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Method for cultivating human umbilical blood stem cell and directionally differentiating the same to dopaminergic nerve cell and application for the obtained dopaminergic nerve cell

A technology for umbilical cord blood stem cells and umbilical cord blood stem cells is applied in the field of culture and differentiation of human umbilical cord blood stem cells, which can solve the problems of difficulty and ethics in obtaining seed cells.

Inactive Publication Date: 2007-10-17
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems such as difficulty in obtaining materials and ethics in the source of seed cells

Method used

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  • Method for cultivating human umbilical blood stem cell and directionally differentiating the same to dopaminergic nerve cell and application for the obtained dopaminergic nerve cell
  • Method for cultivating human umbilical blood stem cell and directionally differentiating the same to dopaminergic nerve cell and application for the obtained dopaminergic nerve cell
  • Method for cultivating human umbilical blood stem cell and directionally differentiating the same to dopaminergic nerve cell and application for the obtained dopaminergic nerve cell

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preparation example Construction

[0056] A. Preparation of striatal conditioned medium:

[0057] Cut the striatal tissue from the brain tissue of mammals (such as rats, mice, rabbits, and monkeys), carefully remove fibrous structures such as meninges and blood vessels under a dissecting microscope, pipette into a single-cell suspension with PBS, and centrifuge and wash for 2 ~3 times, then resuspended with L-DMEM / F12 complete culture medium (containing 10% FBS, 100IU / ml penicillin and 100IU / ml streptomycin), inoculated in a culture bottle placed at 37°C, 5% CO 2 cultured in a humidified incubator. The cell culture fluid was collected every day, centrifuged at 3000r / min×20min, the supernatant was filtered through a 0.22μm microporous membrane, and frozen for future use. Before use, it is mixed with H-DMEM complete culture medium at a ratio of 3 / 2 (v / v) to prepare striatal conditioned medium.

[0058] B. Preparation of conditioned medium for astrocytes:

[0059] The striatal cells were firstly cultured, and w...

Embodiment 1

[0074] Example 1 Isolation, cultivation and identification of umbilical cord blood stem cells

[0075] Separation and culture: collect 40-60ml of umbilical cord blood from healthy newborns, dilute it with sterile PBS in equal volume, then add 0.5% methylcellulose solution at a ratio of 1:4, mix well, let stand for 40-60min, and draw plasma After centrifuging the supernatant, resuspend with PBS to prepare a single cell suspension, then superimpose on an equal volume of lymphocyte separation medium, centrifuge at 1800r / min×20min, carefully absorb the interface cell layer, wash 3 times with PBS, and then Resuspend with H-DMEM complete culture medium (containing 10% FBS, 100IU / ml penicillin and 100IU / ml streptomycin), in 5×10 6 The density of cells / ml was seeded in a 12-well plate coated with poly-lysine containing coverslips, placed at 37°C, 5% CO 2 cultured in a humidified incubator. According to the shape of the cells, replace the culture medium in full for the first 7 days, ...

Embodiment 2

[0078] The preparation of embodiment 2 striatum conditioned medium

[0079] Newborn SD rats (within 24 hours after birth) were killed by neck dislocation, and the brain tissue was completely stripped, placed in PBS at 4°C, and the striatum tissue was cut out. The fibrous structures such as meninges and blood vessels were carefully removed under a dissecting microscope, and blown into PBS The single cell suspension was centrifuged and washed 2 to 3 times, and then resuspended with L-DMEM / F12 complete culture medium (containing 10% FBS, 100IU / ml penicillin and 100IU / ml streptomycin), and mixed with 2.5×10 5 The density of cells / ml was seeded at 25cm 2 Place the flask at 37°C, 5% CO 2 cultured in a humidified incubator. After 3 days, the medium was completely changed, and on the 6th day, the complete H-DMEM medium was added. The cell culture medium was collected every day, centrifuged at 3000r / min×20min, and the supernatant was filtered through a 0.22μm microporous membrane, an...

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Abstract

The present invention relates to method for human navel blood stem cell cultivation and directional differentiation to dopaminergic nerve cell, the dopaminergic nerve cell and its uses. The method comprises the following steps: a) navel chord blood stem cell culture; separating navel chord blood single-nucleus cell, then inoculating it at porous plate with cover glass, culturing at culture box of 36.5-37.5 degree, 3-8% CO2, saturated humidity, the cultured navel blood stem cell being obtained. b) dopaminergic nerve cell induction and differentiation : taking navel blood stem cell of good growth state, changing liquid when the stem cell grows for12-16 days and about 70-90% cell fuse, inducing cell with conditioned medium inductor or growth factor inductor, so dopaminergic nerve cell being obtained. The present invention can provides large dopaminergic nerve cell differentiated from navel chord blood stem cell.

Description

technical field [0001] The invention relates to the cultivation and differentiation of human umbilical cord blood stem cells, in particular to a method for culturing and directional differentiation of human umbilical cord blood stem cells into dopaminergic nerve cells, the obtained dopaminergic nerve cells and applications thereof. Background technique [0002] Dopamine (DA) nerve cells play a very important role in the body's own movement, emotional behavior and cognitive ability. The damage or functional degeneration of DA nerve cells in the substantia nigra will reduce the dopamine content in the striatum. And then lead to Parkinson's disease (Parkinson's disease, PD). At present, cell transplantation is a new therapeutic approach to treat such neurological diseases, so how to obtain a large number of DA nerve cells in vitro is the key to Parkinson's disease cell transplantation and replacement therapy. Studies have found that human or mouse bone marrow mesenchymal stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A61K35/30A61P25/16A61K35/51C12N5/0789C12N5/079
Inventor 季凤清杨慧孙海梅赵春礼赵焕英
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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