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Process for producing nerve cells

A technology of nerve cells and neural stem cells, which is applied in the field of treatment of neurodegenerative diseases or nerve damage, and can solve problems such as being unsuitable for a large number of neural stem cells, difficult for neural stem cells, and slow in proliferation of neural stem cells.

Inactive Publication Date: 2005-08-31
MITSUBISHI TANABE PHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current situation is that it is difficult to maintain neural stem cells in an undifferentiated state, and since the proliferation of the neural stem cells is slow, it is not suitable for purposes requiring a large number of neural stem cells

Method used

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  • Process for producing nerve cells
  • Process for producing nerve cells
  • Process for producing nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1 Preparation of neural stem cells

[0149] As embryonic stem cells, an HK cell line (passage number 10 or less) established from an embryonic primordium of a C57BL / 6 mouse (day 3.5 after confirmation of a vaginal plug) was used by a conventional method. The aforementioned HK cells are cells with a small number of passages and are difficult to undergo spontaneous differentiation.

[0150] Fibroblasts prepared from syngeneic mice on day 14 of pregnancy were cultured to confluence in DMEM medium containing 10% (w / v) fetal calf serum (FCS). Then, mitomycin C (1 µg / ml) was added to the obtained cell culture, and 3 mice were incubated to obtain inactivated cells. Passivated cells were washed with phosphate-buffered saline and then trypsinized. The resulting cells were seeded on gelatin-coated plates to obtain feeder cell layers. A 60mm flat plate (1.5×10 6 cells / plate) and 4-well plates (3×10 5 cells / well).

[0151] Inoculate the above-mentioned HK cell lines o...

Embodiment 2

[0161] Example 2 Differentiation into nerve cells

[0162] Neural stem cells are differentiated and induced to the superficial SCS [SCS (suspension culture for 4 days) obtained in the above-mentioned embodiment 1] in a mixture of astrocyte conditioned medium and astrocyte basal medium (volume ratio 1: 1) in CO 2 In the incubator, 37°C, CO in the air 2 Suspension culture was carried out under 5% concentration and 100% humidified atmosphere.

[0163] In addition, in order to study the time-dependent change of the differentiation state in SCS, the suspension-cultured SCS was fixed in the same manner as in the above operation during 1 to 4-day culture.

[0164] As an indicator of differentiation into neurons, antibody TUJ1 that recognizes class III β-tubulin, a marker of weak neurons, was used. An upright fluorescent microscope (trade name: Eclipse E800) manufactured by Nikon was used for immunofluorescence histochemical observation.

[0165] As a result, it was observed in the i...

Embodiment 3

[0168] Example 3 Improvement of nerve cell differentiation method

[0169] By suspending culture of SCS in a mixture of astrocyte conditioned medium and astrocyte basal medium, neural stem cells can be prepared and induced to differentiate into immature neurons.

[0170] Next, the optimal culture conditions for the differentiation into neurons of the neural stem cells formed on the surface of the SCS were studied.

[0171] For the culture side surface of the culture dish coated with polylysine, further use 0.1mg / ml laminin or MATRIGEL diluted 10-20 times TM [manufactured by BD Bioscience] was treated to prepare an adhesive culture substrate. The 4-day suspension-cultured SCS was transferred to an adherent culture dish pre-added with astrocyte conditioned medium with a glass capillary tube, and stored in CO 2 In the incubator, 37°C, CO 2 Cultivate for several hours under 5% concentration and 100% humidified atmosphere. Results SCS adhered to the culture substrate.

[0172]...

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Abstract

To supply substantially isolated neural cells in a large amount, and to provide an application means for a neurodegenerative medicine or the like for a neurodegenerative disease, a nervous damage or the like. A method for producing a substantially isolated neural cell, comprising the step of carrying out the suspension culture of embryonic stem cells in the presence of an astrocyte conditioned medium or ingredients substantially equivalent to the conditioned medium; and a neural cell obtained thereby; a cell pharmaceutical composition comprising, as an active ingredient, the isolated neural stem cell; and a method for treating a neurodegenerative disease or nervous damage, comprising the step of introducing the neural cell into a neurodegenerative site or a nervous damage site.

Description

technical field [0001] The present invention relates to a method for producing substantially isolated neural cells, substantially isolated neural stem cells, substantially isolated neurons, substantially isolated glial cells, neural cells of cell pharmaceutical compositions, and neurodegenerative diseases or treatments for nerve damage. Background technique [0002] At present, the production of nerve cells from embryonic stem cells is mainly carried out by the following four methods. [0003] ① A method of treating suspension cultures of embryonic stem cell aggregates with retinoic acid and inducing their differentiation into various nerve cells [Bain, G. et al., Dev. Biol., 168: 642-657 ( 1995)] [0004] ② After preparing embryoid bodies [called EBs] and culturing the EBs with serum-free medium to obtain neuroepithelial stem cell protein-positive neural stem cells, in the presence of basic fibroblast growth factor (bFGF), A method for culturing the neural stem cells and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/12A61K35/30A61K35/545A61L27/38A61P25/00C12N5/07C12N5/0735C12N5/079C12N5/0793C12N5/0797
CPCC12N2501/115C12N5/0618C12N2506/02C12N2500/44C12N2501/235C12N2501/06C12N5/0619C12N2502/13C12N5/0623A61P25/00A61P25/16A61P25/28C12N5/00A61K35/30
Inventor 中山孝井上顺雄近藤靖铃木丰
Owner MITSUBISHI TANABE PHARMA CORP
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