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Neural stem cell preparation, preparing method thereof and use of same

A technology of neural stem cells and preparations, applied in the field of neural stem cell preparations, can solve the problems such as the preparation methods of neural stem cell preparations that have not been reported in the literature, and achieve the effects of strong cell proliferation ability, high yield, and simple methods

Inactive Publication Date: 2005-03-23
北京科宇联合干细胞生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But so far there is no published literature reporting neural stem cell preparations and preparation methods thereof

Method used

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  • Neural stem cell preparation, preparing method thereof and use of same
  • Neural stem cell preparation, preparing method thereof and use of same
  • Neural stem cell preparation, preparing method thereof and use of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The subventricular zone (SVZ) was isolated from fetal brains that had died at 11 weeks, digested with 0.01% EDTA and 0.25% trypsin for 3 minutes, and added 50ml of DMEM / F12 medium containing 10% fetal bovine serum to terminate the digestion , centrifuged and pipetted with culture medium to form a single cell suspension, 5% CO 2 , Culture at 37°C.

[0054] Cultured in vitro, screened out neural stem cell spheres, that is, neural stem cells. Neurospheres formed in the subependymal zone (SVZ) in 2-3 days.

[0055] The medium conditions are:

[0056] (1)DMEM / F12 12mg / ml

[0057] (2) bFGF (basic fibroblast growth factor) 10ng / ml

[0058] (3) EGF (epidermal growth factor) 20ng / ml

[0059] (4) transferring (transferrin) 0.1mg / ml

[0060] (5) Sodium Selenite (sodium selenate) 5.2ng / ml

[0061] (6) Putresine (putrescine) 9.6μg / ml

[0062] (7) Insulin (insulin) 0.025mg / ml

[0063] (8) L-Glutamine (Glutamine) 0.3mg / ml

[0064] (9) Progerterone 6.3ng / ml

[0065] (10) Ant...

Embodiment 2

[0068] The hippocampus tissue was isolated from the dead fetal brain at 15 weeks, digested with 0.20% trypsin digestion solution for 4 minutes, added 70ml of DMEM / F12 medium containing 20% ​​fetal bovine serum to terminate the digestion, centrifuged at 1200RPM for 10min, and then added the medium Mechanical pipetting for single cell suspension culture, 5% CO 2 , Culture at 37°C.

[0069] Cultured in vitro, screened out neural stem cell spheres, that is, neural stem cells. Neuron spheres were formed in the hippocampus for 5 days. Passed on for 3 generations.

[0070] The medium conditions are:

[0071] (1) DMEM / F12 (Hyclone) 20mg / ml

[0072] (2) bFGF (basic fibroblast growth factor) 15ng / ml

[0073] (3) EGF (epidermal growth factor) 10ng / ml

[0074] (4) transferring (transferrin) 0.5mg / ml

[0075] (5) Sodium Selenite (sodium selenate) 2ng / ml

[0076] (6) Putresine (putrescine) 7μg / ml

[0077] (7) Insulin (insulin) 0.05mg / ml

[0078] (8) L-Glutamine (Glutamine) 0.1mg / m...

Embodiment 3

[0083] The midbrain tissue was separated from the dead fetus at 14 weeks, digested with 200u / ml collagenase digestion solution for 3 minutes, added 30ml DMEM medium containing 15% fetal bovine serum to stop the digestion, centrifuged at 1200RPM for 10min, and then added the medium Mechanical pipetting for single cell suspension culture, 5% CO 2 , Culture at 37°C.

[0084] Cultured in vitro, screened out neural stem cell spheres, that is, neural stem cells. Neuron spheres were formed in the midbrain tissue at 7 days. 5 generations passed on.

[0085] The medium conditions are:

[0086] (1) DMEM / F12 (Hyclone) 8mg / ml

[0087] (2) bFGF (basic fibroblast growth factor) 20ng / ml

[0088] (3) EGF (epidermal growth factor) 15ng / ml

[0089] (4) transferring (transferrin) 0.05mg / ml

[0090] (5) Sodium Selenite (sodium selenate) 10ng / ml

[0091] (6) Putresine (putrescine) 5μg / ml

[0092] (7) Insulin (insulin) 0.01mg / ml

[0093] (8) L-Glutamine (Glutamine) 0.5mg / ml

[0094] (9) ...

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Abstract

A neural stem cell preparation for treating human myelopathy, especially the motor neuron diseases, is prepared through digesting the brain tissue of human embryo, adding to culture medium, centrifugal processing, mechanical beating to obtain cell suspension, culturing in culture medium in CO2 atmosphere, screening neural stem cell balls, and passage by 3-6 generations.

Description

field of invention [0001] The invention relates to a neural stem cell preparation, its preparation method and its use as a treatment for human spinal cord diseases, belonging to the field of biological products. Background technique [0002] Human spinal cord diseases are mainly divided into two categories, ① Motor neuron disease (Motor neuron disease, MND), motor neuron disease is a group of chronic degenerative diseases that mainly affect the upper and lower motor neurons. Depending on the location of the injury, it can be divided into lower motor neuron type (progressive spinal muscular atrophy and progressive bulbar palsy), upper motor neuron type (primary lateral sclerosis), and mixed type (amyotrophic lateral sclerosis) and other syndromes. The course of disease is generally 1-7 years, with an average of 5 years. ② primary and secondary spinal cord injury. Primary spinal cord injury refers to the damage of gray matter and white matter caused by trauma itself, and th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K35/30A61K35/54A61P21/00A61P25/00A61P43/00C12N5/0797
Inventor 沈丽李凌松张浣清李肖霞
Owner 北京科宇联合干细胞生物技术有限公司
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