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Cell subpopulation with therapeutic effect on brain injury diseases and preparation method thereof

A technology of cell subsets and therapeutic effects, applied in the field of biomedicine, can solve problems such as unreachable treatment and no specific treatment of brain injury diseases

Inactive Publication Date: 2018-09-21
SHANGHAI ANGECON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, all the drugs for the treatment of brain injury mostly provide nutrition and relieve symptoms, but cannot achieve the purpose of treatment
That is to say, there is currently no effective drug for the specific treatment of brain injury diseases on the market

Method used

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  • Cell subpopulation with therapeutic effect on brain injury diseases and preparation method thereof
  • Cell subpopulation with therapeutic effect on brain injury diseases and preparation method thereof
  • Cell subpopulation with therapeutic effect on brain injury diseases and preparation method thereof

Examples

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preparation example Construction

[0034] The present invention provides a method for preparing the above-mentioned cell subpopulation, comprising the following steps:

[0035] 1) inoculating the single cell suspension of adult neural tissue or induced pluripotent stem cells into complete serum-free medium for suspension culture to obtain primary cells;

[0036] 2) Cultivate the primary cells obtained in step 1) for 12 to 16 days to obtain primary nerve cell subpopulations, perform repeated digestion and subculture of the primary neuronal cell subpopulations in sequence, and pass to the second generation neuron cell subgroups. Five passages, P5 generation cells were obtained;

[0037] 3) The P5 generation cells obtained in step 2) are subjected to cell subpopulation detection, and the cells with a qualified positive rate are used as seed cells;

[0038] 4) The seed cells obtained in step 3) are amplified and subcultured to P6 to P9 generations to obtain cell subgroups.

[0039] When preparing the above-mentio...

Embodiment 1

[0058] Put the spinal cord tissue in the nerve tissue buffer solution in a 100mm sterile petri dish, and wash it repeatedly with the buffer solution. Under the dissecting microscope, hold ophthalmic tweezers in one hand and ophthalmic scissors in the other, peel off the connective tissue around the spinal cord tissue and the surrounding vascular membrane, separate the spinal cord with fine surgical forceps, and put it in the buffer solution of a 35mm sterile Petri dish. Then use fine surgical forceps to divide the spinal cord into tissue pieces about 1mm in size.

[0059] Transfer the obtained tissue and buffer solution into one or more 15 / 50ml centrifuge tubes with a sterile disposable 3ml dropper, and let it stand for 5 minutes to allow the tissue pieces to fully settle to the bottom of the tube, and discard the supernatant as much as possible. Then perform sterile filtration with a sterile syringe and a 0.22um filter membrane, and filter directly into a centrifuge tube. Eac...

Embodiment 2

[0068] Use purchased IPSC cells to culture IPSC cells in a 6-well plate. When the density reaches 80%-90%, replace the differentiation medium (N2B27 contains 5mmol / L SB431542, 5mmol / L drosomophorin), and then replace it every other day. 1 culture medium, the cells were scraped off on the 7th day, and transferred to a pretreated 6-well plate for culture, and the medium contained 2% N2 and 2% B-27. Then observe, after the cells adhered to the wall for 2 days, the medium was changed, and the culture was continued, and the medium was changed every other day until the 15th day. The cells were digested, washed once with DMEM / F12 medium, cultured in a 6-well plate with complete serum-free medium for 1 week, digested, centrifuged, and the supernatant was removed, and the cells were blown away to obtain a single cell suspension.

[0069] The steps after obtaining the single cell suspension are exactly the same as in Example 1.

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Abstract

The invention provides a cell subpopulation with a therapeutic effect on brain injury diseases and a preparation method thereof, and belongs to the field of biomedicine. The cell subpopulation is fromadult neural tissue or induced pluripotent stem cells; the cell subpopulation is detected by using specific markers including nidogen, PAX6, SOX2, vimentin, Musashi-1 and NGFR. The cell subpopulationis a single cell subpopulation, when a P5 generation is cultured in vitro, the purity can reach 90% or above, the cell activity is 95% or above, and the cell subpopulation can still keep good drynessand differentiation potential even if a 32nd generation is cultured through continuous passage; a neuronal death and inflammatory response after a brain injury can be reversed by adjusting an NF-kappaB pathway reaction, and the effect of inhibiting the proliferation of lymphocytes and immune cells is achieved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a cell subgroup having a therapeutic effect on brain injury diseases and a preparation method thereof. Background technique [0002] Brain injury usually includes ischemic brain injury (such as stroke), ischemic and hypoxic brain injury (such as cerebral palsy in children) and traumatic brain injury. Brain-injury diseases impose a serious burden on individual health and society. Stroke is the third leading cause of disability and death in adults in developed countries. Cerebral palsy in children usually refers to central movement disorders caused by non-progressive brain damage or abnormal brain development caused by various reasons before birth to one month after birth. Clinically, it is characterized by abnormal posture and muscle tone, muscle weakness, involuntary movement, and ataxia, often accompanied by sensory, cognitive, communication, behavioral and other disorde...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/0797A61P25/00
CPCA61K35/30A61K35/545C12N5/0623C12N5/0696
Inventor 郑佳威赵雄飞黄倩莹王晓明
Owner SHANGHAI ANGECON BIOTECH
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