JPH0561908B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a composition for proliferating lymphoid cells and a method for producing a method for proliferating lymphoid cells. BACKGROUND OF THE INVENTION Research on efficient production of useful physiologically active substances, such as lymphokines such as interferon and monoclonal antibodies, using lymphoid cells is being actively pursued from various aspects. On the other hand, polyvinylpyrrolidone, which is used as a plasma bulking agent, has been shown to be effective against hepatoma cells (The Japanese Journal of Experimental Medicine).
Experimental Medicine) Volume 29, Page 45, 1959
), Fibroblasts (The Japanese Journal of Experimental Medicine 29)
It has long been reported by Katsuta et al. that it promotes the proliferation of healer cells (Vol. 191, 1959) and healer cells (The Japanese Journal of Experimental Medicine, Vol. 30, p. 147, 1960). It was completely unknown that it also affected the proliferation of lymphoid cells. Furthermore, it was completely unknown that physiologically active substances could be efficiently produced by culturing in a medium supplemented with this compound. Conventionally, in the culture of lymphoid cells, a medium supplemented with approximately 10% serum has been mainly used, as in the case of other cells, and a medium containing fetal calf serum (FCS) has been particularly used. Problems to be Solved by the Invention However, since serum is very expensive and there are unexplained differences between lots, there are many problems in culturing cells in large quantities. Furthermore, since serum contains many types of foreign proteins, there are also inconveniences when recovering and purifying the produced useful substances from the culture solution. In an attempt to overcome these disadvantages, various serum-free media (serum-free media) have been developed, but they generally have low versatility and are inferior to serum-containing media in terms of proliferation and physiologically active substance productivity. It is not necessarily sufficient. In addition, in serum-free media, various cell growth factors and hormones are added as substitutes for serum, but many of these factors are expensive, and are often even more expensive than serum-containing media. . In any case, conventionally known media have not always been fully satisfactory for efficiently obtaining large amounts of useful substances through cell culture. Thus, it is desired to develop a medium for lymphoid cell growth that is inexpensive, can be supplied in large quantities, and contains as little protein of unknown nature derived from serum or the like as possible. The ideal medium is one that is serum-free, versatile, and has a high cell proliferation ability, but even in serum-containing media, if the amount of serum used can be greatly reduced, the economic efficiency of the medium will improve. It is possible to greatly improve problems related to impurity content in the culture solution. Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors proceeded with the search for a substance that promotes the proliferation of lymphoid cells, and found that a composition for proliferation of lymphoid cells containing polyvinylpyrrolidone was used. Based on the discovery that when lymphoid cells or lymphoid cells that produce physiologically active substances are cultured in a culture medium, the lymphoid cells proliferate significantly and that this increases the amount of physiologically active substances produced. As a result of further research, the present invention was completed. The present invention provides a method for growing lymphoid cells in a medium containing (1) a composition for proliferation of lymphoid cells containing polyvinyl pyrrolidone, and (2) a composition for proliferation of lymphoid cells containing polyvinyl pyrrolidone. This is a method for propagating lymphoid cells characterized by culturing them. In this specification, polyvinyl pyrrolidone may be abbreviated as PVP. The composition of the invention consists of a basal medium and polyvinylpyrrolidone. The basal medium may be any medium that can be used for culturing lymphoid cells. The basal medium used in the present invention includes, for example, various commercially available basal media [e.g., Eagle's minimum essential medium (MEM) (Science, Vol. 130, p. 432, 1959), Eagle's basal medium (BME) ( Proceedings of
The Society for Experimental Biology and Medicine
Experimental Biology and Medicine) Volume 89
362 p. 1965), Dulbecco's Modified Eagle Medium (DME) (Virology, Vol. 8, 396)
p. 1959), Iscove's Modified Dulbecco Medium (IMDM) (The Journal of Experimental Medicine)
Experimental Medicine) Volume 147, Page 923, 1978
), L-15 medium (American Journal of Hygiene), L-15 medium (American Journal of Hygiene)
Hygiene) Vol. 78, p. 173, 1963), Matsukoi 5a
Culture medium (Proceedings of the Society for Experimental Biology and Medicine, Vol. 100, p. 115, 1959),
Ham F12 medium (Proceedings of the National Academy of Sciences U.S.)
Proceedings of National
Academy of Science.USA) Volume 53, Page 288, 1965
), RPMI1640 medium (Journal of the
Journal of the American Medical Association
199, p. 519, 1967) or a mixture of these. The basal medium contains factors essential for the growth of each cell (auxiliary growth factors), such as hormones (such as insulin, transferrin, steroid hormones, etc.), protein growth factors [such as epidermal growth factor (EGF), platelet-derived growth factors], etc. (PDGF), fibroblast growth factor (FGF), etc.], heavy metals (e.g., sodium selenite, etc.), and phospholipids (e.g., ethanolamine, phosphatidylethanolamine, etc.) are added as necessary. Can be mentioned. In addition, these may be supplemented with serum substitutes [e.g.
GFS (2nd Next Generation Industrial Infrastructure Technology Symposium)
Biotechnology Preliminary Lecture Collection, p. 161, 1984),
NU-Sealum (manufactured by Collaborative Research), Serum Plus (manufactured by KC Biologicals, etc.)], or serum supplemented with less than the usual amount of serum [e.g. fetal calf serum (FCS), newborn calf serum (FCS), etc.]. Examples include medium supplemented with bovine serum, calf serum, pig serum, goat serum, chicken serum, etc.
In addition, commercially available serum-free media [e.g.
1 (manufactured by Nippon Yakuhin Co., Ltd.), HB-102 (manufactured by Hana Media Co., Ltd.), HL-1 (manufactured by Bentored Co., Ltd.)] can also be used as the basal medium of the present invention,
It is also possible to create and use a medium with an optimized concentration of amino acids, etc., based on a commercially available basal medium. The polyvinyl pyrrolidone used in the present invention is

It is a high polymer having the structure of the formula (n represents the degree of polymerization), and commercially available polymers having an average molecular weight of about 10,000 to 1,200,000 are advantageously used. More specifically, PVP K- with an average molecular weight of 25,000
25, 40000 PVP K-30 or 1200000 PVP
K-90 etc. are used. The amount of polyvinyl pyrrolidone used in the present invention is approximately 0.001% (W/V) at the time of use.
from 10% to 10%, more preferably from about 0.01% to 5%
(W/V), more preferably about 0.1% to 5%
(W/V). Polyvinyl pyrrolidone may be mixed into the composition in advance, or may be mixed in when used as a medium. The composition for lymphoid cell proliferation of the present invention may be in either a solid state or an aqueous solution. Those in a solid state are used, for example, by dissolving or suspending them in water. In addition, the composition can be used as a medium for lymphoid cell proliferation, but when used as a medium, it may be used as a medium that does not contain serum, or as a medium that contains serum in an amount less than the amount normally used. good. Here, the usual usage amount is, for example, about 10% (V/V). When using the composition of the present invention as a culture medium,
The medium may contain a serum substitute at or below the amount normally used. Here, for example, in the case of GFS, the usual usage amount is about 3 to 4 g/
(as protein), which is about 10% V/V for NU-Seerum and about 10% V/V for Serum Plus. The lymphoid cells to be cultured by the method of the present invention include lymphocytes or leukemia-derived cells of mammals such as humans, mice, rats, cows, and hamsters, primary cultured cells derived from lymphoma, leukemia, and bone marrow tumors. Examples include cell lines, hybridomas in which one parent cell is a lymphocyte, and lymphocyte cell lines mutated by a virus or the like. More specifically, human lymphoid cells include:
Namalva (ATCC CRL1432) (International Journal of Cancer) Volume 12 396
Page 1973), Raji (ATCC CCL86) (Lancet vol. 1 p. 238 1964), EB-3
(ATCC CCL85) (Lancet Vol. 1, p. 252)
1964), WI-L2 (Cancer) vol. 22
517 pages 1968), Daudi (ATCC CCL213) (Cancer Research, vol. 28)
1300 pages 1968), RPMI 8226 (ATCC CCL155)
(Proceedings of the Society for Experimental Biology and Medicine, Volume 125, Page 1246, 1967
), CCRF-CEM (ATCC CCL119) (Cancer Vol. 18, p. 522, 1965), RPMI 1788 (ATCC
CCL156) (Journal of the National Cancer Institute (United States)
Cancer Institute (United States) Volume 43
1119 pages 1969), CRCF-SB (ATCC CCL120)
(Cancer Research Vol. 27, p. 2479, 1967
), Jurkat (Immunogenetics, Vol. 10, p. 247, 1980), but as mouse lymphoid cells, for example,
MPC-11 (ATCC CCL 167) (The Journal
Of Experimental Medicine Volume 131
515 pages 1970), NS-1 (ATCC TIB18) (European Journal of Immunology) Volume 6
511 pages 1976), P3X63Ag8U・1 (P3U1)
(ATCC CRL1597) (Current Topics of Microbiology and Immunology)
Immunology) Vol. 81, p. 1, 1978), and examples of hybridomas include mouse hybridoma CEA (2nd Next Generation Industrial Infrastructure Technology Symposium - Biotechnology Proceedings, p. 175, 1981), HS- (same as above) E235163 (hybridoma) (Hybridoma) Vol. 4, p. 47, 1985), mouse-human-human heterohybridoma N12-16.
63 (2nd Next Generation Industrial Infrastructure Technology Symposium - Biotechnology Proceedings, p. 175, 1982),
112-22・25 (Biochemical and Biophysical Research Communication)
Communication) Vol. 129, p. 26, 1985), HB
Examples include -43・1 (mutual). Lymphoid cells that produce physiologically active substances used in the method for producing physiologically active substances of the present invention include, for example, CEA, HS-, and E235163 that produce mouse monoclonal antibodies, and N12-16/63 that produces human monoclonal antibodies. , I12−22・
25, HB-43.1, etc., leukocyte interferon-producing Namalva cells, and interleukin-2-producing Jurkat cells. For culturing in the method of the present invention, containers or devices commonly used for culturing are used. For example, multi-well plates, culture flasks, spinner flasks,
Jar fermenters, fermenters, hollow fiber culture devices, culture devices using ceramic matrices, microcapsule culture methods, etc. are appropriately employed. For the culture of the present invention, conditions suitable for culturing the lymphoid cells used are employed. Generally, the culture is carried out at a culture temperature of about 37° C. and a pH of about 6.5 to 7.5 for several days to three months. For example, the culture medium of the present invention
Seed 0.1 to 5 × 10 ⁵ cells/m1, and in the case of multi-well plates or flasks, at about 37℃ and 5%
It is cultured in a carbon dioxide incubator (incubator with a carbon dioxide concentration of 5%) at a pH of about 6.5 to 7.5 for about 1 to 20 days. Aerated agitation culture is carried out in jar fermenters and fermenters. In addition, in cultivation using these culture vessels, hollow fiber ceramic matrices, microcapsules, etc., physiologically active substances can be improved by replacing the medium batchwise or continuously. In the case of continuous perfusion culture, it may continue for one to several months (about 3 months). It is also ventilated if necessary. To collect cells from a culture solution, collect the culture solution by directly passing it through a centrifuge or filter. In addition, when the physiologically active substance produced by culturing lymphoid cells accumulates in the culture medium, a supernatant is obtained by filtration or centrifugation, and the supernatant is collected from the supernatant. In the case of substances that accumulate within cells, cells obtained by filtration or centrifugation can be treated using physical methods (e.g., ultrasound, French press, Dynomill, etc.) or chemical methods (e.g., guanidine hydrochloride, etc.). After processing and extracting the product, a supernatant liquid is obtained. The physiologically active substance can be separated and purified from the supernatant by appropriately combining known separation and purification methods. For example, when the physiologically active substance is a protein or peptide, methods that utilize solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis are mainly used. Methods that utilize differences in molecular weight, methods that utilize differences in charge such as ion exchange chromatography, methods that utilize specific affinity such as affinity chromatography, and hydrophobicity such as reversed phase high performance liquid chromatography. Methods that utilize the difference in isoelectric points, such as methods that utilize the difference in isoelectric points, such as isoelectric focusing, are commonly used. Lymphoid cells grown according to the method of the present invention can contain various lymphokines (e.g., leukemia interferon, immune interferon, interleukin-2, etc.) and various monoclonal antibodies (e.g., mouse monoclonal antibodies, human monoclonal antibodies, etc.). ) etc. In addition, there is an adaptive method in which normal lymphocytes are stimulated with interleukin-2 and proliferated according to the method of the present invention, and the collected cells are directly returned to human blood.
Adoptive Immunotherapy
It can also be used for Since the method of the present invention allows lymphoid cells to proliferate efficiently, it can be advantageously used as a method for industrially proliferating lymphoid cells in large quantities. In the method of the present invention, when lymphoid cells that produce physiologically active substances are cultured, the cells are efficiently proliferated, so that the physiologically active substances can be efficiently produced, which is advantageous in industrial production. . EXAMPLES The present invention will be explained in more detail with reference to Examples below. The addition percentage of PVP represents W/V%. The percentage of serum added represents V/V%. Example 1 IMDM, Ham's F12 and L-15 medium 1:1:
Add 2 mg/insulin, 2 mg/transferrin, 2 x 10 ^-6 M ethanolamine, and 2.5 x 10 ^-8 M sodium selenite (together referred to as ITES) to the medium mixed at a ratio of 2. This was combined with various concentrations of PVP K-90 (average molecular weight
1,200,000) was prepared, and 1 ml of this was dispensed into a 24-well multi-well plate.
Add 1x WI-L2 cells derived from human lymphoblasts to this.
The cells were seeded at a rate of 10 ⁵ cells/ml, and after culturing in a 5% carbon dioxide incubator at 37° C. for 4 days, the number of cells was measured using a Coulter counter, and the results shown in FIG. 1 were obtained. As is clear from this figure, the number of cells in the medium without PVP was 4.2 × 10 ⁵ cells/ml, whereas
When PVP was added, the number of cells increased as the concentration increased, and when PVP was added at 0.2 to 0.8%, the number of cells increased by approximately 1.5
It reached as high as ¹⁰⁶ cells/ml. Example 2 ITES was added to the same basal medium as in Example 1, and various concentrations of PVP K-90 (average molecular weight 1200000) were added.
Serum-free medium supplemented with PVP K at various concentrations
-30 (average molecular weight 40,000) was prepared, and 1 ml of this was added to a 24-well multi-well plate.
Dispense to address. To this, add 1 x 10 ⁵ cells/mouse/human/human heterohybridoma N12-16/63.
The cells were seeded at a rate of 1 ml, and after culturing in a 5% carbon dioxide incubator at 37° C. for 5 days, the number of cells was measured using a Coulter counter, and the results shown in FIG. 2 were obtained. PVP K-
In the case of PVP K-30 (indicated by 〓〓), the optimum concentration was around 0.4%.
It was particularly effective over a wide range of 0.2 to 6.4%. Example 3 Medium 1 was prepared by adding ITES to the same basal medium as in Example 1, and to this was added 0.2% PVP K-30 (average molecular weight
40000) and 0.2% PVP K
Prepare medium 3 supplemented with -90 (average molecular weight 1200000) and add 1 ml of these to a 24-well multi-well plate.
Dispense to address. The various lymphoid cells shown in Table 1 were seeded at a rate of 1 x 10 ⁵ cells/ml, and after culturing for 4 days at 37°C in a 5% carbon dioxide incubator, the number of cells was counted using a Coulter counter. The results were obtained.

[Table] Ridoma

Claims (1)

[Scope of Claims] 1. A composition for proliferation of lymphoid cells containing polyvinylpyrrolidone. 2. The composition according to claim 1, which is in a solid state. 3. The composition according to claim 1, which is an aqueous solution. 4 The components of the basal medium are Iscove's medium, Ham's medium,
The composition according to claim 1, which has the same components as L-15 medium or a mixed medium of two or more thereof. 5. The composition according to claim 1 or 4, to which one or more of insulin, transferrin, ethanolamine, and selenite is added. 6. The composition according to claim 3, which does not contain serum. 7. The composition according to claim 3, which contains serum in an amount less than the amount normally used. 8. A method for propagating lymphoid cells, which comprises culturing lymphoid cells in a medium containing a composition for propagating lymphoid cells containing polyvinylpyrrolidone. 9. The growth method according to claim 8, which is a serum-free medium. 10. The proliferation method according to claim 8, wherein the medium contains serum in an amount less than the amount normally used.