Culture synchrony

The temperature of incubators must be uniform throughout and should not vary more than 0.2 C. The vegetative assay organism must be sensitive to the analyte, be stable (resistant to spontaneous change), be in the logarithmic growth phase (for uniformity of response), and be easily cultured, maintained and standardized. Spores suspension have similar criteria except that the spores must be capable of germinating with reasonable synchrony. 

The added attraction of the transition from myoblast to myotube is the synchrony with which differentiation occurs in vitro. Myogen-esis will occur in primary cultures of skeletal muscle (e.g. 6.12) but can also be induced in diploid myoblast lines (Richter and Yaffe, 1970) which has allowed the selection of mutants (Chapter 13) that exhibit drug resistance or temperature-sensitive differentiation (Loomis et al., 1973 Somers et al., 1975). Holtzer et al. (1975) and Fiszman and Fuchs (1975) have developed a myoblast line transformed with a temperature-sensitive virus. At the permissive temper-... 

Parenchyma cells go into dormancy in the DNA presynthetic phase (Gj) (Adamson, 1962 Mitchell, 1967), and when these cells are held on a culture medium containing 2,4-dichlorophenoxyacetic acid to break or circumvent the dormancy, the cells enter mitosis (Bennici et al., 1982). The first and second divisions are well synchronized (Serafini-Fracassini et al., 1980) however, with further division, synchrony is gradually lost (Yeoman et al., 1965). Likewise, there are marked changes in the timing of the first and second cell cycles with the progression of dormancy (Bennici et al., 1982). 

Response of the organism to copper stress is dependent on the status of the culture. In the absence of synchrony, effects can be masked by the presence of only a fraction of the cells being in a sensitive phase of their development. Application of copper in the dark often elicits responses that are different in intensity or type than those observed in the light88-91. Anaerobic conditions exacerbate the deleterious effects of Cu92. Critical evaluation of these data and data generated in our own work has led us to some hypotheses regarding the systems affected by copper. 

The concept of a structured but distributed model can be carried further than in Section IV. However, we turn instead to the more complicated case of structured and segregated models. In other words, we now acknowledge that the culture is made up of individuals. [It should be remembered that we are speaking about a culture of cells, not about a population of cells forming a multicellular organism or a tissue in the latter case there may be communication between the cells such that intercellular events achieve a degree of synchrony (cf. Gl).]... 

When employing roller-flask cultures to harvest colchicine-arrested metaphases, calcium is omitted from the medium only during the last day. Colchicine (2.5 X 10 m stock) is added at this time in the preparation of 0.3 ml colchicine per 100 ml medium. After 18-24 hr of treatment, followed by several gentle swirls of the medium, up to 50-55 percent of the cells will detach as metaphases. This amounts to approximately 20 x 10 metaphases per flask per 24 hr. Similarly, calcium must be omitted from the medium when harvesting metaphases for synchrony studies without the aid of colchicine. 

When heterokaryons are formed between animal cells the nuclei in the same cytoplasm undergo synchronous initiation of DNA synthesis (Harris and Watkins, 1965) similar synchrony is observed in binucleate cells occurring in mouse embryo cultures (Church, 1967). DNA synthesis has been examined in multinucleate HeLa cells formed by fusion between cells in different phases of the life cycle (Rao and Johnson, 1970). There was a rapid induction of DNA synthesis in G1 phase nuclei following the fusion of G1 and S phase cells. When S phase cells were fused with G2 phase cells, no induction of DNA synthesis was observed in G2 phase nuclei and, no matter what the ratio of G2 S nuclei in the fused cell, the S phase nuclei continued DNA synthesis. 

In bacterial and mammalian cell systems in exponential growth new DNA synthesis occurs in synchrony when the blockage of DNA synthesis by selective thymine starvation is released after a period. The same is true in Tetrahymena cultures. This is seen from Figure 7, in which 2 mM thymidine was added after an exponentially multiplying population had been treated with methotrexate + uridine for 3 hours (3 hours is nearly equal to one generation time). Synthesis of DNA began immediately after thymidine was added, and cell division occurred 110 minutes later. 

Test 3, II. Cultures were synchronized using shock temperatures ranging from 30.8 to 35.8°C. The free-running systems obtained with four shocks are illustrated in Figure 18. The conclusion is that synchrony of good quality is obtained with shock temperatures between 31.8 and 34.3°C. In the following discussions the first chosen shock temperature, 33.8°C, is henceforth fixed (see A). 

One observation of interest has been made and is illustrated in Figure 22. The question is what happens if we synchronize repetitively, using four shocks spaced 150 minutes apart, and then shift to the multiple heat shock procedure. We know by now that the first four shocks can start a train of synchronous, free-running cell divisions, each closely followed by synchronous DNA replication. However, when the shift is made as described to the multiple shock procedure, divisions are suppressed but DNA replication continues as a cyclic, though damped, phenomenon occurring with a period of nearly four hours (lower curve, main culture). Samples were removed at the end of each new heat shock. They displayed synchronous division after 1.5 hours at 28 C. The quality of this synchrony (upper curves) bears a distinct relationship to the phase in the DNA replication cycle at the time the sample was removed from the main culture. 

At the time when the removed samples divide they have had a different fate for the past 40 to 50 minutes than the main culture, which in this period received a new heat shock. Nonetheless, the suggestion can be made that the poorest synchrony of division... 

Two general classes of methods exist for attaining synchrony in batch and/or continuous culture ... 

Synchronous culture a synchronized cell population, in which all cells divide and pass through subsequent phases of the cell cycle at Ae same time. Synchronization can be achieved in various ways, e.g. by nutrient limitation, light stimulation, temperature change, treatmennt with antimetabolites of nucleic acid metabolism. In S.c. the cell count increases stepwise. Synchrony is usually lost after a few synchronous divisions, i.e. the cell count reverts to a continual increase. S.c. techniques have been applied to various bacteria, Chlorella, Euglena gracilis, etc. Light... 

Constabel F, Kurz WGW, Chatson KB, Kirkpatrick JW (1977) Partial synchrony in soybean cell suspension cultures induced by ethylene. Exp Cell Res 105 263-268 Cooke RJ, Kendrick RE (1976) Phytochrome-controlled gibberellin metabolism in etio-plast envelopes. Planta 131 303-307... 

W. G. Thilly, D. I. Arkin, T. S. Nowak, Jr., and G. N. Wogan, Maintenance of perpetual synchrony in HeLa suspension cultures Question of unbalanced growth, Biotechnol Bioeng. 

Pardee I agree. What I was trying to say is that if one wants to know what goes on in a naturally dividing cell in balanced culture, one should look at cells in this state. And if one perturbs the metabolism of a cell as one does by forcing synchrony, one isn t sure that what one sees is the same thing as occurs in a naturally growing cell. 

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