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Preparation method of bone marrow mesenchymal stem cell exosome

A technology of bone marrow mesenchyme and exosomes, which is applied in the field of biomedicine, can solve the problems of low extraction efficiency, high cost, and high extraction efficiency, and achieve the effect of high purity and yield, and increase yield

Inactive Publication Date: 2020-12-08
淄博市中心医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although medical technology is constantly improving, the early diagnosis rate of liver cancer in my country is only about 20%, and the 5-year survival rate of liver cancer patients after treatment is less than 10%.
[0005] At present, the exosome extraction methods mainly include ultra-high-speed centrifugation. The exosomes obtained are of high purity, but the extraction efficiency is too low, and the separation time is long, which requires high equipment requirements; the immunomagnetic bead method can specifically capture Exosomes with protein tags, due to the need for a large number of antibodies, magnetic beads, etc., lead to high costs and extraction efficiency is only higher than ultra-high-speed centrifugation; kit precipitation methods, such as Thermofisher and SBI's related serum exosome extraction Reagents, the extraction efficiency of exosomes in this method is the highest compared with the other two methods, but the purity of exosomes is relatively low. While capturing exosomes, high-abundance proteins in body fluids will also precipitate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] S1: Using serum-free conditioned medium, put the cultured bone marrow mesenchymal stem cells into the medium for 12 hours, then add 5ug production-promoting reagents to the medium, and then continue to culture for 24 hours;

[0026] S2: Centrifuge to remove cells, collect the crude supernatant, centrifuge again, filter, remove residual cells and cell debris, and prepare a supernatant containing exosomes;

[0027] S3: After adding ethylene glycol polyoxyethylene ether and dextran to the solution of exosomes to be extracted in S2, place the solution at 1°C for 35 minutes;

[0028] S4: Centrifuge the solution in S3 to remove cells, collect the crude supernatant, centrifuge again, filter, take the precipitate, collect the precipitate and resuspend in the resuspension reagent to obtain a pre-resuspension;

[0029] S5: After adding ethylene glycol polyoxyethylene ether and dextran to the pre-resuspension in S4, place the pre-resuspension at 0°C for 30 minutes;

[0030] S6: T...

Embodiment 2

[0032] S1: Using serum-free conditioned medium, put the cultured bone marrow mesenchymal stem cells into the medium for 15 hours, then add 5ug production-promoting reagents to the medium, and then continue to culture for 30 hours;

[0033] S2: Centrifuge to remove cells, collect the crude supernatant, centrifuge again, filter, remove residual cells and cell debris, and prepare a supernatant containing exosomes;

[0034] S3: the solution of exosomes to be extracted in S2, after adding ethylene glycol polyoxyethylene ether and dextran, reacting the solution at 5°C for 55 minutes;

[0035] S4: Centrifuge the solution in S3 to remove cells, collect the crude supernatant, centrifuge again, filter, take the precipitate, collect the precipitate and resuspend in the resuspension reagent to obtain a pre-resuspension;

[0036] S5: After adding ethylene glycol polyoxyethylene ether and dextran to the pre-resuspension in S4, place the pre-resuspension at 3°C ​​for 45 minutes;

[0037] S6...

Embodiment 3

[0039] S1: Using serum-free conditioned medium, put the cultured bone marrow mesenchymal stem cells into the medium for 18 hours, then add 5ug production-promoting reagents to the medium, and then continue to culture for 36 hours;

[0040] S2: Centrifuge to remove cells, collect the crude supernatant, centrifuge again, filter, remove residual cells and cell debris, and prepare a supernatant containing exosomes;

[0041] S3: After adding ethylene glycol polyoxyethylene ether and dextran to the solution of exosomes to be extracted in S2, place the solution at 9° C. for 85 minutes;

[0042] S4: Centrifuge the solution in S3 to remove cells, collect the crude supernatant, centrifuge again, filter, take the precipitate, collect the precipitate and resuspend in the resuspension reagent to obtain a pre-resuspension;

[0043] S5: After adding ethylene glycol polyoxyethylene ether and dextran to the pre-resuspension in S4, place the pre-resuspension at 5°C for 65 minutes;

[0044] S6:...

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PUM

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Abstract

The invention discloses a preparation method of a bone marrow mesenchymal stem cell exosome. The method comprises the following preparation steps of: S1, by using a serum-free condition culture medium, putting cultured bone marrow mesenchymal stem cells into the culture medium for 12 hours, adding 5ug of yield promoting reagent into the culture medium, and then culturing for 24 hours; S2, removingcells through centrifugation, collecting a supernatant crude product, and performing centrifugation and filtering to remove residual cells and cell debris to obtain an exosome-containing supernatant;and S3, adding ethylene glycol polyoxyethylene ether and glucan into the solution containing the exosome to be extracted in the step S2. According to the method for extracting the exosome, the yieldof the exosome is greatly improved, and the purity and yield of the obtained bone marrow mesenchymal stem cell exosome are very high after two times of resuspension treatment while the yield is improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing bone marrow mesenchymal stem cell exosomes. Background technique [0002] Liver cancer is a common malignant tumor, which can be divided into two categories: primary and secondary. Primary liver cancer originates from liver epithelium or mesenchymal tissue; secondary or metastatic liver cancer refers to multiple organs throughout the body. The malignancy of origin invades the liver. Whether it is the generation of new cases or the number of deaths, the number of people suffering from liver cancer in developing countries is far more than that in developed countries, and the number of incidences and deaths of men is more than that of women worldwide. [0003] At the same time, studies have confirmed that hepatitis B virus (HBV) is the main pathogen that causes liver cancer, and liver cancer caused by HBV infection accounts for about 80% of all liver cancer cases. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2500/90C12N2500/30C12N2500/32C12N2501/58
Inventor 翟俏丽王连庆郑林林
Owner 淄博市中心医院