Download Growth of 293 Cells in Suspension Culture

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
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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.
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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.
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(Received 8 September 1986)
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