[56] Cell synchronization

592

ISOLATION AND CULTURE OF CELLS

[56]

forming assay. While the sample of cells is tested the main lot can be stored frozen as described by Dougherty and Rasmussen 16 or held in sparsely seeded petri dishes. Plate about 1-2 × 106 cells in plastic 50-mm dishes in Eagle's or NCI medium supplemented with 2-fold vitamins and amino acids and containing 10% tryptose phosphate broth plus 7% calf serum. Make dilutions of RSV in the same medium and add 100-1000 focusforming units (FFU) in 0.1 ml to the cell suspensions in each dish. Incubate the cultures overnight in 10% COs/air to permit the cells to spread out into a sparse monolayer. Aspirate the medium and replace with the same medium containing 0.5% agar. Continue incubation for a further 5-6 days. If the cultures turn yellow, the COs should be reduced to 5%. On day 5, the cultures should be fed by addition of 2.5 ml of the same medium. On day 10, foci of ceils showing morphological alterations and different growth properties will be visible at 100x by phase contrast illumination. These foci can be picked by aspiration through the agar layer or counted by staining the monolayer with Giemsa after removal of the agar overlays. Finally, it is important to realize that the agents used to cause transformation of cells in culture also produce tumors in experimental animals and should be treated with caution. Stringent safety precautions, such as those described in Hellman's safety booklet published by NCI should be enforced. Most of the rules are no more than common sense. I~R. M. Dougherty and R, Rasmussen, Nat. Cancer Inst. Monogr. 17, 337 (1964).

[56]

Cell S y n c h r o n i z a t i o n

By

ELLIOTT ROBBINS

It is possible to obtain large numbers of relatively well synchronized cells in the G1, S, and G2 and mitotic phases of the cell cycle, thus allowing biochemical and morphological studies which potentially may elucidate the mechanisms that regulate cell division. Factors that must be considered in the choice of a particular method for synchronization are: the number of cells required, the specific phase in which optimal synchrony is desired, and the possible interference of chemical synchronizing drugs with projected analyses. The two basic methods most commonly used for cell synchronization are: (1) chemical blockage of the cell's progression through its cycle at a

[56]

CELL SYNCHRONIZATION

593

selected phase1-6; (2) physical separation of mitotic cells from interphase cellsY ,8 In certain instances an appropriate combination of these two techniques is efficacious in improving either the degree of synchrony or the yield2, TM Methods M i t o t i c Cells. The most effective method available for obtaining cells, most of which are in mitosis, is based upon two long-known facts of cell monolayer behavior: (1) the tenacity of cell attachment to substrate is Ca 2÷ dependent, and (2) the typical mammalian cell rounds up when it enters mitosis relinquishing much of its substrate attachment. By maintaining monolayer cultures in medium containing no Ca z÷ other than that normally present in the serum supplement, those entering mitosis become so tenuously attached that relatively gentle shearing forces are sufficient to detach them preferentially. Monodisperse suspensions obtained from continuous suspension cultures or by enzymatic detachment of monolayer cultures are planted on Blake (or any flat, large surface) bottles 1-2 days prior to synchronization. The medium used for H e L a cells is that described for suspension cultures 11 supplemented with 7% fetal calf serum plus nonessential amino acids and is commercially available from any medium supply house. While this medium contains only the calcium in the serum, all cells on which we have used it attach to glass and propagate with a normal generation time; this includes HeLa, Chinese hamster lung, KB, and various diploid cell lines. Optimum cell number per bottle varies with different cell types, but it is generally desirable to have only a partially confluent culture at synchronization. Before collecting synchronized cells, the bottles are shaken vigorously to detach loosely adherent cells and debris. After rinsing the monolayer

1D. Bootsma, L. Budke, and O. Vos, Exp. Cell Res. 33, 301 (1964). 2R. R. Rueckert and G. C. Mueller, Cancer Res. 20, 1584 (1960). 3G. C. Mueller, K. Kajiwara, E. Stubblefield, and R. R. Rueckert, Cancer Res. 22, 1084 (1962). ~W. K. Sinclair, Science 150, 1729 (1965). 5G. C. Mueller and K. Kajiwara, "Developmental and Metabolic Control Mechanisms and Neoplasia," p. 452. Williams & Wilkins, Baltimore, Maryland, 1965. E. Stubblefield, R. Klevecz, and L. Olaven, 1. Cell. Physiol. 69, 345 (1967). ~E. Robbins and P. I. Marcus, Science 44, 1153 (1964). ST. Terasima and L. J. Tolmach, Exp. Cell Res. 30, 344 (1963). E. Robbins and M. D. Scharff, in "Cell Synchrony Studies in Biosynthetic Regulation" (I. L. Cameron and G. Padilla, eds.), p. 353. Academic Press, New York, 1966. 1°T. Pederson and E. Robbins, J. Cell Biol. 49, 942 (1971). 11H. Eagle, Science 130, 423 (1959).

594

ISOLATION AND CULTURE OF CELLS

[56]

thoroughly, 30 ml of fresh medium are added to each bottle to cover the cell sheet. Thirty to 60 minutes later, mitotic cells are harvested by serial transfer of the collecting medium as follows: Medium from bottle No. 1 is carefully decanted to avoid disturbance of the monolayer and 30 ml of fresh medium are added; the bottle is rocked 20 times in the horizontal plane with the medium flowing back and forth over the cell sheet. Bottle No. 2 is decanted, and the medium containing the harvested cells from bottle No. 1 is transferred to bottle No. 2 which is then shaken as described. Bottle No. 3 is treated as bottle No. 2, and so on. In this manner, two workers can collect mitotic cells from 40 bottles in 15 minutes with an average yield of 3 x 10 ~ cells per bottle (for HeLa). Microscopic examination of collected cells from a single bottle reveals that about 50% are in metaphase, 40% are in anaphase or telophase, and the remainder are either in early G~ or are interphase contaminants at an unknown stage of the life cycle. When several dozen bottles are collected, many of the first cells shaken off will obviously pass through mitosis while the collection is in progress so that the final population will contain cells in anaphase and G1 with relatively fewer in metaphase. A few simple manipulations can improve the percent of cells in metaphase if this particular stage is the desired one. The most obvious is harvesting the ceils at low temperature, either in a conventional walk-in cold room or by keeping the bottles in a shallow ice bath; the former being the only practical way with large numbers of bottles. A second perhaps more physiological method simply requires shortening the length of time between when the monolayers are rinsed free of debris and when the mitotic cells are harvested. The initial rinse removes all mitotic cellsImetaphase, anaphase, and telophase inclusive. It is clear that if mitotic cells are harvested within 30 minutes of this initial rinse they must be largely in metaphase. This follows from the fact that metaphase lasts approximately 30 minutes; therefore, a 30-minute interval between rinsing and harvesting will not be sufficient for the new crop of metaphase cells appearing subsequent to the rinse to enter anaphase. Since prophase cells are not detached by the collection procedure, the preparation will be largely metaphase. If chilling cells is inconsistent with experimental design, speed is of the essence in obtaining a metaphase-rich population, especially when large numbers of bottles are involved. GI and S Phase Cells. The method described for obtaining mitotic ceils yields a population which enters G1 (postmitotic) phase and the S phase (DNA replication) in good synchrony. However G2 (premitotic phase) and the immediately following mitosis show that as the population has proceeded through one cycle the inherent cellular variability has resulted in a significant loss of synchrony. No more than 10-15% of the ceils re-

[56]

CELL SYNCHRONIZATION

595

enter mitosis simultaneously even though all of them started out together. The method of selective detachment of mitotic cells gives adequate preparations of G1 and, as noted, S phase cells. However, synchronization with metabolic inhibitors such as high concentrations of thymidine, hydroxyurea, fluorodeoxyuridine, amethopterin, adenosine, deoxyguanosine, etc., is simpler, less time consuming, and more readily provides gram quantities of cells when these are desired. The important disadvantage to be considered when these drugs are used is that during exposure the cells are in a state of unbalanced growth; DNA synthesis is inhibited but RNA and protein synthesis are either unaffected or only slightly depressed. Treated cells that are not synthesizing DNA progress to the G1-S boundary and there they remained blocked. When the drug is removed they all progress synchronously through S. The state of unbalanced growth that occurs during blockade at the G1-S boundary is reversible for about 24 hours. After this period, cells that remained blocked show signs of degeneration. An unanswered question is whether there are subtle functional effects that accrue even if the time of exposure does not exceed 24 hours. This possibility must be considered when results obtained with drug synchronized cells are interpreted. The methodology currently used for synchronization with metabolic inhibitors simply entails exposure of the cells (either in monolayer or suspension culture) to a drug concentration that blocks replication of DNA. After 12-16 hours the medium containing the inhibitor is modified (see below) or replaced by fresh medium. The majority of the cells then proceed to synthesize DNA synchronously. The final concentrations of the various agents most commonly used are: thymidine, 2 mM; hydroxyurea, 1 mM; fluorodeoxyuridine, 1 ~M; amethopterin, 1 ~,M + adenosine 50 td~/; deoxyguanosine, 0.2 mM, deoxyadenosine, 1 mM. The effects of fluorodeoxyuridine and amethopterin in conjunction with adenosine have the advantage of being readily reversed without a medium change simply by adding 4 ~M thymidine which counteracts the nucleotide deficiency. We have found, however, that in general 2 mM thymidine is the most useful and least toxic inhibitor for HeLa cells even though in this case the media must be changed to effect reversal. G.,_ Cells. The G2 phase of the cell cycle is only 2-4 hours long. The methodology thus far described does not yield adequately synchronized G~ populations, which are important for study of premitotic events. In the case of selectively detached cells we have already alluded to the deterioration of synchrony that accrues by the time the cells reach G2. In the case of metabolic inhibitors, about 30-40% of the cells in any random population are scattered throughout the S phase. Thus when the inhibitors are added these S phase cells are arrested. When the inhibitors are removed,

596

ISOLATION AND CULTURE OF CELLS

[56]

these cells resume DNA synthesis and reach G2 before the bulk of the synchronized population, and significantly affect the purity of the G2 population. By combining the method of selective detachment of mitotic cells with metabolic DNA inhibitors, a usable G2 phase may be obtained. The goal here is a population that shows a very low level of thymidine incorporation and a low but gradually increasing mitotic index. These requisite characteristics follow from the fact that cells in G2 have completed S but have not yet entered mitosis. The procedure is as follows: cells in mitosis are collected by selective detachment, and 2 mM thymidine is immediately added. When the cells arrive at the G1-S boundary, they are all arrested here. Thus the decay in synchrony that normally occurs during the 5-7 hours of G1 following selective detachment is eliminated, as is the scatter of cells throughout the S phase that characterizes cells synchronized with DNA inhibitors alone. When the thymidine is removed, the improved synchrony yields a G2 population where DNA synthesis is only about 10% of peak levels and mitotic index is very low for about 2 hours. DN,4 Inhibitors with Selective Detachment. The milligram yield of synchronized mitotic cells can be greatly increased by combining DNA inhibitors with the selective detachment technique. When monolayers are exposed for 15 hours to 2 mM thymidine, decanted, and fresh thymidine-free medium added for 7-10 hours before collecting, the yield of synchronized cells is increased 10-fold. These cells complete mitosis in the usual time and clone with a normal plating efficiency. Spindle Inhibitors with Selective Detachment. Selective detachment, in conjunction with the use of spindle inhibitors, such as colchicine or vinblastine, is another useful combination, and may be profitably employed for biochemical characterization of cells specifically in metaphase since the cells cannot progress into anaphase. By treating monolayer cultures with colchicine (0.1 ~g/ml) and then preferentially detaching the mitotic cells as already described, it is possible to acquire pure populations of metaphase-arrested cells which have been exposed to the drug for an hour or even less and which remain metabolically active for 8-10 hours. This is in contrast to a generation time in colchicine required by a random culture to arrive at a point where the population is predominantly metaphase. Perhaps more important than the self-evident advantages of the short drug exposure is the fact that the metaphase-arrested population may be compared with interphase cells incubated with the drug for the same short length of time thus providing a reasonable control on the effects of the treatment per se. The above techniques provide a means of obtaining workable quantities of mitotic G,, S, and G2 cells. Prophase, anaphase, and telophase cells are much more difficult to obtain, and no general method applicable to all or even most cell types is available. Theoretically the use of a re-

[57]

MEASUREMENT OF CELL--CELL INTERACTIONS

597

versible spindle inhibitor, e.g., Colcemid, with the appropriate cell strain, such as Chinese hamster ovary, should supply a population of metaphase cells which can then be transformed into an anaphase or telophase population when the drug is removed. Unfortunately Colcemid is not reversible in many cell types. In addition to the methods of synchronization described, there have been reports of other approaches such as starving cells for isoleucine or starving cells in general until they reach a stationary state and then feeding them whereupon they show a certain degree of synchronization. Either of these methods would seem significantly more traumatic than those already detailed, and it is not clear that they have any singular advantage.

[57] Measurement

of C e l l - C e l l I n t e r a c t i o n s

By SAUL ROSEMAN, W. ROTTMANN, B. WALTHER, R. OHMAN, and J. UMBREIT I. Cell Adhesion

By SAUL ROSEMAN Celia -t- cellA--~ cellA -- cellA CellA -{- cella --* celia -- cella CellA d- substratum --* cellA- substratum

(~) (b) (e)

Definitions and Assay Procedures Attempts to identify the molecular events involved in cell adhesion have been severely limited by lack of suitable quantitative methodology. In fact, there is no general agreement on even the definitions for specific and nonspecific intercellular adhesion; each investigator employs a definition that is intrinsically limited to, and circumscribed by, the method used to study the phenomena. It appears probable that different methods are measuring different processes, since a variety of reactions may take place when cells come into contact with one another. For example, cells from higher animals may form electrical junctions, some cells secrete intercellular matrices containing collagen and connective tissue polysaccharides, and other cells secrete substances that may promote or inhibit adhesion after they come into contact with each other. In view of these complications, it is not surprising that conflicting results have been published when different methods for assaying and defining "cell adhesion" have been used.