for finite cell lines.
3. Aneuploid (between In and 4n) chromosome number showing
considerable variation among cells of a cell line.
4. Reduced serum dependence.
5. Increased cloning efficiency.
6. Reduced dependence on substrate adhesion (anchorage) and
increased tendency to grow in suspension.
7. Many of the cell lines become malignant, but some cell lines are
normal and nonmalignant. The changes in growth control are under
distinct positive (oncogen) and negative (tumour suppressor genes)
control genes.
8. Ability to grow up to higher cell densities, i.e., growth is less
dependent on cell density.

© 2009, Maulik Suthar

 Cryopreservation

• Freeze preservation of animal cells is now routine in all

cell line banks. Acryoprotective agent like DMSO or
glycerol is generally added to minimize injury to cells
during freezing and thawing.
• Frozen ampoules are generally stored in liquid nitrogen
refrigerators which are rather convenient and quite safe.
• Following appropriate protocols most animal cells can be
stored for quite long periods.

© 2009, Maulik Suthar

“Transformed” (or continuous) cell lines

• Large-scale production of bio-pharmaceuticals normally

utilizes “transformed” (or continuous) cell lines. These
grow faster than ordinary cells and can be grown at
greater densities, so they give higher yields in
bioreactors.
• They are also easier to maintain in simple culture media.
Like cancer cells, “transformed” cell lines do not stop
dividing to repair any damage, instead, they keep
dividing as long as there are sufficient nutrients and that
other suitable chemical and physical conditions for
survival are maintained.

© 2009, Maulik Suthar

Applications of Flow Cytometry to Cell
Culture Identification and Control

© 2009, Maulik Suthar

Flow Cytometry to Cell Culture Identification
and Control

© 2009, Maulik Suthar