A Review report On CELL SYNCHRONIZATION
Submitted to Department of Biotechnology
For the partial fulfillment of the degree of MASTER OF SCIENCE IN BIOTECHNOLOGY (2010-2012)
Submitted by Heena Shukla Under the guidance of Mrs. Payal Mehtani Assistant professor Department of Biotechnology International International college for Girls THE IIS University Jaipur (Rajasthan) (Rajasthan)
ACKNOWLEDGMENT
I express my deep sense of gratitude to Dr. Ashok Gupta, Vice Chancellor, The IIS University and Dr. Raakhi Gupta, Rector and Registrar, The IIS University, who provide me an excellent and healthy environment as well as equipped lab facilities. With humble request, gratitude and honour, I would like to give my sincere thanks to Professor Pradeep Bhatnagar, Dean of the Life Sciences, the IIS University, for acting as the constant pillar of support and guidance throughout my endeavour.
I also extend my heartfelt gratitude to Dr Sreemoyee Chatterjee, Head of Department of Biotechnology The IIS University, who constantly guided and motivated me to explore new heights in this field. I have had the advantage of her critical advice from her profound knowledge of biotechnology.
I place on record my indebtedness and profound sense of gratitude to Mrs. Payal Mehtani, Assistant professor of Department of Biotechnology, International College for Girls for her support and encouragement she provide me during my entire work. She showed me the path and helps us to choose the interesting topic. Her word always gave me a lot of enthusiasm and spirit to complete my work. I have been extremely motivated by her critical appraisals throughout the work. Without her guidance the completion of this work was unimaginable.
CONTENT Cell cycle G1-phase Variability Cell Synchronization Criteria for Synchronization Synchronous Growth Cell separation
Cell separation by physical means Centrifugal separation Fluorescence-activated cell sorting
Cell separation by chemical blockade Blocking of Cell Cycle (Inhibition of DNA synthesis)by an Inhibitory
Compound Nutritional deprivation
Population Synchronization Low-temperature procedure Thymidine and double-thymidine Block Colcemid Arrest
Various techniques which are used to identified the cell stages
Cell-cycle Mapping
Radioactive Thymidine Incorporation
Autoradiography
Karyotype Analysis
Chromosome Staining
G-Banding with Trypsin
Banding with Quinacrine Mustard Cell cycle synchronization uses References
Cell synchronization Cell cycle: The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. Non-dividing cells not considered to be in the cell cycle. The stages, pictured, are G1-S-G2-M. The G1 stage stands for "GAP 1". The S stage stands for "Synthesis". This is the stage when DNA replication occurs. The G2 stage stands for "GAP 2". The M stage stands for "mitosis", and is when nuclear (chromosomes separate) and cytoplasmic (cytokinesis) division occur.
Current view of the Mammalian Division Cycle
•
Regulatory controls are in G1-phase
•
Preparations for S phase occur in G1-phase
•
Cells arrest in G1-phase
•
Cells differentiate from G1-phase
•
Cells die or apoptose from G1 phase
•
Cells regulate division cycle length in G1 phase
•
Specific biochemical events in G1 phase
G1-phase Variability
•
G1-phase is most variable phase
•
Long G1 phase associated with, or produced, slow growing cells
•
Short G1 phase associated with, or produced, fast growing cells
•
Was concluded that G1 phase controlled growth rate and interdivision time
Cell Synchronization
Cell Synchronization is a process by which cells at different stages of the cell cycle in a culture
are brought to the same phase. "Cell synchrony" is required to study the progression of cells through the cell cycle. The types of synchronizations are broadly categorized into two groups: "Physical Fractionation" and "Chemical Blockade." A synchronized culture is one where cells pass through the division cycle as a relatively uniform cohort and represent, at different time points, cells of different cell cycle ages. In passing through the cell cycle a new born eukaryotic cell first passes through the G 1 phase (absence of DNA synthesis), then S phase (period of DNA synthesis), then G 2 phase (absence of DNA synthesis), with division occurring at M phase (mitosis). If many cells in a culture approximate this pattern as a group, these cells would be called a synchronized culture.
“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 identically 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 synchronized culture. One common and often-described synchronization method involves placing growing mammalian cells in a low serum medium 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:
Hydroxyl urea to inhibit DNA replication
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. Results of W hole-culture Synchronization (A)
(B)
Steady-State Growth
Inhibition of Mass 0
1
2 3 G1 G2 G1 G2 G1 G2 G1 G1 S S S S S M
4
0
1
2
3
4
2
3
4
G1 G1 G1 G1 G1
(C)
(D)
Inhibit DNA Synthesis
Inhibit Mitosis 0
1
2
3
4
0
S SS S S
1
M M M M M M
Criteria for Synchronization
•
Cells should exhibit synchronized divisions!
•
Cells should have proper DNA contents through sequential division cycles
•
Cells should have narrower size distribution throughout synchronous growth
•
Cells should be prepared by selective, not “whole-culture”, synchronization (not criterion, more of a fact)
Criteria for Synchronization Doubling time equals exponential growth
Number doubles
2 No artistic enhancement
Lag 1
Results repeat
Short rise time
These criteria are rarely met by Synchronization methods
Whole Culture Synchronization Cannot Work
•
Whole-culture Synchronization is most widely used approach to cell-cycle analysis
•
Whole culture Synchronization cannot, in theory, produce synchronized cells
•
Both cytoplasm and DNA amounts vary during division cycle
•
Whole-culture synchronization methods cannot produce cells with both specific DNA content and narrow size distribution for a particular cell age
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 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 synthesized in mid interphase, and is separated by two gaps occurring before and after mitosis. Howard and Pelc had previously
recognized 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 generation time; the M stage occupies 3 to 4 percent of the division cycle. It appears that mammalian cells have different generation time. Synchronous Growth 5
n o i t a r t n e c n o C l l e C e v i t a l e R
Huma n U-937 Huma n M OLT-4 1
Mouse L1210 E.col i B/r
0.5
0
1
2
3
Generations
Two general procedures are employed to obtain synchronously growing populations 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.
“Synchronization” by Arrest
with G1-phase DNA 1.0 0.7
Arrest with G1-DNA
Starve
1.4
Refeed
Arrest with G1-phase DNA, But No Synchronization 0.999
1.001
0.7
~0.5
Arrest with G1-DNA0.999
Starve
1.4
Refeed
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 observation 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.
(b) Separation of uniformly sized cells by gravity
Synchronous cells have been separated by centrifuging exponentially growing populations in linear 2 to 10 percent (w/v) sucrose gradients made up in a complete 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 seated 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.
Cell separation
Cell separation can takes place by two methods
Cell separation by physical means Cell separation by chemical blockade
(A) Cell separation by physical means
Physical fractionation or cell separation techniques, based on the following characteristics are in use.
Cell density
Cell size
Affinity of antibodies on cell surface epitopes.
Light scatter or fluorescent emission by labeled cells.
The two commonly used techniques are: (1) Centrifugal separation
The physical characteristics, cell size and sedimentation velocity are operative in the technique of centrifugal elutriation . Centrifugal elutriator (from Beckman) is an advanced device for increasing the sedimentation rate so that the yield and resolution of cells is better. The cell separation is carried out in a specially designed centrifuge and rotor. Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counter flow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei
of murine pre-B cells. The installation and elutriation by collecting 16 – 30 synchronized fractions, including particle size analysis, can be achieved in 4 – 5 h.
(2) Fluorescence-activated cell sorting
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 and antigens). The procedure involves passing of a single stream of cells through a laser beam so that the scattered light from the cells can be detected and recorded. There are two instruments in use based on its principle: a)
Flow cytometer
b)
Fluorescence-activated cell sorter
(B) Cell separation by chemical blockade
The cells can be separated by blocking metabolic reactions.Two types of metabolic blockades are in use: (1) Blocking of Cell Cycle (Inhibition of DNA synthesis)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 procedure 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 37°C. 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.
During the S-phase of cell cycle, DNA synthesis can be inhibited by using inhibitors such as thymidine, aminopterin, hydroxyurea and cytosine arabinoside. The effects of these inhibitors are variable. The cell cycle is predominantly blocked in S phase that results in viable cells. Hydroxyurea and thymidine are two compounds very often employed to induce partial synchrony at the G1/S boundary. Their use, however, is not without problems. Hydroxyurea at low concentration does not lead to uniform synchrony at the G1/S boundary and both high concentrations and increased duration of exposure are toxic to "S" phase cells (2, 3). Thymidine block also does not completely arrest cells at the G1/S interphase and it induces both chromosome aberrations and cell-cycle & dependent alterations in metabolism .For the reasons cited above and because nucleoside disulphate reductase seems to be the common target for both hydroxyurea and thymidine, it is highly desirable to find another inhibitor lacking the side effects just mentioned and acting on another step of DNA replication.
Mode of action of HU
Aphidicolin treatment appears to be a suitable method for synchronization of large quantities of suspension and monolayer (Pedrali-Noy and Spadari, unpublished) cell cultures. The drug seems less toxic than the available ones since it does not reduce cell viability and does not interfere with the synthesis of deoxyribonucleoside triphosphate or polymerases required for rapid cell division after reversal of the block. Synchronization Patterns
Hela cells synchronized by thymidine block followed by Aphidicolin block
(2) Nutritional deprivation
Elimination of serum 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) or low serum concentration is believed to arrest cells at a particular point in the cell cycle. The arrested cells with a G phase amount of DNA are proposed to be arrested at this point in the G 1 phase or in what is generally called the G 0 phase. Upon restoration of serum, these arrested cells are assumed to pass synchronously through the cell cycle.
At 30-40% cell confluency wash twice with 1xPBS and add DMEM (1% Pen-Strep, 1% Glutamine) w/o Serum after 72h restimulation with 10-15% Serum
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 4°C for a 1- hour time period. When the cultures were incubated at 37°C, 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
1) Double Thymidine block (early S-phase block)
At 25-30% confluency of HeLa cell culture wash twice with 1xPBS and add DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) + 2mM Thymidine for 18 h (first block)
after first Thymidine block: remove Thymidine by washing with 1xPBS;
add fresh DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) for 9h to release cells
after releasing: add DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) + 2mM Thymidine for 17 h (second block)
after second block: remove Thymidine by washing with 1xPBS; release cells by adding fresh DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine)
cells progress synchronously through G2- and mitotic phase
Figure A. Synchrony of HeLa cells
2) Thymidine-Nocodazole block (mitotic block)
at 40% confluency of HeLa cell culture add DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) + 2mM Thymidine for 24 h (S-phase block)
after Thymidine block: remove Thymidine by washing with 1xPBS;add fresh DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) for 3h to release cells
after releasing of the cells add 100ng/ml Nocodazole to the Media for 12h (mitotic block)
remove Nocodazole by washing with 1xPBS and add fresh DMEM (10%FCS, 1% Pen-Strep, 1% Glutamine) to release cells
cells progress synchronously through G1- and S-phase
Figure B.
Cells were arrested in mitosis by blocking first in thymidine followed by release and then blocking in nocodazole. After release from the nocodazole block, most of the cells (>75%) divided synchronously within 2 h of release from the arrest, entered S phase by 10-12 h after release, and completed the next synchronous mitosis by 18-20 h, ultimately completing two full cell cycles .
(c) Colcemid Arrest
Synchronization of animal cells can be achieved using Colcemid (iV-desacetylethylcolchicine), 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 concentration of 0.02 mg per ml and the loosely
attached arrested cells are removed with a low trypsin concentration combined with vigorous shaking at 6 hours .
Various techniques which are used to identified the cell stages: 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 37°C. 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 temperature. It is then dried at 110°C 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 a particles from [3H] , because of their short range, are ideal for studying the localization of the actual biosynthetic event in the cell.
Karyotype Analysis
The chromatin in the nucleus of a eukaryotic cell begins to condense into chromosomes with the onset of mitosis. In the metaphase, as the nuclear membrane fragments, the chromosomes further condense, and are clearly visible when mitotic cells are viewed
through a light microscope. A metaphase chromosome 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.
Chromosome Staining
The technical aspects important for obtaining good chromosome 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.
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 chromosomal 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 heterochromatin 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 trypsinversene (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 30°C.
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 fluorescence 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 Macllvaine’s 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.
Cell cycle synchronization uses
Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phasedependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations
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