Method for cultivating insect cells

Cultivation and reproduction of insect cells in a nutrient medium in which fetal calf serum is partially or completely replaced by egg yolk.

In connection with the general discussion on the environmental hazard of 
chemical pesticides, biological methods for combating pests have gathered 
momentum. One of the most promising methods is the use of highly specific 
insect pathogenic viruses, especially nucleopolyhedrosis viruses (NPV) and 
granulosis viruses (GV) from the group of baculoviruses. These viruses are 
applied to the environment in the same manner as chemical pesticides, 
where they are picked up by their specific hosts in which they cause a 
virus infection killing the hosts within a short period of time. A number 
of NPV and GV products are already commercialized or are tested for 
practical use (see Shieh and Bohmfalk, Production and Efficiency of 
Baculoviruses, Biotechnology and Bioengineering Vol. XXII, 1357 et seq. 
(1980)). 
However, the production of sufficient quantities of virus material meets 
with practical problems which have not found a satisfactory solution as 
yet. In principle, there are two methods available (loc. cit., 1361 et 
seq.): 
(1) reproduction of large host numbers, artificial infection with virus and 
isolation of virus from the dead animals; 
(2) cultivation of individual insect cells in vitro in a suitable nutrient 
medium, infection of the cells and work-up of the virus material. 
The method described, sub (1), although supplying large amounts of virus 
material, has the following disadvantages: the host animals are available 
only during a limited period of the year, and the virus material is 
contaminated with insect residues and microbial impurities. Although 
purification is possible, it would greatly raise the cost of production. 
The method described sub (2) avoids these difficulties; however, it depends 
fully on the availability of a suitable culture medium. The compositions 
proposed or used for this purpose contain, in addition to inorganic salts, 
vitamins, amino acids, sugars and a number of other organic compounds 
important for cell physiology, about 5 to 20% of fetal calf serum (FCS) 
which is indispensable for cell growth. Typical compositions are for 
example described in Nature 195, 4843 (1962) and J. Invertebrate Pathology 
25, 363-370 (1975). 
FCS is the limiting factor due to its high price and because it is 
available in relatively small amounts only. Production of insect cells on 
an industrial scale required for an industrial manufacture of insect 
pathogenic viruses is therefore not possible in this way. There is thus 
the need of replacing FCS by a cheaper component available in sufficient 
quantities. 
Surprisingly, it has now been found that instead of FCS, egg yolk can be 
advantageously used. 
Subject of the invention is therefore a method for cultivation and 
reproduction of cells, especially insect cells, with the use of known 
nutrient media containing inorganic salts, amino acids, sugars, vitamins 
and other organic substances, which comprises adding egg yolk to these 
nutrient media. 
The content of egg yolk is from 0.1 to 5, preferably 0.5 to 2, % by weight 
of the nutrient solution. It may replace FCS completely or partially. 
For preparing a nutrient medium suitable for cell culture, only about 5 to 
20% of the FCS amount of egg yolk is required. The egg yolk must not 
necessarily be separated from the egg white; suitable are therefore also 
whisked eggs, for example. Preferably, however, commercial egg yolk 
emulsion is used. 
The origin of the egg yolk, too, is of secondary importance only. For 
reasons of availability, egg yolk of hen's eggs is particularly suitable, 
but duck and goose eggs may also be used as source of egg yolk. 
The method of the invention is suitable for the cultivation of numerous 
insect cell lines, for example from the orders of diptera and lepidoptera. 
There may be mentioned for example cell lines of Spodoptera frugiperda, 
Lymantria dispar, Mamestra brassicae, Trichoplusia ni, Aedes albopictus 
and Aedes aegypti, which in turn are suitable for the reproduction of 
numerous insect-pathogenic viruses such as parvoviruses, pox viruses, 
baculoviruses and rhabdoviruses, of which especially nucleopolyhedrosis 
viruses such as those from Autographa spp., Spodoptera spp. and Lymantria 
spp. are of interest.

The process is subsequently described using the cultivation of an 
established cell strain of Spodoptera frugiperda (Sf) as example. 
To a commercial TC 10 culture medium (consisting of a salt solution, 
glucose, defined amounts of amino acids and vitamins and a protein 
hydrolysate (tryptose broth)) 5% by weight of FCS (instead of the usual 
10%) and 0.5% by weight of commercial egg yolk emulsion were added. In the 
culture medium thus modified the cells were cultivated using monolayer 
flasks (3 ml), shaker cultures (20 ml) and flasks with stirrer (500 ml). 
The addition of egg yolk increased the cell yield by 100% (from 
1.4.times.10.sup.6 to 2.8.times.10.sup.6 cells/ml) as compared with a 
control batch containing 10% of FCS only. 
In further tests, FCS was completely omitted and varying egg yolk 
concentrations were used. At a rate of 1% of egg yolk FCS could be 
completely replaced; the cell yield was in the same range as that obtained 
when using 10% of FCS (Table 1). 
TABLE 1 
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Cell yield at addition of FCS or egg yolk emulsion 
in monolayer system after 50 passages 
cell yield (cells/ml) on use of 
TC 10 
+ 1% egg yolk 
+ 10% FCS 
emulsion 
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Test 1 1.4 .times. 10.sup.6 
1.4 .times. 10.sup.6 
Test 2 2.0 .times. 10.sup.6 
1.7 .times. 10.sup.6 
Test 3 1.4 .times. 10.sup.6 
1.8 .times. 10.sup.6 
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Comparable results were obtained when cultivating the cells in shaked or 
stirred solutions. Long duration tests proved that the cell growth in the 
culture media of the invention was stable over more than 60 passages. 
The cells cultivated in a FCS-free medium with addition of egg yolk were 
regularly used as substrate for the production of insect pathogenic 
nucleopolyhedrosis viruses. The virus yield remained unchanged as compared 
to control batches with addition of FCS (Table 2). 
TABLE 2 
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Polyhedrosis virus yield in monolayer system 
TC 10 
+ 10% CFS 
+ 1% egg yolk 
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PIB/ml 1.3 .times. 10.sup.7 
1.2 .times. 10.sup.7 
(polyhedral in- 
1.9 .times. 10.sup.7 
1.6 .times. 10.sup.7 
clusion bodies) 1.3 .times. 10.sup.7 
(2 tests) (3 tests) 
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Electron-optical examinations and infection tests in vitro proved that the 
morphology and the biological activity of the viruses produced were 
unchanged.