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Preparation method of brain acellular matrix

A decellularized matrix and decellularized technology, applied in the field of biomaterials, can solve the problems of difficult preparation of acellular matrix, no recognized method, damage, etc.

Inactive Publication Date: 2022-03-18
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing decellularization methods will cause some damage to the tissue, which needs to be minimized as much as possible
Due to the fragility of brain tissue, the preparation of its acellular matrix is ​​difficult, and there is no recognized method

Method used

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  • Preparation method of brain acellular matrix
  • Preparation method of brain acellular matrix
  • Preparation method of brain acellular matrix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Preparation of acellular matrix from C57 mouse brain

[0052] C57BL / 6 mice aged 6-8 weeks were sacrificed by cervical dislocation, soaked in 75v / v% ethanol solution for 2min, and the brain was removed by craniotomy. The extracted brain was removed from the cerebellum and cut into two halves of equal quality along the coronal plane, soaked in PBS solution added with 1% (v / v) double antibody and washed.

[0053] The first step in decellularizing brain tissue is a freeze-thaw cycle. The brain tissue was placed in a 1.5mL EP tube, and was repeatedly frozen and thawed with liquid nitrogen for 3 times: freezing for 10 minutes each time, and then thawing and warming to room temperature for about 20 minutes. Then rinse with PBS solution for 5 to 10 minutes.

[0054] The second step is to transfer the brain tissue to a 24-well plate, and carry out cycle decellularization under the shaking condition of 80 rpm on the shaker, and all the reagents used are added with 1% (v / v) doub...

Embodiment 2

[0057] Evaluation of C57 Mouse Brain Decellularized Matrix

[0058] 1. Histological evaluation

[0059] The decellularized brain obtained in Example 1 (ie, brain decellularized matrix) was evaluated for the effect of removing nuclei. For this, decellularized and non-decellularized brain sections were fixed with 4% paraformaldehyde at room temperature, stained with 4',6-diamidino-2-phenylindole (DAPI), and visualized with a confocal microscope . DAPI staining picture as figure 2 As shown, the results showed that no cell nuclei remained in the decellularized brain tissue.

[0060] In addition, HE staining was used to verify the effect of decellularization, such as image 3 As shown, it shows that the decellularization method of the present invention has good decellularization effect and less nucleus residue.

[0061] Quantitative detection of DNA content in brain decellularized matrix. DNA was purified using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and then th...

Embodiment 3

[0077] C57BL / 6 mice aged 6-8 weeks were sacrificed by cervical dislocation, soaked in 75% ethanol solution for disinfection for 2 minutes, and the brain was removed by craniotomy. The extracted brain was removed from the cerebellum and cut into two halves of equal quality along the coronal plane, soaked in PBS solution added with 2% (v / v) double antibody and washed.

[0078] To decellularize the brain tissue, the first step is freeze-thaw cycle treatment, which is the same as in Example 1, and three freeze-thaw cycles are performed.

[0079] The second step is to carry out cycle decellularization under the shaking condition of 60 rpm on the shaker, and all the reagents used are added with 2% (v / v) double antibody. First at 25°C with dH 2O was washed for 7 hours, treated with 4w / v% sodium deoxycholate (SDC) for 12 hours, and then washed with PBS solution for 30 minutes; then treated with 40kU / ml DNase I at 37°C for 1h, and then washed with PBS solution at 25°C Wash for 30 min...

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Abstract

The invention discloses a preparation method of a brain acellular matrix. The preparation method comprises the following steps: i) extracting the brain of a mouse as a raw material of the acellular matrix; and ii) carrying out decellularization treatment on the brain of the mouse by adopting a physical method, a chemical method and a biological method to obtain the brain decellularization matrix. According to the method, the biological activity and integrity of the biological tissue can be well kept, multiple key proteins and factors are reserved, and the decellularization rate is high. The brain acellular matrix prepared on the basis of the method can be used for constructing a composite biological scaffold and culturing stem cells, and is applied to the aspects of repairing and regenerating traumatic brain injury.

Description

technical field [0001] The invention belongs to the technical field of biological materials, and in particular relates to a preparation method of brain decellularized matrix. Background technique [0002] Traumatic brain injury (TBI) is a common central nervous system disease, which is difficult to cure clinically. Because the brain belongs to the central nervous system, and the neurons in the central nervous system can hardly regenerate due to their weak regenerative potential and the inhibitory effect of the damaged environment on neuron regeneration. Traumatic brain injuries can severely affect the nervous system, causing functional impairment in patients and even threatening their lives. For such diseases, stem cell therapy has been widely used, but the traditional infusion of neural stem cells is not effective. Therefore, in recent years, researchers have turned their attention to tissue engineering therapy, that is, culturing cells isolated from the body in vitro and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36
CPCA61L27/3633A61L27/3675A61L27/3691A61L27/3687A61L2430/40A61L2430/32
Inventor 刘昌胜陈曦张雪薇王碧雪
Owner EAST CHINA UNIV OF SCI & TECH
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