WO1997047733A1 - Liquid medium for nerve cells, process for producing the same and method for incubating nerve cells with the use of the same - Google Patents

Liquid medium for nerve cells, process for producing the same and method for incubating nerve cells with the use of the same Download PDF

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WO1997047733A1
WO1997047733A1 PCT/JP1996/001765 JP9601765W WO9747733A1 WO 1997047733 A1 WO1997047733 A1 WO 1997047733A1 JP 9601765 W JP9601765 W JP 9601765W WO 9747733 A1 WO9747733 A1 WO 9747733A1
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cells
culture
medium
nerve cells
culture solution
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PCT/JP1996/001765
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French (fr)
Japanese (ja)
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Yoshiaki Watanabe
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Sumitomo Bakelite Co., Ltd.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system

Definitions

  • the present invention relates to a culture solution for use in culturing nerve cells, a method for producing the culture solution, and a method for culturing nerve cells using the same.
  • neural growth factor was the first discovery of a factor that acts specifically on nerve cells and has had a major impact on the field of neurobiology.
  • NGF neural growth factor
  • CNTF ciliary neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophic factor-1
  • NT-4 neurotrophic factor-1
  • NT-5 neurotrophic factor-1 5
  • GDNF glial cell line-derived neurotrophic factor
  • N 2 additives from Bottenstein et al. [Insulin (5 ⁇ g ⁇ ), transferrin (100 ⁇ g / mi), progesterone (200 ⁇ ), putrescine (100 ⁇ ⁇ ), and selenite (30 ⁇ )] [Proceeding of National Academy of Science, USA, 76: 514 (1979) ))
  • Bottenstein et al. Insulin (5 ⁇ g ⁇ ), transferrin (100 ⁇ g / mi), progesterone (200 ⁇ ), putrescine (100 ⁇ ⁇ ), and selenite (30 ⁇ )]
  • a culture solution (Brain. Esearch, 65: 494 (1989)) formulated by Brewer et al. Can be used for short-term culture of central nervous cells, When cultured over a period of time, cell function cannot be maintained stably.
  • a method in which a cultured glial cell or a primary glial cell culture supernatant is used for culturing nerve cells.
  • the culture supernatant of established glial cells when used, it generally acts on glial cells at the same time as acting on neurons, and promotes proliferation. In other words, it has an effect not only on the survival of nerve cells but also on glial cells.
  • Factors called glial cell growth factors have been purified from these culture supernatants. (Journal of Biological Chemistry, 268: 2857 (1993, etc.) ].
  • Japanese Patent Application Laid-Open No. 7-110990 discloses that a concentrated glial cell, astrocyte, which is a concentrated culture of astrocytes, has an activity increasing effect of 30 to 50% on the culture of neurons. It has been reported that It has also been shown to suppress the growth of neuroblastoma. In this method, the culture supernatant was concentrated to remove some of the active ingredients in order to suppress the growth of glial cells, which is a drawback when using the culture supernatant of established glial cells. However, to cultivate neurons stably, it is necessary to use not only a single substance but also a total effect of multiple components. If the effect of increasing the activity is about 30% compared to that without using this concentrate, a stable neuronal cell culture system cannot be constructed.
  • a normal serum-free culture medium or a culture medium containing serum is used to obtain a culture supernatant.
  • the usual method of collecting a culture supernatant using a serum-free culture solution does not provide a sufficient culture solution for the survival of glial cells, so that stable culture cannot be performed.
  • the amount of the factor group acting on the nerve cells contained in the culture supernatant is small, and the effect on the culture of the nerve cells is low.
  • there is a limit in collecting a culture supernatant and it is difficult to collect a culture supernatant having a stable effect in about several times.
  • a stable culture supernatant can be collected.However, when nerve cells are cultured, growth factors act on glial cells that coexist with nerve cells, and cell growth is increased. Promoted. As a result, the stable culture of nerve cells is impaired.
  • a method of adding trophic factors such as hormones to the serum-free culture solution is employed.
  • a culture medium containing Dulbecco's modified Eagle's medium hereinafter abbreviated as DMEM
  • insulin 5 ⁇ g / mi
  • transphenylene 1 g / ml
  • Hydrocotisizone 2 OnM
  • 3,3 ', 5-Triyodo L-thyronine 0.3 nM
  • 2-macroglobulin also designated as a neurite outgrowth factor, has a low effect on stable culture in central nervous system culture, and plays only an auxiliary role, not a major one. Things.
  • microglial cells have an effect on the extension of process of nerve cells.
  • Microglial cells are said to exhibit the function of immune system cells such as macrophages (phagocytic cells), and are activated to a high degree when a tissue is damaged.
  • macrophages phagocytic cells
  • the ratio of macroglial cells containing astroglial cells is high in living organisms, and it is considered that macroglial cells mainly maintain homeostasis except in the case of inflammation. .
  • the present invention has been made in view of such a current situation in the culture of nerve cells, and has as its object to provide a culture solution capable of stably culturing nerve cells for a long period of time. Disclosure of the invention
  • the present inventors considered that various trophic factors are necessary for stable culture of nerve cells, and examined the effects of these trophic factors on the culture of nerve cells.
  • the conventional culture supernatant of primary astroglial cells was also considered as one of the trophic factors, the culture supernatant of conventional primary astroglial cells has been reported for stable neuronal cultures. It proved ineffective.
  • the present inventors have conducted intensive studies on the culture conditions of primary ostial glial cells, and found that the culture supernatant obtained by culturing with the addition of a specific additive has an excellent effect. Thus, the present invention has been completed.
  • the present invention relates to insulin, transferrin, selenite or a salt thereof, albumin, progesterone, superoxide dismutase and catalase, or insulin, transferrin, selenite or a salt thereof, prodisterone, albumin And a culture solution for nerve cells, which comprises a culture supernatant obtained by culturing primary astroglial cells in a nutrient medium supplemented with tocopherols.
  • the present invention provides a method for culturing and growing primary ostial glial cells in a medium supplemented with animal serum, followed by prodisterone, superoxide dismutase and dexamethasone, and lime for insulin and transferrin. Culturing in a nutrient medium supplemented with selenous acid or a salt thereof, prodiesterone, albumin and glucose, and collecting a supernatant thereof, wherein the method for producing a culture solution for nerve cells is provided. Is what you do.
  • the present invention provides a method for culturing a nerve cell, which comprises culturing the nerve cell in the culture solution for a nerve cell.
  • glial cells of the ostium of the mouth are collected from the brain of the animal, and the glial cells of the primary ostium are grown to obtain a sufficient amount of the glial cells of the primary ostium.
  • a newborn animal 1-2 days
  • the animal for collecting astroglial cells is preferably a rat or a mouse such as a rat, a mouse, a mouse, a mouse, a pig, a monkey, a rabbit, a chicken, and the like.
  • the cerebrum is cut out from the neonatal brain, the brain membrane is removed, and the cells are dispersed using enzymes such as trypsin, dispase, collagenase, and papain.
  • enzymes such as trypsin, dispase, collagenase, and papain.
  • trypsin 0.05 to 0.35 w / v% (hereinafter simply indicated by%).
  • deoxyribonuclease 0.01% (100-500 J / m6), ethylenediamine N, N, N ', N'-tetraacetic acid (EDTA) 0.01
  • EDTA ethylenediamine N, N, N ', N'-tetraacetic acid
  • Culture and propagation of the collected astroglial cells are preferably performed in a medium supplemented with animal serum.
  • animal serum used here, male serum is preferable, and fetal serum, baby serum, and neonatal maggot serum are particularly preferable. Further, the amount of animal serum to be added is preferably 5 to 20%.
  • the medium is not particularly limited as long as it is a nutrient medium for culturing animal cells, and Eagle's minimum essential medium (hereinafter abbreviated as MEM), Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM), DMEMZ ham F- Medium such as 12 medium (hereinafter abbreviated as F-12), F-12, and ham F-10 medium (hereinafter abbreviated as F-10) can be used.
  • MEM Eagle's minimum essential medium
  • DMEM Dulbecco's modified Eagle medium
  • F-12 DMEMZ ham F- Medium
  • F-12 12 medium
  • F-12 ham F-10 medium
  • the DMEM / F-12 mixed medium may be used at various mixing ratios, or may be mixed in the range of about 60/40 to 40/60 (weight ratio), which has the characteristics of both mediums. Medium is particularly preferred.
  • the astroglial cells dispersed in the culture solution are confluent (density over the entire culture surface) using a cell culture flask, dish, plate, or a flask coated with polylysine, a dish, a plate, or a microcarrier. Incubate and grow until complete.
  • the culture area is preferably 10 to 100 cm 2 for one newborn baby. Then, further subculture is performed, and the culture area is suitably about 2 to 10 times. Similarly, culture until confluent.
  • the confluent cells are mainly classified as type 1 astroglia (or type 1 astrosite). Type 1 astroglia can be confirmed by immunocytochemical staining.
  • the primary astroglial cells thus obtained can be treated with insulin, transferrin, selenite or a salt thereof, progesterone, albumin, superoxide dismutase and lyase, or insulin, transferrin, selenite.
  • culture the cells in a nutrient medium supplemented with a salt thereof, progesterone, albumin, and tocopherols and collect the supernatant.
  • primary astroglial cells are supplemented with insulin, transferrin, selenite or a salt thereof, progesterone, albumin, superoxide dismutase, catalase, and yeast cells. More preferably, the cells are cultured in a nutrient medium prepared in the above manner, and the supernatant is collected.
  • the nutrient medium used here may be one or more selected from MEM, DMEM, F-10 and F-12, and may be MEM, DMEM, DMEM / F-10 or DMEMZF- 12 is preferred, and DMEM / F-12 force is particularly preferred. Further, DMEMZF-12 preferably has a mixing ratio of 60/40 to 40/60 (weight ratio).
  • Examples of the salt of selenious acid added to these nutrient media include sodium selenite, potassium selenite, and the like.
  • insulin so as to have a concentration of 1 to 100 1g Zm, particularly 3 to 20 ⁇ g ⁇ ⁇ .
  • the transphenylene to a concentration of 1 to 100 g / mL, particularly 3 to 20 g ⁇ ⁇ .
  • selenous acid or a salt thereof is added so as to have a concentration of 1 to 100 nl'l, particularly 3 to 5 ⁇ .
  • albumin is added to a concentration of 0.5 to 2.5 rngZ.
  • progesterone so as to have a concentration of 1 to 10 ⁇ .
  • the preferred concentration of superoxide dismutase is 1 to 100 g / TO, and the preferred concentration of TO enzyme is 1 to 100 g / m. g Z ⁇ .
  • the monotocopherols include monotocopherol esters such as monotocopherol, tocopherol acetate, and tocopherol succinate. Insulin, transferrin, and selenite are all water-soluble and may be added as they are, but it is also preferable to prepare a high-concentration solution in which each component is mixed and add a fixed amount thereof. Is the way.
  • a water-soluble protoester is used as the progesterone and a water-soluble heart tocopher is used as the monoprotolol
  • a method of adding each component as a high concentration solution in the same manner as described above can be applied.
  • water-insoluble prodisterone and water-insoluble tocopherol are used, those dissolved in ethanol in advance may be used.
  • the cultivation period may be about one day. If the supernatant is collected and the culture is performed again with a new medium and additives, the supernatant can be collected repeatedly. If the supernatant is collected every day in this way, it is possible to collect the supernatant up to about 20 to 25 times by one preparation operation of the primary ostial glial cells, but preferably 1 0 to 15 times.
  • the collected culture supernatant is preferably sterilized by filtration through a filter having a pore size of from 0.02 to 0.45 m, and the cells are preferably used without removing cell fragments.
  • the culture supernatant can be stably stored by freezing. Therefore, when collecting the supernatant repeatedly every day as described above, freeze it and store it.Thaw the frozen culture supernatant at the time when the collection of the supernatant for a plurality of times has been completed. If they are mixed uniformly, a more homogeneous culture solution for nerve cells can be obtained.
  • the optimal freezing and storage temperature of the culture supernatant is -10 to 180 ° C, and long-term stable storage at refrigerated temperatures (4 to 8 ° C) is difficult. It is preferable that the freeze-thaw operation is not repeated.
  • the above culture medium may be newly added to the culture supernatant as a nutrient source for nerve cells, and a more preferable medium is a DMEM / F-12 mixed medium.
  • the addition amount of the newly added medium is preferably 0 to 75%, particularly preferably 0.1 to 50%.
  • nerve cells may be added to the culture solution and cultured according to a conventional method.
  • Neurons can be prepared from animals similar to primary astroglial cells. Culture can be performed using an individual after birth, but generally the survival rate of neurons is higher when a fetus is used. In rats, embryos of about 15 to 20 days are suitable or it is possible to use immature fetuses. is there. Culturing limited to a specific region of the brain, for example, hippocampus, striatum, septum, or midbrain or cerebellum is also possible. In the case of the cerebellum, effective culture is possible by using individuals about one week after birth according to the differentiation of neurons.
  • glial cells and the like When the brain is enzymatically treated and the nerve cells are dispersed, glial cells and the like (primary astroglial cells and the like) are usually contaminated in addition to the nerve cells.
  • the primary astroglial cells are of type 1 and type 2, and in the case of serum-supplemented culture medium, type 1 astual glial cells proliferate in this medium, causing a problem.
  • the culture solution of the present invention has an effect of suppressing the growth of the cells. It is not reliable to distinguish type 1 and type 2 from the morphology of primary astroglial cells, and a method using immunocytochemical staining is used.
  • Anti-GFAP antibody stains both types 1 and 2, while anti-A2B5 antibody stains only type 2. This difference in staining makes it possible to distinguish between type 1 and type 2.
  • the culture solution of the present invention contains oligodendroglia (or oligodend mouth site) which is one of the undifferentiated stem cells—type 2 astroglia (or type 2 fast mouth site) stem cells (hereinafter referred to as ⁇ -2A stem cells). It has the effect of inducing proliferation and differentiation. If the culture is continued, the culture system will be able to detect oligodendroglia, which was not initially recognized. This is the result of the proliferation and differentiation of 0-2A stem cells.
  • the fact that the cells are oligodendroglia can be applied by the same method of immunocytochemical staining as in the case of identifying astroglial cells. For example, a clear distinction can be made by staining with an anti-GC (galactocereb mouth side) antibody, an anti-MBP (myelin basic protein) antibody, or the like.
  • a specific method for culturing nerve cells is to first cut out the brain in the same manner as described above. Then, a nerve cell dispersion is prepared using enzymes such as trypsin, papain, and dispase.
  • enzymes such as trypsin, papain, and dispase.
  • papain 10 to 50 U /; ⁇
  • L-cysteine 0.5-5 m
  • glucose 5-5 5 ⁇
  • the nerve cells are dispersed by carefully stirring and mixing the dispersed enzyme solution. Addition of deoxyribonuclease (0.01%) can prevent cell aggregation due to leaked nucleic acid.
  • the cells were separated by a centrifuge, and the culture solution of the present invention prepared as described above was used.
  • the plate, dish, etc. used for the culture can be of any material such as glass and plastic. It is preferable to use a single layer or a multi-layer coated layer.
  • the collected culture supernatant was sterilized by filtration with a 0.22 m filter (manufactured by Millipore), frozen and stored. After collecting the supernatant for 10 times, thawed and the whole was uniformly mixed and mixed. Stored at 70 ° C.
  • the nerve cells used as samples were cut out of the cerebrum from a 17-day-old rat embryo and used as a phosphate buffer containing ImM cysteine, 25 mM glucose, and lmgZ albumin (manufactured by Sigma) at 20 UZ? ⁇ .
  • the enzyme was treated with papain (manufactured by Worthington) at 37 for 45 minutes. After removing the enzyme solution and dispersing the cells with a DMEMZF-12 mixed culture solution, the cells were separated under centrifugation at 700 rpm for 5 minutes.
  • a solution having a cell concentration of 2500,000 c / was prepared, and a polylysine-coated 24-well plate (manufactured by Sumitomo Bei-Client) was used. And cultured at 0.5? ⁇ / ⁇ .
  • cultivation was performed under the same conditions using DMEM / F-12 two-component equal-density mixed culture to which the culture solution of the present invention was not added.
  • Example 2 Primary astroglial cells were cultured in the same manner as in Example 1, and the culture supernatant was collected. Using the obtained culture supernatant, the ratio of the culture supernatant to the DMEM / F-12 isosteric mixed culture solution was 75./25, 50/50, 25/75, and 10/9. Each was prepared to be 0. Further, as Comparative Example 3, a solution prepared by adding 10% of fetal calf serum (manufactured by Hyclone) to a DMEM F-12 equimolar mixture was used. In the same manner as in Example 1, the nerve cell fluid was added to 200,000 Culture was performed in the same manner as in Example 1.
  • the culture supernatant After culturing for 4 days and observing under a microscope, the culture supernatant with the ratio of the culture supernatant to the DMEMZF-12 mixture of 75/25. Neurite extension was observed. In the culture medium of 25/75, the number of surviving cells was slightly lower, and in the culture medium of 10/90, viable cells and neurite outgrowth were both inferior. In the culture solution using the fetal calf serum of Comparative Example 3, the number of surviving neurons was small, and the presence of glial cells was recognized.
  • the ABC immunohistochemistry kit manufactured by Vecto
  • the DAB substrate kit Manufactured by Vector
  • Example 2 a culture solution for nerve cells was prepared. Nerve cells as samples were prepared in the same manner as in Example 1 from the hippocampus of a Wistar rat fetus (embryon day 16). 250,000 c-nodule cells were added to a 24 ⁇ Erla minin coat plate (manufactured by Sumitomo BeiClient) and cultured for 14 days. Between 3 and 5 days of culture, 5 M of cytosine arabinofuranoside (Sigma) was added. For the exchange of the culture solution, the 1/2 volume was exchanged twice a week.
  • a culture solution was prepared in the same manner as in Example 1.
  • the culture solution of Comparative Example 3 was used.
  • a nerve cell solution was prepared in the same manner as in Example 1, and cultured at a culture density of 1,200 eel I s / mm 2 for 8 days using a laminin-core 12-well plate (manufactured by Sumitomo Belite).
  • the culture solution of the present invention in addition to the neurons stably cultured, cells that were judged to be oligodendroglia from their morphology were observed.
  • the number of nerve cells was small, and most of the cells were judged to be astroglial cells based on their morphology.
  • anti-GC antibody manufactured by Boehringer Mannheim, adjusted to 5 g
  • anti-GFAP antibody manufactured by Boehringer Mannheim, adjusted to 8 g Z
  • anti-A2B 5 Antibody was used for immunocytochemical staining. After removing the culture solution and washing with a phosphate buffer, paraformaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) is used for 20 minutes in a 4% phosphate buffer.
  • the culture of central nerve cells can be performed stably, and in low-density culture, extensibility of neurites is excellent and rapid synapse formation is possible.
  • density culture cells that have formed a neural network have excellent long-term stability. This makes it possible to carry out neuropharmacological tests, neuronal information transmission tests, etc. with high accuracy, and is useful for research on dementia, neurological diseases, neurotoxicity, etc. in the fields of neuropharmacology and sanitary chemistry, and for elucidation of pathological conditions.

Abstract

A liquid medium for nerve cells which contains the culture supernatant of primary astroglia cells obtained by incubating the astroglia cells in a nutritional medium containing insulin, transferrin, selenious acid or its salt, progesterone and albumin together with either superoxide dismutase and catalase or α-tocopherols. By using the liquid medium, central nerve cells can be stably incubated. Thus, in low density incubation, synapses excellent in the elongation of axons can be quickly formed, while, in high density incubation, it is possible to provide cells forming a nervous network and being excellent in the stability over a prolonged storage.

Description

明 細 書 神経細胞用培養液、 その製造方法及びこれを用いる神経細胞の培養方法 技術分野  Description: Culture solution for nerve cells, method for producing the same, and method for culturing nerve cells using the same
本発明は、 神経細胞の培養に用いるための培養液、 当該培養液の製造方法、 及 びこれを用いる神経細胞の培養方法に関する。  The present invention relates to a culture solution for use in culturing nerve cells, a method for producing the culture solution, and a method for culturing nerve cells using the same.
'景技術 '' Jing Technology
神経細胞を生体外で培養するために、 古くから幾多の検討かなされてきた。 神 経成長因子 （NGF) の発見は、 神経細胞に特異的に働く因子のはじめての発見 であり、 神経生物学の分野に大きな影響を与えた。 神経細胞の培養に於いても、 神経繊維の成長が誘導できるようになり、 より生体内に近し、条件で培養できるよ うになった。  Many studies have been made since ancient times to culture neurons in vitro. The discovery of neural growth factor (NGF) was the first discovery of a factor that acts specifically on nerve cells and has had a major impact on the field of neurobiology. In the cultivation of nerve cells, the growth of nerve fibers can be induced, and the cells can be cultured closer to the living body under conditions.
最近になって、 毛様体神経栄養因子 （CNTF) 、 脳由来神経栄養因子 (BDNF) 、 神経栄養因子一 3 (NT - 3) 、 神経栄養因子一 4 (NT- 4) , 神経栄養因子一 5 (NT- 5) 、 グリア細胞株由来神経栄養因子 （GDNF) と、 次々に新しレ、神経成長因子が発見されてきた。 これらは培養神経細胞によって、 その効果や作用メカニズムが検討され、 更には実用化されている遺伝子工学の手 法により、 疾病治療のための医薬としての利用も検討されるまでになっている。 しかし、 これらの因子を通常の中枢神経細胞の培養系で調べてみると、 単一物 質の添加だけで各種の神経細胞に対して著しい効果を示すとは言いにく く、 特定 の細胞に対してのみ効果を示すという場合が多レ、。  Recently, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-1 (NT-3), neurotrophic factor-1 (NT-4), neurotrophic factor-1 5 (NT-5), a glial cell line-derived neurotrophic factor (GDNF), and one after another, new nerve growth factors have been discovered. The effects and mechanism of action of these cells have been studied by cultured neurons, and furthermore, their use as drugs for the treatment of diseases has been studied by means of practical genetic engineering techniques. However, when examining these factors in a normal central nervous cell culture system, it is difficult to say that the addition of a single substance has a remarkable effect on various types of neurons. In many cases, it is only effective against
中枢神経細胞の培養に関しては、 これらとは別の流れとして、 ホルモン （イン シュリ ン、 サイロキシン、 プロジェステロン等） 、 ビタミ ン、 不飽和脂肪酸、 紬 胞成長因子 （塩基性繊維芽細胞増殖因子等） などが検討され続けてきており、 こ れらの因子を種々組み合わせた無血清培養液が報告されている 〔ジャーナル .ォ ブ ·ニューロサイエンス · メッツ ド （Journal of Neuroscience Methods, 23:75 (1988) 等〕 。 しかしこれらのものも、 株化細胞 （株化グリア細胞等） には効果的であるが、 神経細胞に対しては安定培養ができない場合や、 神経細胞には効果があるものの、 同時にグリア細胞の増殖が促進されてしまい、 いわゆるグリア細胞との混合培養 系になってしまう場合など、 神経細胞に対する薬理作用を検討する場合には不適 切なものが多い。 例えば、 ボテンシュ夕イン （Bot tenstein) らの N 2添加物 〔 インシュリン （ 5〃g τηβ) 、 トランスフエリン （ 1 0 0〃g /mi) 、 プロジェ ステロン （ 2 0 ηΜ) 、 プトレシン （ 1 0 0〃Μ ) 、 及び亜セレン酸塩 （ 3 0 ηΜ) 〕 〔プロスィ一ディング ·ォブ ·ナショナル ·アカデミー ·ォブ ·サイェン ス (Proceeding of Nat i onal Academy of Sci ence, U. S. A. ) ， 76 : 514( 1979) ) は、 株化ダリ了細胞の培養には適している力 初代神経細胞の場合は安定した紬 胞の生存維持が図れない。 また、 ブリュヮー （Brewer) らの処方の培養液 〔ブレ イン . リサーチ (Brain. esearch) , 65 : 494(1989)) も、 中枢神経細胞に対して 短期間の培養は可能であるが、 長期間に渡って培養した場合、 細胞機能を安定し て維持することができない。 Regarding the culture of central nervous cells, there are other flows such as hormones (insulin, thyroxine, progesterone, etc.), vitamins, unsaturated fatty acids, cell growth factors (basic fibroblast growth factors, etc.). And serum-free culture solutions in which these factors are variously combined have been reported [Journal of Neuroscience Methods, 23:75 (1988) etc〕 . However, these are also effective for established cells (such as glial cells), but when stable culture is not possible for nerve cells, or when they are effective for nerve cells, they are also effective for glial cells. Many cases are inappropriate when examining the pharmacological effects on nerve cells, such as when proliferation is promoted and a so-called mixed culture system with glial cells is used. For example, N 2 additives from Bottenstein et al. [Insulin (5〃g τηβ), transferrin (100〃g / mi), progesterone (200 ηΜ), putrescine (100〃 Μ), and selenite (30 ηΜ)] [Proceeding of National Academy of Science, USA, 76: 514 (1979) )) Is a force suitable for cultivation of established cell lines. Primary neurons cannot maintain stable survival of cells. In addition, a culture solution (Brain. Esearch, 65: 494 (1989)) formulated by Brewer et al. Can be used for short-term culture of central nervous cells, When cultured over a period of time, cell function cannot be maintained stably.
また、 神経細胞の培養に株化グリァ細胞ないしは初代グリァ細胞の培養上清を 用いる方法が知られている。 しかし、 株化グリア細胞の培養上清を用いた場合は、 一般的に、 神経細胞に対しても作用するカ 同時にグリア細胞にも作用し増殖が 促進される。 つまり神経細胞の生存に対する効果だけでなく、 それ以上にグリア 細胞に対して効果を示す。 グリア細胞増殖因子と称呼される因子は、 これらの培 養上清より精製されている 〔ジャーナル ·ォブ 'バイオロジカル 'ケミストリ一 (Journal of Bi ol ogi cal Chemi stry) , 268 : 2857( 1993 など〕 。  Also, a method is known in which a cultured glial cell or a primary glial cell culture supernatant is used for culturing nerve cells. However, when the culture supernatant of established glial cells is used, it generally acts on glial cells at the same time as acting on neurons, and promotes proliferation. In other words, it has an effect not only on the survival of nerve cells but also on glial cells. Factors called glial cell growth factors have been purified from these culture supernatants. (Journal of Biological Chemistry, 268: 2857 (1993, etc.) ].
また特開平 7 - 1 0 1 9 9 0号公報には株化グリア細胞であるァストロサイト 一マの培養上淸を濃縮したものが神経細胞の培養に 3 0〜5 0 %の活性増加効果 を示すことが報告されている。 また、 神経芽細胞腫の増殖を抑制することが示さ れている。 これは株化グリァ細胞の培養上清を用いた場合の欠点であるグリァ紬 胞の増殖を抑えるため、 培養上清を濃縮し一部の有効成分をとりだしたものであ る。 しかし神経細胞を安定に培養するためには単一物質ではなく複数の成分によ る総合的な作用が必要である。 この濃縮物を用いないものに比べた活性増加効果 が 3 0 %程度では、 安定な神経細胞の培養系を構成することはできなし、。 初代グリア細胞を用いる方法では、 培養上清を得るために、 通常の無血清培養 液ないしは血清が含まれている培養液を用いる。 しかし、 通常の無血清培養液を 用いる培養上清の採取方法では、 グリァ細胞の生存に対して充分な培養液とはな らず、 安定培養ができない。 このため培養上清に含まれる神経細胞に作用する因 子群の量が少なく、 神経細胞の培養に対して効果が低し、。 また培養上清の採取に は限度があり、 数回程度で、 安定した効果を持つ培養上清の採取は難しくなる。 血清含有培養液を用いた場合には、 安定した培養上清の採取は行えるが、 神経細 胞を培養する際に、 神経細胞よりも共存するグリァ細胞に対して増殖因子が作用 し細胞増殖が促進される。 その結果、 神経細胞の安定培養が障害される。 Japanese Patent Application Laid-Open No. 7-110990 discloses that a concentrated glial cell, astrocyte, which is a concentrated culture of astrocytes, has an activity increasing effect of 30 to 50% on the culture of neurons. It has been reported that It has also been shown to suppress the growth of neuroblastoma. In this method, the culture supernatant was concentrated to remove some of the active ingredients in order to suppress the growth of glial cells, which is a drawback when using the culture supernatant of established glial cells. However, to cultivate neurons stably, it is necessary to use not only a single substance but also a total effect of multiple components. If the effect of increasing the activity is about 30% compared to that without using this concentrate, a stable neuronal cell culture system cannot be constructed. In the method using primary glial cells, a normal serum-free culture medium or a culture medium containing serum is used to obtain a culture supernatant. However, the usual method of collecting a culture supernatant using a serum-free culture solution does not provide a sufficient culture solution for the survival of glial cells, so that stable culture cannot be performed. As a result, the amount of the factor group acting on the nerve cells contained in the culture supernatant is small, and the effect on the culture of the nerve cells is low. In addition, there is a limit in collecting a culture supernatant, and it is difficult to collect a culture supernatant having a stable effect in about several times. When a serum-containing culture solution is used, a stable culture supernatant can be collected.However, when nerve cells are cultured, growth factors act on glial cells that coexist with nerve cells, and cell growth is increased. Promoted. As a result, the stable culture of nerve cells is impaired.
通常、 無血清培養液にはホルモン等の栄養因子を加える方法が採られる。 例え ば、 特開平 3 - 6 6 7 0 0号公報には、 ダルべッコ改変イーグル培地 （以下、 D M E Mと略す） 培養液にインシュリン （5〃g /mi) 、 トランスフエリ ン （ 1 g / ) 、 ハイ ドロコ一チゾン （2 O nM) 、 及び 3， 3 ' , 5 — 卜リヨ一ドー Lーチロニン （ 0 . 3 nM) を添加する培養法が開示されている。 しかしこの培養 上清を用いて神経細胞を培養しても数日間の培養しか行えず安定した培養を行う ことができない。 また、 同じく神経突起伸展因子として明示されているひ 2—マ クログロブリンは、 中枢神経の培養においては安定培養の効果が低く、 主要な役 割を果たすものではなく補助的役割を果たすにすぎないものである。  Usually, a method of adding trophic factors such as hormones to the serum-free culture solution is employed. For example, Japanese Unexamined Patent Publication (Kokai) No. 3-66070 discloses that a culture medium containing Dulbecco's modified Eagle's medium (hereinafter abbreviated as DMEM) contains insulin (5 μg / mi) and transphenylene (1 g / ml). ), Hydrocotisizone (2 OnM), and 3,3 ', 5-Triyodo L-thyronine (0.3 nM) are disclosed. However, even if nerve cells are cultured using this culture supernatant, only a few days can be cultured, and stable culture cannot be performed. In addition, 2-macroglobulin, also designated as a neurite outgrowth factor, has a low effect on stable culture in central nervous system culture, and plays only an auxiliary role, not a major one. Things.
更に、 特開平 3 - 1 5 5 7 7 7号公報には、 ミクログリア細胞の産生する因子 力 \ 神経細胞の突起伸展に効果があることが示されている。 ミクログ'リア細胞は、 マクロファージ （貧食細胞） のような免疫系細胞の機能を示すといわれ、 組織か 障害を受けたときに活性化の度合いが高くなる。 し力、し、 一般的に生体内では、 ァストログリァ紬胞を含むマク口グリァ細胞の比率が高く、 炎症などの場合を除 き、 主としてマクログリァ細胞が恒常性を維持していると考えられている。  Furthermore, Japanese Patent Application Laid-Open No. H3-15557777 discloses that microglial cells have an effect on the extension of process of nerve cells. Microglial cells are said to exhibit the function of immune system cells such as macrophages (phagocytic cells), and are activated to a high degree when a tissue is damaged. In general, the ratio of macroglial cells containing astroglial cells is high in living organisms, and it is considered that macroglial cells mainly maintain homeostasis except in the case of inflammation. .
上記のような従来の培養液群を中枢神経細胞の培養に用いた場合、 その効果は 低く、 神経細胞の安定した培養を行うことができない。 すなわち、 神経薬理試験 等を行った場合に於いて、 期待する充分な結果が得られない。 従って本発明は、 神経細胞の培養におけるこのような現状に鑑みてなされたもので、 神経細胞を長 期間安定に培養できる培養液を提供することを目的とするものである。 発明の開示 When the conventional culture solution group as described above is used for culturing central nervous cells, the effect is low, and stable culturing of nerve cells cannot be performed. In other words, the expected results cannot be obtained in neuropharmacology tests. Therefore, the present invention has been made in view of such a current situation in the culture of nerve cells, and has as its object to provide a culture solution capable of stably culturing nerve cells for a long period of time. Disclosure of the invention
本発明者らは神経細胞を安定に培養するためには種々の栄養因子が'必要である と考え、 これらの栄養因子に関して神経細胞の培養に与える効果を検討した。 初 代ァスト口グリァ細胞の従来の培養上清も栄養因子の一つとし検討したが、 従来 の初代ァストログリア細胞の培養上清は、 神経細胞の安定培養に対して、 これま で報告されて来た程の効果がない事が明らかになった。 そこで本発明者らは、 初 代ァスト口グリァ細胞の培養条件について鋭意研究を進めた結果、 ある特定の添 加剤を加えて培養して得た培養上清に優れた効果があることを見出し、 本発明を 完成するに至ったものである。  The present inventors considered that various trophic factors are necessary for stable culture of nerve cells, and examined the effects of these trophic factors on the culture of nerve cells. Although the conventional culture supernatant of primary astroglial cells was also considered as one of the trophic factors, the culture supernatant of conventional primary astroglial cells has been reported for stable neuronal cultures. It proved ineffective. Accordingly, the present inventors have conducted intensive studies on the culture conditions of primary ostial glial cells, and found that the culture supernatant obtained by culturing with the addition of a specific additive has an excellent effect. Thus, the present invention has been completed.
すなわち、 本発明はインシュリン、 トランスフェリ ン、 亜セレン酸若しくはそ の塩、 アルブミン、 プロジェステロン、 スーパーォキシドジスムターゼ及びカタ ラーゼ、 又はインシュリン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プ ロジヱステロン、 アルブミン及び トコフエロール類を添加した栄養培地中で 初代ァストログリァ細胞を培養して採取した培養上清を含有することを特徴とす る神経細胞用培養液を提供するものである。  That is, the present invention relates to insulin, transferrin, selenite or a salt thereof, albumin, progesterone, superoxide dismutase and catalase, or insulin, transferrin, selenite or a salt thereof, prodisterone, albumin And a culture solution for nerve cells, which comprises a culture supernatant obtained by culturing primary astroglial cells in a nutrient medium supplemented with tocopherols.
また、 本発明は初代ァスト口グリァ細胞を動物血清を添加した培地中で培養、 増殖させた後、 プロジヱステロン、 スーパーォキシドジスム夕ーゼ及び力タラ一 ゼ、 乂はインシュリ ン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プロジ エステロン、 アルブミン及びひ一 トコフヱロール類を添加した栄養培地中で培養 し、 その上清を採取することを特徴とする上記の神経細胞用培養液の製造方法を 提供するものである。  In addition, the present invention provides a method for culturing and growing primary ostial glial cells in a medium supplemented with animal serum, followed by prodisterone, superoxide dismutase and dexamethasone, and lime for insulin and transferrin. Culturing in a nutrient medium supplemented with selenous acid or a salt thereof, prodiesterone, albumin and glucose, and collecting a supernatant thereof, wherein the method for producing a culture solution for nerve cells is provided. Is what you do.
更にまた、 本発明は、 上記神経細胞用培養液中で神経細胞を培養することを特 徴とする神経細胞の培養方法を提供するものである。 発明を実施するための最良の形態  Furthermore, the present invention provides a method for culturing a nerve cell, which comprises culturing the nerve cell in the culture solution for a nerve cell. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の神経細胞用培養液を調製するには、 まず動物の脳よりァスト口グリァ 細胞を採取し、 初代ァスト口グリァ細胞を増殖させて充分量の初代ァスト口グリ ァ紬胞を得る。 動物の脳よりァストログリア細胞を採取するには、 動物の新生仔 ( 1〜2日） の大脳より採取するのがよい。 ここでァストログリア細胞採取用の 動物としては、 ラッ ト、 マウス、 ゥシ、 ゥマ、 ブタ、 サル、 ゥサギ、 ニヮトリ等 が挙げられる力 \ ラッ ト又はマウスが好ましい。 In order to prepare the culture solution for nerve cells of the present invention, first, glial cells of the ostium of the mouth are collected from the brain of the animal, and the glial cells of the primary ostium are grown to obtain a sufficient amount of the glial cells of the primary ostium. To collect astroglial cells from animal brain, a newborn animal (1-2 days) It is better to collect from the cerebrum. Here, the animal for collecting astroglial cells is preferably a rat or a mouse such as a rat, a mouse, a mouse, a mouse, a pig, a monkey, a rabbit, a chicken, and the like.
具体的には、 新生仔の脳より大脳を切り出し、 脳膜を除いた後、 トリプシン、 ディスパ一ゼ、 コラゲナーゼ、 パパイン等の酵素を用いて細胞を分散させる。 こ れらの中でも特にトリプシン 0. 0 5〜0. 3 5 w/v % (以下、 単に％で 示す） を用いるのが好ましい。 この酵素に更に、 デォキンリボヌクレアーゼ 0. 0 1 % ( 1 0 0〜 5 0 0 J/m6) や、 エチレンジァミ ン一 N， N， N' ， N' —四酢酸 （EDTA) 0. 0 1 %を加える方法も効果的である。  Specifically, the cerebrum is cut out from the neonatal brain, the brain membrane is removed, and the cells are dispersed using enzymes such as trypsin, dispase, collagenase, and papain. Among these, it is particularly preferable to use trypsin 0.05 to 0.35 w / v% (hereinafter simply indicated by%). In addition to this enzyme, deoxyribonuclease 0.01% (100-500 J / m6), ethylenediamine N, N, N ', N'-tetraacetic acid (EDTA) 0.01 The method of adding% is also effective.
採取されたァストログリア細胞の培養、 増殖は動物血清を添加した培地中で行 うのが好ましい。 ここで用いられる動物血清としては、 ゥシ血清が好ましく、 ゥ シ胎児血清、 仔ゥシ血清、 新生仔ウジ血清が特に好ましい。 また、 動物血清の添 加量は 5〜20 %となる量が好ましい。 また、 培地としては、 動物細胞培養用の 栄養培地であれば特に制限されず、 イーグルの最小必須培地 （以下 MEMと略 す） 、 ダルベッコ改変イーグル培地 （以下 DMEMと略す） 、 DMEMZハムの F- 1 2培地 （以下、 F - 1 2と略す） 、 F— 1 2、 ハムの F— 1 0培地 （以下 F- 1 0と略す） などの培地を用いることができる。 DMEM/F— 1 2混合培 地は混合比を各種変えたものが用いられるか、 両培地の特徴を合わせ持つ、 6 0 /4 0〜4 0/6 0 (重量比） 程度の範囲の混合培地が特に好ましい。  Culture and propagation of the collected astroglial cells are preferably performed in a medium supplemented with animal serum. As the animal serum used here, male serum is preferable, and fetal serum, baby serum, and neonatal maggot serum are particularly preferable. Further, the amount of animal serum to be added is preferably 5 to 20%. The medium is not particularly limited as long as it is a nutrient medium for culturing animal cells, and Eagle's minimum essential medium (hereinafter abbreviated as MEM), Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM), DMEMZ ham F- Medium such as 12 medium (hereinafter abbreviated as F-12), F-12, and ham F-10 medium (hereinafter abbreviated as F-10) can be used. The DMEM / F-12 mixed medium may be used at various mixing ratios, or may be mixed in the range of about 60/40 to 40/60 (weight ratio), which has the characteristics of both mediums. Medium is particularly preferred.
培養液に分散したァストログリア細胞は、 細胞培養用のフラスコ、 ディ ッシュ、 プレート、 もしくはポリリジンコートを施したフラスコ、 ディ ッシュ、 プレー卜 やマイクロキャリア一などを用いて、 コンフルェントに （培養面全体に密に） な るまで培養し、 増殖させる。 培養面積は、 新生仔 1に対して 1 0〜1 0 0cm2 と するのが好適である。 そして、 更に継代培養を行うが培養面積は 2〜 1 0倍程度 が適当である。 同様にコンフルェントになるまで培養する。 このコンフルェント になった細胞は主として 1型ァストログリア （又は 1型ァストロサイ ト） に分類 されるものである。 1型ァストログリアであることは免疫細胞化学染色を行うこ とにより確認できる。 この細胞は抗 GFAP (グリア繊維性酸性蛋白質） 抗体で 染色され、 抗 A 2 B 5抗体 （抗シアル酸含有糖蛋白質抗体） で染色されない。 細胞がコンフルェントの状態になつた時点で培養液を除去し、 リン酸塩緩衝液 等で洗浄する。 The astroglial cells dispersed in the culture solution are confluent (density over the entire culture surface) using a cell culture flask, dish, plate, or a flask coated with polylysine, a dish, a plate, or a microcarrier. Incubate and grow until complete. The culture area is preferably 10 to 100 cm 2 for one newborn baby. Then, further subculture is performed, and the culture area is suitably about 2 to 10 times. Similarly, culture until confluent. The confluent cells are mainly classified as type 1 astroglia (or type 1 astrosite). Type 1 astroglia can be confirmed by immunocytochemical staining. These cells are stained with anti-GFAP (glial fibrillary acidic protein) antibody and not with anti-A2B5 antibody (anti-sialic acid-containing glycoprotein antibody). When the cells become confluent, remove the culture solution and wash with phosphate buffer or the like.
かく して得られた初代ァストログリア細胞を、 インシュリ ン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プロジェステロン、 アルブミン、 スーパーォキ シドジス厶夕ーゼ及び力夕ラーゼ、 又はインシュリン、 トランスフェリン、 亜セ レン酸若しくはその塩、 プロジェステロン、 アルブミン及びひ一トコフエロール 類を添加した栄養培地中で培養し、 その上清を採取する。 ここで、 本発明におい ては、 初代ァストログリア細胞を、 インシュリン、 トランスフェリ ン、 亜セレン 酸若しくはその塩、 プロジェステロン、 アルブミン、 スーパ一ォキシドジスム夕 —ゼ、 カタラーゼ及びひ—トコフヱ口一ル類を添加した栄養培地中で培養し、 そ の上清を採取するのがより好ましい。  The primary astroglial cells thus obtained can be treated with insulin, transferrin, selenite or a salt thereof, progesterone, albumin, superoxide dismutase and lyase, or insulin, transferrin, selenite. Alternatively, culture the cells in a nutrient medium supplemented with a salt thereof, progesterone, albumin, and tocopherols, and collect the supernatant. Here, in the present invention, primary astroglial cells are supplemented with insulin, transferrin, selenite or a salt thereof, progesterone, albumin, superoxide dismutase, catalase, and yeast cells. More preferably, the cells are cultured in a nutrient medium prepared in the above manner, and the supernatant is collected.
ここで用いられる栄養培地としては、 MEM、 DMEM, F- 1 0及び F— 1 2から選ばれる 1種又は 2種以上が举げられるが、 MEM、 DMEM、 DMEM/F- 1 0又は DMEMZF - 1 2が好ましく、 DMEM/F— 1 2力 特に好ましい。 更に DMEMZF— 1 2は 60/40〜4 0/ 60 (重量比） の 混合比のものが好ましい。  The nutrient medium used here may be one or more selected from MEM, DMEM, F-10 and F-12, and may be MEM, DMEM, DMEM / F-10 or DMEMZF- 12 is preferred, and DMEM / F-12 force is particularly preferred. Further, DMEMZF-12 preferably has a mixing ratio of 60/40 to 40/60 (weight ratio).
これらの栄養培地に添加される亜セレン酸の塩としては、 亜セレン酸ナ卜リウ ム、 亜セレン酸力リゥ厶等が挙げられる。 ここでィンシュリンは 1〜 1 00〃g Zm 特に 3〜20〃g Ζτ^の濃度となるように添加するのが好ましい。 トラン スフエリ ンは 1〜 1 00〃g /mL 特に 3〜20 g Ζττ^の濃度となるように添 加するのが好ましい。 また、 亜セレン酸又はその塩は 1〜 1 0 0nl'l、 特に 3〜 5 ΟηΜの濃度となるように添加するのか好ましい。 また、 アルブミンは 0. 5〜 2. 5 rngZ の濃度となるように添加するのが好ましい。 プロジェステロンは 1 〜 1 0 ΟπΜの濃度となるように添加するのか好ましい。 またスーパ一ォキシドジ スム夕ーゼの好ましい濃度は 1〜 1 O O g /TO 力夕ラーゼの好ましい濃度は 1〜 1 00〃g /m ひ一トコフヱ口一ル類の好ましい濃度は 1〜 1 00〃g Z τηβである。 ここで 一 トコフエロール類としては 一 トコフエロール、 酢酸トコ フエロール、 コハク酸トコフヱロール等のひ一トコフエロールエステルが挙げら れる。 インシュリ ン、 トランスフェリ ン、 亜セレン酸塩はいずれも水溶性であり、 そ のまま添加してもよいが、 各成分を混合した高濃度溶液を調製しておき、 その一 定量加えるのも好ましい方法である。 またプロジェステロンとして水溶性プロジ エステ口ン、 ひ一トコフヱロールとして水溶性ひ—トコフヱロールを用いれば、 前記と同様に各成分を混合した高濃度溶液として添加する方法が適用できる。 ま た、 非水溶性プロジヱステロン、 非水溶性ひ一トコフエロールを用いる場合は、 エタノールにあらかじめ溶解したものを用いればよい。 Examples of the salt of selenious acid added to these nutrient media include sodium selenite, potassium selenite, and the like. Here, it is preferable to add insulin so as to have a concentration of 1 to 100 1g Zm, particularly 3 to 20〃gΖτ ^. It is preferable to add the transphenylene to a concentration of 1 to 100 g / mL, particularly 3 to 20 g {ττ ^}. It is preferable that selenous acid or a salt thereof is added so as to have a concentration of 1 to 100 nl'l, particularly 3 to 5 {η}. Preferably, albumin is added to a concentration of 0.5 to 2.5 rngZ. It is preferable to add progesterone so as to have a concentration of 1 to 10 {π}. The preferred concentration of superoxide dismutase is 1 to 100 g / TO, and the preferred concentration of TO enzyme is 1 to 100 g / m. g Z τηβ. Here, the monotocopherols include monotocopherol esters such as monotocopherol, tocopherol acetate, and tocopherol succinate. Insulin, transferrin, and selenite are all water-soluble and may be added as they are, but it is also preferable to prepare a high-concentration solution in which each component is mixed and add a fixed amount thereof. Is the way. If a water-soluble protoester is used as the progesterone and a water-soluble heart tocopher is used as the monoprotolol, a method of adding each component as a high concentration solution in the same manner as described above can be applied. When water-insoluble prodisterone and water-insoluble tocopherol are used, those dissolved in ethanol in advance may be used.
培養期間は 1 日程度でよく、 上清を採取した後、 再び新しい培地及び添加剤を 加えて培養を行えば、 上清を繰り返し採取することが出来る。 このようにして 1 日毎に上清を採取すれば、 初代ァスト口グリァ細胞の 1回の準備操作で、 2 0〜 2 5回程度まで上清を採取することが可能であるが、 好ましくは 1 0〜 1 5回で める。  The cultivation period may be about one day. If the supernatant is collected and the culture is performed again with a new medium and additives, the supernatant can be collected repeatedly. If the supernatant is collected every day in this way, it is possible to collect the supernatant up to about 20 to 25 times by one preparation operation of the primary ostial glial cells, but preferably 1 0 to 15 times.
採取した培養上清は、 孔径 0 . 0 2〜 0 . 4 5 m のフィル夕一により濾過滅 菌し、 紬胞片などを除いて用いるのが好ましい。  The collected culture supernatant is preferably sterilized by filtration through a filter having a pore size of from 0.02 to 0.45 m, and the cells are preferably used without removing cell fragments.
培養上清は凍結することにより安定に保存することか可能である。 そこで前記 のように 1 日毎に繰り返して上清を採取する場合は、 凍結して保存しておき、 複 数回分の上清の採取が終わつた時点で凍結した培養上清を融解し、 全体を均一に 混合すれば、 より均質な神経細胞用培養液を得ることができる。 培養上清の凍結 保存温度は— 1 0〜一 8 0 °Cが適しており、 冷蔵温度 （4〜 8 °C) では長期間の 安定保存は難しい。 また凍結融解の操作は繰り返さないことが好ましい。  The culture supernatant can be stably stored by freezing. Therefore, when collecting the supernatant repeatedly every day as described above, freeze it and store it.Thaw the frozen culture supernatant at the time when the collection of the supernatant for a plurality of times has been completed. If they are mixed uniformly, a more homogeneous culture solution for nerve cells can be obtained. The optimal freezing and storage temperature of the culture supernatant is -10 to 180 ° C, and long-term stable storage at refrigerated temperatures (4 to 8 ° C) is difficult. It is preferable that the freeze-thaw operation is not repeated.
また、 前記培養上清には、 神経細胞の栄養源として新たに前記の培地を添加し てもよく、 より好ましい培地としては D M E M/ F— 1 2混合培地が挙げられる。 新たに加える培地の添加量は 0〜 7 5 %、 特に 0 . 1 〜 5 0 %になる量が好まし い。  In addition, the above culture medium may be newly added to the culture supernatant as a nutrient source for nerve cells, and a more preferable medium is a DMEM / F-12 mixed medium. The addition amount of the newly added medium is preferably 0 to 75%, particularly preferably 0.1 to 50%.
本発明の培養液を用いて神経細胞の培養を行うには、 この培養液に神経細胞を 添加し常法に従い、 培養すればよい。 神経細胞は、 初代ァストログリア細胞と同 様の動物から細胞を調製することが可能である。 生後の個体を用いても培養は可 能であるが、 一般的に胎児を用いた方が神経細胞の生存率は高くなる。 ラッ 卜で は胎生 1 5〜 2 0日程が好適であるか、 より未熟な胎児を使用することも可能で ある。 脳の特定領域、 例えば、 海馬、 線条体、 中隔野あるいは中脳、 小脳などに 限定しての培養も可能である。 小脳の場合は神経細胞の分化にあわせた生後 1週 間ほどの個体を用いると効果的な培養が可能である。 In order to culture nerve cells using the culture solution of the present invention, nerve cells may be added to the culture solution and cultured according to a conventional method. Neurons can be prepared from animals similar to primary astroglial cells. Culture can be performed using an individual after birth, but generally the survival rate of neurons is higher when a fetus is used. In rats, embryos of about 15 to 20 days are suitable or it is possible to use immature fetuses. is there. Culturing limited to a specific region of the brain, for example, hippocampus, striatum, septum, or midbrain or cerebellum is also possible. In the case of the cerebellum, effective culture is possible by using individuals about one week after birth according to the differentiation of neurons.
脳を酵素処理し神経細胞を分散した場合には、 通常、 神経細胞の他にグリア細 胞等 （初代ァス卜ログリア細胞など） が混入してくる。 初代ァストログリア細胞 には 1型と 2型がある力く、 血清添加培養液の場合には、 このなかで 1型ァスト口 グリァ細胞が増殖し問題となる。 しかし本発明の培養液にはこの細胞の増殖を抑 える効果がある。 初代ァス卜ログリア細胞の形態からの 1型、 2型の判別は確実 とはいえず、 免疫細胞化学染色法による判別方法を用いる。 抗 G F A P抗体では 1型、 2型両方が染色されるが、 抗 A 2 B 5抗体では 2型のみが染色される。 こ の染色性の違いにより 1型と 2型の判別が可能である。  When the brain is enzymatically treated and the nerve cells are dispersed, glial cells and the like (primary astroglial cells and the like) are usually contaminated in addition to the nerve cells. The primary astroglial cells are of type 1 and type 2, and in the case of serum-supplemented culture medium, type 1 astual glial cells proliferate in this medium, causing a problem. However, the culture solution of the present invention has an effect of suppressing the growth of the cells. It is not reliable to distinguish type 1 and type 2 from the morphology of primary astroglial cells, and a method using immunocytochemical staining is used. Anti-GFAP antibody stains both types 1 and 2, while anti-A2B5 antibody stains only type 2. This difference in staining makes it possible to distinguish between type 1 and type 2.
胎児の脳を用いた場合は未分化細胞の混人の比率が高くなる。 本発明の培養液 は、 この未分化な幹細胞の一^ ^であるオリゴデンドログリア （又はォリゴデンド 口サイト） — 2型ァストログリア （又は 2型ァスト口サイ ト） 幹細胞 （以下〇— 2 A幹細胞） の増殖分化を誘導する効果がある。 培養を継続すると、 培養系に、 当初認められなかったォリゴデンドログリァを認めることができるようになる。 これは 0— 2 A幹細胞が増殖、 分化したものである。 オリゴデンドログ'リアであ ることは、 ァストログリァ細胞を判別する場合と同様の免疫紬胞化学染色の方法 で適用できる。 例えば抗 G C (ガラクトセレブ口シド） 抗体、 抗 M B P (ミエリ ン塩基性蛋白質） 抗体などを用いて染色すると明確な判別が可能である。  When the fetal brain is used, the ratio of mixed undifferentiated cells increases. The culture solution of the present invention contains oligodendroglia (or oligodend mouth site) which is one of the undifferentiated stem cells—type 2 astroglia (or type 2 fast mouth site) stem cells (hereinafter referred to as 〇-2A stem cells). It has the effect of inducing proliferation and differentiation. If the culture is continued, the culture system will be able to detect oligodendroglia, which was not initially recognized. This is the result of the proliferation and differentiation of 0-2A stem cells. The fact that the cells are oligodendroglia can be applied by the same method of immunocytochemical staining as in the case of identifying astroglial cells. For example, a clear distinction can be made by staining with an anti-GC (galactocereb mouth side) antibody, an anti-MBP (myelin basic protein) antibody, or the like.
具体的な神経細胞の培養方法は、 先ず前記と同様にして脳を切り出す。 そして トリプシン、 パパイン、 ディスパ一ゼなどの酵素を用いて神経細胞分散液を調製 する。 好ましくはパパイン （ 1 0〜 5 0 U /;^) を用いる方がよい。 ノ パインを 溶解したリン酸塩緩衝液に L一システィン （0 . 5〜5 m ) 、 グルコース （5〜 5 Ο πΛΙ) を加え、 3 7 °Cで 3 0〜 1 2 0分、 脳組織を酵素処理する。 分散酵素液 を丁寧に攪拌、 混和することにより神経細胞を分散させる。 デォキシリボヌクレ ァ―ゼ （0 . 0 1 %) を更に添加した方が漏出した核酸による細胞の凝集を防ぐ ことができる。  A specific method for culturing nerve cells is to first cut out the brain in the same manner as described above. Then, a nerve cell dispersion is prepared using enzymes such as trypsin, papain, and dispase. Preferably, papain (10 to 50 U /; ^) is used. L-cysteine (0.5-5 m) and glucose (5-5 5πΟ) are added to the phosphate buffer in which nopain is dissolved, and the brain tissue is removed at 37 ° C for 30-120 minutes. Enzyme treatment. The nerve cells are dispersed by carefully stirring and mixing the dispersed enzyme solution. Addition of deoxyribonuclease (0.01%) can prevent cell aggregation due to leaked nucleic acid.
次に、 遠心分離機により細胞を分離し、 前記のように調製した本発明の培養液 を用いて 1 0， 0 0 0〜2, 0 0 0, 0 0 Ocells/ の細胞液を調製し、 培養 用のプレート、 ディッシュなどを用いて、 37°Cの 5 %炭酸ガスインキュベータ 一で培養する。 培養に用いるプレート、 ディッシュ等は、 ガラス、 プラスチック 等材質は問わないが、 ポリリジシン、 ポリオル二チン、 ポリアリルァミン、 プロ 夕ミ ン、 ラミニン、 コラーゲン、 ゼラチン、 フイブロネクチン、 ビト αネクチン、 テネイシン、 及びこれらを混和したものを、 単層ないしは複層コートしたものを 用いるのが良い。 Next, the cells were separated by a centrifuge, and the culture solution of the present invention prepared as described above was used. Prepare a cell solution of 100,000 to 2,000,000 Ocells / using a plate, and culture it in a 5% CO 2 incubator at 37 ° C using a culture plate, dish, etc. I do. The plate, dish, etc. used for the culture can be of any material such as glass and plastic. It is preferable to use a single layer or a multi-layer coated layer.
実施例 Example
以下、 実施例に基づいて本発明を具体的に説明するが、 本発明はこれら実施例 に限定されるものではない。  Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples.
実施例 1、 比較例 1〜 2 Example 1, Comparative Examples 1-2
( 1 ) 培養液の調製  (1) Preparation of culture solution
ウィスター系ラッ 卜の新生仔 （生後 1 日） 3匹の脳より大脳を切り出し、 0. 2 5 %のトリプシン Ζリン酸塩緩衝液 （フロウ ·ラボラトリ一社製） を用い て 37°Cで 30分酵素処理した。 酵素液を除き、 1 0%牛胎児血清 （ハイク口— ン社製） 及びゲンタマイシン （シグマ社製） 5 011% ^を含む DMEMZF— 1 2等比混合培養液 （ライフ ·テクノロジ一社製） で組織を分散させ、 細胞分離 用遠心機 （クボタ社製） により 9 0 0回転、 5分の条件で細胞を遠心分離した。 分離した細胞は、 1 5^の同じ培養液を加え 75cm2 の培養フラスコ （住友べ 一クライ ト社製） を用いて、 37°C、 5 %の炭酸ガスインキュベータ一内で 1 0 日間培養した。 培養液を除き、 フラスコ内にコンフルェントに増殖した細胞を、 リン酸塩緩衝液で洗浄した後、 0. 25 %のトリプシン/リン酸塩緩衝液で、 37 °Cで 5分問酵素処理した。 ここで再び同じ培養液 1 5 0 ττ^を加えて細胞を分 散させ、 同様に遠心分離し 1 5 Ora の培養液に再分散させた後、 これを 2 2 5 cm2 の培養フラスコ （住友べ一クライト社製） 3個に分けて、 1 0日間培養した。 なお、 培養液は 2〜3日に 1回新しいものと交換した。 Newborn pups of Wistar rats (1 day after birth) The cerebrum was excised from the brains of three rats, and thawed at 37 ° C using 0.25% trypsin-phosphate buffer (Flow Laboratories, Inc.). The enzyme treatment was performed. Except for the enzyme solution, use a DMEMZF-12 isosteric mixture culture solution (manufactured by Life Technology Inc.) containing 10% fetal bovine serum (manufactured by Hike Corporation) and 5011% ^ of gentamicin (manufactured by Sigma). The tissue was dispersed, and the cells were centrifuged at 900 rpm for 5 minutes using a cell separation centrifuge (manufactured by Kubota Corporation). Separated cells 1 5 ^ with the same culture medium were added 75 cm 2 culture flasks (manufactured by Sumitomo base one Cry preparative Inc.) of and incubated for 1 0 days at 37 ° C, 5% of the inner carbon dioxide gas incubator one . After removing the culture medium, the cells grown confluently in the flask were washed with a phosphate buffer, and then subjected to an enzyme treatment with 0.25% trypsin / phosphate buffer at 37 ° C for 5 minutes. Here is dispersed cells divided by adding the same culture 1 5 0 ττ ^ Again, similarly after redispersed in broth centrifuged 1 5 Ora, which 2 2 5 cm 2 culture flasks (Sumitomo (Manufactured by Beclite Co., Ltd.) and cultured for 10 days. The culture medium was replaced with a new one once every 2-3 days.
培養液を除去し、 リン酸塩緩衝液で 2回洗浄した後、 インシュリ ン （5 g Z Ώの 、 トランスフエリ ン （ 5〃g /m£) 、 亜セレン酸ナトリウム （ 1 0 ΟηΜ) 、 スーパーォキシドジスム夕一ゼ （ 2. 5〃g 、 力タラ一ゼ （ 2. 5 zg / md) 、 プロジヱステロン （2 OnM) 、 酢酸トコフヱロール （ 1 /m ) (いず れもシグマ社製） 及びアルブミ ン （ALBUMAXTX I， ライフテクノロジ一社 製、 2. SmgZ^) を添加した DMEM/F - 1 2等比混合培養液を加えて、 1 日間培養し、 その培養上清を全量採取して、 再び同じ培養液を加えた。 同様の操 作を 1 0日間 （ 1 0回） 繰り返した。 比較例 1 として、 上記の添加物を加えずに、 同様に操作して培養上淸を採取した。 尚、 培養液の量は、 培養器の形態により適 宜増減することか可能であるが、 プレート、 フラスコ、 ディッシュ、 トレーなど では、 培養面積当たりでは 0. 1 5〜0. 3 5 mi /cm2 の範囲とするのか適切で ある。 After removing the culture medium and washing twice with a phosphate buffer, insulin (5 g ZΏ), transphenylene (5 g / m £), sodium selenite (10ΟηΜ), super Oxidism (2.5〃g), power (2.5 zg / md), prodosterone (2 OnM), tocoprol acetate (1 / m) (all manufactured by Sigma) and albumin (ALBUMAX TX I, manufactured by Life Technology, 2. SmgZ ^) -12 Equivalent mixed culture was added and cultured for 1 day. The whole culture supernatant was collected, and the same culture was added again. The same operation was repeated for 10 days (10 times). As Comparative Example 1, a culture supernatant was collected in the same manner without adding the above additives. The volume of the culture solution can be appropriately increased or decreased depending on the form of the incubator.However, for plates, flasks, dishes, trays, etc., 0.15 to 0.35 mi / cm / culture area The range of 2 is appropriate.
採取した培養上清は、 0. 22 m フィルター （ミ リポア社製） で濾過滅菌し て凍結保存し、 1 0回分の上清を採取した後、 解凍し全体をまとめて均一に混合 し、 一 7 0°Cで保存した。  The collected culture supernatant was sterilized by filtration with a 0.22 m filter (manufactured by Millipore), frozen and stored. After collecting the supernatant for 10 times, thawed and the whole was uniformly mixed and mixed. Stored at 70 ° C.
( 2 ) 神経細胞の培養試験  (2) Nerve cell culture test
試料となる神経細胞は、 胎生 1 7日のラッ 卜より大脳を切り出し、 ImMシステ イン、 2 5mMグルコース、 lmgZ アルブミン （各シグマ社製） を含有するリン 酸塩緩衝液で、 2 0 UZ?^に調製した、 パパイン （ワージン トン社製） を用いて 37でで 4 5分酵素処理した。 酵素液を除き、 DMEMZF - 1 2混合培養液で 钿胞を分散させた後、 700回転、 5分の遠心分離条件で細胞を分離した。  The nerve cells used as samples were cut out of the cerebrum from a 17-day-old rat embryo and used as a phosphate buffer containing ImM cysteine, 25 mM glucose, and lmgZ albumin (manufactured by Sigma) at 20 UZ? ^. The enzyme was treated with papain (manufactured by Worthington) at 37 for 45 minutes. After removing the enzyme solution and dispersing the cells with a DMEMZF-12 mixed culture solution, the cells were separated under centrifugation at 700 rpm for 5 minutes.
( 1 ) で得られた本発明の培養液を用いて、 25 0, 0 0 0 c/ の細胞濃度 の溶液を調製し、 ポリ リジンコート 2 4ゥヱルプレート （住友べ一クライ ト社 製） を用いて 0. 5?^/ゥエルで培養した。 比較例 2としては、 本発明の培養液 を加えない DMEM/F - 1 2液等比混合培養を用いて、 同じ条件で培養を行つ た。  Using the culture solution of the present invention obtained in (1), a solution having a cell concentration of 2500,000 c / was prepared, and a polylysine-coated 24-well plate (manufactured by Sumitomo Bei-Client) was used. And cultured at 0.5? ^ / ゥ. As Comparative Example 2, cultivation was performed under the same conditions using DMEM / F-12 two-component equal-density mixed culture to which the culture solution of the present invention was not added.
4曰間培養した後、 顕微鏡下で細胞の形態を観察した。 本発明の培養液を用い たものは、 良好な生存と長い神経突起の伸展が観察され、 シナプスを形成する突 起間のコンタク トがたいへん多く認められた。 しかし、 比較例 1の培養液を用い たものでは、 神経突起の伸展が著しく劣っており、 死細胞と思われる形態をして いるものが多く認められた。 また、 比較例 1、 2の培養液では、 生存細胞は認め られなかった。 更に、 生細胞と死細胞の比率を求めるため、 リン酸塩緩衝液に溶解し培養液に 加えた二酢酸フルォレセイン （シグマ社製） 1 0〃g Ζττ^とヨウ化プロビジゥム (シグマ社製） 1 5 ノ^とを反応させた。 蛍光顕微鏡 （オリンパス光学社 製） で異なった蛍光を発する生細胞と死細胞の比率を測定したところ、 本発明の 培養液で培養したサンプルでは、 生存率は 9 0%以上の良好な値を示した。 After incubation for 4 weeks, the morphology of the cells was observed under a microscope. In the culture using the culture solution of the present invention, good survival and long neurite outgrowth were observed, and very many contacts between protrusions forming synapses were observed. However, in the case of using the culture solution of Comparative Example 1, neurite outgrowth was remarkably inferior, and many of the morphologies considered to be dead cells were observed. In the culture solutions of Comparative Examples 1 and 2, no viable cells were observed. Furthermore, in order to determine the ratio of living cells to dead cells, fluorescein diacetate (Sigma) dissolved in phosphate buffer and added to the culture solution was added with 10〃g Ζττ ^ and probidium iodide (Sigma) 1 5 No ^ and reacted. When the ratio of living cells to dead cells emitting different fluorescence was measured with a fluorescence microscope (Olympus Optical Co., Ltd.), the sample cultured in the culture solution of the present invention showed a good survival rate of 90% or more. Was.
実施例 2、 比較例 3 Example 2, Comparative Example 3
実施例 1 と同様にして初代ァストログリア細胞を培養し、 培養上清を採取した。 得られた培養上清を用いて、 培養上清と DMEM/F - 1 2等比混合培養液の比 率が 7 5./ 25、 5 0 / 5 0、 25ノ 75、 及び 1 0 / 9 0になるようにそれぞ れ調製した。 更に、 比較例 3として、 DMEM F - 1 2等比混合液にゥシ胎児 血清 （ハイクローン社製） 1 0 %を加えたものを用いた。 実施例 1 と同様にして 神経細胞液を 20 0, 00 0
Figure imgf000013_0001
実施例 1 と同様にして培養 を行った。 Primary astroglial cells were cultured in the same manner as in Example 1, and the culture supernatant was collected. Using the obtained culture supernatant, the ratio of the culture supernatant to the DMEM / F-12 isosteric mixed culture solution was 75./25, 50/50, 25/75, and 10/9. Each was prepared to be 0. Further, as Comparative Example 3, a solution prepared by adding 10% of fetal calf serum (manufactured by Hyclone) to a DMEM F-12 equimolar mixture was used. In the same manner as in Example 1, the nerve cell fluid was added to 200,000
Figure imgf000013_0001
Culture was performed in the same manner as in Example 1.
4 日間培養した後、 顕微鏡下で観察したところ、 培養上清と DMEMZF— 1 2混合液の比を 75/25. 及び 5 0/50とした培養液では、 神経細胞の良 好な生存維持と神経突起の伸展が観察された。 2 5 /7 5の培養液では、 生存細 胞数がやや少なく、 1 0/9 0の培養液では、 生存細胞、 神経突起の伸展のいず れも劣っていた。 また、 比較例 3のゥシ胎児血清を用いた培養液では、 生存して いる神経細胞の数が少なく、 グリァ細胞の存在が多く認められた。  After culturing for 4 days and observing under a microscope, the culture supernatant with the ratio of the culture supernatant to the DMEMZF-12 mixture of 75/25. Neurite extension was observed. In the culture medium of 25/75, the number of surviving cells was slightly lower, and in the culture medium of 10/90, viable cells and neurite outgrowth were both inferior. In the culture solution using the fetal calf serum of Comparative Example 3, the number of surviving neurons was small, and the presence of glial cells was recognized.
培養上清と DMEMZF - 1 2混合液の比が 75 /2 5の培養液で培養したも の、 及び比較例 3の血清添加で培養したものについて、 神経細胞であることを確 認するため、 抗 MAP 2抗体 （ベーリンガーマンハイム社製） による免疫組織化 学染色を行った。 培養液を除きリン酸塩緩衝液で洗浄した後バラフオルムアルデ ヒド （和光純薬社製） 4 %のリン酸塩緩衝液で 20分、 トリ トン X— 1 0 0 (ベ 一リ ンガーマンハイム社製） の 0. 1 %リン酸塩緩衝液を 20分、 ヒッジ血清 1 %のリン酸塩緩衝液を 20分、 順次反応させ、 更に、 抗 MAP 2抗体をリン酸塩 緩衝液で 5 g
Figure imgf000013_0002
調製し、 3 0分間反応させた。 なお、 反応はいずれも室温 で行い、 各反応後にはリン酸塩緩衝液で洗浄した。 In order to confirm that the culture supernatant and the DMEMZF-12 mixture were cultured in a culture medium with a ratio of 75/25, and the culture in which serum was added in Comparative Example 3 were neurons, Immunohistochemical staining was performed with an anti-MAP2 antibody (Boehringer Mannheim). After removing the culture solution and washing with a phosphate buffer, Baraformaldehyde (manufactured by Wako Pure Chemical Industries) with 4% phosphate buffer for 20 minutes, Triton X—100 (Belinger Mannheim) Of 0.1% phosphate buffer solution for 20 minutes and 1% phosphate buffer solution of hidge serum for 20 minutes. Then, anti-MAP2 antibody was added with 5 g of phosphate buffer solution.
Figure imgf000013_0002
Prepared and reacted for 30 minutes. All reactions were performed at room temperature, and after each reaction, the cells were washed with a phosphate buffer.
次に、 ABC免疫組織化学キッ ト （ベクタ—社製） 、 及び DAB基質キッ ト (ベクター社製） で染色を行った。 その結果、 前記の形態観察から神経細胞と判 断された細胞は大半が染色され、 神経細胞であることが確認された。 し力、し、 比 較例 3の血清添加で培養した方の紬胞は染色される細胞が少なく、 形態からグリ ァ細胞と判断されるものは染色されなかった。 Next, the ABC immunohistochemistry kit (manufactured by Vecto) and the DAB substrate kit (Manufactured by Vector). As a result, most of the cells judged to be nerve cells from the morphological observation were stained, and it was confirmed that the cells were nerve cells. In the cells cultured in Comparative Example 3 with the addition of serum, the cells that had been stained had few cells stained, and those that were judged to be glial cells from the morphology were not stained.
実施例 3、 比較例 4 Example 3, Comparative Example 4
実施例 1 と同様にして神経細胞用培養液を調製した。 試料となる神経細胞は、 ウィスター系ラッ トの胎児 （胎生 1 6日） の海馬より、 実施例 1 と同様にして調 製した。 2 4ゥエルラ ミニンコートプレート （住友べ一クライ ト社製） に 2 5 0 , 0 0 0 cノウヱルの細胞を加え、 1 4日間培養した。 培養 3〜 5日の間 は、 シトシンァラビノフラノシド （シグマ社製） を 5 M加えた。 培養液の交換 は 1 / 2量を週 2度交換した。 また、 比較例 4として、 D M E M/ F— 1 2等比 混合培養液に、 インシュリ ン （ 5〃g /md) 、 トランスフヱリ ン （ 5〃g /m&) 、 プロジェステロン （ 2 0 nM) 、 及び亜セレン酸ナトリウム （2 0 n ) を加えたが、 アルブミンを加えないものを用いて、 同様にして培養を行った。  In the same manner as in Example 1, a culture solution for nerve cells was prepared. Nerve cells as samples were prepared in the same manner as in Example 1 from the hippocampus of a Wistar rat fetus (embryon day 16). 250,000 c-nodule cells were added to a 24 ゥ Erla minin coat plate (manufactured by Sumitomo BeiClient) and cultured for 14 days. Between 3 and 5 days of culture, 5 M of cytosine arabinofuranoside (Sigma) was added. For the exchange of the culture solution, the 1/2 volume was exchanged twice a week. Also, as Comparative Example 4, insulin (5 μg / md), transfrin (5 μg / m &), progesterone (20 nM), and Culture was carried out in the same manner using sodium selenate (20 n) but without albumin.
培養 1 4日の時点で観察したところ、 比較例 4の培養液を用いた方は細胞が全 て死滅していたが、 本発明の培養液を用いた場合では、 神経細胞間の緊密なネッ トワーク形成が観察された。  Observation on the 14th day of culture revealed that all the cells died when using the culture solution of Comparative Example 4, but when the culture solution of the present invention was used, the tight network between nerve cells was observed. Network formation was observed.
実施例 4、 比較例 4 Example 4, Comparative Example 4
実施例 1 と同様にして培養液を調製した。 比較例として比較例 3の培養液を使 用した。 実施例 1 と同様に神経細胞液を調製し 1， 2 0 0 eel I s/mm2の培養密度 でラミニンコ一ト 1 2ゥヱルプレート （住友べ一クライト社製） を用い、 8日間 培養した。 A culture solution was prepared in the same manner as in Example 1. As a comparative example, the culture solution of Comparative Example 3 was used. A nerve cell solution was prepared in the same manner as in Example 1, and cultured at a culture density of 1,200 eel I s / mm 2 for 8 days using a laminin-core 12-well plate (manufactured by Sumitomo Belite).
本発明の培養液を用いたものは、 安定に培養された神経細胞の他に、 形態から オリゴデンドログリアと判断される細胞が認められた。 また比較例の方は神経細 胞はわずかであり、 形態からァストログリア細胞と判断される細胞が大半であつ た。  In the case of using the culture solution of the present invention, in addition to the neurons stably cultured, cells that were judged to be oligodendroglia from their morphology were observed. In the comparative example, the number of nerve cells was small, and most of the cells were judged to be astroglial cells based on their morphology.
グリア細胞の種類を確認するため、 抗 G C抗体 （ベ—リンガーマンハイム社製、 5 ノ に調製） 、 抗 G F A P抗体 （ベ一リンガ一マンハイム社製、 8〃g Z に調製） 、 抗 A 2 B 5抗体 （ベーリンガ—マンハイム社製、 5 u Ζτηβに 調製） を用いて免疫細胞化学染色を行った。 培養液を除きリン酸塩緩衝液で洗浄 した後パラフォルムアルデヒド （和光純薬社製） 4 %のリン酸塩緩衝液で 2 0分、 トリ トン X— 1 0 0 (ベ一リンガ一マンハイ厶社製） の 0 . 1 ?4リン酸塩緩衝液 を 2 0分、 ヒッジ血清 1 %のリン酸塩緩衝液を 2 0分、 順次反応させ、 更に上記 抗体をそれぞれ 3 0分間反応させた。 各抗体の反応は比較例、 実施例各 1ゥエル ずつ計 6ゥエル、 いずれも室温で行い、 各反応液にはリン酸塩緩衝液で洗浄した。 次に、 A B C免疫組織化学キッ ト （ベクター社製） 、 及び D A B ( 3 , 3 ' 一 ジァミノべンジジン） 基質キッ ト （ベクター社製） で染色を行った。 本発明の培 養液を用いたもので、 形態からオリゴデンドログリアと判断される細胞はこの紬 胞と反応する抗 G C抗体で染色された。 To confirm the type of glial cells, anti-GC antibody (manufactured by Boehringer Mannheim, adjusted to 5 g), anti-GFAP antibody (manufactured by Boehringer Mannheim, adjusted to 8 g Z), anti-A2B 5 Antibody (Boehringer Mannheim, 5 u Ζτηβ Preparation) was used for immunocytochemical staining. After removing the culture solution and washing with a phosphate buffer, paraformaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) is used for 20 minutes in a 4% phosphate buffer. Was reacted for 20 minutes with a 0.1-4 phosphate buffer solution (manufactured by K.K.) and 20 minutes with a phosphate buffer solution containing 1% of hedged serum, and then the above antibodies were reacted for 30 minutes. The reaction of each antibody was performed at room temperature for a total of 6 wells, 1 well each for the comparative example and the working example. Each reaction solution was washed with a phosphate buffer. Next, staining was performed with an ABC immunohistochemistry kit (manufactured by Vector) and a DAB (3,3'-diaminobenzidine) substrate kit (manufactured by Vector). Cells that were determined to be oligodendroglia from the morphology using the culture solution of the present invention were stained with an anti-GC antibody that reacts with the cells.
一方、 比較例の形態からァストログリア細胞と判断される細胞は、 抗 G F A P 抗体で染色され、 抗 A 2 B 5抗体には染色されないことから 1型ァス卜ログリア 細胞と判断された。 産業上の利用可能性  On the other hand, cells determined to be astroglial cells from the morphology of the comparative example were stained with an anti-GFAP antibody and not stained with an anti-A2B5 antibody, and thus were determined to be type 1 astroglial cells. Industrial applicability
本発明による神経細胞用培養液を用いることにより、 中枢神経細胞の培養を安 定に行うことができ、 低密度培養においては神経突起の伸展性に優れ迅速なシナ ブス形成が可能であり、 高密度培養においては神経ネッ トワークを形成した細胞 の長期安定性に優れている。 これにより神経薬理試験、 神経情報伝達試験等を確 度高く実施することができ、 神経薬理、 衛生化学などの分野で痴呆症、 神経疾患、 神経毒性などの研究、 病態の解明等に役立つ。  By using the culture solution for nerve cells according to the present invention, the culture of central nerve cells can be performed stably, and in low-density culture, extensibility of neurites is excellent and rapid synapse formation is possible. In density culture, cells that have formed a neural network have excellent long-term stability. This makes it possible to carry out neuropharmacological tests, neuronal information transmission tests, etc. with high accuracy, and is useful for research on dementia, neurological diseases, neurotoxicity, etc. in the fields of neuropharmacology and sanitary chemistry, and for elucidation of pathological conditions.

Claims

請 求 の 範 囲 The scope of the claims
1 . インシュリ ン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プロジエス テロン、 アルブミ ン、 スーパ一ォキシドジス厶ターゼ及ぴ力夕ラーゼ、 又はイン シュリ ン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プロジェステロン、 アルブミン及びひ—トコフヱロール類を添加した栄養培地中で初代ァス卜口グリ ァ紬胞を培養して採取した培養上清を含有することを特徴とする神経細胞用培養 液。 1. Insulin, transferrin, selenite or a salt thereof, progesterone, albumin, superoxide dismutase and phosphatase, or insulin, transferrin, selenite or a salt thereof, or a project. A culture solution for nerve cells, comprising a culture supernatant obtained by culturing primary astrocyte glial cells in a nutrient medium supplemented with sterone, albumin and heart coproles.
2 . 栄養培地中に、 インシュリン、 トランスフヱリ ン、 亜セレン酸若しくはその 塩、 プロジェステロン、 アルブミ ン、 スーパ一ォキシドジスム夕ーゼ、 力夕ラー ゼ及びひ - トコフエロール類を添加するものである請求項 1記載の神経細胞用培 養液。  2. The nutrient medium is supplemented with insulin, transfusin, selenous acid or a salt thereof, progesterone, albumin, superoxide dismutase, lipase lase and sodium tocopherol. The culture solution for nerve cells as described.
3 . 栄養培地が、 イーグルの基本培地 （M E I) 、 ダルベッコ改変イーグル培地 ( D M E M) 、 ハムの F— 1 2培地及びハムの F _ 1 0培地から選ばれる 1種又 は 2種以上である請求項 1又は 2記載の神経細胞用培養液。  3. The nutrient medium is one or more selected from Eagle's basic medium (MEI), Dulbecco's modified Eagle medium (DMEM), Ham's F-12 medium and Ham's F_10 medium. Item 3. The culture solution for nerve cells according to Item 1 or 2.
4 . 初代ァス卜ログリア細胞を動物血清を添加した培地中で培養、 増殖させた後、 インシュリン、 トランスフェリ ン、 亜セレン酸若しくはその塩、 プロジエステ口 ン、 アルブミ ン、 スーパ一ォキシドジス厶ターゼ及び力タラ一ゼ、 又はインシュ リ ン、 トランスフェリン、 亜セレン酸若しくはその塩、 プロジェステロン、 アル ブミン及びひ一トコフヱロール類を添加した栄養培地中で培養し、 その上清を採 取することを特徴とする神経細胞用培養液の製造方法。  4. After culturing and growing primary astroglial cells in a medium supplemented with animal serum, insulin, transferrin, selenite or a salt thereof, progestestin, albumin, superoxide dismutase Culture in a nutrient medium supplemented with insulin, transferrin, insulin, transferrin, selenite or a salt thereof, progesterone, albumin, and glucose, and collecting the supernatant. A method for producing a culture solution for nerve cells.
5 . 栄養培地中に、 インシュリ ン、 トランスフヱリ ン、 亜セレン酸若しくはその 塩、 プロジェステロン、 アルブミ ン、 スーパーォキシドジスム夕一ゼ、 力夕ラー ゼ及びひ -トコフエロール類を添加するものである請求項 4記載の神経細胞用培 養液の製造方法。  5. The nutrient medium is supplemented with insulin, transfusin, selenous acid or its salt, progesterone, albumin, superoxide dismutase, lipase lase, and o-tocopherols. A method for producing the culture solution for nerve cells according to claim 4.
6 . 栄養培地が、 イーグルの基本培地 （M E M) 、 ダルベッコ改変イーグル培地 ( D M E M) 、 ハムの F— 1 2培地及びハムの F - 1 0培地から選ばれる 1種又 は 2種以上である請求項 4又は 5記載の神経細胞用培養液の製造方法。  6. The nutrient medium is one or more selected from Eagle's basal medium (MEM), Dulbecco's modified Eagle medium (DMEM), Ham's F-12 medium and Ham's F-10 medium. Item 6. The method for producing a culture solution for nerve cells according to Item 4 or 5.
7 . 請求項 1 〜 3のいずれか 1項記載の神経細胞用培養液中で神経細胞を培養す ることを特徴とする神経細胞の培養方法。 7. The method for culturing nerve cells in the culture solution for nerve cells according to any one of claims 1 to 3. A method for culturing a nerve cell, comprising:
PCT/JP1996/001765 1996-06-10 1996-06-26 Liquid medium for nerve cells, process for producing the same and method for incubating nerve cells with the use of the same WO1997047733A1 (en)

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JPH05111381A (en) * 1991-10-22 1993-05-07 Bio Material Kenkyusho:Kk Medium for neutral nerve cell
JPH07227278A (en) * 1994-02-18 1995-08-29 Kyorin Pharmaceut Co Ltd Serum-free medium for mature central neurocyte

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JPH07227278A (en) * 1994-02-18 1995-08-29 Kyorin Pharmaceut Co Ltd Serum-free medium for mature central neurocyte

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