CELL SYNCHRONIZATION (PHYSICAL SEPARATION AND CHEMICAL BLOCKADE)

Cell Synchronization
Cell cycle
Cell division
Checkpoints of cell cycle
History of cell Synchronization
Needs for Synchronization
Principle and types
Physical separation
Cell density, cell size, antibodies
Centrifugal elutriation
Flow cytometry
FACS
Chemical blockage
G1 phase lovastatin
G2 phase RO3306
S phase double thymine block
M phase nocodazole
Other methods
Conformation of Synchronization

1. IAR UNIVERSITY
ICROBIAL BIOTECHNOLOGY
BIOTECHNOLOGY

ell
Synchronization

Presented To: Presented By:
. BRINDHA PRIYADARISINI SABARIDARAN.B 23MBTB18
te Professor IM. Sc. Microbiology

2. Introduction

Cell cycle

History and Needs
Principle Criteria

Types of Synchronization

Agenda

y” N

Physical Fractionation

Chemical Blockade

Other methods of Synchronization
Conformation of Synchronization

Summary and Reference

22

29

31

32

3. Introduction

+ Cell Synchronisation is process by which
cells at different stages of cell cycle ina
culture are brought to the same phase.

+ Cell Synchrony is required to study the
progression of cells through the cell cycle.

+ Synchronization literally means to make
two or more things happen exactly,
simunltaneously

« For instance two or more watches can be
synchronized to show exactly the same
time

+ The cells at different stages of cell cycle in
a culture can be synchronizer so that the
cells will be at the same phase

4. The Cell Cycle

The cell grows and copies its DNA
© G1: Cell growth
+ S: DNA synthesis
* G2: More growth,
preparation for mitosis

The cell divides its DNA and
cytoplasm, forming two new cells

+ Prophase

+ Metaphase

+ Anaphase

* Telophase
Go: Resting state where the cell
performs its functions and is not
preparing to divide

5. CELL DIVISION

Centrosomes

Spindle poles Kinetochore

CHA

chromatids

7. History

Sedimentation at unit gravity by Shail in 1971, q CI NS

centrifugal elutriation by Mikulits In 1997 cA

Fluorescence activated cell sorting by Hoffman
& Houck in 1997

A number of cell types have been seperated by
centrifugation elutriations method, as have
cells of different phases of the cell cycle

Cell cycle progression in G1 (lovastatin), S À
(mimosine) and G2/M (nocodazole)

Nutritional deprivation (G1 phase) Serum is removed
from the medium for 24hrs and then restored where
upon transit through the cycle is resumed in synchrony 7

8. require cell cycle Synchronization of
cell populations

Studies examining cell cycle regulatory
© mechanisms and progression invariably

Need of
Synchronization (9) en

cellular growth

en |

[ $ o
Investigation of regulatory events
that most often occur in a cell
cycle phase dependent fashion

8

9. There are severally principle criteria for

Synchronization that should be met:

Both normal and tumor cells should be arrested at the
same specific phase of the cell cyle

Synchronization must be non cytotoxic and
reversible

The metabolic block should be targeted to a specific
reaction and must be reversible

Large quantities of synchronous cell populations should be obtained
The synchronization must be medium independent

Synchrony should be maintained for more than one cell cycle to =
study biochemical processes taking place in cycling cells

6 06005 + © 2

Synchronized cells should exhibit uniform size

10. 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
synchronizer.

For example: for photosynthetic cells, light

can be eliminated for several hours and
then reintroduced. 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

For example: Nocodazole has been
used in biological research for
synchronization, although some
evidence suggest it may lack such

ability to synchronize cells en

11. Types of Synchronization

Chemical Physical
Blockade Fractionation

Nocodazole Cell Density
RO-3306 Centrifugal
Double elutriation
thymidine block Cell size
Other methods Ellorescence
Antibody emission

Lovastatin pos
specific

12. Physical Separation

Physical fractionation is a process by which continuously 0

dividing cells are separated into phase-enriched populations
based on characteristics such as the following: A o
Pr face} o

Cell density

Cell size

The presence of cell surface epitopes marked by antibodies

Light scatter y
Fluorescent emission by labeled cells.

Given that cells take on varying morphologies and surface

markers throughout the cell cycle, these traits can be used to

separate by phase.

The commonly used methods are. 12

13. = D-PBSA Cell
O Density

n Ei Ts
De ee ie LEI

LA

14. Rotor Gentrituge tid Viewing port From reservoir

15. p-—— Paramagnetic
(2 Antibody to cell type

O-nm microsphere

= specific surface antigen Piston ———>|
(a) (b) (c)
Magnet
y “Column

Ferromagnetic
spheres Tagged cells
released and
flushed out by

syringe piston —

__ Unlabeled cells
Tagged cells © do not adhere
drawn to walls © and are eluted
by magnet from column

Surface epitope marked antibodies

16. n . .
Centrifugal Elutriation o

Previously called (counter streaming centrifugation) Centrifugal elutriation can be
used to separate cells in different phases of the cell cycle based on their size and

sedimentation velocity (related to sedimentation coefficient).

Because of the consistent growth patterns throughout the cell cycle, centrifugal

elutriation can separate cells into G1, S, G2, and M phases by increasing size (and
increasing sedimentation coefficients) with diminished resolution between G2 and
M phases due to cellular heterogeneity and lack of a distinct size change

Larger cells sediment faster, so a cell in G2, which has experienced more growth
time, will sediment faster than a cell in G1 and can therefore be fractionated out.
Cells grown in suspension tend to be easier to elutriate given that they do not
adhere to one another and have rounded, uniform shapes.

17. Pressure
guage Elutriating centrifuge

Peristaltic
pump

Bubble
trap

+, Fraction
*, collectior

Reservoir

EG] Centrifugal force
a eS Counter flow

18. Flow Cytometry

Cytometry allows for detection, counting, and measurement of the physical and
chemical properties of cells. Cells are suspended in fluid and put through the flow
cytometer.

Cells are sent one at a time through a laser beam and the light scatter is measured
by a detector.

Cells or their components can be labeled with fluorescent markers so that they
emit different wavelengths of light in response to the laser, allowing for additional
data collection.

For quantitative cell cycle analysis, cells are usually fixed with ethanol and stained

with DNA-binding dyes like propidium iodide, 18

19. To Waste Filters

Container
ee)
Fluidic System Optical System Electronic System

19

20. FACS

+ Flow cytometers can be used to collect multiparameter
cytometry data, but cannot be used to separate or purify
cells.

+ Fluorescence-activated cell sorting (FACS) 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 penetrated DNA, RNA, proteins or antigens).

+ The system works much like flow cytometry, but will also 6
charge each cell droplet after it has been measured based on =>
a defined parameter. . Can

+ The charged droplet will then encounter an electrostatic
deflection system that will sort the cell to a different container
based on that charge.
+ This allows cells to be separated on the basis of fluorescent
content or scatter. 20

22. Chemical
blockade

The addition of exogenous substrates can be used to
block cells in certain phases of the cell cycle and
frequently target cell cycle checkpoints.

These techniques can be carried out in vitro and do
not require removal from the culture environment.

The most common type of chemical blockade is
arrest-and-release, which involves treatment of a
culture with a chemical block and subsequent
release by washing or addition of a neutralizing agent
for the block.

22

23. Arrest in G1 phase
Lovastatin

A single commonly-used chemical method exists for synchronization of cells in G1.

It involves Lovastatin, a reversible competitive inhibitor of 3-hydroxy-3-
methylglutaryl-coenzyme A reductase, an enzyme vital in the production of
mevalonic acid.

Mevalonic acid is a key intermediate in the mevalonate pathway responsible for
synthesis of cholesterol.

Addition of cholesterol to Lovastatin-treated cells does not undo the arrest affect,
so Lovastatin appears to inhibit the formation of some early intermediate in the

pathway that is essential for progression through early G1 23

24. Arrest in S phase

Double thymidine block

Arrest in S phase typically involves inhibition of DNA synthesis as the genome is
being replicated. Most methods are reversible through washing.

Double thymidine block:
High concentrations of thymidine interrupt the deoxynucleotide metabolism
pathway through competitive inhibition, thus blocking DNA replication.

A single treatment with thymidine arrests cells throughout S phase, so a double
treatment acts to induce a more uniform block in early S phase.

The process begins with a treatment with thymidine, washing of the culture,

followed by another thymidine treatment.
24

25. Double thymidine
block

To synchronise cells at the G1/S
border a freshly prepared thymidine
solution (16 mM) was made up in
complete medium and filter-
sterilised using a 0.22 um filter disc.

Cells (1 X 107) were cultured in 60 ml
complete medium containing
thymidine (2 mM) for 16 h in a 175
cmécell culture flask.

The cells were then resuspended in
complete medium (52.5 mi) for 8 h
to allow cells to reenter the cell cycle.

Cells were harvested by
centrifugation at 400 x g for 5 min
and washed X 2 in complete medium
(10 mi). Cells (1.x 107) were cultured
in 60 mi complete medium
containing thymidine (2mM) for 16 h
to synchronise cells at the Gl/S
border.

Cell synchronisation was confirmed
by flow cytometry.

26. The G2 checkpoint prevents cells
from entering mitosis when DNA is
damaged, providing an opportunity

Arrest in G2 for repair and stopping the

proliferation of damaged cells

p hase RO-3306-induced G2 arrest

RO 3306 prevented cells with DNA double-
strand breaks from transitioning into

the M-phase and that the cells

maintained their DNA repair capacity

in G2-phase, even under RO-3306

dose-dependent DNA repair
inhibition.

26

27. Arrest in M Phase
Nocodazole

Mitotic arrest can be achieved through many methods and at various points within M-
phase, including the G2/M transition, the metaphase/anaphase transition, and mitotic
exit.

Nocodazole is a rapidly-reversible inhibitor of microtubule polymerization that can be
used to arrest cells before Anaphase at the spindle assembly checkpoint in the
metaphase/anaphase transition.

The microtubule poison works by blocking the formation of the mitotic spindles that
attach to and pull apart sister chromatids in dividing cells. Cells will remain arrested
until the nocodazole has been washed out. Nocodazole does not appear to disrupt
interphase metabolism, and released cells return to normal cell cycle progression.
27

28. Nocodazole

Roscovitine
Nocodazole |
| cs

| Gus

Microtubule |

ir

G,

M (MITOSIS)

G,/G,

Cells (1x 10”) were synchronised in
mitosis by treating cells for 18 h with
Nocodazole (100 nM) and incubated
at 37 °C in a 175 cm?

Cells were washed with complete
medium X3 and re-plated in
complete medium and samples
were taken at 0, 30, 60, 120 and 180
min post-release.

As a control, one sample was re-
plated in the presence of
Nocodazole (100 nM) to maintain
cells in mitosisand a sample was
take 180 min post-release.

Cells were lysed in RIPA buffer and
whole cell extracts were prepared.
Samples were stored at -20 °©g

29. Other methods of

synchronization

Mitotic selection is a drug-free procedure for the selection
of mitotic cells from a monolayer undergoing exponential A
growth.

During mitosis, cells undergo changes in morphology,
and mitotic selection takes advantage of this in adherent B
cells grown in a monolayer.

The cells become more spherical, decreasing the surface
area of cell membrane attached to the culture plate. c

Mitotic cells can therefore be completely detached by Al
gently shaking and collected from the supernatant. :

30. Contact inhibition occurs when cells are allowed to grow to high or full emo
confluence, maximizing cell-to-cell contact. This triggers arrest in early G1
in normal cells. Arrest is reversed by replating cells at a lower density.

Because of the proliferation-promoting mutations intrinsic to cancer, tumor
cell lines are not usually able to undergo contact inhibition, though there

are exceptions.

Elimination of serum from the culture medium for about 24 hours
results in the accumulation of cells at the transition between GO
quiescence and early G1.

This arrest is easily reversible through addition of serum or the
deprived nutrient. Upon release, progression through the cell cycle is
variable, as some cells remain quiescent while others proceed through
the cell cycle at variable rates

31. Conformation of cell

Synchronization

microscopy or flow cytometry.

Microscopy allows you to see what is
actually going on inside you cells.

Flow cytometry enables you to compare
your treated synchronised cells against a

asynchronous control.

Briefly the protocol is as follows:

Procedure

Fix and permeabilize your cells in 70
% ethanol

Stain with 40 pg/ml propidium
iodide, and include 25 ug/mi of
RNase (to degrade RNA and ensure
that you stain DNA only).

Run your samples on the flow
cytometer. 31

32. Su mma ry er \ Double thymidine

\ block

Centrifugal -~ Flow AC

N
elutriation cytometry FACS 39

Conformation of cell
Synchronization

33. Reference

6 + + + +

https://www.assaygenie.com/blog/cell-
synchronisation-methods

https://en.m.wikipedia.org/wiki/Cell_
synchronization

https://www.slideshare.net/6263234147/cell-
synchronization-234020996

Basic cell culture — j.m. davis

Some contents were taken from google, internet

and images were taken in net AN 1,7)

34. Thank
You