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Extracellular matrix gel carrying mesenchymal stem cell exosome and preparation method thereof

A technology of stem cells and extracellular matrix, applied in the fields of prosthesis, medical science, tissue regeneration, etc., can solve the problems of insufficient treatment safety guarantee, failure to carry exosome natural cell-derived repair materials, etc., and achieve good tissue repair. Effect

Inactive Publication Date: 2021-10-15
THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent with the application number CN201811029396.7 discloses a decellularized scaffold solution-GelMA hydrogel composite material and its preparation method, but it is limited to drug mixing, and does not realize the repair materials derived from natural cells such as exosomes , making its treatment security insufficient

Method used

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  • Extracellular matrix gel carrying mesenchymal stem cell exosome and preparation method thereof
  • Extracellular matrix gel carrying mesenchymal stem cell exosome and preparation method thereof
  • Extracellular matrix gel carrying mesenchymal stem cell exosome and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Preparation of mesenchymal stem cell exosomes and cartilage extracellular matrix gel / GelMA mixed light-cured gel by differential centrifugation + ultrafiltration

[0039] 1. When the confluence of bone marrow mesenchymal stem cells (BMSCs) reaches 50%, they are washed and cultured in serum-free medium for another 48 hours.

[0040] 2. Collect the medium, and centrifuge in a gradient, at 200×g for 15 minutes, and at 2500×g for 15 minutes.

[0041] 3. Take the supernatant and filter the supernatant with a 0.22 μm filter to remove residual cells and debris.

[0042] 4. Transfer the solution using the ultracentrifuge filter unit. Centrifuge the solution at 4,000 x g until the volume in the upper compartment is concentrated to approximately 200 μL.

[0043] 5. Wash the ultrafiltration solution with phosphate buffered saline, and repeat the centrifugation 3 times.

[0044] 6. Ultracentrifuge the washed ultrafiltration liquid containing exosomes for 1 hour. Exos...

Embodiment 2

[0049] Example 2. Preparation of Mesenchymal Stem Cell Exosomes and Periosteal Extracellular Matrix Gel / GelMA Hybrid Photocurable Gel by Density Gradient Method

[0050] 1. When the confluence of bone marrow mesenchymal stem cells (BMSCs) reaches 55%, they are washed and cultured in serum-free medium for another 48 hours.

[0051] 2. Collect the culture medium and centrifuge at 1000×g with Percoll density gradient centrifugation medium for 20 min.

[0052] 3. Aspirate the exosome layer liquid at the interface of the upper layer, add 2 times the volume of PBS and mix well.

[0053]4. Filter using a 0.22 μm filter to remove residual cells and debris, and transfer the solution using an ultracentrifugal filter device. Centrifuge the solution at 4,000 x g until the volume in the upper compartment is concentrated to approximately 200 μL.

[0054] 5. Wash with phosphate buffered saline and repeat centrifugation 3 times.

[0055] 6. Resuspend the pellet with 200 μL PBS for quantifi...

Embodiment 3

[0060] Example 3. Preparation of Mesenchymal Stem Cell Exosomes and Tendon Extracellular Matrix Gel / GelMA Hybrid Photocurable Gel by Differential Centrifugation

[0061] 1. When the confluence of bone marrow mesenchymal stem cells (BMSCs) reaches 60%, they are washed and cultured in serum-free medium for another 48 hours.

[0062] 2. Collect the culture medium, centrifuge in a gradient at 300×g for 10 minutes, and take the supernatant.

[0063] 3. Centrifuge at 2000×g for 10 minutes, and take the supernatant.

[0064] 4. Centrifuge at 10,000×g for 30 minutes, and take the supernatant.

[0065] 5. Continuously centrifuge at 100,000×g for 90 minutes at 4°C, remove the supernatant, resuspend the remaining pellet in PBS, and centrifuge again at 100,000×g for 90 minutes.

[0066] 6. Resuspend the pellet with 200 μL PBS for quantification of exosome protein.

[0067] 7. Freezing and thawing the tendon three times, and eluted with 1% Triton X-100 for 24 hours.

[0068] 8. The elu...

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Abstract

The invention discloses extracellular matrix gel carrying mesenchymal stem cell exosomes and a preparation method thereof. The mesenchymal stem cell-derived exosome is extracted and is mixed with the extracellular matrix and the methylacryloylated gel, so that the mesenchymal stem cell-derived exosome has uniformity and formability, the biological activity of the mesenchymal stem cell is retained, and a new thought is provided for a biological material tissue repair preparation.

Description

technical field [0001] The invention belongs to the field of biomaterial tissue repair preparations, and in particular relates to an extracellular matrix gel loaded with mesenchymal stem cell exosomes and a preparation method thereof. Background technique [0002] Mesenchymal stem cell exosomes are a type of extracellular vesicles secreted by mesenchymal stem cells, with a particle size of about 60-150nm, usually carrying bioactive molecules such as protein, mRNA, miRNA and lipid, which can promote the growth of tissue cells , differentiation and tissue repair. Therefore, MSC exosomes have been extensively studied in the field of tissue repair. [0003] The extracellular matrix is ​​a complex structural network composed of polysaccharides, proteins, proteoglycans and other biomacromolecules secreted by cells, supporting cell growth and regulating the biological behavior of cells. Extracellular matrix has been widely used in the field of tissue repair. [0004] Methacrylat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36A61L27/38A61L27/50A61L27/54A61L27/22
CPCA61L27/3633A61L27/3834A61L27/50A61L27/54A61L27/222A61L2300/412A61L2430/40A61L2430/06
Inventor 陈鹏飞林贤丰顾辰辉范顺武
Owner THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
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