Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-02-27
2002-04-23
Wityshyn, Michael G. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C424S093700, C424S520000, C424S570000, C435S404000, C435S407000, C435S408000
Reexamination Certificate
active
06376238
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a culture media for culturing neurons, to methods for preparing the culture media, and to methods for culturing neurons by use of the culture media.
DESCRIPTION OF THE RELATED ART
In order to culture neurons ex vivo, many studies have been conducted since long ago. The discovery of the nerve growth factor (NGF), being the first discovery of a factor that specifically functions on neurons, was epoch-making in the field of neurobiology. In culturing neurons, NGF has made it possible to induce growth of nerve fibers, and accordingly, neurons have come to be cultured under conditions more closely resembling in vivo.
Recently, new nerve growth factors such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-5 (NT-5), and glial cell line derived neurotrophic factor (GDNF) have been discovered one after another. These factors have been studied with respect to their effects and functional mechanisms by use of cultured neurons, and have now come to the stage where use thereof as medicines for the treatment of diseases is being considered through the application of genetic engineering techniques that have been put into practice.
However, when these factors are investigated in an ordinary culture system for central nerve cells, a disadvantage is encountered in that a plurality of substances must be added in order for a significant effect to be obtained for various neurons; i.e., when a single substance is added, effect is observed on only certain specific cells.
Regarding culturing of central nerve cells, there has been an another approach in which use of hormones (insulin, thyroxine, progesterone, etc.), vitamins, unsaturated fatty acids, and growth factors (such as basic fibroblast growth factor) has been studied. Serum-free culture media in which these substances are combined differently have heretofore been reported [Journal of Neuroscience Methods, 23:75 (1988), etc.]. Although these culture media are effective toward certain cell lines (such as the glial cell line), there are cases in which neurons cannot be stably cultured, or in which the culture media are effective toward neurons but proliferation of glial cells is simultaneously stimulated, to thereby render the culture system a so-called mixed culture system involving glial cells in addition to neurons. This is detrimental, particularly when pharmacological action on neurons is to be studied. For example, although the N2 supplement of Bottenstein et al. [insulin (5 &mgr;g/ml), transferrin (100 &mgr;g/ml), progesterone (20 nM), putrescine (100 &mgr;M), and selenious acid salts (30 nM); Proceeding of National Academy of Science, U.S.A., 76:514(1979)] is suitable for culturing glial cell lines, it cannot stably maintain viable primary neurons. Also, the culture medium disclosed by Brewer et al. [Brain Research, 65:494(1989)] cannot stably maintain cellular functions when used for long-term culturing, although it permits short-term culturing of central nerve cells.
In the meantime, it is known that a culture supernatant of a glial cell line or primary glial cells is used for culturing neurons. When a culture supernatant of a glial cell line is used, it is generally unavoidable that the supernatant acts not only on neurons but also on glial cells to thereby stimulate the proliferation of glial cells. In other words, although the culture supernatant is effective in maintaining survival of neurons, it exhibits much greater effects on glial cells. A so-called glial cell growth factor has been purified from these culture supernatants [Journal of Biological Chemistry, 268: 2857 (1993), etc.].
In addition, Japanese Patent Application Laid-Open (kokai) No. 7-101990 discloses that a concentrated culture supernatant of astrocytoma, which is a type of glial cell lines, exhibits 30-50% elevation in activity of cultured neurons. In that publication, it is also disclosed that the concentrate suppresses proliferation of neuroblastoma. The technique of that publication is significant in that a culture supernatant is concentrated to take up part of active components so as to suppress proliferation of glial cells, which proliferation is the drawback involved in use of a culture supernatant of a glial cell line. However, in order to stably and consistently culture neurons, collective action of a plurality of components is required rather than the action of a single substance. A stable culture system for neurons cannot be established through elevation in activity as low as 30% relative to the case in which the concentrate is not used.
In a method in which primary glial cells are used, an ordinary serum-free culture medium is employed, or serum-containing culture medium is used to obtain a culture supernatant. When an ordinary serum-free culture medium is used to obtain a culture supernatant, the culture medium does not satisfactorily promise viability of glial cells, and therefore, stable culturing cannot be realized. Consequently, the amount of factors (substances) that act on the neurons present in the culture supernatant becomes small, to thereby exhibit only an insignificant effect in culturing of neurons. Moreover, collection of a culture supernatant may be performed in limited numbers, and after several times of collection, it becomes difficult to collect a culture supernatant that provides a stable effect. On the other hand, when a serum-containing culture medium is used, a culture supernatant can be collected in a stable manner. However, when neurons are cultured, the growth factor acts on glial cells rather than on co-existing neurons, to thereby stimulate proliferation of cells. As a result, stable culturing of neurons is hampered.
Generally, serum-free culture media are supplemented with trophic factors such as hormones. For example, Japanese Patent Application Laid-Open (kokai) No. 3-66700 discloses a culturing method in which Dulbeccol's modified Eagle medium (hereinafter referred to as DMEM) is supplemented with insulin (5 &mgr;g/ml), transferrin (1 &mgr;g/ml), hydrocortisone (20 nM), and 3,3′,5-triiodo-L-thyronine (0.3 nM). However, when this culture supernatant is used for the culturing of neurons, stable culturing cannot be achieved beyond a period of several days. Also, &agr;2-macroglobulin—which is explicitly described in that publication as a neurite-outgrowth promoting factor—exhibits insignificant effect on achieving stable culturing for central nerve cells, and thus plays only an auxiliary role, not a principal role.
Japanese Patent Application Laid-Open (kokai) No. 3-155777 discloses that a certain factor produced by microgliacytes is effective for neurite-outgrowth. Microglia is said to exhibit a function analogous to lymphocyte such as macrophage, and their activity elevates when tissue is injured. However, it is accepted that macroglias—including astroglial cells—are predominantly present in living bodies in general, and that it is principally macroglias that maintain homeostasis except in special cases such as inflammation.
The above-described conventional culture media are not sufficiently effective when they are used to culture central nerve cells, and therefore, consistent culturing of neurons cannot be achieved. In other words, satisfactory results as expected cannot be obtained through use of conventional culture media in neuropharmacological tests. Accordingly, the present invention was made in view of the foregoing circumstances in relation to culturing of neurons, and objects of the invention are to provide culture media that are capable of consistently culturing neurons for long periods.
DISCLOSURE OF THE INVENTION
Based on the idea that the presence of a variety of trophic factors must be required for consistent culturing of neurons, the present inventors investigated how trophic factors affect culturing of neurons. They also examined a culture supernatant of primary astroglial cells as one of the trophic f
Sumitomo Bakelite Co. Ltd.
Ware Deborah K.
Wityshyn Michael G.
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