CA1198701A - Animal cell culture medium containing cyclodextrin - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Cyclodextrin shows no or low cytotoxicity on cell growth and lipophilic substances such as unsaturated fatty acids and lipophilic vitamins when present together with, or included in, cyclodextrin have a cell growth promoting effect and accelerating effect on the production of valuable products; a serum-free or serum-reduced culture medium or a substitute composition for serum for culture medium comprises cyclodextrin and at least one lipophilic nutrient substance, the cyclodextrin and the lipophilic nutrient substance being preferably in the form of inclusion complex between them.

Description

This invention relates to improvements in culture media~
In accordance with the invention there is ~xovided a serum-free culture medium, a substitute composition for serum for culture media, a means of reducing the serum concentration or the serum moiety in a culture medium for mammalian cells, and a better use of the serum moiety con-tained in a culture medium.
Culture media for the proliferation of mammalian cells are generally prepared with the addition of a large amount of serum and, consequently conventional culture media usually contain a large amount of serum The following problems are experienced with conventional culture media:
(a) for the production of interferon by human cells, or for the propagation of viruses in order to prepare vaccines, for example, fetal bovine serum or calf serum is usually added in an amount of about 10% to the culture media. Therefore, more than half of the cost of such media relates to such sera, a~d these media axe expensive, (b) there is the troublesome necessity of checking serum lots in advance, before the sera are used to prepare culture media, because these sera might be contaminated with mycoplasma or viruses, and because the quality of the sera often differs from lot to lot, (c) the sera contain various and unidentified proteins derived from bovine or horse, and this makes it difficult to isolate such products as interferon from the remainder of the culture medium.
~he inventors attempted to decrease the serum concentration in a culture medium or to deve]op serum-Eree culture media, and found that serum albumin may substitute 7~

for sera and that mammalian ce]ls grow as well in a culture medium containing serum albumin without any other serum components as in conventional serurn-containing culture rnedi,a.

The inventors also tried to develop a serurn-free cu]ture medium, which would be easier to prepare or handle and accordingly less expensive.
The inventors were finally successful in preparing a new serum-free culture medium which contains a cyclodextrin and a lipophilic substance or a cyclodextrin-lipophilic substance inclusion compound.
This new serum~free culture medium has been found to allow mammalian cells to grow without the need for serum albumin or serum as usually employed in a conventional culture in media and thus provides a serum-free culture medium.
A particular problem with culture media is that water-insoluble or sparingly soluble unsaturated or saturated fatty acids and other water-insoluble or sparingly soluble nutrients are very difficult to introduce into the culture media without introducing their cytotoxic effect.
The inventors have been successful in introducing these lipophilic nutrients into a serum-free culture medium by using a cyclodextrin as a vehicle.
In accordance with this invention it has been found that a cyclodextrin is not cytotoxic or is less cytotoxic for mammalian cell growth and is also an appropriate additive to facilitate the introduction of various lipophilic substances, for example saturated or unsaturated fatty acids and lipophilic ~' ,.. . ..

-37~3~ ' vitamins to a culture medium, in other words, use of a cyclodextrin makes it easier to add lipophilic substance nutrients to a serum-free culkure medium.
There are three cyclodextrins, namely, ~-, ~-, and ~-cyclodextrin, and each of the cyclodextrins can be used in accordance with this invention. Among them, ~-cyclodextrin is preferred for the purpose of the invention because of its non-cytotoxicity for mammalian cell growth. ~-Cyclodextrin is generally employed in drug preparation, but shows some cytotoxic effect on the growth of mammalian cells, Therefore, ~-cyclodextrinis less preferred than ~-cyclodextrin for that purpose at its higher concentrations.
In this invention, cyclodextrin is used for the addition of lipophilic substance ingredients which are to be added as nutrients in a culture medium or to decrease the cytotoxicity of such ingredients against m~mma 1 i an cells.
The lipophilic substances include unsaturated fatty acids, for example linoleic acid, linolenic acid, oleic acid, arachidonic acid ,and erucic acid and their glycerides;
saturated fatty acids, for example palmitic acid and stearic acid and their glycerides, and lipophilic vitamins, for example vitamins A, D and E.
The cyclodextrin and lipophilic substance may be added individually, or as a mixture of the cyclodextrin and lipophilic substance, but preferably there are added to a culture medium after being reacted with one another.
The reaction is, for example, carried out, as follows: a cyclodextrin aqueous solution and a solution of a lipophilic substance dissolved in a hydrophilic organic solvent, for example, an alcohol or dioxane are first prepared, then both solutions are rnixed and, for example, heated up to ~L~L9~ 3~

75C or heated to 70C and maintained at this ternperature -for 5 minutes The reaction product is, for example, preci-pitated by cooling the reaction mixture and it i.5 easily separated in such precipita-te form from the reaction mixture.
The reaction product thus obtained can be introduced into the culture rnediurn~
The reaction product is usually in the form of an inclusion complex in which cyclodextrin includes the lipo-p~ilic substance inside, but it may be in a form in which cyclodextr7n is bound to the lipophilic substance in some other way.
The amount of cyclodextrin to be used depends on the purposes of its use. When it is used in the form of its inclusion complex with a lipophilic substance, an inclusion complex i9 preferably used which results from reacting cyclodextrin with a lipophilic substance in a cyclodextrin/-lipophilic substance molar ratio of 3 to 500:1, and usually 10 to 100:1.
For the preparation of a culture medium for mammal-ian cells, the addition of serum has been thought to beessential.

However, according to this invention, for example, in a serum-free culture medium which contains a reaction product of linoleic acid with ~-cyclodextrin, instead oE serum or serum albumin, mammalian cells can grow as well as in a conventional serum-containing medium and can produce physiologically active substances such as interferon.
Cell growth and production of physiologi~ally -- 4 ~

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active substance can further be improved when other unsatura-ted fatty acids,-for example oleic acid and linolenic acid, and/or vitamin E axe added as their respective inclusion complexes with cyclodextrin, together with the linoleic acid/
cyclodextrin inclusion complex.
An inclusion complex prepared from vitamin E and cyclodextrin, even when singly used, can also improve cell growth or enhance productivity of interferon, as can an inclusion complex of cyclodextrin with one of the o'cher unsaturated fatty acids, for example oleic acid.
When aninclusion complex prepared under the above-mentioned conditions from linoleic acid and ~-cyclodextrin is used as a substitute for serum or serum albumin, it is sultably introduced in an amount of about 0.1 to about 5 mg per liter of culture medium, preferably about 0.5 to about 2 mg per liter of culture medium, e~pressed in terms of the linoleic acid moiety. Usually, larger amounts of the linoleic acid moiety than the above will have a deterimental effect on cell growth, i.e., cytotoxicity. The lower limit of the concentration of the linoleic acid moiety which will have a cytotoxic effect on cell growth can however be increased up to 10 mg per liter of culture medium, by the addition of ~-cyclodextrin into the medium in an amount of about 300 to about 1000 mg per liter of culture medium.
An inclusion complex of ~-cyclodextrin with fatty acids other than linoleic acid, for example oleic acid, linolenic acid, ~almitic acid and stearic acid, is preferably introduced in an amount of about 0.1 to about 1 mg/l expressed in terms of the fatty acid moiety.

In case of an inclusion complex prepared frorn vitamin E and ~-cyclodextrin added to the rernainder of a serum-free or serum albumin-free culture medium, its optimal con-centration is about 0.5 to about 10 mg/l, pre~erably about 1 to about 5 mgjl, as expressed in terms of the concentration of vitamin E.
A serum- or serum albumin-free culture medium of the invention may suitably contain other ingredients, for example cell growth factors, for e~ample insulin and human trans~errin and nucleic acid precursors, for example hypo-xanthine, thymidine, deoxyadenosine and deoxycytidine, together with a carbon source for example glucose, amino acids, vitamins, minerals and other nutrients which are contained in a conventional serum-supplemented medium or a known serum-free medium for m~mm~ 1; an cell culture.
A cell growth factor is preferably used in a concentration of about 1 to about 100 mg per liter of culture medium, and a nucleic acid precursor is preferably used in a concentration of about 0.01 to about 50 mg per liter of culture medium. Two or more cell growth factors are used simultaneously or in combination, depending on the kind of mammalian cells. Two or more nucleic acid precursors may be used in the same manner.
Glucose is usually employed as the carbon source in an amount of about 0.5 to about 10 g per liter of culture medi-lm. Other carbonsources, for example pyruvate may be added in adequate amounts.
Amino acids, for exam~le L-alanine, L-arginine, L-glutamine, L-methionine, L-threonine, L-lysine, L-valine and L-phenylalanine are also used, such amino aclds beiny the components of protein. Into a serum-containing culture medium, essentiaL amino acids are mainly introduced, but various non-essential amino acids as well as essential amino acids are preferably introduced into a serum-free culture medium according to this invention. Amino acids are preferably added in total amounts of about 0.5 to about 5 g per liter of culture medium. Other amino acids are preferably supplemerlted, in consideration of usual serum-containing medium.
Vitamins, for example ascorbic acid, riboflavin, thiamine-HCl, Ca-pantothenate, nicotic acid amide, pyridoxal-HCl, i-inositol, folic acid, vitamin B12 and biotin may be added, together with minerals for exarnple as NaCl, KCl, CaC12, MgSO4, NaH2PO4, FeSO4, ZnSO4, and ~aSeO3, in adequate amounts.
In addition, such metabolic intermediates as choline fitartrate, glutathione and putrescine 2HCl, and buffering substances, for example ~aHCO3, ~-glycerophosphate-2~a, and/or ~ 2-hydroxyethylpiperazine-~-2-ethane sulfonate are suitably employed as supple~ents.
In addition to the above ingredients and additives, the following basal media are referred to, in determining the composition of a culture medium according to this invention Dulbecco's Modified Eagle Medium, RPMI-1640 Medium, Eagle Minimum Essential Medium and Ham F-12 Medium.
The method of preparing a culture medium according to the invention is not critical. It may be prepared, for example, by dissolving all the ingredients and additives in water in their respective appropriate concentrations, and then filtering the solution on a membrane filter under pressure to obtain a sterilized culture medium. It will be apparent from the above disclosure that cyclodextrin and a lipophilic substance are preferabl~ used in the form of an inclusion complex between them.

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Mammalian cells which can grow in a serum-free culture medium of this invention are not limited to specified cells, but a wi~e variety of cells, for example lyrnphocyte~, fibroblastes, epithelial cells, and the;r transforrned cells, various neoplastic cells, and hybridorn~s der.ived therefrom can grow therein.
Examples of such mammalian cells include Epstein-Barr Virus (RBV)-trans~ormed human lymphoblastoid cell lines such as UMCL and C5180Y, human Burkitt's lymphoma-derived Namalwa cells, murine lymphoid cell-derived myeloma SPI
cells, human fibroblast cells such as ~EL and IMR-90 human tumor-derived epithelial cells such as Heha-S3, Hep-2 and KB, human primary cultured cells, rat Yoshida sarcoma cells, hamster fibroblast cells BHK-21, murine fibroblast cells 3T3, and murine lymphoma cells YAC-l.
The method of culturing mammalian cells with the use of a culture medium of this invention is not critical, either. Mammalian cells are cultured in a serum-free culture , medium of the invention under the same or almost the same conditions as those for a conventional serum-containing medium; for example, cells are cultured in a culture medium at an initial cell density of 10 ~10 cells/ml and 35~37C
under supply of 4~6 % (V/v) C02-containing sterilized air into the culture vessel. Fibroblast cells are preferably cultured under lower 2 supply (5~10 % V~v) than atmosphere, especially in a serum-free medium.
Serum~free media of this invention are employable not only for the cell growth of mammalian cells but also for the production of useful physioloc~ically act.ive substances, for example inter~erons, lymphokines, and antibodies.

Serum-free media of the invention have been established on the basis of the inventors' findings that cyclodextrin does not show any cytotoxic effect on cell growth and that lipophilic substances included in cyclodextrin still show such effects as a cell growth promotiny effect and an accelerating effect on the productivity of valuable products.
This invention has also been established on the findiny that the solubility and stability in water of the culture medium inyredients are improved by the use of cyclodextrîn as a vehicle.
In accordance with this invention, those substances which are water-insoluble, unstable in culture medium or cytotoxic as such and which were accordingly considered as being unemployable as culture medium ingredients can now be em~loyed. In other words, this invention enlarges the availability of use of various ingredients and the ranges of research on the developments of new culture media.
It will be apparent from the foregoing disclosure, especially in connection with the effect which cyclodextrin has on cell growth and/or some culture medium ingredients, that a conventional serum culture medium may be made more effective ~hen it is added with cyclodextrin because some lipophilic substances originating from serum ~re contained in such a conventional medium. A culture medium containing serum albumin at its lower concentrations instead of serum may also be made more effective when added with cyclodextrin because commercial serum albumin products, for example crystalline albumin and Cohn's Fraction V are usually accompanied by lipophilic substances. An animal cell culture meclium comprisiny cyclodextrin as well as ordinary nutrients and additives incl~lding serum or serum albumin is accordinyly in accordance with this invention.

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It will also be apparent that the serum moiety or the serum albumin moiety in a conventional serum culture medium may be partially replaced with at least one lipophilic substance nutrient and cyclodextr:in, in which the lipophilic substance and cyclodextrin are preferably in the form of an inclusion complex between them. A conventional animal cell culture medium in which the ~erum moiety or the serum albumin moiety is partially replaced with at least on lipophilic substance nutrient and cyclodextrin also forms an aspect of the invention.
An animal cell culture nutrient substitute composi-tion ~or serum comprising at least one lipophilic substance nutrient and cyclodextrin may be put on the market as such and also represents an aspect of the invention.
Concerning the use of a lipid as an ingredient, for example linoleic acid, linoleic acid shows almost no gro~lth promoting effect at a concentration lower than 1 mg/l, when linoleic acid itself is directly added to a culture medium, while at a concentration of more than 1 mg/l this fatty acid displays a cytotoxic effect on the cell growth.
However, if it is added in the form of an inclusion comp ~ with ~-cyclodextrin into culture medium, the upper limit of the fatt~ acid concentration which shows cytotoxicity is increased to about 10 mg per liter of culture medium and at a lower concentration than this concentration, mammalian cells grow well and produce a satisfactory productivity of physiologically active substances, such as interferon.
Based on these new findings, the inventors have been successful in dispensing with, or reducing the content of serum which had been considered essential ~or cell growth but was very expensive and appeared to be dificult to obtain;

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in addition the inventors have established a new culture medium which contains no or less proteinaceous eubstances.
At the same time the inven-tors have provided the possibility of sterilizing culture medium b~ means of ~n autoclave~ Use oE a culture mediutn of this i.n~ention makes it easier to isolate and purify the product accumulated in the culture medium. Mass production of a culture medium is also made easier, according to this invention.
It is necessary to add serum albumin or an expensive cell-attachment factor such as fibronectin to the medium when fibroblast cells are cultivated in ordinary serum albumin-containing medium. ~o fibronectin, however, needs to be added if an inclusion complex prepared from ~-cyclodextri.n and an unsaturated fatty acid for example linoleic acid is added to the medium.
Further, there have not previously been known any satis~actory means of introducing lipophilic vitamins, for example vitamin E into culture medium, without an organic solvent though such vitamins have been considered as essential for cell growth, but now these nutrients may be added in sufficient amounts into a culture medium according to this invention.
The invention is illustrated by reference to the accompanying drawings in which:
Figures 1 to 4 show graphically the cell density and interferon production for a cultured medium employing di:Eferent inclusion cGmplexes and ~-cyclodextrin, in accordance wilh the invention, Figure 5 illustrates graphically the relationship belweell cell concentration and inclusion complex in the cuLture medium, and ~3Lg~3~7l.r3~

Figure 6 illustrates graphically the cytoto~icity of cyclodextrins for mammalian cells.
With further reference to the drawings, Figure 6 demonstrates that ~-cyclode~trin is more cytoto~ic than ~-cyclode~trin, particularly at higher concentrations.
Figure 1 demonstrates that mammalian cells grow as well in a culture medium of the invention as in a conventional serum-containing medium, and that a culture medium of the invention can produce physiologically active substances such as interferon.
Figures 2, 3, and 5 demonstratethat cell growth and production of physiologically active substance can he improved with the use of inclusion complex-containing culture media of the invention.
Figure 4 demonstratesthe decrease of the cytotoxic effect of linoleic acid obtained by the use of ~-cyclodextrin.
The invention is explained in greater detail by reference to the following examples.
Interferon activity produced was determined in terms of anti-viral activity using F~ cells and Vesicular Stomatits Virus in the following examples.
EX~MPLE 1 (Propagation of UMCL cells) Fresh heparinized umbilical cord blood, 20 ml, was obtained from a new born infant. The cord blood lymphocytes were quickly isolated by the Ficoll isopaque density gradient method. The cord blood lymphocyte fraction was mixed with 3 times its volume of Eagle's Minimum Essential Medium (Nissui Pharmaceutical Co.) and the mixture was centrifuged. The supernantant was discarded. After repeating this treatment 3 times, the washed lym~hocytes were suspended at a cell dcnsity of 3 x 10 /ml in Culture ~edium RPMI-1640 - 12 ~

7~3: L

(Nissui Pharmaceutical Co,) containing 10 /v % fetal bovine serum.
To ~his lymphocytes suspension, EBV (Epstein Barr Yirus) grown in the ~-95-8 cells was added in a concentration of 5 x 10 TD50/ml, and this mixture was incubated at 37C
for 2 hours, followed ~y harvesting of the lymphocytes with a centrifuge. After washing these lymphocytes with Eagle's MEM mediurn 3 times, the lymphocytes were resuspended at a cell density of 3~10 /ml in Culture Medium RPMI-1640 contain-ing 10 v/~ % fetal bovine serum and cultured at 36~37C for 1.5 months in a humidified atmosphere containing 5 % C02 in air. Durlng this cultivation, fresh RPMI-16~0 medium contain-ing 10 V/v % fetal bovine serum was added in an equal or a half volume of the culture medium was exchanged with this fresh medium every 5 days.
An additional two samples of umbilical cord blood from new-born infants were processed in the same way and 3 samples in total of lymphoblastoid cells, i.e., UMCL-l, UMCL-2 and UMCL-3, were prepared.
The spontaneous interferon productivity of these transformed cells was assayed and the results are shown in Table 1.
The assay was carried out as ~ollows: each lymphoblastoid cell line was cultured at an initial cell density of 5 x 105fml in Culture Medium RPMI-1640 containing 10 V/v % fetal bovine serum at 37C for 5 days in a humidified atmosphere containing 5 ~/O CO2 in air and the interfero activity in the supernatant was measured.

Cell LineInt~rferon Activity UMCL-l 2.5 (10 U/ml) UMCL-2 6~1 UMCL-3 4.5 In connection with this example, reference is made to T~Sato et al., Exp. Cell ~es., 138, 127 (1982).
EX~MPLE 2 A culture medium identified as RITC 56-1 was prepared by dissolving in a Dulbecco t S Modified Eagle Medium ha.ving the composition shown under A the additional nutrients and additives in the given amounts shown under B.
A (Dulbecco's Modified Eagle Medium):
NaCl 6400.0 (mg/l) KCl 400 0 CaC12 (non hydrate) 200.0 MgS0~ (non-hydrate) 97.7

2 4~ H20 125,0 ( 3~3 9~2 0.1 Glucose 1000.0 Na pyruvate 110.0 L-arginine HCl 84.0 L-Cystine 2HC1 62.6 glycine 30.0 L-glutamine 584.0 L-histidine HCl~H20 42.0 L-isoleucine 104.8 (mg/l) L-leucine 104.8 L-lysine HCl 146.2 L-Methionine 30.0 L-phenylalanine 66.0 L-serine 42.0 L-threonine 95.2 I.-tryptophan 16.0 L-tyrosinate 2Na 89.5 L-valine 93.6 chol.ine bitartrate 7.2 folic acid 4.0 nicotinic acid amide 4.0 Ca pantothenate 4.0 pyridoxal.HCl 4.0 riboflavin 0.4 thiamine-HCl 4.0 i-inositol 7.2 Phenol Red 5.0 B (Additional Nutrients):
insulin 10 (mg/l) human transferrin 5 hypoxanthine 4 ,.-thymidine 0.7 deoxycytidine 0.03 deoxyadenosine 1.0 (mg/l) 6.8-dihydroxypurine 0.3 glucose 1000 mannose 500 yalactose 500 lecithin 2.5 cholesterol L-alanine 20 L-asparagine.H20 56 I,-aspartic acid 20 L-cysteine-HCl~H20 40 ~3~37~

L-glutamic acid 20 L-proline 20 ascorbic acid 10 biotin 0.2 folinic acid 0.01 vitamin B12 0.1 FeSO~-7H20 0.8 Z S04 7H20 0.02 Na2SeO3 0.004 cacl2 100 glutathione 1.0 putrescine-2HCl 0.1 ~-glycerophosphate-2Na 1500 NaHC03 1300 An inclusion complex between ~-cyclodextrin (~-CD) and linoleic acid, oleic acid or vitamin E (VE) was prepared as follows;
1 g of ~-CD was dissolved in 7 ml of distilled water, and 10 mg of linoleic acid, oleic acid or vitamin E was dissolved in 7 ml of ethanol. The aqueous and ethanolic solutions were mixed and the mixture was heated to, and maintained at, 70ac under bubbling with nitrogen gas until the mixture became opalescent. The opalescent solution was irnrnediately left at room temperature, and then maintained at 4C for an additional 20 hours after it had been cooled to room temperature. The precipitate formed, i.e., inclusion complex, was collected by centrifuge, lightly washed once with 10 ml of ethanol and dried in vacuo. The resulting powder was washed two tlmes wlth petroleum-ether and dried in vacuo.

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Figures 1 to 4 show the cell density and interferon production in the cultured medium when an UMCL-3 cell line was cultured in the RITC 56-1 culture medium supplernented w:ith one or more inclusion complexes (Figures 1 to 3) and further with ~-cyclodextrin (Figure 4).
The procedures are as follows, RITC 56 1 culture medium was supplemented with one or more inclusion complexes and further ~-cyclodextrin, as the case may be, given in Figures 1 to 4 in the respective amounts given in the same figures. r~he resulting solution was sterilized by filtering with the use of membrane filter. In the resulting medium UMCL-3 cells were inoculated at an initial cell density of 5 x 105 cells/ml, and cultured for 5 days at 37~C under 5% C02-95% air. The cell density and interferon production in the cultured medium were measured~
In connection with Figure 4, the culture media were RITC 56-1 culture media supplemented with 300 mg/l of the inclusion complex of ~cyclodextrin with linoleic acid and further with ~-cyclodextrin in the amounts given in the figure.
~he same cell line was cultured in RPMI-1640 culture medium supplemented with 10 V/v % fetal bovine serum, and the results are shown on the right side in all the figures for the purpose of comparison.
In all the fi~ure$, the closed circles and open circles indicate the concentrations of viable cells and inteferon production, respectively.

~ culture medium identified as RITC 80-7 was prepared by using and MEM medium and some other nutrients and additives.
The composi-tion of the RITC 80-7 medium is, as follows, MEM medium 9400 (mg/l) (Nissui PharmaCeUtical Co~) L--aspartic acid 13.3 L-glutamine 292 g]ycine 7.5 L-glutamic acid 0.15 (mg/1) L-proline 3.5 L-serine 10.5 folinic acid 0.00005

3,3',5-triido-L-thyronine 0.0002 mouse-EGF 0.01 human transferrin 10 insulin vitamin B12 0.02 biotin 0.02 putrescine 2HC1 0.02 Na pyruvate 110 Choline chloride 16 thymidine 0.07 hypoxanthine 0.24 CuS04-5H2o 0.0000025 FeS04 7H20 0 . 8 ~nS04 7H20 0.0000024

4~6M724 H20 0.0012 NiC12 6H2o o.000012 NH4V03 0.000058 2 3 0.00039 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid 3300 NaOH 300 NaIIC03 1400 (mg/l) 7~3L

In connectiOn with the above RITC ~0-7 culture medium, reference is made to I.Yamane et al., Exp. Cell Res., 134, 470(1981).
The inclusion complexes given in Figure 5 of ~-cyclodextrin and unsaturated fatty acids or vitamirl E
prepared by the same method as in EXAMPLE 2 were added in the amounts given in the same figure to RITC 80-7 medium, and the resulting culture medium solution was sterilized by filtration on a membrane filter.
1.5 ml of the sterile medium was pipetted into each culture dish with 3.5 cm diameter (Lux Co.). Human embryonic lung diploid fibroblast cells cultured in the MEM medium supplemented with 10 V/v % fetal bovine serum were plated in the above dishes at an intial concentration of 2 x 104 cells/-dish, and cultured at 37C for 5 days in air containing 5 vol.
% C2 and 7 vol. % 2 The results of cell counting in each culture dish are shown in Figure 5. In this figure, FA means a 1:1 mixture of linoleic acid and oleic acid. As a control examination, the result of cel.l counting obtained when the same cells were cultured in MEM medium supplemented with 10 V/v % of fetal bovine serum are shown on the right side of the same figure.
EX~MPLE 4 RITC 55-9 culture medium, of which the composition was the same as that of the RITC 56-1 medium except that the former contains no mannose, galactose, lecithin or cholesterol, was added with ~- or ~-cyclodextrin the amounts given in Figure 6 and the resulting solution was sterilized by filtering on a membrane filter.
In order to examine the cytotoxicity of cyclo-dextrins, UMCI.-3 cells were inoculated at an initial cell density of 5 x 105 /ml to RITC 55-9 medium containing various amounts of ~- and ~-cyclodextrin as shown in Figure 6, and cultured at 37C for 4 days in air containing 5% CO2.
The resulting cell concentrations in the cultured media were measured and are shown against the cyclodextrin concentrations in the same figure. In this figure the closed circles and the closed squares indicate the data obtained in connection with ~- and ~-cyclodextrin, respectively.
It is-evident from the figure that ~-cyclodextrin is more toxic than ~-cyclodextrin and is more toxic at its higher concentrations.

Claims (24)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An animal cell culture medium comprising cyclodextrin and nutrients, said nutrients comprising at least one of serum and serum albumin.

2. An animal cell culture medium comprising nutrients in-cluding at least one lipophilic substance nutrient, and cyclodextrin, said medium being free of serum and serum albumin.

3. An animal cell culture medium according to claim 2 in which said at least one lipophilic substance nutrient and said cyclodextrin are in the form of an inclusion complex between them.

4. An animal cell culture medium according to claim 2 or 3 in which said cyclodextrin is ?-cyclodextrin and said at least one lipophilic substance nutrient is selected from the group consisting of linoleic acid, oleic acid and vitamin E.

5. An animal cell culture medium in which the serum moiety or the serum albumin moiety is partially replaced by at least one lipophilic substance nutrient and cyclodextrin.

6. An animal cell culture medium according to claim 5 in which said at least one lipophilic substance nutrient and said cyclo-dextrin are in the form of an inclusion complex between them.

7. An animal cell culture medium according to claim 5 or 6 in which said cyclodextrin is ?-cyclodextrin and said at least one lipophilic substance nutrient is selected from the group consisting of linoleic acid, oleic acid and vitamin E.

8. An animal cell culture nutrient substitute composition for serum comprising at least one lipophilic substance nutrient and cyclodextrin.

9. An animal cell culture nutrient substitute composition for serum according to claim 8 in which said at least one lipophilic substance nutrient and said cyclodextrin are in the form of an inclusion complex between them.

10. An animal cell culture nutrient substitute composition for serum according to claim 8 or 9 in which said cyclodextrin is ?-cyclodextrin and said at least one lipophilic substance nutrient is selected from the group consisting of linoleic acid, oleic acid and vitamin E.

11. A culture comprising a culture medium as defined in claim 1 and mammalian cells.

12. A culture comprising a culture medium as defined in claim 2 or 3 and mammalian cells.

13. A culture comprising a culture medium as defined in claim 5 or 6 and mammalian cells.

14. In a culture medium comprising nutrients for mammalian cell growth, the improvement wherein said medium contains cyclodextrin and at least one lipophilic substance nutrient.

15. The culture medium of claim 14, wherein said cyclodextrin is ? -cyclodextrin.

16. In a culture medium comprising nutrients for mammalian cell growth, the improvement wherein said medium is free of serum and serum albumin and contains cyclodextrin and at least one lipophilic substance as a nutrient.

17. A culture medium according to claim 16, wherein said cyclodextrin and said at least one lipophilic substance are present as an inclusion complex.

18. A culture medium according to claim 17, wherein said inclusion complex is a reaction product of said cyclodextrin and said at least one lipophilic substance in a cyclodextrin/-lipophilic substance molar ratio of 10 to 100:1.

19. A culture medium according to claim 16, wherein said cyclodextrin is ?-cyclodextrin and said at least one lipophilic substance is selected from the group consisting of saturated and unsaturated fatty acids, glycerides of saturated and unsaturated fatty acids and lipophilic vitamins.

20. A culture medium according to claim 18, wherein said cyclodextrin is ?-cyclodextrin and said at least one lipophilic substance is selected from the group consisting of saturated and unsaturated fatty acids, glycerides of saturated and unsaturated fatty acids and lipophilic vitamins.

21. A culture medium according to claim 19, containing up to 10 mg/liter of said lipophilic substance and about 300 to about 1000 mg/liter of ?-cyclodextrin, based on the volume of culture medium.

22. A method of reducing the content of serum moiety or serum albumin moiety of culture medium for mammalian cells which comprises replacing a part of the serum or serum albumin content with cyclodextrin and at least one nutrient lipophilic substance.

23. In a method of producing physiologically active substances selected from the group consisting of interferons, lymphokines and antibodies in a culture medium, the improve-ment wherein the medium is as defined in claim 1, 2 or 5.

24. In a method of culturing mammalian cells in a culture medium, the improvement wherein the culture medium is as defined in claim 1, 2 or 5.