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Separating and culturing process of human amnion mesenchyme stem cell and its medical composition

A technology of stromal stem cells and culturing methods, applied in the field of separation and culturing of mesenchymal stem cells, can solve the problems of limited number of stem cells due to ethical restrictions, and achieve the effect of being free from ethical restrictions, having a wide range of sources and a wide range of applications

Active Publication Date: 2006-08-02
SHENZHEN BEIKE BIOTECH +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first purpose of the present invention is to provide a method for isolating and culturing human amniotic mesenchymal stem cells that is not subject to ethical restrictions and has a wide range of sources in view of the existing source of stem cells that is either limited by ethics or limited in number

Method used

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  • Separating and culturing process of human amnion mesenchyme stem cell and its medical composition
  • Separating and culturing process of human amnion mesenchyme stem cell and its medical composition
  • Separating and culturing process of human amnion mesenchyme stem cell and its medical composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Isolation, culture, expansion and purification of human amniotic mesenchymal stem cells

[0020] 1. Isolation of human amniotic mesenchymal cells:

[0021] Under sterile conditions, human placenta from normal full-term caesarean section fetuses was taken, and the amniotic membrane on the umbilical cord surface of the placenta was bluntly separated, washed with phosphate buffered solution (PBS), and the amniotic membrane was cut into pieces with a size of 1.0cm×1.0cm. Add 2 ml of 0.25% trypsin to 1 gram of tissue, digest at room temperature for 30 minutes, 3 times in total, then stop trypsin with DMEM / F12 culture medium containing 10% fetal bovine serum (FBS) by volume, To remove as much epithelial cells as possible. Then the amniotic membrane was cut into pieces as much as possible, and V containing 1.0g / L collagenase and 0.10g / L deoxyribonuclease was added per gram of tissue. DMEM :V F12 = 2ml of 1:1 DMEM / F12 culture solution, digested at 37°C for 1 hour ...

Embodiment 2

[0028] Example 2: Directional induction of HADMSCs to differentiate into neuron-like cells

[0029] 1. Orientation induction: take the HADMSCs passed to the third generation and press 4×10 5 / L density was seeded in a six-well plate with sterilized coverslips placed in advance to prepare cell slides. When the cells reached 80% confluence, the cells were mixed with 30 μmol / L all-trans retinoic acid (Retinic acid, purchased from sigma company), DMEM / F12 medium with 20ng / mlbFGF and 10% FBS was induced for 7 days, and no inducer was added to the control group. Results: The cultured human amniotic mesenchymal stem cells expressed neural stem cell marker human nestin (nestin), and the induced cells expressed nestin and neuron marker human neuron-specific enolase (NSE). Expression of glial cell marker glial fibrillary acidic protein (GFAP).

[0030] 2. Observation under an inverted microscope: HADMSCs are induced to differentiate into nerve cells. Two days after adding the inductio...

Embodiment 3

[0033] Example 3: Application of the composition containing HADMSCs in the treatment of nervous system diseases

[0034] HADMSCs can differentiate into nerve cells, opening up a new cell source for nerve transplantation. HADMSCs can be transplanted into human body through local application or lumbar puncture and intravenous infusion to treat neurological diseases such as brain injury, Parkinson's syndrome, stroke, spinal cord injury and peripheral nerve injury.

[0035] 1. Animal grouping and model making:

[0036] 80 Wistar rats, male or female, were randomly divided into 4 groups: Group A was the injury group (Sham), 20 rats, with only craniotomy and drilling without hitting brain tissue and transplanting cells, as a negative control group. Group B is the sham transplantation group (TBI+NS), 20 rats were injected with 10 μl of normal saline 1 day after craniotomy and drilling, as an intervention control. Group C is the transplantation group (TBI+HADMSCs), 20 rats were inje...

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Abstract

The present invention is separating and culturing process of human amnion mesenchyme stem cell and its medical composition. The separating and culturing process includes digesting human amnion successively with trypsin, collagenase and deoxyribonuclease, and filtering to prepare single cell suspension; culturing in DMEM / F12 culture medium with VDMEM and VF12 in the equal ratio and containing ox embryo blood serum in 10-20 vol% and basic fibroblast growth factor of ultimate concentration 10-20 ng / ml inside a culture box at 37 deg.c, saturated humidity and CO2 in 5 vol%; and replacing liquid and culture passage to proliferate and purify human amnion mesenchyme stem cell. The process has wide material source no ethnic limitation and wide application foreground. The medical composition may be used in various kinds of treatment.

Description

technical field [0001] The invention belongs to a method for separating and culturing mesenchymal stem cells, in particular to a method for isolating and in vitro culturing human amniotic mesenchymal stem cells, and a medical composition containing human amniotic mesenchymal stem cells as an active ingredient. Background technique [0002] In recent years, major breakthroughs have been made in the research of stem cells. In 1999 and 2000, the world's most authoritative American magazine "Science" listed stem cells and the Human Genome Project as the top 10 scientific breakthroughs of the year for two consecutive years, especially in 1999 and even Put stem cell research first. Stem cells are likely to lead to revolutionary progress in the medical field, and thus have immeasurable medical value and have attracted widespread attention and research around the world. The American "Time" magazine believes that stem cells and the Human Genome Project will simultaneously become the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A61K35/50C12N5/074C12N5/0775
Inventor 胡祥杨波
Owner SHENZHEN BEIKE BIOTECH
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