Composition for cell cultivation, production and use thereof

The present inventiona is directed to a mammalian serum-originated growth factor-containing composition useful in cell cultivation. The composition of the present invention is substantially free of active microorganisms and other harmful substances which would otherwise interfere with cell cultivation. Preferably the composition of the present invention is totally free of contaminant microorganisms, i.e., is sterile. The present invention is also directed to a method of producing the composition of the invention. One step of this method comprises the inactivation of any contaminant microorganisms present in the starting serum. Another step comprises the salting out and desalting of the starting serum to obtain a fraction of the serum which contains the desired cell growth factor. Either step may be conducted first. Finally the present invention provides a medium for cell cultivation which medium contains the present composition together with a basal medium, as well as a cultivation composition which contains the medium and the cells.

FIELD OF THE INVENTION 
This invention relates to a composition for cell cultivation, production 
and use thereof. 
BACKGROUND OF THE INVENTION 
For the cultivation of animal cells or animal tissues, it has been found 
that the addition of animal serum, as a cell growth factor, to the basal 
medium is essential. With the advances in recent years in cytology and 
immunology as well as in large-scale animal cell culture techniques, the 
demand for serum is markedly increasing. 
Important criteria in using animal sera as a cell growth additive include 
the species and age of the animals whence the sera is derived, as well as 
any presence of contaminant microorganisms, cytotoxic substances, 
antibodies, growth-inhibiting substances, and the like. The cost and labor 
involved in obtaining such information are considerable and, moreover, the 
number or quantity of serum lots which can meet the high purity 
requirements is limited in many cases. 
Among various sera, fetal bovine serum and neonatal bovine serum are being 
used with increasing frequency because of their superiority to other sera 
with respect to their cell growth promoting effect and their low content 
of undesired substances, among others. However, current problems with such 
sera include the difficulty in obtaining them, especially due to limited 
source availability and high costs. 
In view of these circumstances, the present inventors conducted intensive 
studies and have now created a composition for cell cultivation which has 
excellent cell growth promoting effect and which contains no significant 
amounts of useless or harmful substances. The composition may be produced, 
with ease and at low cost, by using not only fetal or neonatal bovine 
serum as the starting material, but also adult bovine serum, or serum of 
some other animal species which is readily available for large quantity 
blood collection, such as horse, sheep or swine. 
SUMMARY OF THE INVENTION 
The present invention provides a mammalian serum-originated growth 
factor-containing composition for cell cultivation, the composition being 
substantially free of active microorganisms and other harmful substances 
and preferably being totally free of active microorganism (i.e., sterile. 
The present invention also provides a method of producing the composition 
which comprises subjecting a mammalian serum to treatments comprising a 
step of inactivating contaminated microorganisms and a step of salting out 
and desalting to obtain a fraction which contains cell growth factor and 
is free of other harmful substances. Finally, the present invention 
provides a medium for cell cultivation which contains said composition 
together with a basal medium, and a cultivation composition which contains 
the medium and cells. 
The mammalian serum to be used in the practice of the invention may be 
derived from any species, although bovine, equine, ovine and swine sera, 
among others, may be advantageously used for reasons of their ready 
availability. 
The mammals from which the serum is derived may be at any age, e.g., 
fetuses, newborns, youngs or adults. That sera of adult animals can also 
be used is a characteristic feature of the method of the present invention 
.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The first step of the method of the present invention, i.e., the step of 
inactivating contamined microorganisms, is conducted for the purpose of 
inactivating animal-derived microorganisms as well as microorganisms 
possibly coming into the blood or serum, either at the time of, or 
sometime after the blood collection. Since such microorganisms are 
generally viruses, mycoplasmas and the like, such treatment is preferably 
carried out by adding an inactivating agent highly capable of inactivating 
viruses, mycoplasmas and so on but poorly capable of affecting cell growth 
promoting substances in serum. Effective inactivating agents include 
C.sub.2 to C.sub.4 alkenyl oxides such as ethylene oxide and propylene 
oxide and dialdehydes such as glyoxal (C.sub.2) and glutaraldehyde 
(C.sub.5) and, among the effective agents, ethylene oxide, particularly 
ethylene oxide in liquid form, is especially advantageous from the 
viewpoints of inactivating capacity and influence on growth promoting 
substances, among others. 
When liquid ethylene oxide is used, it is added in an amount of about 0.1-5 
percent by volume, preferably about 1-3 percent by volume and the serum is 
allowed to stand at about 0.degree. to 30.degree. C., preferably about 
5.degree. C. to room temperature, for about 1-7 days, preferably about 2-5 
days, during which inactivation is effected. The above conditions are also 
applicable to the cases where other inactivating agents are used. 
Generally, no particular treatment is required for the removal of the 
inactivating agent added for the purpose of inactivating contaminant 
microorganisms. Said agent is eliminated during standing or during other 
procedures involved in the normal handling of the growth medium. If 
desired, however, a positive measure for removal of the inactivating 
agent, such as dialysis, may be taken in a manner known per se in the art. 
The second step of the method of the present invention, i.e., the step of 
salting out and desalting in accordance with the invention are carried 
out, for instance, in the following manner: 
For salting out, a salt, for example an inorganic salt, is used. The 
inorganic salt includes, among others, ammonium salts (ammonium sulfate, 
ammonium chloride, etc.), sodium salts (sodium chloride, etc.) and 
potassium salts (potassium carbonate). Among these, preferred are ammonium 
salts, in particular ammonium sulfate. 
In the practice of the method of the present invention, the salting out is 
performed in a conventional manner. Thus, the raw material serum or the 
serum from the above-mentioned step of inactivating contaminant 
microorganisms is dissolved or suspended in a solvent (e.g., water, 
ethanol, aqueous ethanol), and the salt is added thereto until a 
predetermined lower limit concentration of the salt is reached. The 
resulting precipitate is removed. To the supernatant, there is further 
added a salt to a predetermined upper limit concentration and the 
resulting precipitate is collected. This is the desired fraction. 
In a preferred embodiment, i.e., when ammonium sulfate is used as the salt, 
the salting out is preferably conducted by using said salt at a lower 
limit concentration of not less than 40% saturation, preferably not less 
than 50% saturation, most preferably 55% saturation, and at an upper limit 
concentration of not higher than 80% saturation, most preferably 70% 
saturation. When other salts are used, the salting out can be effected at 
predetermined concentrations corresponding to the above-mentioned ammonium 
sulfate concentrations. The precipitate can be separated from the 
supernatant advantageously by centrifugation, for instance. 
The precipitate thus obtained is dissolved in physiological saline, for 
instance, and desalted by procedures know per se, e.g., by dialysis or 
ultrafiltration. 
Dialysis may be carried out in a conventional manner, for example, by using 
a membrane dialyzer. Ultrafiltration may likewise be effected in a 
conventional manner, e.g., by filtration under pressure using an 
ultrafiltration membrane permeable to substances not higher than 1,000 in 
molecular weight. 
The thus-obtained composition for cell cultivation is generally adjusted to 
a concentration of from about 20 to 80 mg/ml with physiological saline or 
the like, followed by bacterial filtration using a membrane filter or the 
like and, as necessary, further by freezing or lyophilization. Thereafter, 
the composition can be stored. 
In producing the composition for cell cultivation in accordance with the 
present invention, the step of inactivating contaminant microorganisms can 
precede or follow the steps of salting out and desalting of the 
composition. 
The composition of the present invention contains a salting-out fraction 
corresponding to the ammonium sulfate concentration range of 40-80% 
saturation, which fraction is preferably soluble at the concentration of 
not less than 40% saturation, preferably not less than 55% saturation, and 
is insoluble at the concentration of not higher than 80% saturation, 
preferably not higher than 70% saturation of inorganic salt solution, all 
of these values being in terms of the preferred embodiment, i.e., sulfate 
solution. The composition of the present invention is free of harmful 
substances such as cytotoxic substances and growth inhibiting substances. 
Said composition is also free of useless substances which are otherwise 
contained in mammalian serum such as immunoglobulins. The composition of 
the present invention and contains proteins in the molecular weight range 
of from 60,000 to 80,000, mostly albumin. As compared with the bovine 
serum albumin of the prior art, the present composition has good cell 
growth promoting effect in a greater number of cell species, especially 
animal cell species. Furthermore, the composition of the present invention 
can also be used for the passaging of animal cells in tissue culture. The 
composition for cell cultivation of the present invention is highly 
sterile. Use of the term "highly sterile" means that the present 
composition is free of filtrable microorganisms, the contamination with 
which has been a matter of concern in the prior art of serum preparation, 
and can be handled in safety. Moreover, said composition is at least 
comparable in its cell growth promoting effect to various known animal 
sera, such as fetal or neonatal bovine serum, and bovine serum albumin, 
and said composition can be used advantageously in culturing various 
cells, especially animal cells such as myelomas, hybridomas, monolayer 
cells and other animal cells. In such use, said composition can be added 
to a basal medium in a concentration of 1-10 mg/ml either alone or in 
admixture with trace growth promoting substances such as insulin. 
Most monolayer or attached cultured cells can grow to a sufficient extent 
in a basal medium plus the present composition alone. With most myeloma 
cells, a satisfactory extent of cell proliferation can be achieved when 
said composition is used in admixture with trace growth promoting 
substances. Animal cell subculture using a medium containing the present 
composition is also possible. 
The basal medium to which the composition is added may be a single medium 
or a mixed medium containing from 2 to 4, preferably 2 basal media, for 
example Iscove/F12, Iscove/Serumless Medium, F12/Serumless Medium, or 
alpha-MEM/Serumless Medium. The ratios(V/V), for example, of two basal 
media are in the range of 1:1-1:15. 
By using the medium of the present invention containing the composition for 
cell cultivation of the present invention together with a basal medium, an 
excellent cell proliferation promoting effect is attained, not only in 
stationary culture, but also in roller bottle culture. 
The basal media described in the present specification are publicly 
available. The following is a list of reference works directed to such 
media, with both the abbreviated and full names thereof identified: 
Iscove: Iscove's medium [J. Exp. Med., 147, 923 (1978)] 
F12: Ham's F12 medium [P.N.A.S.(USA), 53, 288 (1965)] 
Serumless Medium: 
Gibco's Serumless Medium 
[P.S.E.B.M., 104, 252 (1960)] 
alpha-MEM: MEM alpha medium [Nature, 230, 310 (1971)] 
DME: Dulbecco's modified Eagle Medium [Virology, 8, 396 (1959)] 
The following examples further illustrate the present invention. It is to 
be noted however that they are by no means limitative of the scope of this 
invention. 
EXAMPLE 1 
(Selective separation of effective fraction) 
Calf serum was fractionated using ammonium sulfate and each fraction was 
examined for cell growth promoting effect. Thus, the salting out was 
performed in a conventional manner and salted-out fractions corresponding 
to the stepwise ammonium sulfate concentration ranges of 0-52%, 52-57%, 
57-62%, 62-67%, 67-72% and 72-80% saturation were collected. Each 
precipitate fraction was dissolved in physiological saline and dialyzed 
against physiological saline. 
After bacterial filtration using a membrane filter (Mirex-GV, 0.22 .mu.m; 
Millipore), each dialyzate was added to a 1:1 mixture of DME medium 
(Nippon Suisan) and F12 medium (Flow) (hereinafter referred to as 
DME/F12), together with four trace growth promoting substances, namely 10 
.mu.g/ml insulin (Sigma), 20 .mu.g/ml transferrin (Green Cross), 2 .mu.M 
ethanolamine (Wako Pure Chemical) and 2.5.times.10.sup.-6 M sodium 
selenate (Wako Pure Chemical). [The mixed additive consisting of the above 
trace growth promoting substances at the respective indicated 
concentrations is hereinafter referred to as ITES; Murakami et al., 
Proceedings of the National Academy of Sciences USA, 79, 1158-1162 
(1982)]. The media thus prepared were comparatively examined for cell 
proliferation rate. As the controls, there were used three groups in which 
(i) the serum before salting out, (ii) fetal bovine serum (5 mg/ml), and 
(iii) bovine serum albumin (5 mg/ml) were respectively added. The cells 
used were NGE-41 cells obtained by cloning U266 cells [IgE-producing human 
myeloma cells; Journal of Clinical and Experimental Immunology, 7, 477 
(1970)], and anti-human IgE antibody-producing hybridoma I-63 cells (cf. 
Japanese Patent Unexamined Publication No. 96028/1983). Each medium 
prepared was distributed in 1 ml/well portions into the wells of a 24-well 
multidish. To each well, there was added 0.1 ml of a NGE-41 or I-63 cell 
suspension (5.times.10.sup.5 to 1.5.times.10.sup.6 cells/ml). After 4-7 
days of incubation in a 5% CO.sub.2 incubator at 37.degree. C., the cells 
in each well were counted using a Coulter counter (Nippon Kagaku Kikai). 
The results thus obtained are summarized in Table 1. The cell growth 
promoting effect was expressed in terms of cell proliferation rate (cell 
count after incubation.div.cell count at start of incubation). 
TABLE 1 
__________________________________________________________________________ 
Ammonium sulfate-salted out fractions 
from calf serum Fetal 
Bovine 
Calf % saturation bovine 
serum 
serum 
0-52 
52-57 
57-62 
62-67 
67-72 
72-80 
serum 
albumin 
__________________________________________________________________________ 
amount 7.0 5.0 
0.8 3.0 5.0 3.4 0.6 5.0 5.0 
added (mg/ml) 
.sup. (5.6)*.sup.1 
(2.2) 
(4.8) 
(3.8) 
NGE-41 
cell line 
- 3.8 1.3 
2.1 4.6 1.8 1.3 1.4 7.3 3.0 
ITES (Death) (5.5) 
(1.9) 
(1.6) 
+ 5.6 1.3 
4.3 5.9 5.1 3.5 1.4 -- 4.0 
(Death) (6.8) 
(5.9) 
(2.3) 
I-63 
cell line 
- 13.6 1.0 
2.0 8.5 5.5 4.4 1.3 16.0 
3.0 
ITES (Death) (5.7) 
(5.1) 
(3.3) 
+ 14.3 1.2 
5.1 9.3 9.5 9.9 1.3 -- 7.0 
(Death) (6.6) 
(9.2) 
(6.4) 
__________________________________________________________________________ 
The values in the table each indicates the cell proliferation rate (cell 
count after incubation/cell count at start of incubation). 
*.sup.1 The data in the parentheses are for the cases where a calf serum 
showing cytotoxicity was used. 
When added alone, the salted out fraction at 57-62% ammonium sulfate 
saturation gave a medium proliferation rate in NGE-41 cells, and those at 
57-62% and 62-67% saturation each gave a medium proliferation rate in I-63 
cells when compared with the proliferation obtained with BSA. When added 
together with ITES, those at 57-62% and 62-67% saturation gave 
considerably high cell proliferation rates in NGE-41 cells whereas, in 
I-63 cells, those at 57-62%, 62-67% and 67-72% saturation gave fairly high 
proliferation rates. These high cell proliferation rates, though yet 
inferior to those attainable with fetal bovine serum, indicate that the 
fractions mentioned above can be used to satisfaction. The fractions were 
superior in cell proliferation rate to bovine serum albumin hitherto in 
use. When a calf serum showing cytotoxicity was subjected to salting out 
with ammonium sulfate, the cytotoxicity was eliminated. 
EXAMPLE 2 
(Search for cell growth promoting substances in each animal serum) 
With the purpose of discovering further serum sources, neonatal bovine 
serum, calf serum and adult bovine serum as well as equine and ovine sera 
were also made subjects of investigation. For each serum species, in the 
same manner as in Example 1, the serum before salting out and an ammonium 
sulfate-salted out fraction were added either alone or in combination with 
ITES and examined for growth promoting effect in each cell line. The 
results thus obtained are summarized in Table 2. 
Based on the results of Example 1, fetal bovine serum, neonatal bovine 
serum, calf serum, adult bovine serum, equine serum and ovine serum were 
each fractionated by 45-80% or 57-70% ammonium sulfate saturation and each 
fraction salted out was dialyzed against physiological saline and then 
filtered for removal of bacteria. For each serum species, the serum before 
salting out and the ammonium sulfate-salted out fraction were added to 
DME/F12 medium at a concentration, as protein, of 5 mg/ml (for myelomas) 
or to MEM medium (Nissui Seiyaku) at a concentration of 3 mg/ml (for 
monolayer cells), in each case either alone or in combination with ITES, 
and examined for cell growth promoting effect in each cell line as 
expressed in terms of cell proliferation rate. The cell lines used as 
myelomas were IgE-producing human myeloma NGE-41 and mouse myeloma MPC11 
(purchased from Dainippon Pharmaceutical) and the cells used as monolayer 
cells were monkey kidney-derived Vero cells (purchased from Flow Lab. 
Inc., USA) and swine kidney-derived PS cells (available from Kyoto 
University, Institute for Virus Research). 
With the myelomas, incubation and cell proliferation ratio measurement were 
conducted in the same manner as in Example 1. 
With the monolayer cells, each medium prepared was distributed in 1 ml/well 
portions into the wells of a 24-well multidish and then a suspension of 
Vero cells or PS cells (5.times.10.sup.5 to 1.5.times.10.sup.6 cells/ml) 
was distributed in 0.1-ml portions thereinto. After 5 days of incubation 
in a 5% CO.sub.2 incubator at 37.degree. C., the supernatant was 
discarded, 0.25% trypsin was newly added in 1-ml portions and the cells 
were scraped off from the multidish to give a cell suspension, which was 
then subjected to cell counting using a Coulter counter. 
TABLE 2 
______________________________________ 
Salting 
out with 
ammo- Addi- 
nium tion of Cell line 
sulfate*.sup.1 
ITES NGE-41 MPC-11 Vero PS 
______________________________________ 
Fetal Not done - 11.0 13.0 5.3 3.7 
bovine + -- -- 4.3 3.6 
serum Done - 8.0 11.0 3.7 3.7 
+ 10.5 13.0 3.9 3.6 
Neo- Not done - 7.0 15.0 -- -- 
natal + 11.0 21.0 -- -- 
bovine 
Done - 5.5 11.0 -- -- 
serum + 7.0 21.0 -- -- 
Calf Not done - 7.0 14.0 3.2 3.0 
serum + 9.0 15.0 3.4 3.4 
Done - 6.0 8.0 3.6 4.3 
+ 9.0 13.0 3.9 4.1 
Adult Not done - Death 3.0 Death Death 
bovine (8.0)*.sup.2 
(9.0) 
serum + Death 3.0 Death Death 
(9.0) (11.0) 
Done - 5.0 4.0 2.8 2.8 
(5.0) (5.0) 
+ 7.0 10.0 4.0 2.5 
(9.0) (14.0) 
Equine 
Not done - 6.5 17.0 3.4 3.2 
serum + 7.0 19.0 3.6 3.6 
Done - 4.5 11.0 3.5 3.6 
+ 5.0 15.0 3.7 3.8 
Ovine Not done - 5.0 5.0 4.0 3.6 
serum + 5.0 10.0 3.9 3.4 
Done - 5.0 5.0 3.8 4.2 
+ 7.5 10.0 3.9 4.6 
Bovine 
Not done - 3.0 4.0 2.5 2.5 
serum + 5.0 16.0 2.5 3.4 
al- 
bumin 
No Not done - 2.5 2.5 2.2 2.0 
addi- + 2.5 4.0 2.5 3.3 
tion 
______________________________________ 
The values in the table each indicates the cell proliferation rate. 
*.sup.1 Fraction salted out by 45-80% or 57-70% ammonium sulfate 
saturation 
*.sup.2 The values in the parentheses are for the cases where an adult 
bovine serum lot lacking in cytoxicity was used. 
For the myeloma cell lines, sera other than ovine serum, when added alone 
after salting out, each gave a reduced cell proliferation rate as compared 
with the rate before salting out. However, when added in combination with 
ITES, each serum fraction gave a cell proliferation rate comparable to or 
rather higher than the cell proliferation rate obtained with the 
corresponing serum before salting out. 
In the case of ovine serum, the single addition of the serum before salting 
out and the ammonium sulfate-salted out fraction resulted in little 
difference in cell proliferation rate. Combined addition of the ammonium 
sulfate-salted out fraction with ITES increased the cell proliferation 
rate to a considerable extent and, in some kinds of cells, good cell 
proliferation rates were obtained. 
On the other hand, for the monolayer cells, the use of the ammonium 
sulfate-salted out fractions of sera other than fetal bovine serum gave 
cell proliferation rate values comparable to or slightly higher than the 
values obtained with the sera before salting out. In particular, the 
addition of the ammonium sulfate-salted out fractions in combination with 
ITES gave good cell proliferation rates. 
As in Example 1, a bovine serum showing cytotoxicity was freed of toxic 
substances by salting out with ammonium sulfate. 
As compared with bovine serum albumin, higher cell proliferation rates were 
obtained, in both the cell lines, with the ammonium sulfate-salted out 
fractions. The differences were significant in particular when the 
fractions were added together with ITES. Thus, also in the monolayer 
cells, good cell growth promoting effect was attained by adding various 
salted-out serum fractions in combination with ITES. 
EXAMPLE 3 
(Effect of inactivating agents on contamined microorganisms in serum) 
It is a matter of concern that animal sera are sometimes contaminated with 
microorganisms, mainly animal-derived viruses and mycoplasmas. If such 
contaminated sera are used as cell growth promoting substances, cells may 
be infected with such microorganisms, in particular those filtrable 
microorganisms, whereby serious obstacles such as cell growth inhibition 
and death of cells will be encountered. 
However, it is impossible to sterilize sera by heating. For the time being, 
there is no alternative but to sterilize sera by bacterial filtration. 
Filtrable microorganisms such as viruses cannot be removed by such method, 
however. To cope with this situation, an inactivating agent was sought 
which could inactivate microorganisms occurring in serum completely but 
would not inhibit cell growth promoting substances contained in serum. 
Mycoplasma, vaccinia virus and Japanese encephalitis virus were separately 
suspended, as contaminant microorganisms, in microorganism-free, normal 
calf serum having good cell growth promoting effect. The concentrations of 
microorganisms were: 10.sup.7 PFU/ml for mycoplasma, 10.sup.8 ICID.sub.50 
/ml for vaccinia virus and 10.sup.6 TCID.sub.50 /ml for Japanese 
encephalitis virus. Following addition of each of the inactivating agents 
given in Table 3 to each microorganism suspension in serum, inactivation 
treatment was performed under varied conditions. After inactivation 
treatment, each serum was dialyzed against physiological saline overnight 
to thereby remove the inactivating agent, and then the remaining 
microorganisms in each serum were counted and compared with the count of 
microorganisms remaining in the serum treated in the same manner without 
adding the inactivating agents. 
Furthermore, these inactivating agents were examined for their effect on 
cell growth promoting substances. Thus, each inactivating agent was added 
to normal calf serum, followed by inactivation treatment under the same 
conditions as mentioned above. Part of the serum was dialyzed against 
physiological saline overnight and the dialyzate was used as the serum 
before salting out. The remaining serum portion was salted out by 57-80% 
ammonium sulfate saturation and then dialyzed against physiological saline 
overnight and the dialyzate was used as the ammonium sulfate-salted out 
fraction. Each serum material was added to DME/F12 medium to a 
concentration of 5 mg/ml as protein, either alone or in combination with 
ITES, followed by incubation of NGE-41 cells by the procedure of Example 
1. The cell proliferation rates thus obtained were compared. 
The results obtained in the above are shown in Table 3. 
TABLE 3 
__________________________________________________________________________ 
Cell proliferation rate*.sup.1 
Microorganism inactivating 
effect 
Ammonium Japanese 
sulfate- Vaccinia 
encephalitis 
Serum before 
salted out 
Mycoplasma 
virus 
virus 
concen- 
Treatment 
duration 
salting out 
fraction 
Number of 
Tissue culture 
tration 
tempera- 
of treat- 
ITES ITES colonies 
infectious dose 
Inactivating agent 
(vol. %) 
ture (.degree.C.) 
ment (days) 
- + - + (log.sub.10 PFU/ml) 
(log.sub.10 TCID.sub.5 
0 /ml) 
__________________________________________________________________________ 
Liquid 1.0 5 7 4.5 4.7 4.7 5.0 &lt;0 &lt;0 &lt;0 
ethylene oxide 
3.0 5 7 4.0 4.0 4.8 4.8 -- -- -- 
1.5 5 3 -- -- 3.8 5.2 &lt;0 &lt;0 &lt;0 
1.5 18-22 3 -- -- 3.2 4.8 &lt;0 &lt;0 &lt;0 
Glutaraldehyde 
0.1 5 3 -- -- 2.5 4.2 6 &lt;0 &lt;0 
0.1 18- 22 
3 -- -- 2.9 4.5 6 &lt;0 &lt;0 
Phenol 0.5 5 7 5.0 5.3 4.0 4.3 6.5 8 5 
Formalin 0.1 5 7 Death 
Death 
Death 
Death 
-- -- -- 
Sodium azide 
0.1 5 7 1.3 1.3 1.3 2.0 7 8 6 
Chlorhexidine 
0.007 
5 6 -- -- 1.0 1.0 -- -- -- 
Sodium hypochlorite 
0.02 5 6 -- -- 3.7 5.0 -- 7 6 
No addition 
-- 5 7 5.0 5.5 4.5 5.0 7 8 6 
__________________________________________________________________________ 
*.sup.1 Cell proliferation rate in cell line NGE41. 
Sera or their fractions were use at the concentration of 5 mg/ml. 
EXAMPLE 4 
To 1 liter of adult bovine serum, there was added dropwise 15 ml of liquid 
ethylene oxide with adequate stirring. After addition, the mixture was 
allowed to stand at 5.degree. C. for 5 days. Thereto was added portionwise 
350 g of ammonium sulfate. After dissolution of the ammonium sulfate, the 
solution was allowed to stand at 5.degree. C. overnight. The resulting 
precipitate was removed by centrifugation (9,000.times.g, 20 minutes). To 
the supernatant thus obtained, there was added portionwise 140 g of 
ammonium sulfate. After dissolution of the ammonium sulfate, the solution 
was allowed to stand as it was at 5.degree. C. overnight. The precipitate 
formed was collected by centrifugation (9,000.times.g, minutes) and 
dissolved in about 100 ml of physiological saline. The solution was placed 
in a dialyzing membrane (Visking) and dialyzed against 15 liters of 
physiological saline at 5.degree. C. overnight. The dialyzing fluid was 
then replaced with fresh 15 liters of physiological saline and the 
dialysis was further continued at 5.degree. C. overnight. 
The dialyzate was taken out and adjusted to a protein content of 60 mg/ml 
with physiological saline and filtered for removal of bacteria through a 
membrane filter (0.22 .mu.m, Millipore) to give 500 ml of a composition 
for animal cell culture. 
To 300 ml of a basal medium composed of DME/F12 (1:1), there was added the 
above composition for cell culture to a concentration of 3 mg/ml as 
protein, followed by further addition of 3 mg of insulin, 6 mg of 
transferrin, 36.6 .mu.g of ethanolamine and 1.4 .mu.g of sodium selenate 
and the subsequent bacterial filtration through a membrane filter 
(Millipore). There was thus obtained a medium for cell cultivation. 
EXAMPLE 5 
A swine serum fraction obtained by salting out with ammonium sulfate was 
examined for cell growth promoting activity. The results obtained are 
shown in Table 4. Thus, 7.5 ml of liquid ethylene oxide was added dropwise 
to 1 liter of swine serum with good stirring. The mixture was allowed to 
stand at 25.degree. C. for 2 days. To each of 3 lots (A, B, C) obtained in 
this manner, there was added portionwise 350 g of ammonium sulfate. The 
resultant solution was allowed to stand at 5.degree. C. overnight. The 
resultant precipitate was removed by centrifugation (9000 xg, 20 minutes). 
To the supernatant obtained was further added portionwise 140 g of 
ammonium sulfate. After dissolution of the ammonium sulfate, the solution 
was allowed to stand at 5.degree. C. overnight. The resultant precipitate 
was collected by centrifugation (9000 xg, 20 minutes) and then dissolved 
in about 100 ml of physiological saline. This solution was placed in a 
dialyzing membrane (Visking) and dialyzed against 15 liters of 
physiological saline at 5.degree. C. overnight. The dialyzing fluid was 
then exchanged for fresh 15 liters of physiological saline and the 
dialysis was further conducted at 5.degree. C. overnight. 
The dialyzate was taken out and the protein concentration was adjusted to 
60 mg/ml with physiological saline. The dialyzate was filtered through a 
membrane filter (0.22 .mu.m; Millipore) for removal of bacteria. Thus was 
obtained 500 ml of a composition for cell cultivation. 
The above composition was added, in an amount of 2 mg/ml, to Iscove/F12 
basal medium, together with ITES and, following the procedure of Example 
1, the cell growth promoting effect was investigated in a total of 6 
myeloma and hybridoma cell lines. As controls for comparison with respect 
to cell growth promoting activity, there were used fetal bovine serum and 
ammonium sulfate fractions from calf serum and adult bovine serum. No 
difference in cell growth promotion was noted among the 3 lots of swine 
serum-drived ammonium sulfate fraction, and said lots were comparable or 
rather superior in cell growth promoting activity to ammonium 
sulfate-salted out fractions from calf and adult bovine sera. 
TABLE 4 
__________________________________________________________________________ 
Cell 
Material I-63 
CEA HS-11 
MPC-11 NGE-41 
NGE-44 
__________________________________________________________________________ 
Fetal bovine serum 
100* 
100 100 100 100 100 
Ammonium sulfate- 
salted out fraction 
from 
Swine serum 
A 72 94 70 96 79 141 
B 84 98 80 111 86 136 
C 66 60 67 76 75 135 
Fetal bovine serum 
61 88 73 69 77 111 
(obtained in 
Example 2) 
Adult bovine serum 
80 102 78 93 84 112 
(obtained in 
Example 2) 
__________________________________________________________________________ 
*The values in the table each indicates the relative cell growth promotin 
activity (the activity of fetal bovine serum being 100) after 3 
subcultures in each medium. 
Cells were cultured in Iscove/F12 with fetal bovine serum (6 mg/ml) or 
with each ammonium sulfatesalted out fraction (2 mg/ml) plus ITES 
EXAMPLE 6 
(Experiments in which the composition for animal cell culture according to 
the invention was applied to various basal media) 
Generally, the growth of cells largely depends on the basal medium and the 
kind of proliferation promoting additive. Therefore, a search was 
conducted to identify which basal media could be used with the composition 
of the present invention to achieve the maximum growth promoting effect. 
In the medium search, 14 media were used, namely Serumless Medium (GIBCO; 
liquid; Neuman & Tytell formulation) and the following 13 publicly 
available basal media: Iscove [Boehringer Mannheim-Yamanouchi; power; made 
ready for use in situ (i.e. dissolved in water followed by bacterial 
filtration in accordance with the directions for use; the same shall apply 
hereinbelow)], F12 (Nissui; powder; made ready for use in situ); MEM, 
William-D, William-E, Waymouth-MB752/1, Fischer, RPMI-1640, 199 (each from 
GIBCO; liquid); DME, NCTC-109, McCoy 5A and alpha-MEM (each from MAB; 
liquid). Cell proliferation experiments were conducted using the above 14 
media either alone or in combination with one another. Thus, the 
composition of the invention as produced by the procedure of Example 4 was 
added, in an amount of 2 mg/ml and together with ITES, to each of the 14 
media and 91 mixed media prepared by mixing two of the media in a 1:1 
ratio (105 media in total). Cells were cultured following the procedure of 
Example 1 and the cells were counted for the 3rd subculture. 
The cells used were I-63 (mouse hybridoma), CEA (mouse hybridoma) and 
HL15-10 (human hybridoma). 
The results obtained are shown in Table 5. 
TABLE 5 
__________________________________________________________________________ 
basal medium*.sup.1 
abcdefghijklmn*.sup.2 
__________________________________________________________________________ 
cell: HL15-10 
a ISCOVEb F12c DMEd NCTC-109 
##STR1## 
e McCoy-5Af MEMg alpha-MEMh William-D 
##STR2## 
i William-Ej Waymouth MBk Fisherl RPMI-1640 
##STR3## 
m 199n Serumless MediumRef. fetal bovine serumRef. bovine serum 
##STR4## 
cell: CEA 
a ISCOVEb F12c DMEd NCTC-109 
##STR5## 
e McCoy-5Af MEMg alpha-MEMh William-D 
##STR6## 
i William-Ej Waymouth MBk Fisherl RPMI-1640 
##STR7## 
m 199n Serumless MediumRef. fetal bovine serumRef. bovine serum 
##STR8## 
cell: I-63 
a ISCOVEb F12c DMEd NCTC-109 
##STR9## 
e McCoy-5Af MEMg alpha-MEMh William-D 
##STR10## 
i William-Ej Waymouth MBk Fisherl RPMI-1640 
##STR11## 
m 199n Serumless MediumRef. fetal bovine serumRef. bovine serum 
##STR12## 
__________________________________________________________________________ 
##STR13## 
composition of the invention from bovine serum, in an amount of 2 mg/ml, 
together with ITES. 
*.sup.2 Each value in the table indicates the score assigned, according t 
the range conversion given below, to the number of cells after 3 
subcultures in each medium: 
For HL1510:For CEAFor I63 
0: Death0: Death0: Death 
1: less than 50 .times. 10.sup.4 cells/ml1: less than 15 .times. 10.sup.4 
cells/ml1: less than 15 .times. 10.sup.4 cells/ml 
2: 50 to 100 .times. 10.sup.4 cells/ml2: 15 to 25 .times. 10.sup.4 
cells/ml2: 15 to 30 .times. 10.sup.4 cells/ml 
3: more than 100 .times. 10.sup.4 cells/ml3: more than 25 .times. 10.sup. 
cells/ml3: more than 30 .times. 10.sup.4 cells/ml. 
Fetal bovine serum: As a control, fetal bovine serum was added to each 
individual basal medium to a concentration of 10%. 
Bovine serum albumin: As a control, bovine serum albumin was added to eac 
individual basal medium to a concentration of 5 mg/ml, together with ITES 
As for the single media, good cell growth was noted with Iscove in I-6 
and with F12 in CEA, while the cell proliferation was poor with any of 
other media in these cell lines. In HL15-10, all the single media failed 
to achieve good proliferation. The same phenomena were encountered also 
with bovine serum albumin used as a control. Even with fetal bovine serum, 
which is said to be capable of promoting good proliferation, only a fairly 
limited number of media could attain good proliferation in all the above 
three cell lines equally. 
With regard to the mixed media, on the other hand, Iscove- or Serumless 
Medium-containing mixed media allowed good proliferation of I-63 and 
HL15-10. In CEA, few mixed media afforded good proliferation. Among 
others, Iscove and Serumless Medium produced the desired mixture effect 
with a number of other media. In view of factors such as cell 
proliferation, general usefulness, and the like, it was generally 
considered that mixtures of basal media produced a much better growth 
enhancement effect than did the use of a single medium. Preferred mixed 
media are, for example, Iscove/F12, Iscove/Serumless Medium, F12/Serumless 
Medium, and alpha-MEM/Serumless Medium. 
Next, the effect of the mixing ratio between two media was studied for the 
combination of Iscove and F12. Thus, Iscove (Boehringer 
Mannheim-Yamanouchi) and F12 (Nissui) were mixed in the ratios of 1:0, 
1:1, 1:1, 1:3, 1:4, 1:7, 1:15 and 0:1, and, following addition of 2 mg/ml 
of the composition of the invention as obtained in Example 4 and ITES, 
cultivation was performed in these mixed media. After three subcultures, 
the cells were counted. The cell lines used were NGE-44 (derived from the 
above-mentioned NGE-41 by adaptation to a medium containing said 
composition), CEA (mouse hybridoma) and I-63 (mouse hybridoma). 
The results obtained are illustrated in FIG. 1. In all the cell lines used, 
the mixed media each brought about fairly good cell proliferation as 
compared with the single medium Iscove or F12. In the Iscove-to-F12 mixing 
ratio of 1:1 to 1:7, the cell proliferation was good, and it was concluded 
that the mixing ratio between the media in preparing mixed media 
supplemented with the composition according to the invention can be 
selected within a fairly wide range. 
EXAMPLE 7 
(Effect of the composition for animal cell culture according to the 
invention in roller bottle culture) 
The applicability of the composition of the present invention to roller 
bottle culture was investigated in connection with the kind of basal 
medium employed. As the media, there were used Iscove/F12 (1:1) and 
DME/F12 (1:1), each supplemented with the composition obtained in Example 
4 (2 mg/ml) and ITES. I-63 cells were cultured in a 1-liter jar fermenter 
(Mitsuwa Rika model KMJ-2). In a control run, a suspension of the same 
cells were distributed in about 6-ml portions into 25-cm.sup.2 tissue 
culture flasks (Falcon) and stationary culture was conducted. The results 
thus obtained are shown in FIG. 2. 
As is evident from FIG. 2, the composition according to the present 
invention exhibited excellent cell proliferation promoting effect in both 
the roller bottle culture and stationary culture. 
In roller bottle culture, the Iscove/F12 mixed medium gave excellent 
results, with the final number of cells amounting to 80.times.10.sup.4 
cells.