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Preparation method and application of adipose-derived mesenchymal stem cell exosome

A mesenchymal stem cell and adipose-derived technology, which is applied in the field of preparation of adipose-derived mesenchymal stem cell-like exosomes, can solve the problems of uncertain factors in clinical application, difficulty in realizing large-scale preparation of exosomes, etc., and achieve good therapeutic effect

Active Publication Date: 2019-06-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the curative effect of AMSC-Exo can be improved through genetic modification and other methods, there may be potential uncertainties in its clinical application due to genetic manipulation
However, traditional exosome preparation techniques, including ultracentrifugation, ultrafiltration, magnetic bead immunization, PEG precipitation, etc., are still difficult to achieve large-scale preparation of exosomes.
Therefore, the promotion of its clinical application still depends on improving its preparation technology and increasing its preparation amount to meet the clinical treatment dosage requirements.

Method used

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  • Preparation method and application of adipose-derived mesenchymal stem cell exosome
  • Preparation method and application of adipose-derived mesenchymal stem cell exosome
  • Preparation method and application of adipose-derived mesenchymal stem cell exosome

Examples

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preparation example Construction

[0027] The method for preparing exosomes from adipose-derived mesenchymal stem cells provided by the present invention at least includes the following steps:

[0028] (1) Dilute the adipose-derived mesenchymal stem cells and extrude to obtain an extruded liquid;

[0029] (2) The extruded liquid is placed in the gradient separation liquid for centrifugation, and the speed is reduced without braking to obtain a layered mixed liquid. The layered mixed liquid includes the upper layer component, the middle layer component and the lower layer component ;

[0030] (3) The middle layer fraction was collected, mixed with adipose-derived mesenchymal stem cell exosomes (AMSC-Exo), centrifuged, and the resulting precipitate was adipose-derived mesenchymal stem cell exosomes (AMSC-miExo).

[0031] In one embodiment, the reagent for diluting the adipose-derived mesenchymal stem cells is selected from physiological saline, PBS, D-PBS, Hanks, D-Hanks, HEPES buffer, Earle's Balanced Salt Solu...

Embodiment 1

[0068] Example 1 AMSC-miExo was prepared using 1×10 7 Preparation of AMSC cells for AMSC-miExo and AMSC-Exo:

[0069] According to the method mentioned in the patent ZL 201310390760.3, adipose-derived mesenchymal stem cells (AMSCs) were prepared. When the AMSCs proliferated close to 80% confluence, they were digested and passaged. For the 3rd to 8th generation AMSCs, the 3rd to 8th generation AMSCs were replaced. Liquid culture, the culture solution (the culture solution includes the following components: no phenol red DMEM low-sugar medium, 1mM L-glutamine, 1% non-essential amino acids, 1% penicillin and streptomycin, and the solvent is water) Serum without exosomes was added. After culturing with the culture medium for 48 hours, the cell supernatant was collected, and AMSC-Exo was prepared by ultracentrifugation. At the same time, the AMSCs were collected with a cell scraper, and after washing with PBS, the cell suspension (5×10 6 cells / mL). Then use the Mini-extruder eq...

Embodiment 2

[0072] Example 2 Curative effect of AMSC-miExo on acute liver failure model in mice

[0073]C57 mice were intraperitoneally injected with LPS / GalN (10 μg / kg of LPS+400 mg / kg of D-GalN) to establish a semi-lethal dose of acute liver failure model. Gradient doses (5 mg / kg, 20 mg / kg) of AMSC-miExo and AMSC-Exo were infused into the tail vein immediately after modeling, and the corresponding volume of PBS was injected into the tail vein as the control group (Vehicle). Six hours after modeling, the mice were sacrificed, and serum and liver tissue were collected for detection of liver function, liver pathology, inflammatory factors, and necroptosis of liver tissue. Serum ALT and AST were detected using FUJI DRI-CHEM Slide GFP / ALT-PIII and GOT / AST-PIII kits, respectively, and measured with DRI-CHEM 4000ie (FUJIFILM). Liver pathology was detected by HE. Serum inflammatory factors were detected using corresponding ELISA kits. The levels of related molecules (p-RIP1, p-RIP3, p-MLKL) ...

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Abstract

The invention provides a preparation method and an application of an adipose-derived mesenchymal stem cell exosome. The preparation method of the adipose-derived mesenchymal stem cell exosome is characterized in that the preparation method at least includes the following steps: (1) diluting adipose-derived mesenchymal stem cells and extruding to obtain an extrusion liquid; (2) placing the extrusion liquid into a gradient separation liquid for centrifugation, and reducing speed without braking to obtain a layered mixed liquid, wherein the layered mixed liquid comprises an upper layer component,an intermediate layer component and a lower layer component; and (3) collecting the intermediate layer component, mixing with adipose-derived mesenchymal stem cell exosome, and centrifuging to obtaina precipitate which is the adipose-derived mesenchymal stem cell exosome. The preparation amount of the exosome prepared by the preparation method of the adipose-derived mesenchymal stem cell exosomeis much higher than that of the traditional exosome; and the exosome has a better liver disease treatment effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of adipose-derived mesenchymal stem cell-like exosomes. Background technique [0002] With the advancement of stem cell therapy research, mesenchymal stem cells (MSCs) have achieved positive curative effects in both basic research and clinical trials for the treatment of liver diseases. The main sources of MSCs are bone marrow, umbilical cord blood, adipose tissue, synovium and so on. We also isolated and cultured mesenchymal stem cells (AMSC) from adipose tissue, and their properties were similar to those derived from bone marrow, and the frequency of MSC in fat was nearly 100 times that in bone marrow. There are abundant sources of adipose tissue, low donor discomfort, no ethical restrictions, and can be induced to differentiate into hepatocytes in vitro. We have confirmed in a variety of liver disease models that adipose tissue-derived MSCs (...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P1/16
Inventor 刘艳宁楼国华郑敏
Owner ZHEJIANG UNIV
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