Cell synchronization

CELL
SYNCHRONIZATION
5/15/2020
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

SYNOPSIS
 INTRODUCTION
 HISTORY
 NEED OF SYNCHRONIZATION
 SYNCHRONOUS CULTURES CAN BE OBTAINED IN
SEVERAL WAYS:
 Physical fractionation .
 Chemical appro ach
 CENTRIFUGAL ELUTRIATION
 Inhibition of DNA synthesis
 Nutritional deprivation
 SYNCHRONIZATION AT LOW TEMPERATURE
 CELLULAR TOTIPOTENCY
 SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
 REFERENCES

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HISTORY
 Cells separation by blocking metabolic reactions has been
reviewed by Merrill in 1998.
 Sedimentation at unit gravity by[Shall & McClelland, 1971;
Shall, 1973], but centrifugal elutriation by [Mikulits et al., 1997]
 Fluorescence-activated cell sorting by Hoffman & Houck in 1997
 Nutritional deprivation (G1 phase): Serum [Chang & Baserga,
1977] [Ley & Tobey, 1970] is removed from the medium for 24 h
and then restored, where upon transit through the cycle is
resumed in synchrony [Yoshida & Beppu, 1990; Jackman &
O’Connor, 2001]
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NEED OF SYNCHRONIZATION
 Understanding the molecular and biochemical basis
of cellular growth
 Investigation of regulatory events.
 Studies examining cell-cycle regulatory
mechanisms and progression invariably require
cell-cycle synchronization of cell populations
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There are several principal criteria for synchronization
that should be met:
(a) Both normal and tumor cells should be arrested at the
same specific phase of the cell cycle,
(b) Synchronization must be noncytotoxic and reversible,
(c) The metabolic block should be targeted to a specific
reaction and must be reversible,
(d) Large quantities of synchronous cell populations
should be obtained,
(e) The synchronization must be medium independent,
(f) Synchrony should be maintained for more than one cell
cycle to study biochemical processes taking place in cycling
cells,
(g) Synchronized cells should exhibit uniform size,
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SYNCHRONOUS CULTURES CAN BE
OBTAINED IN SEVERAL WAYS:
 External conditions can be changed, so as to arrest growth of
all cells in the culture, and then changed again to resume
growth. The newly growing cells are now all starting to grow at
the same stage, and they are synchronized. For example,
for photosynthetic cells, light can be eliminated for several hours
and then re-introduced. Another method is to eliminate an
essential nutrient from the growth medium and later re-introduce
it.
 Cell growth can also be arrested using chemical growth
inhibitors. After growth has completely stopped for all cells, the
inhibitor can be removed from the culture and the cells then
begin to grow synchronously.Nocodazole, for example, has
been used in biological research for synchronization, although
some evidence suggests it may lack such ability to synchronize
cells.
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THE MOST WIDELY USED METHODS OF CELL CYCLE
SYNCHRONIZATION ARE BASED ON TWO DISTINCT
STRATEGIES:
 PHYSICAL FRACTIONATION
 CHEMICAL APPROACH
 The separation of cells by physical means is based
on cell density , cell size, antibody binding to cell
surface epitopes, fluorescent emission of labeled
cells and light scatter analysis.
 The two most often used methods of biophysical
fractionation are the centrifugal elutriation and
fluorescent activated cell sorting .
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CENTRIFUGAL ELUTRIATION
 Is an advanced centrifugation device that uses an increasing
sedimentation rate to yield a better separation of cells in a specially
designed centrifuge and a rotor containing the elutriation chamber.
The advantages of centrifugal elutriation are as follows:
 Differences in sedimentation velocity are exploited to isolate various types
of cells from various in homogeneous cell suspensions,
 Different subpopulations representing different stages of the cell cycle of
the same cell type can be separated.
 The isolated cells or subpopulations of cells can be used in clinical
experiments.
 Centrifugal elutriation fulfills the three principal criteria for
synchronization. Autoradiographic data indicated that fractions containing
≥ 97 % G1 cells, > 80 % S cells, and 70–75 % G2 cells could be routinely
recovered with centrifugal elutriation.
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There are two instruments in use based on the principle of
fluorescent-activated cell sorting
•1 . Flow cytometer : This instrument is capable of sorting out cells (from a
population) in different phases of the cell cvcle based on the measuremenrs of
a combination of cell size and DNA fluorescence.
•Most often used light sources in flow cytometry are argon-, krypton-, helium-
neon-, helium cadmium lasers and mercury lamp. Detectors are focused to the
interrogation point where the light beam (regularly laser beam) passes through
the fl uid stream.
Advantages and disadvantages of flow cytometry:
•The simultaneous physical and/or chemical analysis of thousands of particles
per second.
•Routinely used diagnostic tool in health disorders (especially cancers) and has
many applications in both research and clinical practice.
• Data of samples can be stored in computer as listmode and/or histogram files.
•The disadvantage of cell sorting is that it exhibits limitations in sample size
and time required for synchronization
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2. Fluorescent-activated cell sorter (FACS) : in this
instrument, the emission signals from the cells are measured,
and the cells sorted out into collection tubes.
•Fluorescence-activated cell sorting is a technique for sorting
out the cells based on the differences that can be detected by
light scatter (e.g.cell size) or fluorescence emission (by
pretreated DNA, RNA, proteins, antigens).
•The procedure involves passing of a single stream of cells
through a laser beam so that the scattered light
•When the cells are pretreated with a fluorescent stain (e.g.
chromomycin A for DNA), the fluorescent emission excited by
the laser can be detected.
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Comparison between physical methods
•For separation of a large number of cells,
centrifugal elutriator is preferred.
•On the other hand, fluorescent-activated cell
sorting is mostly used to obtain high grade pure
fraction of cell from small quantities of cells.
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CHEMICALAPPROACH
•The cells can be separated by blocking metabolic reactions. Two types
of metabolic blockades are in use - inhibition of DNA synthesis and
nutritional deprivation.
• The idea behind in vitro chemical synchronization is the exposure of a
random population of cells to agents that interfere with speciﬁc
biosynthetic processes, such as DNA replication.
1. Mitotic Arrest: Colchicine has been widely used to arrest cells at
metaphase. Suspension cultures in exponential growth are supplied
with 0.02% (w/v) colchicine for 4-8 hr in order to inhibit spindle
formation.
• Colchicine . One of the best known examples of chemical
synchronization is the addition of colchicine ( colcemid ) which
causes cell cycle arrest in metaphase by depolymerizing tubulin in
microtubules . Other example is- Roscovitin and momosine etc.
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2.Inhibition of DNA synthesis
•One method that is generally used is the exposure of randomly proliferating cells to
agents that interfere with speciﬁc biosynthetic activities, e.g., DNA replication.
•The cells blocked in the S phase are subsequently released by washing in control
media, followed in some cases by the addition of exogenous tracers such as nucleotides.
•The removal of the blocking agent permits cells to move into succeeding segments of
the cell cycle.
•Hydroxyurea. Hydroxyurea is used to treat certain types of cancer or blood
disorders.
•This medication may also be useful for chronic urinary tract infections or certain cases
of psoriasis.
•In cell biology hydroxyurea synchronization increases mitotic yield of cell lines.
• Large quantities of synchronized cells have been isolated in late G1 by growth in
isoleucine-deﬁcient medium followed by resuspension in fresh, complete medium
containing either hydroxyurea (to 10 −3 M) or cytosine arabinoside (to 5 μg/ml)
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3.Nutritional deprivation
•Elimination of serum or isoleucine from the culture medium for about 24 hours results
in the accumulation of cells at G1 phase.
•This effect of nutritional deprivation can be restored by their addition by which time
the cell synchrony occurs.
•Serum starvation (G0/G1 block) . Removal of serum from a rapidly growing cell
culture for about 24 h results in the accumulation of cells in G1 phase.
•Synchronized cells can then be released into S phase by the addition of serum.
•Nutritional serum starvation has been widely used for synchronizing cells by arresting
them in the G0/G 1 phase of the cell cycle, but it often reduced cell survival and
increased DNA fragmentation.
•Which caused high- embryonic losses after NT. Regularly only non-tumor cells can be
synchronized in G 0 /G 1 by removal of growth factors (“serum starvation”, amino-acid
depletion).
• Other inhibitors . It has been reported that roscovitine, a specific cyclin-dependent
kinase (CDK) 2 inhibitor more efficiently synchronized cells in the G0/ G1 phase of the
cell cycle than serum starvation , and resulted in an increase in cloning efficiency as
defined in terms of survival of fetuses and calves following embryo transfer.
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4. Temperature Shock: Low temperature
shocks combined with nutrient
starvation are reported to induce
synchronization of suspension culture.
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5.Cellular Totipotency
 Cellular Totipotency is the ability of a single cell to produce all cell types and to
organise them into an entire organism when cultured in a suitable culture medium at
appropriate temperature and aeration conditions.
Spores and Zygote are examples of totipotent cells.
 In more or less suitable medium, the totipotent cells of the callus tissue give rise to
meristematic nodules or meristemoids by repeated cell division.
This may subsequently give rise to vascular differentiation or it may form a
primordium capable of giving rise to a shoot or root.
Sometimes the totipotent cell may produce embryoids through sequential stages of
development such as globular stage, heart shaped stage and torpedo stage etc.
After prolonged culture, it has been observed that calluses in some species (e.g.
Ntcotiana tabacum, Citrus aurantifolia etc.) maybe- come habituated. This means that
they are now able to grow on a standard maintenance medium which is devoid of
growth hormones.
The cells of habituated callus also remain totipotent and are capable to regenerate a
plant without any major manipulation.
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SOME HIGHLIGHTS OF CELL
SYNCHRONIZATION
•Cell separation by physical methods is more effective than
chemical procedures
•Chemical blockade is often toxic to the cells.
•Transformed cells cannot be synchronized by nutritional
deprivation. .
•A high degree of cell synchrony (>80%) can be obtained in
the first cycle, and in the second cycle it would be <6o%. The
cell distribution may occur randomly in the third cycle.
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Cell synchronization

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