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J. gen. Virol. (1987), 68, 937-940. Printedin Great Britain 937 Key words: adenovirus/permissive/cell line Growth of 293 Cells in Suspension Culture By F. L. G R A H A M Departments o f Biology and Pathology, McMaster University, 1280 Main Street West, Hamilton, Ontario L 8 S 4K1, Canada (Accepted 28 October 1986) SUMMARY A subline of 293 cells able to grow in suspension culture has been developed by passage of 293 cells through nude mice. This new line, designated 293N3S, grows with a doubling time of approximately 30 h, continues to express adenovirus 5 early region 1 (E 1) antigens, and remains permissive for adenovirus 5 host range mutants defective in E1 functions. The human adenovirus 5 (Ad5)-transformed human embryonic cell line 293 (Graham et al., 1977) has proven useful for a number of purposes. Because the cells contain the left end (the transforming region) of the Ad5 genome and express early region 1 (E 1) they are permissive for growth of Ad2 and Ad5 mutants that are defective in E 1 functions. Thus 293 cells are extensively used for the isolation and propagation of E1 mutants (for review, see Young et al., 1984). Because they permit the growth of mutant viruses from which the entire E1 region has been deleted, thus providing a non-essential region for DNA insertion as well as increasing the potential size of DNA inserts (Haj-Ahmad & Graham, 1986), 293 cells are likely to be extensively used in the development of adenovirus as helper-independent cloning and expression vectors in mammalian cells (see Berkner & Sharp, 1984; Van Doren et al., 1984; HajAhmad & Graham, 1986). It has also been reported that genes transfected into 293 cells are often efficiently transcribed in transient expression assays (Treisman et al., 1983; Imperiale et al., 1983; Green et al., 1983; Gaynor et al., 1984), an effect which is apparently related to the endogenous E1A gene products of these cells. Some additional useful properties of 293 cells which may or may not be related to their expression of E1 are enhanced sensitivity in assays of infectious adenovirus DNA (Graham et al., 1977) and the ability to support the replication of socalled fastidious adenovirus serotypes which cannot be grown in other established human cell lines (Takiff et al., 1981; Brown et al., 1984). It is also interesting that 293 cells have recently been found to be highly permissive for replication of simian virus 40-based vectors (Lebkowski et al., 1985; Lewis & Manley, 1985). Because growth of cells in suspension offers certain advantages over growth in monolayer, particularly for large scale production of virus or other products, attempts were made early on to adapt 293 cells directly to suspension culture, without success (F. L. Graham, unpublished). Since it is well established that the ability of cells to induce tumours often correlates with anchorage independence (Freedman & Shin, 1974; Shin et al., 1975) an alternative strategy was devised based on the assumption that cells selected for increased tumourigenicity might simultaneously acquire the ability to grow in suspension. This approach was successful and resulted in the development of the cell line described below. Tumourigenicity of 293 cells in nude mice is extremely low and tumours are rarely obtained even several months after injection of as many as 107 cells (F. L. Graham, unpublished). In the experiments whose results are given in Table 1, a single small tumour was obtained in one of four mice injected with 293 cells in two doses 8 weeks apart (expt. 1). This tumour was excised and explanted into culture to generate 293N1 cells which were found to have enhanced tumourigenicity (Table 1, expts. 2 and 3) relative to 293 cells. A second passage through nude 0000-7445 © 1987 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 14:54:53 Short communication 938 Fig. 1. Indirect immunofluorescentstaining of E1 tumour antigens in 293 (a) or 293N3S (b) cells. Cells were fixed and stained with 14b hamster tumour antiserum as described previously (Rowe et al., 1983). This serum reacts predominantly with the E1B 58000 mol. wt. protein but has low avidity for the E1B 19000 mol. wt. protein and for E1A products as well (Rowe et al., 1984). The bar marker in (a) represents 10 Mm. Table 1. Tumour induction in nude mice by 293 cells and sublines Cell line 293 293N1 Fraction positive (time after injection)* Expt. l ExpL 2 Expt. 3 0/4 (8 weeks)t 0/4 (4 weeks) 0/4 (3 weeks) 1/4 (12 weeks):~ 0/4 (12 weeks) 0/4 (8 weeks) 2/4 (4 weeks) 2/4 (3 weeks) 2/4 (12 weeks)§ 3/4 (8 weeks) 2/4 (3 weeks) 2/4 (8 weeks)ll - 293N2 - * Nude mice (6 to 8 weeks old) were injected subcutaneously with 107 cells. t Mice were re-injected at 8 weeks with a second dose of 293 cells. $§11Tumourcells were explanted into culture to generate, respectively, 293N1, 293N2 and 293N3 cell lines. mice yielded 293N2 cells which were still more tumourigenic: they induced tumours with approximately the same frequency as did 293N 1 cells, but the resulting tumours grew much more rapidly (Table 1, expt. 3 and unpublished observations). Finally, 293N3 cells were obtained from a tumour induced by 293N2 cells. After this third passage through a nude mouse it was observed that the explanted cells attached very poorly to the culture dishes but grew in suspension. Following a period of rather slow growth at 37 °C the cells ultimately achieved a doubling time of approximately 30 h and are now routinely maintained as spinner cultures in Joklik's modified minimal essential medium supplemented with 1 0 ~ horse serum. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 14:54:53 939 Short communication l0 1 -~ 8 I I J J / o2 Z ~ 7 6 5 J I I I I 10 20 30 40 50 Time after infection (h) Fig. 2. Virus production in Ad5-infected 293N3S cells. Exponentially growing spinner cells (2 x 10s cells/ml) were infected at a multiplicityof infection of 10 plaque-forming units/cell with either wild-type (0), hrl (O) or hr6 (A). At the indicated times, aliquots of cell suspension were removed and virus titres were determined by plaque assay on 293 monolayer cells as described previously (Harrison et al., 1977). The curve has been drawn through the average of all three titres. 0 The 293N3S cell line was not subcloned following its establishment from a nude mouse tumour. However it is unlikely the line is contaminated with mouse cells. K a r y o t y p i c analysis has revealed no cells containing mouse chromosomes (less than one cell per 1000). Also, several lines of evidence indicated that certain essential properties of 293 cells were unchanged in 293N 3S ceils after nearly a year in suspension culture. Firstly, the suspension cells still contained Ad5 D N A and the integration pattern was identical to that of the parental line (Ruben, 1984). Secondly, they continued to express E1 antigens as indicated by fluorescent antibody staining with an El-specific tumour antiserum (Fig. 1). A n d finally, and most important, 293N3S cells were permissive for growth of Ad5 mutants defective in E 1 functions: yields of infectious virus were similar for wild-type Ad5 and hrl, an E1A mutant, or hr6, an E1B m u t a n t (Harrison et al., 1977) (Fig. 2) and not greatly different from the yields of wild-type virus routinely obtained following infection of HeLa or KB cells. For large scale production of E1 m u t a n t viruses or viral D N A the use of suspension cultures has obvious advantages over monolayer cultures in terms of efficiency and economy as well as a potential for automation. A litre of infected 293N3S cells has been found to produce as much virus as approximately 30 to 40 150 m m dishes of 293 cells infected with Ad5 host range mutants. Equivalent yields would be expected for other kinds of E 1 mutants and perhaps also for those adenovirus serotypes which, as mentioned above, grow poorly in most established human cell lines but well in 293 cells. Whether or not 293N3S cells have retained other useful properties of the parental line remains to be determined. This cell line has been deposited with and will be available from the American Type Culture Collection, Rockville, Md., U.S.A. This work was supported by the Medical Research Council and the National Cancer Institute of Canada and was carried out with the technical assistance of J. Rudy. The author is a Terry Fox Cancer Research Scientist of the National Cancer Institute. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 14:54:53 940 Short communication REFERENCES BERKNER, I¢. L & SHARE,1'. A. (1984). Expression of dihydrofolate reductase, and of the adjacent E lb region, in an Ad5-dihydrofolate reductase recombinant virus. Nucleic Acids Research 12, 1925-1941. BROWN, M., PETRIC, M. & MIDDLETON, P. J. (1984). Diagnosis of fastidious enteric adenoviruses 40 and 41 in stool specimens. Journal of Clinical Microbiology 20, 334-338. 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Infection of eukaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA. Journal of Virology 50, 606-614. YOUNG,C. S. H., SHENK,T. & GINSBERG,H. S. (1984). The genetic system. In TheAdenoviruses, pp. 125-172. Edited by H. S. Ginsberg. New York & London: Plenum Press. (Received 8 September 1986) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 14:54:53