JPS6251983A - Serum-free culture medium for cultivating human/human hybridoma - Google Patents

Abstract

PURPOSE:To obtain the titled culture medium, obtained by incorporating L- arginine within a specific high concentration range as one component of an amino acid component in a complete culture medium for cultivating an animal cell and applicable to practical use without requiring blood serum albumin component. CONSTITUTION:A serum-free culture medium obtained by incorporating L- arginine in an amount of >about 1,000mg/l complete culture medium - <about 4,500mg/l complete culture medium concentration in the complete culture medium containing (A) a basic culture medium for cultivating an animal cell, (B) insulin and/or transferring, (C) ethanolamine, (D) mercaptoethanol and (E) a selenite. Thereby, the growth and multiplication of human/human hybridoma are satisfactorily and smoothly carried out and the antibody productivity having practicality is also exhibited.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a serum-free medium useful for culturing human/human hybridomas, and is useful for, for example, elucidating the cell proliferation mechanism, elucidating the antibody production mechanism, and furthermore for producing antibodies and other useful substances in the bioindustrial field. In the biotechnology field such as manufacturing,
It is freed from the inconvenient effects of the various mixed foreign factors in serum and serum albumin components, which were previously essential, and can be used for growth, proliferation, antibody production, etc. of humans/human hybridomas. The present invention relates to a serum-free medium for human/human hyturidoma culture that is highly suitable for culture.

More specifically, the present invention provides (1) an animal cell culture basal medium, (2) at least one growth factor selected from the group consisting of insulin and transferrin, (3) ethanolamine, (4) mercaptoethanol, and (5) )
A complete animal cell culture medium containing selenite, as an amino acid component, exceeding about 100° ray/l - more than the complete medium, about 4500 ray/l! The present invention relates to a serum-free medium for human/human hybridoma culture, characterized in that it contains L-arginine at a high concentration of less than one complete medium.

A large number of animal cell culture basal media (mediums that do not contain serum components such as serum and serum albumin components) are known, and about 50 types of basal media are available on the market. When culturing animal cells, in order to maintain the growth and proliferation ability of the cells that can be used for practical purposes, serum albumin and its complexes are added to these basic media, such as serum such as calf serum and human serum, and serum albumin separated from serum. Serum components such as physical strength, for example, about 5 to about 10% (vol/vol)
It is common to mix some of the ingredients and use it as a complete medium.

On the other hand, serum contains interstitial factors such as interstitial lipids, high-density ribolipids (HDL), and low-density lipolipids (LDL), and contamination with these undesirable foreign factors cannot be avoided. Furthermore, purification of serum albumin and its complexes is complicated, and even if careful purification is performed, it is extremely difficult to completely avoid contamination with undesirable foreign factors derived from serum. This poses an obstacle in elucidating the physiological and chemical mechanisms described above, and also in producing bioproducts useful for medical purposes such as diagnosis, prevention, and treatment.
This causes technical troubles such as the inability to easily produce high-quality useful substances free from the contamination of undesirable foreign factors.

In order to avoid such troubles, various attempts have been made to develop a "serum-free medium" that does not require the addition of serum. However, it is a serum-free medium that can be practically used for culturing human/human hyperridomas, and does not require the addition of foreign factor-containing components derived from serum components such as serum albumin or its complexes. A serum-free medium for culturing No. 2019-2012 is completely unknown so far.

For example, the Journal of Immunological Methods
ical Meth-ods], 39t369-375
(1980) described the use of a serum-free/serum-free albumin medium containing insulin and transferrin in the basal medium RPMI-1640 or MEM when culturing mouse/mouse hybridoma cells, and also described the use of a serum-free/serum-free albumin medium containing insulin and transferrin in the basal medium RPMI-1640 or MEM. National Academy of Sciences U, S, A (proc, NatL,
Acad, Sci, U, S, A), Yuj.

1158-1162 (1982) used a serum-free/serum-free albumin medium containing insulin, transferrin, sodium selenite, and ethanolamine in the same basal medium as above in mouse/mouse hyperidoma cell culture. It is stated that.

However, according to studies by the present inventors, although such serum-free/serum-free albumin medium can be used for mouse/mouse hybridoma cell culture, it cannot be used for human/human hybridoma cell culture. As a result, it was found that it was not possible to maintain growth and proliferation ability that could be used for practical purposes.

Furthermore, JP-A-58-63385 proposes a medium for growing cultured cells in a serum-free or low-serum state. Although this proposal does not provide any specific examples, it states that it can also be used for serum-free culture of human hybridoma cell lines.

However, in the proposed medium for growing cultured cells under serum-free or low-serum conditions, groups such as arachidonic acid, oleic acid, linoleic acid, normitic acid, stearic acid, lylunic acid, and lumitoleic acid are used. at least one fatty acid selected from 1:0.5 to 1
: about 100 to 1000, combined at a molar ratio in the range of 5.
μ? The inclusion of bovine serum albumin (BSA) is essential. Although serum albumin separated from serum has a reduced amount of interspecific factors compared to serum, it cannot be avoided that it still contains non-negligible interspecific factors. Therefore, it still cannot be freed from the unfavorable influence of these foreign factors.

The present inventors have devised a method that not only is serum-free but also does not require such a serum albumin component, and the growth and proliferation of human/human hybridomas that can be used for practical purposes can be performed smoothly and smoothly. We have been conducting research to provide a genuine serum-free medium that can also exhibit practical antibody production ability.

As a result, by containing amino acid components as at least one type of amino acid component at a considerably high concentration amount exceeding the amino acid concentration that has conventionally been commonly used, and in particular, L-arginine at a specific high concentration range. Particularly, it is a serum-free/serum-free albumin medium and is capable of retaining human/human hybridoma growth, proliferation, and even antibody production ability, thus eliminating the need for a medium derived from the use of serum and/or serum albumin. It has now been discovered that it is possible to provide an authentic serum-free complete medium for human/human hybridoma culture that is free from the undesirable effects of factors.

According to the research of the present inventors, (1) an animal cell culture basal medium, (2) at least -N, (31 ethanolamine), and (4) mercagtoethanol, a growth factor selected from the group consisting of insulin and transferrin. and (5
) A complete animal cell culture medium containing selenite, as an amino acid component, a high concentration of L-arginine exceeding about 1000 Ql/l-complete medium and below about 4500 Ql/l-complete medium. Although the medium containing L-arginine is free of serum and serum albumin components, by containing the above-mentioned high concentration of L-arginine,
Human/comparable to serum and serum albumin-containing media
It is completely predicted that it will enable the growth and proliferation of human hybridomas to proceed smoothly and smoothly, and will be able to exhibit a practical antibody production ability comparable to or clearly higher than that of the serum and serum albumin component-containing medium. I discovered an outside fact.

L-15 is a basal medium that is known to have a high amount of amino acids and that essential amino acids are added to the maximum concentration that can be added.
Medium (L-15MedilLfrL) (La1bov
itz, A,, Amar, /, Hyg, 73
.

173 (1963)), L-arginine (
Considering that the concentration of free base) is 500 μ/l-medium, the high-concentration L-arginine-containing medium specified in the present invention does not contain serum or serum albumin components. / Functions as a complete medium for the growth and proliferation of human hybridomas, and exhibits performance comparable to or superior to a complete medium containing serum and serum albumin components, without the troubles caused by the interstitial factors mentioned above. This was a completely unexpected result.

According to the research conducted by the present inventors, as will be explained later with reference to FIG.
Although the arginine-containing medium of the present invention does not contain serum or serum albumin components, it is comparable to or clearly superior to a medium containing normal amounts of L-arginine that contains serum or serum albumin components. - It was found that the growth, proliferation ability, and antibody production ability of hybridomas were shown, and that these abilities were reduced if the L-arginine concentration was too low or too high. Furthermore, as shown later in FIG. 2, it was found that using the above-mentioned medium of the present invention, it was possible to subculture for a long period of time without any substantial decrease in antibody production ability. Thus, according to our research, it is possible to provide a genuine serum-free complete medium for human/human hybridoma culture that can be completely free from the undesirable effects of foreign factors, such as in the use of serum and serum albumin. I understand.

Therefore, an object of the present invention is to provide a serum-free medium for human/human hybridoma culture.

The above objects and many other objects and advantages of the present invention will become more apparent from the following description.

The serum-free medium for human/human hybridoma culture of the present invention comprises (1) an animal cell culture basal medium, (2) at least one growth factor selected from the group consisting of insulin and transferrin, (3) ethanolamine, ( 4)
A complete animal cell culture medium containing mercagtoethanol and (5) selenite, the amino acid component of which exceeds about 1000111 v/j' - the complete medium and about 4,500 v/l - the high level of the complete medium. * Contains a certain amount of L-arginine, and there is no need to incorporate serum and serum albumin components.

(1) Various animal cell culture basal media are known, and are described in known literature (for example, "Cell Culture Manual", 3rd edition, 1
Published by Kodansha Scientific on July 20, 1984)
Many of these are commercially available and can be used in the present invention.

Examples of such basal media include the following known basal media and known modified media.

BME medium (Ba5al bhdium Eagle
) (Eagle,
H,,5cien-c,e)+122:501 (1
955); E1” le.

Eagle, H, J, Ezp, Mad,
).

102.37 (1955) and 102,595 (195
5); Eagle H, Zoyanal Op Bioloncal Chemistry (Eagltt.

H,, J, Biol, Ch#m,) +
2 14 t 839 (1955); Eagle
H, Etoard.

Size x:ysu (Eagle, H,, 5science
), 123.

845 (1956); Hanks Soei H.

Wallace R.E., Proceedings of the Society for Experimental Biolozo and Medicine (Hanks, 1.H,
,Wallace, R.E., ,proc.

Soc, Ezp, Eiol, Med), 71
, 196 (1949);
Mane.

/,, proc, Sac, Ezp, Biol,
Mad ) + 127 + 335 (1968);
Morton H. Soei.

In vitro (Morton, H, /, In V
zetro).

6.89 (1970); Eagle H.
le, H,, proc, Soc, Er;p.

Biol, Med), 89. 362 (1955); et al.], HEM medium (Minimmsrn Es5ent i
Al Medium) [For example, Eagle H.Science (Eagle.

H., Schittncg), 130 + 43
2 (1959); Stoker M, Mag 7 Arson I.

Byloroso (5toker, kf, Macph
ertton, 1. .

Viroloytt), 14, 359 (1961); Macphtrson, 1., 5toker,
M.

+ 16, 147 (196
2); Stoker M, MacFarnon Eye, Nature (5toker, lK, Macp
herson, 1. , Natur-re, 203.
1355 (1964); Dulbetsko R.
Freeman No, Pyro Rosso (Dwlbecco,
R., Freeman, G., Virology
).

8.396 (1959) i Smith zei diraet Earl, pie oo no (Sy+zzth+ 7゜D
,, at atlVirology) + 12 +
185 (1960), Stanners C.P., Etoile, Nature New Bioroso (5ta
ners, C,p,, et aL,, Natu
re New Biotogy)s 230152 (1
971>: Stanners C.B., Stuart C., Personal Communication (Stα
ners.

C,p,, Stewart, C,,peτ5ona
l Corwnunic α-tio 9, (1972) i
et al.], 199 medium [Morgan Soei F, Et.
Earl, Journal Op Cancer Institute (Morgan.

J,F,,at al,,J,Hat,Cancer
In5t).

16.557 (1955) iMorton H-7x
I, in vitro (Morton, H, J,, In
Vitro), 6, 89 (1970); et al.], L-15 Medium (L-15 Medium);

A, America 7 (Lgibwitz, A,, A
mer, J.

H1/Q>・, 78.173 (1963) i, etc.], Ham's MediSm [: Ham・
R-no.

Experimental Cell Research (Ham, R.

C,′,,E:tp,CettlRes),29,5
15 (1963); H.R. No. Grosseing Op National Academy of Sciences
Op the United States Op America (H
am, 7? , G,, proc, Hat, Ac
ad.

5ci), 53, 288 (1965); etc.], Matsukoi 5. Neumann, R.E., Matsukoi, Teini, N.I., Proceedings Op Experimental Bioloso and Medicine (Neuman
, R.E., JfcCoy, T.A.

proc, Ezp, Biol, Med) +
98 + 303 (195B) i Matsukoi T.A., Etoile, Proceaging Op Experimental Piorros and Medicine (Mc
Coy, T. A., et at, proc.
Ezp, Biol.

M'd), 100+ 115 (1959) H No T C, Kellogg T Nis, (Hsx.

T, C,, Kellogg, D, S, /,
Nat, CafLCerInst,,25,221
(1960); et al.], RP MI medium (Rp MI
Mgdiutn ) (Mu-7-no-i,et
R, Soei N.M.

x −(Moore, G, E,, etc c!,,
7. A, M, A,) 199.519 (1967
) i move no e.

EtR, National Cancer Institute (Moors, G, E
, .

at al,, J, Hat, Cancer In
5t ) + 36 + 405 (1966) i etc.]
, Williams' E medium
Medium E) (Williams No.M,
Weisberger E.K., Weisberger G.H. Experimental Cell Research (Wi
l l iams, G, M, , Vle i
sbu-rgttr, E, K,, Wgis
bryger, J. H., Ezp.

Ce11. Re5), 69, 106-11
2 (1971); etc.].

These +1) animal cell culture basic media can also be used by blending a plurality of types in an appropriate ratio.

Furthermore, in the present invention, in addition to (1) the animal cell culture basal medium as described above, (2) at least one growth factor selected from the group consisting of insulin and transferrin, +31 ethanolamine, and (4) mercapto. Contains ethanol and (5) selenite salts such as sodium and potassium selenite salts.

The blending amounts of these components (2) to (5) can be selected and changed as appropriate. For example, preferred blending amounts can be easily selected and set experimentally depending on the purpose of culturing and the human/human hybridoma to be cultured. About 2 to about 50 mol/l - Component (2) of the complete medium, about 10-8 mol - about 10゛4 mol/l
E - component (3) of complete medium, about 10-8 mol - about 10-
Examples of the blending amount include component (4) for 11 minutes of 4 mol/l medium and component (5) for about 10 minutes, Y/l, and complete medium.

The serum-free medium for culturing human/human hybridomas of the present invention has an amino acid component of more than about 1000 μ/l-complete medium and less than about 4500 μ/l-complete medium, preferably about 1100 to about 4000 ml1/l- Contains a highly concentrated amount of L-arginine in the medium. L-arginine is available in the form of free base, acid salts such as hydrochloride and mixtures thereof. In the present invention, the concentration of L-fulginine expressed in my/l complete medium means the concentration expressed in the form of a free base.

Further, the concentration is a concentration based on the sum of (1) L-arginine and/or its acid salt contained in the animal cell culture basal medium and the additional amount further blended. As mentioned above, (i) as a basal medium, L-15 medium, which is special in that it has a large amount of amino acids, has a concentration of at most 500 w/l.
of L-arginine, generally up to about 200 μ/l.

As mentioned above, the serum-free medium for culturing human/human hybridomas of the present invention has a high concentration of L as an amino acid component of more than about 1000 ml in one complete medium and less than about 4500 μ/l in complete medium. - By containing arginine, there is no need to combine serum and serum albumin components.
It shows high growth and proliferation ability of human/human hybridoma, and also shows high antibody production ability. In addition to the components (1) to (5) and the above-mentioned high concentration of L-arginine, the serum-free medium for culturing human/human hybridomas of the present invention optionally contains:
It can contain any other components for human/human hybridoma culture media known per se.

Examples of such components include other culture medium amino acids, vitamins or minerals, nucleic acid derivatives, saccharides, coenzymes, fatty acids, etc., which are known per se.

Furthermore, there are no particular restrictions on human/human hybridomas that can be cultured using the serum-free medium for culturing human/human hybridomas of the present invention.
No. 01994, JP-A-59-135898, JP-A-5
It can be used to culture human/human hybridomas that can be obtained by the method disclosed in No. 9-137497. Examples of such human/human hybridomas include human/human hybridoma CLN/5UZ
H5 [Notification number of refusal of microtechnical deposit acceptance, 57 microbacteria 63
No. 7; human/human hybridoma CLNE5, ATC
CHB8206], human/human hybridoma CLN
/5UZI111 C Microtechnology Institute Deposit Rejection Notification Number, 5
7 Weiyobun No. 638; Human/Human Heituridoma CLN
I111, ATCCHBB507]. Furthermore, although the culture medium of the present invention can be advantageously used to culture human/human hybridomas, it can also be used to culture hybridomas other than human/human hybridomas. Although not necessary, serum or serum albumin may be appropriately added and used as desired.

The serum-free medium for human/human hybridoma culture of the present invention comprises (1) an animal cell culture basal medium, (2) at least one growth factor selected from the group consisting of insulin and transferrin, (3) ethanolamine, ( 4)
mercaptoethanol, (5) selenite and about 10
0CIW1y/l - more than complete medium approximately 4500η/l -
It may be a dosage form containing L-arginine in an amount less than that of a complete medium, or it may be a dosage form consisting of an appropriate combination thereof, which is mixed and used at the time of use.

Hereinafter, according to Examples, preparation of a serum-free medium for human/human hybridoma culture of the present invention and human/human hybridoma culture using the medium.
An example of human hybridoma culture will be illustrated in more detail. Of course, the present invention is not limited to these examples.

Known RPMI-164 as animal cell culture basal medium
0, Dulbetsko MEN (D MEN) and Ham F-
12 (Ham's F-12) was used.

The amount of L-arginine contained in these known basal media is as follows.

The above known animal cell culture basal medium RPMI-1640,
D MEN and Ham's F-12, 2:1:
Mix at a ratio of 1 part, add sterilized distilled water to make 11, add sodium bicarbonate 2219 cape, and stir well (mixed basal medium). After treatment, insulin 10w
j/J, transferrin 10/'IN ethanolamine 10-M, sodium selenite IM'M, mercaptoethanol 10-''M and appropriate amounts of L-arginine (amounts that do not satisfy the requirements of the present invention and amounts that do). In addition, a serum-free medium was prepared by adjusting Hl-7.2 with 1N salt powder.Furthermore, a control medium without L-arginine and a control medium with calf serum added to the mixed basal medium were prepared. The prepared control medium, comparative medium, and medium of the present invention are as follows.

(Control) Medium style 1 The above mixed basal medium (L-arginine derived from the basal medium is 1
61■/J) + 5% calf serum.

(Control) Medium style 2 The above mixed basal medium (L-arginine derived from the basal medium is 1
61■/l).

(Control) Medium No. 3 The above mixed basal medium (L-arginine derived from the basal medium is 1
61η/l) Insulin, transferrin,
Ethanolamine, sodium selenite and mercaptoethanol.

(Comparative) Medium Nl14 The above mixed basal medium + the above insulin, transferrin, ethanolamine/, sodium selenite and mercaptoethanol + L-arginine (L from the basal medium)
-661 towns/A' including arginine).

(This invention) Medium No. 5 The above mixed basal medium + the above insulin, transferrin, ethanolamine, sodium selenite and mercaptoethanol + L-arginine (L derived from the basal medium)
-1161"!9/l) including arginine.

(This invention) Medium volume 6 The above mixed basal medium + the above insulin, transferrin, ethanolamine, sodium selenite and mercazotoethanol + L-arginine (L derived from the basal medium)
-1661m including arginine? /l).

(This invention) Medium N11L7 The above mixed basal medium + the above insulin, transferrin, ethanolamine, sodium selenite and mercaptoethanol + L-arginine (L derived from the basal medium)
-3161q/l including arginine).

(Comparative) Medium N [L8 The above mixed basal medium + the above insulin, transferrin, ethanolamine, sodium selenite and mercaptoethanol + L-arginine (L from the basal medium)
-5161~/j) including arginine.

(Comparative) Medium N119 The above mixed basal medium + the above insulin, transferrin, ethanolamine, sodium selenite and mercaptoethanol + L-arginine (L from the basal medium)
-16261 mg/l including arginine).

Human/human hybridoma CLNI11'1 (ATC
CHB8307; Microtechnical Deposit Rejection Notification No. 57 Microbiology No. 638) was grown in a medium prepared by adding 10% calf serum to the above-mentioned mixed basal medium, and the cells were collected by low-speed centrifugation. do. The above medium Na was added to the collected cells.
After adding the complete serum-free medium from step 7 and loosening the cell clumps, the cells are collected by centrifugation at low speed. CL collected by repeating this washing operation with serum-free medium twice.
N H11 cells were added to the above-mentioned medium Arashi 1 to Arashi 9 so that the number of cells was 5 x 10' cells in the medium Irnt, and 37
Culture was carried out for 6 days in an environment of 597 °C; C'Ot.

Cell number after 6 days of culture (measure the number of living cells with a hemocytometer)
The concentration of the antibody (CLN-IQG) produced in the culture medium (measured by enzyme antibody method) was actually measured, and the results are shown in the attached FIG. 1 as a one-line graph.

In FIG. 1, the white bar line indicates the cell number (cell number/-), and the black frame line indicates the antibody concentration (CLN-1t) Gμ2/-). Medium positive, 1 (serum-containing medium) and medium NIIL5~
As is clear from comparing the results with No. 7 (serum-free medium of the present invention), although the cell number is not as good as that of the serum-containing medium, the results are sufficiently good, while the antibody production amount is low. It can be seen that in this case, a surprising result was obtained in which the concentration was clearly increased compared to the case of a serum-containing medium. This also meant that the amount of antibody produced per unit cell was significantly improved, which was completely unexpected. This outstanding action and effect is compared to other comparative medium Nct4. Nn8゜teeth 9
It can be seen that the effect is remarkable and specific in medium sizes 5 to 7 (serum-free medium of the present invention).

Example 2 The amount of L-arginine, including L-arginine derived from the basal medium, is 2 oaOw/l! A serum-free medium of the present invention was prepared in the same manner as in Example 1, except that

CLN H11 cells washed with the above-mentioned serum-free medium of the present invention in the same manner as in Example 1 were transplanted into this medium 1-1 to give 1 x 10 s cells in the above-mentioned serum-free medium 1-1.
The cells were cultured for 6 days at 5% CO7. Next, the cells separated from the first generation culture are transplanted into the same fresh serum-free medium at 1×10' cells per rnl of serum-free medium, and second generation culture is carried out under the same conditions.

The results of repeated subculturing for 8 generations in this manner are shown in the attached FIG. 2.

In FIG. 2, according to the serum-free medium of the present invention, subculturing can be carried out for a long period of time without a decrease in cell proliferation ability or a drop in antibody production ability, and as shown in Example 1. It can be seen that unexpected and surprising effects can also be achieved by subculture using the serum-free medium of the present invention.

[Brief explanation of drawings]

The attached FIG. 1 is a graph showing the results of Example 1, and the attached FIG. 2 is a graph showing the results of Example 2.

Claims (1)

[Claims] 1. (1) Animal cell culture basal medium, (2) at least one growth factor selected from the group consisting of insulin and transferrin, (3) ethanolamine, (4)
A complete animal cell culture medium containing mercaptoethanol and (5) selenite, the amino acid component of which exceeds about 1000 mg/l of the complete medium and about 4500 mg/l.
A serum-free medium for human/human hybridoma culture, characterized in that it contains L-arginine at a high concentration of mg/l or less of a complete medium. 2. The L-arginine concentration is 1100 mg to 4000 m
g/l - serum-free medium according to claim 1, characterized in that it is a complete medium.