Established cell line of microglia

Abstract

A subcultivatable, established microglia having the following properties. (a) Form: Both or either of a macrophage-like or spherical form in the presence of a granulocyte-macrophage colony stimulation factor and a branched form similar to branched microglia present in the brain in the absence of the factor. (b) Functional characteristics: specific affinity for the brain highly poor phagocytic action. (c) Cell proliferation: proliferative depending upon a granulocyte-macrophage colony stimulation factor. Preparation, separation, and screening methods of the microglia, a pharmaceutical composition using the microglia, and a method for treatment of cerebral diseases using the composition.

Description

[0001] The present invention relates to a subcultivatable, established cell line of microglia, a method of separating the same, and use thereof as a pharmaceutical carrier.[0002] Further, the present invention relates to a microglia into which an extraneous gene or a drug was introduced, a method of introducing it, and a pharmaceutical composition comprising the same.[0003] There are quite a number of hereditary diseases in the nervous system, which occur due to various causative factors, for example by defect of a single enzyme, etc. or by unknown reasons. Under these circumstances, supplementary therapy is used to cope with a large number of such diseases.[0004] A large number of studies have been made worldwide on the system of selective delivery to the brain. For introducing a gene into the brain in an animal, a method of using neuronphilic viruses as adenovirus vectors is devised, and a system for introducing a gene specifically into neurons is known (Kozarsky, K. F and Wilson,...

AI Technical Summary

Benefits of technology

[0033] The established cell line of microglia of the present invention can be purified from mouse or rat brain cells after primary culture and then separated by the following means from this purified microglia. In addition, because the established cell line of microglia of the present invention is easily handled and has affinity for the brain, a gene or drug can be introduced into the established cell line of microglia and injected into peripheral blood vessels to express the gene in the brain or to deliver the drug to the brain specifically.

[0040] The adherent cells which do not float by the above mechanical stimulation are treated with trypsin-EDTA, then divided into single cells, plated on a non-treated plastic dish (non-coat plastic vessel), and allowed to adhere to it. Generally, a conventional culture vessel is treated with chemicals so as to be positively charged, but a dish not subjected to this treatment should be used in order to obtain the adherent cells. After incubation in a CO.sub.2 incubator at 37.degree. C. for 1 hour, cells floating in the medium after mechanical stimulation are removed, and cells capable of proliferation on the vessel are recovered by Rubber Policeman, etc., and the same procedure is repeated twice to obtain cells to be used in establishing a cell line of microglia. Although the purified microglia thus obtained is of adequate purity, cell sorter, etc. can also be used to further improve purity.

[0054] As a means of efficiently introducing a gene into the established cell line of microglia, there is for example a method of using DOTAP (Boehringer-Mannheim Co.). That is, there is a method of culturing the established cell line of microglia together with a desired gene in a gene-introducing medium containing DOTAP. They are cultured in a CO.sub.2 incubator at 37.degree. C. for 16 to 24 hours and further cultured together with rGM-CSF for 30 to 72 hours (preferably for 48 hours) in a medium for culturing microglia.

[0057] When this screening method is applied to the microglia, the microglia coming to express the introduced gene stably and constantly recognize dead cells thus expressing a strong phagocytic ability and simultaneously undergoing activation to lose their cell growth ability. Accordingly, the method of introducing a gene in the present invention is preferably a method wherein an expression vector modified so as to be capable of expressing a fluorescent protein, preferably green florescent protein (GFP) derived from an aurelia, in higher animals is used in place of a drug resistance gene, and by using a difference in fluorescence intensity, the microglia is separated as the desired cells exhibiting stable and constant expression.

[0059] The cells can also be confirmed using magnetic resonance image (MRI), positron emission tomography (PET), etc. For example, contrast media, etc. for MRI may be incorporated into the cells which are then injected into an animal so that the cells can be monitored in the animal. According to these methods, it is not necessary to kill the animal, and the cells can be monitored easily in a non-invasive manner.

Problems solved by technology

In the brain, however, the blood-brain barrier is present, so it is difficult to conduct supplementary therapy and to introduce an effective drug, and even if a substance (e.g. anticancer drug, DNA, etc.) is introduced from a peripheral position, it cannot be introduced specifically into the brain.

Therefore, there was no method other than direct injection of the substance by surgical operations.

However, even in this method too, incorporation of liposomes into the brain is as low as about 1% based on the injection amount, so this method cannot be said to be specific for the brain.

As described above, the blood-brain barrier is present in the brain, so the brain is almost free of infiltration with cells or a substance from the periphery, thus making it difficult to introduce a drug or a gene into the brain.

For this primary culture, however, the brain should be excised and purified for use, and primary culture usually requires a period of about 2 weeks, so the procedures are cumbersome.

Further, the cells are difficult to proliferate during culture and hard to subculture, so after primary culture, it is extremely difficult to introduce a gene into the microglia to express it therein.

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