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ANIMAL CELL CULTURE

LESSON 13 CELL- SYNCHRONIZATION


Hai all! today we will see an important aspect,t he cell synchronization. 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

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 experimental 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 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 lowserummedium 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 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.
Synchronous Growth

(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. 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. 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. 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. 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.

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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 celldivision cycle. One of the first studies showed that DNA is

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(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 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. 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). The column is placed into the incubator at 37 C for 50 minutes, and is tightly capped to prevent the loss of CO2. 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.

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Cells growing in the exponential phase are treated with 0.3 Mg per ml of vinblastine sulfate and incubated at 37C. After 16 hours, the arrested cells are washed off the surface and discarded. A fresh prewarmed or a conditioned medium is added to the cultures. A burst of cell division occurs in the next 5 hours. 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.

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Population Synchronization

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(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 1hour 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-TdRblock 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-monophosphate (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 synchronizing KB cells. 1. 2. 3. Exponentially growing cells in suspension culture are treated with 2 mAf TdR for 18 to 20 hours. The cells are pelleted by centrifuging at M60Xg for 5 minutes, and resus-pended in fresh prewarmed media. 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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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
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After 18 to 20 hours, the cells are again pelleted, suspended in a complete medium, and grown in full synchrony at 37C.

Autoradiography

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(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 concentration 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. Colcemid is added to the culture at a concentration of 0.02 Mg per ml. 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. The cells are collected by centrifugation at room temperature and pooled into a fresh medium containing Colcemid. 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. 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.

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. 2. The cover slips are washed in PBS and fixed in glutaraldehyde for 15 minutes. 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. 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. 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. 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.

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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. 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. 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.

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Cell-cycle Mapping

Karyotype Analysis

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.

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 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. When the centromere is in the middle, the chromosomes are called metacentric; at metaphase these are Xshaped. 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 chromosomes. 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 spermatozoon. 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 characteristics 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

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The cells are repelleted at 200X# for 5 minutes at 4C and resuspended in 3.0 ml of fresh fixative. 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. 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.

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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

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. The following procedure is a modification of that described by Moorhead etal. 1. 2. Cultured cells are treated with Colcemid at a concentration of 0.4 jug per ml for 3 hours at 37C. If the cells are growing in surface cultures, they are harvested at this time with 0.25 percent trypsin in CMFPBS. The suspension is centrifuged at 200X# for 5 minutes to pellet the cells. After aspirating the supernatant fluid, the cell pellet is resuspended in 5.0 ml of hypotonic potassium chloride (0.075 M). 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. 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. After 30 minutes at room temperature, the cells are resuspended and dis-persed by pipetting.

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 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. 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. 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.

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Banding with Quinacrine Mustard

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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.

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