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Hai all! today we will see an important aspect,t he cell synchroni-
zation.
Introduction
Whole-culture methods of eukaryotic cell synchronization are
the most widely used approaches to cell-cycle analysis. Despite
this wide-spread use, theoretical considerations and experimen-
tal evidence indicate that whole-culture methods cannot
synchronize cells. Why, despite these clear experimental and
theoretical critiques, are these methods still so widely used?
Reasons for the persistence of whole-culture synchronization
methods are explored.
It is generally believed that it is possible to synchronize cells by
whole-culture treatment of growing cells.
Definition
Whole-culture synchronization means that the cells in a
previously unsynchronised culture are induced to form a
synchronized culture by applying a common treatment to all
cells. When cells of various cell-cycle ages are all treated identi-
cally and growth arrested, it is generally presumed that the cells
arrest at a common cell-cycle age. It is further believed that upon
release from growth arrest these cells can generate a synchro-
nized culture.
One common and often-described synchronization method
involves placing growing mammalian cells in a low-
serummedium producing growth arrest. The arrested cells are
assumed to enter a G0 or a G0/ G1 phase, or to arrest at a
restriction point within the cell cycle. Upon resumption of
growth by addition of normal serum concentrations, the cells
are believed to move as a synchronized cohort through the cell
cycle.
Other treatments such as hydroxyurea to inhibit DNA replica-
tion, nocodazole to inhibit mitosis, or mimosine inhibition, are
also proposed as synchronizing agents.
Treatment of a growing culture with the cholesterol-lowering
drug lovastatin has even been suggested as a synchronizing
agent.
Synchronous Growth
The sequential order in which biosynthetic events occur during
the cell-division cycle can be ideally studied by following the
activities in a single cell by cytochemical, autoradiographic, and
spectrophotometric methods. Since the same cell cannot be
used to follow the events throughout, populations are
synchronized, so that representative samples can be removed
from the culture at various points in the division cycle. During
the past 20 years, some very successful methods have been
developed for synchronizing populations of mammalian cells
in culture, which, in turn, have led investigators to attempt to
map the order of various events occurring during the cell-
division cycle. One of the first studies showed that DNA is
synthe-sized in mid-interphase, and is separated by two gaps
occurring before and after mitosis. Howard and Pelc had
previously recog-nized four stage in their studies with bean
roots, and named them mitosis (M), first gap (Gl), DNA
synthesis (S), and second gap (G2). In mammalian cells, the S
stage most often occupies about 7 hours of the division cycle,
regardless of the genera-tion time; the M stage occupies 3 to 4
percent of the division cycle. It appears that mammalian cells
have different generation time.
Two general procedures are employed to obtain synchronously
growing popula-tions of mammalian cells in vitro:
(1) A small fraction of the cells in a population can be
selectively isolated at a certain point in the division cycle, or
the undesired cells can be preferentially destroyed.
(2) All the cells, or at least a large fraction, can be blocked at a
specific point in the division cycle by using an inhibitory
com-pound, or by withholding an essential nutrient.
Procedures representing both approaches are outlined.
Selective Isolation of Synchronously Growing Cells
(a) Collection of loosely attached mitotic cells
Terasima and Tolmach introduced a simple procedure for the
selective isolation of dividing cells; they exploited the observa-
tion that cells growing atatched to a surface round up during the
mitotic period and can be dislodged by using a gentle shearing
force. The detached cells are pelleted and resuspended in a
complete medium in which they grow in synchrony for one
division cycle. A limit-ation of this method is that only about 4
percent of the cells are in the mitotic stage when the population
is growing at an exponential rate, and only about one fourth of
these can be obtained. The following procedure can be used to
collect dividing cells.
1. Between 1 and 2 X 106 -cells in 10.0 ml of a complete
medium are cultured in a 100-mm plastic petri dish at 37C
in an atmosphere of 5 percent CO2 in air.
2. The medium is discarded after 18 hours and replaced with
fresh prewarmed medium of the same composition. This
step is carried out to remove any dead cells that have
become detached from the surface.
3. After 6 hours, the medium is again removed and discarded,
and 5.0 ml of fresh prewarmed medium is forcibly ejected
from a 10.0-ml pipette to wash off the loosely attached
dividing cells. The force necessary to release the cells must
be determined empirically.
4. The suspension of detached cells is pelleted and
resuspended in a fresh or a conditioned medium. Since
only small numbers of cells are obtained, the suspen-sion
is usually replicated into Leighton tubes containing cover
slips.
LESSON 13
CELL- SYNCHRONIZATION
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(b) Separation of uniformly sized cells by gravity
Synchronous cells have been separated by centrifuging exponen-
tially growing populations in linear 2 to 10 percent (w/ v)
sucrose gradients made up in a com-plete growth medium .
Shall and McCleUand found that cultured animal cells would
also stratify according to size in a complete medium under the
natural force of gravity. In the latter procedure, the cells se-ated
for their size uniformity had a doubling time of 22 hours,
whereas these cells normally have a doubling time of 28 hours
when growing in the exponential phase. The procedure of Shall
and McClelland is presented.
1. A sterile, cylindrical, screw-capped tube, 120 by 15 mm, is
filled with 12.0 ml of a chemically defined medium
containing 5 percent calf serum. The medium in the tube is
equilibrated at 37C for 30 minutes.
2. A population of 30 X 106 cells growing in the exponential
phase is pelleted at lOOXg for 5 minutes. The growth
medium is removed to another sterile tube and saved, and
the cells are resuspended in 0.75 ml of a serum-free
medium. The suspen-sion is layered onto the column with
a wide-mouth pipette by slowly traversing around the
inside wall, just above the liquid-air interface, so that the
cells spread over the surface to the center. The layer of cells
is gently stirred with the tip of the pipette (a streamer of
cells forms just beneath the layer, but does not influence
the separation).
3. The column is placed into the incubator at 37 C for 50
minutes, and is tightly capped to prevent the loss of CO2.
4. The top 1.0 ml, which contains about 2.25 percent of the
cells, is collected. These cells are uniform in size and
represent a population that can be grown syn-chronously.
The bottom 11.0 ml of cell suspension containing
exponential-phase cells is used as a control population.
Both suspensions are centrifuged and resus-pended in the
conditioned-used medium at comparable cell
concentrations for growth.
Blocking of Cell Cycle by an Inhibitory Compound
(a) Isolation of non-DNA synthesizing cells by the selective
destruction of S-phase cells with radioactive thymidine
Tritium-labeled thymidine with a specific activity of M3.7
Ci per mmol at a concentration of 1 MQ per ml of culture
medium has been used to selectively kill cells that are
synthesizing DNA. A lethal burden of isotope is
incorporated in about 30 to 60 minutes. After several
hours, only cells in the latter part of the Gl stage are still
viable. At this point, the isotope is diluted out and the cells
proceed to grow synchronously. The disadvantage of the
pro-cedure is mat the cultures are contaminated with dead
or damaged cells, which makes biochemical analyses almost
impossible .
(b) Isolation of non dividing cells by discarding mitotic
arrested cells induced with Vinblastine sulphate
Vinblastine sulfate is obtained from an alkaloid extract of
Vinca rosea (peri-winkle). The compound causes
metaphase arrest and can be used in the following way to
select the unarrested cells growing in a surface culture
1. Cells growing in the exponential phase are treated with 0.3
Mg per ml of vinblastine sulfate and incubated at 37C.
2. After 16 hours, the arrested cells are washed off the surface
and discarded.
3. A fresh prewarmed or a conditioned medium is added to
the cultures. A burst of cell division occurs in the next 5
hours.
4. Vinblastine sulfate is added back to the culture at a
concentration of 0.3 pg per ml for another 8-hour time
period, and the newly accumulated metaphase cells are
washed away, which leaves cells growing in synchrony in Gl.
Population Synchronization
(a) Low-temperature procedure
Newton and Wildy introduced a cold-shock method for
synchronizing cultured mammalian cells. In the procedure, cells
growing in the exponential phase were cooled at 4C for a 1-
hour time period. When the cultures were incubated at 37C,
the cells failed to divide for 16 to 20 hours, and then about 80
percent divided within 4 hours. The cold shock appears to force
every cell to move into the Gl stage. The method has only
achieved limited success because of the complex and variable lag
phase occurring when the temperature is changed .
(b) Thymidine and double-thymidine Block
The use of excess thymidine (TdR) to block the synthesis of
DNA reversibly was introduced by Xeros. At a concentration of
2 to 10 mAf TdR, expo-nentially growing cells can be blocked at
any point in the S stage of the division cycle. When the block is
released, the cells start to synthesize DNA at the point of
inhibition, and proceed to move into the G2 stage. Since the S
stage requires 6 to 7 hours, the cell population is only partially
synchronized. To improve synchroni-zation, a double-TdR-
block method has been developed. In this procedure, an excess
of TdR is added back to a culture about 8 hours after the first
reversal when all the cells are in G2. The cells proceed to move
through M and Gl but cannot enter the S stage. Therefore, the
cells accumulate at the Gl * S interphase. Removal of TdR at
this point permits all the cells in the population to move in full
synchrony through the S stage. The biochemical blockage occurs
because the synthesis of other nucleoside triphosphates is
inhibited within cells treated with thymidine. Essentially, TdR is
taken up by cells and phosphorylated to thymidine-monophos-
phate (IMP) and thymidine-triphosphate (TIP). The
accumulation of TIP results in the feedback inhibition of other
nucleotide triphosphates, especially deoxycytidine-triphosphate
(dCTP). The following procedure has been used for synchroniz-
ing KB cells.
1. Exponentially growing cells in suspension culture are
treated with 2 mAf TdR for 18 to 20 hours.
2. The cells are pelleted by centrifuging at M60Xg for 5
minutes, and resus-pended in fresh prewarmed media.
3. The cells are allowed to grow at 37C for 9 to 12 hours. At
this time, TdR is again added to a final concentration of 2
mAf.
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4. After 18 to 20 hours, the cells are again pelleted, suspended
in a complete medium, and grown in full synchrony at
37C.
(c) Colcemid Arrest
Synchronization of animal cells can be achieved using Colcemid
(iV-desacetyl-ethylcolchicine), which induces a nonlethal
metaphase arrest. Colcemid is believed to prevent the centrioles
from organizing the microtubules, which arc necessary for
chromosome migration to the poles.Its removal allows normal
mitosis to proceed in 5 minutes. Thymidine blockage has been
used in conjunction with colcemid arrest to synchronize cells in
one cycle.
In the following procedure, Colcemid is used to arrest cells in
metaphase. Since Colcemid at a concentration of 0.06 jug per ml
induces the formation of binucleate cells after 4 hours the cells
are arrested at a concentra-tion of 0.02 ng per ml and the loosely
attached arrested cells are removed with a low trypsin concentra-
tion combined with vigorous shaking at 6 hours .
1. Approximately 1.5 X 106 cells in 25.0 ml of a complete
medium are cultured in 32-oz prescription bottles for 48
hours at 37C.
2. Colcemid is added to the culture at a concentration of 0.02
Mg per ml.
3. After 6 hours, the loosely attached, arrested, dividing cells
are detached by adding 2.5 ml of 0.5 percent trypsin and
rapping the bottle firmly onto a padded surface about 30
times.
4. The cells are collected by centrifugation at room
temperature and pooled into a fresh medium containing
Colcemid.
5. When a sufficient quantity has been obtained, the cells are
suspended in a fresh complete medium, and inoculated
into culture vessels at a concentration of ^50,000 cells per
ml. This is considered to be the 0 hour of the cell cycle.
After 1.5 hours at 37C, 86 to 96 percent of the cells are in
mitosis.
6. Cover slips are placed into the culture vessels, so that
samples of cells at-tached to these glass slides can be
removed for pulsing with [3 H] -thymidine to deten-nine
the stage of the cell cycle.
Cell-cycle Mapping
The stages of the cell cycle are mapped by analysis of TdR
incorporation. Cover slips with appropriately attached cells are
exposed to 1.0 ml of medium containing 1.25 MCi per ml of
[3II]-TdR (specific activity of 11 Ci per mmol) for 30 minutes at
37C. The reaction is stopped by rinsing the slides in 0.85
percent saline.
Radioactive Thymidine Incorporation
The cell sheet is dissolved in 30 seconds by adding 0.3 ml of 1
percent sodium dodecylsulfate (SDS). A 0.25-ml portion is
placed onto a glass filter disk and dried immediately. The disk is
extracted by dipping it into cold 5 percent TCA for 10 minutes,
and rinsed in 95 percent ethanol at room tem-perature. It is
then dried at 110C and placed into a scintillation vial with an
appropriate scintillation fluor.
Autoradiography
The site of synthesis of nucleic acids in an animal cell can be
determined by autoradiography. In the procedure, radioactively
labeled precursors that have been incorporated can be detected
by placing fixed preparations in direct contact with a layer of
photographic emulsion. As the radioactive atoms decay, they
emit rays that activate silver halide grains in the emulsion. The -
particles from [3H] , because of their short range (1 fim), are
ideal for studying the localization of the actual biosynthetic
event in the cell. A procedure for autoradiography follows:
1. The cover slips are washed in PBS and fixed in
glutaraldehyde for 15 minutes.
2. After thorough washing with water, the cover slips are
twice dipped into cold 2 percent perchloric acid (PCA)
for 5 minutes to remove unincorporated precursors.
3. The cover slips are washed again with water, the backs are
dried, and they are mounted onto a glass slide, cell side up,
with permount.
4. Kodak NTB-3 emulsion is stored refrigerated as a gel in a
screw-cap bottle inside a double light-light box. In the
darkroom, a small amount of gel is trans-ferred to a
suitable vessel and slowly melted.
5. The slide is dipped into the emulsion, momentarily
drained, and set verti-cally in a rack in an oven set at 28C
for at least 1 hour in the dark.
6. The slides are men placed in a light-tight box containing
desiccant and stored at 4C.
7. The photographic emulsion on the slide is developed with
a stock solution of Dektol (D-72) as directed on the
package.
8. The cells are stained with Giemsa solution diluted 1:30 for
10 minutes. The excess stain is washed off with distilled
water. The slides are dipped briefly in 70 percent ethanol,
dehydrated in 95 and 100 percent alcohol, cleared with
xylene, and mounted with permount.
9. The cells are examined with the low-power objective, and
the percentage of cells with label is determined. Under
higher power, the number of grains per cell can be
counted.
Karyotype Analysis
The chromatin in the nucleus of a eucaryotic cell begins to
condense into chromosomes with the onset of mitosis. In the
metaphase, as the nuclear membrane fragments, the chromo-
somes further condense, and are clearly visible when mitotic cells
are viewed through a light microscope. A metaphase chromo-
some is composed of two equivalent segments called
chromatids, which are joined at one point by a centromere. The
position of the centromere varies in different chromosomes,
which gives them a characteristic shape. Chromosomes having a
centromere at one end are called telocentrics, and at metaphase
are V-shaped. When the centromere is in the middle, the
chromosomes are called metacentric; at metaphase these are X-
shaped. If the centromere lies between the middle and one end,
the chromsomes are called acrocentric; these are X-shaped at
metaphase, but with one long and one short pair of arms.the
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somatic cells of eucaryotes contain two sets of paired chromo-
somes. Each organism has a characteristic number of
chromosomes, one half of which 1 from the female parent in
the ovum, and the other half inherited from the spermato-
zoon. Two chromosomes in each set constitute a pair, which
have identical size and shape, but with one exception. Whereas
females have two identical sex chromsomes (XX), males have
only one X chromosome, which is maternally inherited and
paired with a paternally inherited Y chromosome. The sum of
the characteristics defined by chromosome number, size, shape,
and pairing regularity is called the karyotype. In somatic cells, the
karyotype is pre-cisely defined at the diploid level. For humans,
a diploid number of 46 has been established. When the
chromosome set is identical to a standard set, the karyotype is
said to be euploid. At the cellular level, chromosomal character-
istics can serve as an index of the homogeneity and stability of
cell lines growing in vitro. At the level of the organism, genetic
diseases are known and char-acterized by recognizable chemical
syndromes, as well as distinct chromosomal abnormalities.
Abnormal cells containing the wrong number of chromosomes
are aneuploid. When the number of chromosomes varies from
cell to cell, the karyo-type is said to be heteroploid.
Chromosome Staining
The technical aspects important for obtaining good chromo-
some preparations include the following:
(1) the cells must be incubated in the presence of a mitotic
inhibitor (colchicine, Colcemid, or vinblastine sulfate) to
arrest cells in metaphase;
(2) the cells must be swollen in a hypotonic solution to
separate the chromosomes;
(3) the cells must be dried on a slide to spread the
chromosomes in a flat plane; and
(4) the chromosomes must be stained for maximum contrast.
The following procedure is a modification of that described by
Moorhead etal.
1. Cultured cells are treated with Colcemid at a concentration
of 0.4 jug per ml for 3 hours at 37C.
2. If the cells are growing in surface cultures, they are
harvested at this time with 0.25 percent trypsin in CMF-
PBS. The suspension is centrifuged at 200X# for 5
minutes to pellet the cells.
3. After aspirating the supernatant fluid, the cell pellet is
resuspended in 5.0 ml of hypotonic potassium chloride
(0.075 M).
4. The cells are allowed to swell for 8 minutes at room
temperature, at which point one drop of cold fixative
consisting of absolute methanol and glacial acetic acid (3:1
v/ v) is added. The settled cells are gently resuspended, and
then pelleted at 200X,? for 5 minutes at 4C.
5. The supernate is completely removed at this point, and 5.0
ml of fresh fixative is slowly added down the inside wall
of the tube so as not to disrupt the button of cells.
6. After 30 minutes at room temperature, the cells are
resuspended and dis-persed by pipetting.
7. The cells are repelleted at 200X# for 5 minutes at 4C and
resuspended in 3.0 ml of fresh fixative.
8. The cells are again pelleted, and this time are resuspended
in only 0.25 to 0.50 ml of fixative to obtain a fairly dense
suspension.
9. Two or three drops of the cell suspension are placed onto a
wet slide that has been precleaned in absolute ethanol and
left in distilled water for 24 hours at 4C. The slide is tilted
lengthwise, and the excess is drawn off by holding the
edge of the slide on a blotter.
10. The slide is ignited to remove the remaining fixative, and
immediately fanned to prevent it from becoming hot.
11. The slide is air dried for from 5 to 24 hours, and then
stained for 12 min-utes with Giemsa stain. (Five milliliters
of stock Giemsa is mixed with 50.0 ml of distilled water
and 1.5 ml of 0.1 M citric acid adjusted to pH 7.0
with 0.2 M
Na2HP04.)
12. The slides are washed in distilled water, air dried for 24
hours, cleared in xylene, and permanently mounted with
permount.
G-Banding with Trypsin
Chromosomes can be treated with trypsin prior to staining with
Giemsa to pro-duce a characteristic banding pattern, which most
probably reflects the underlying organization of the chromo-
somal DNA. It has been suggested that the enzyme hydrolyzes
the protein component of nucleo-protein, which has been
denatured by the fixation procedure, allowing the stain to react
with DNA exposed in hetetochromatin regions. The following
procedure can be used to obtain the G-banding pattern.
1. Cells are arrested with 0.4 Mg of Colcemid per ml, fixed
with methanol-acetic acid (3:1 v/ v) as above, and flame
dried.
2. The slide is treated with trypsin (0.025 to 0.05 percent in
CMF-PBS), or with trypsin-versene (1 part 0.025 to 0.05
percent trypsin and 1 part 0.02 percent EDTA, pH 7.0) for
10 to 15 minutes at 25 to 30C.
3. The slides are rinsed with 70 to 100 percent ethanol, air
dried, stained for 1 to 2 minutes with Giemsa, rinsed
twice with distilled water, air dried, and mounted.
Banding with Quinacrine Mustard
A fluorescent banding pattern of chromosomes can be viewed
with a fluores-cence microscope after staining with quinacrine
mustard. It is also believed to be a heterochromatin pattern. In
the procedure, air-dried slides containing chromosome spreads
are transferred to Macllvaines buffer (6.5 ml of 0.2 A/ Najlim,,
43.6 ml of 0.1 M citric acid, and H20 to a final volume of 100
ml, pll 7.0). An aliquot of quinacrine mustard dihydrochloride
in aqueous solution is then added to the buffer to give a final
concentration of 50 fig per ml of the fluorochrome. After 20
minutes, the slides are washed three times with the same buffer,
and then sealed with some buffer entrapped.