Cell synchronization

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

1. CELL SYNCHRONIZATION 08/05/2020 1 By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. 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   08/05/2020 2

3. INTRODUCTION 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. Synchronization literally means to make two or more things happen exactly simultaneously. For instance, two or more watches can be synchronized to show exactly the same time. The cells at different stages of the cell cycle in a culture can be synchronized so that the cells will be at the same phase. Cell synchrony is required to study the progression of cells through cell cycle. Several laboratory techniques have been developed to achieve 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. Nocodazole is one of anticancer drugs that synchronize cells at M-phase. It can interfere with the structure and function of microtubules in interphase and mitotic cells. 08/05/2020 3

4. 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] 08/05/2020 4

5. 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 08/05/2020 5

6. 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, 08/05/2020 6

7. 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. 08/05/2020 7

8. 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 . 08/05/2020 8

9. 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. 08/05/2020 9

10. 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 08/05/2020 10

11. 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. 08/05/2020 11

12. 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. 08/05/2020 12

13. CHEMICAL APPROACH 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. 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. 08/05/2020 13

14. 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) 08/05/2020 14

15. 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. 08/05/2020 15

16. 4. Temperature Shock: Low temperature shocks combined with nutrient starvation are reported to induce synchronization of suspension culture. 08/05/2020 16

17. 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. 08/05/2020 17

18. 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. 08/05/2020 18