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Preparation method of exosome and stem cell proliferation reagent containing exosome

A technology of stem cell proliferation and exosomes, which is applied in the field of bioengineering, can solve the problems of no large-scale cell expansion, etc. It is easy to meet quality standards, and the effect of definite absorption and easy absorption

Inactive Publication Date: 2021-01-29
广东佰鸿干细胞再生医学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exosomes have also been previously reported as important components of stem cell secretions, but have not been used to sustain large-scale cell expansion

Method used

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  • Preparation method of exosome and stem cell proliferation reagent containing exosome
  • Preparation method of exosome and stem cell proliferation reagent containing exosome
  • Preparation method of exosome and stem cell proliferation reagent containing exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of bone marrow mesenchymal stem cell exosomes

[0036] 1. Preparation of bone marrow mesenchymal stem cell supernatant:

[0037] The bone marrow mesenchymal stem cells of the third generation in the logarithmic phase of growth were digested, and an appropriate amount of medium without exosome serum was added to make a cell suspension, which was inoculated in a T25 cell culture flask, and the cell culture medium was harvested after 60 hours. The supernatant was collected to reach more than 200ml, and the exosomes were extracted.

[0038] 2. Isolation and storage of exosomes from bone marrow mesenchymal stem cells:

[0039]Centrifuge the supernatant collected above at 4°C, 400×g for 10 minutes, transfer the supernatant to remove cell debris; centrifuge at 4°C, 2000×g for 10 minutes, transfer the supernatant to remove dead cells; Filter the supernatant to further remove impurities; 4°C, 120,000×g, ultracentrifuge for 150 minutes to precipitate exoso...

Embodiment 2

[0044] The isolation and purification of umbilical cord mesenchymal stem cells

[0045] First, discard the excess preservation solution in the fresh umbilical cord obtained from the hospital, pour 75% alcohol into it for disinfection for three minutes, then take out the umbilical cord, put it into normal saline with double antibody and wash it repeatedly to remove the residual alcohol on the surface. Cut off the ligated part and discard it. Divide the remaining umbilical cord evenly into small pieces for cleaning, wash the residual blood repeatedly, remove the umbilical vein and umbilical artery, peel off the Wharton glue, and cut it to 1mm 3 The size of the organization block. According to the amount of tissue pieces, add an appropriate amount of conventional medium, mix evenly, spread it on a 15cm petri dish, and place it at 37°C, 5% CO 2 And culture in a humidity-saturated incubator for 24 hours, and add 8ml of culture solution to each dish. Replenish the fluid on the 5th...

Embodiment 3

[0046] Example three cell function test

[0047] 1. Preparation of exosome culture medium:

[0048] After the exosomes obtained above were taken out from -80°C and thawed, they were added to the culture medium of human umbilical cord mesenchymal stem cells at a final concentration of 20 μg / ml, and stirred evenly.

[0049] Second, the cell proliferation test steps are as follows

[0050] Human umbilical cord mesenchymal stem cells in the logarithmic growth phase of 10 passages were digested, resuspended in human umbilical cord mesenchymal stem cell culture medium supplemented with bone marrow mesenchymal stem cell exosomes, and mixed with 2×10 4 Inoculate in a 24-well plate, 500L / well, at 37°C, 5% CO 2 , 95%O 2 Cultured in an incubator, set up a blank control group. After culturing for 24 hours, trypsinize the cells in the three wells for 2 minutes at room temperature, collect the cells in each well, centrifuge at 1200r for 5 minutes, resuspend with a small amount of medium...

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Abstract

The invention discloses a preparation method of an exosome and a stem cell proliferation reagent containing the exosome. The preparation method of the exosome comprises the following steps: step 1, adding a culture medium without exosome serum into bone marrow mesenchymal stem cells, diluting the bone marrow mesenchymal stem cells into a cell suspension, inoculating the cell suspension into a cellculture bottle for culture, collecting cultured supernatant, and extracting the exosome; and step 2, removing cell debris, dead cells and impurities in the supernatant, and after removal of the supernatant, adding a phosphate buffer salt solution (PBS) to dissolve and collect an exosome precipitate, conducting filtering by using a sterile filter membrane, sub-packaging the obtained exosome precipitate into sterile centrifugal tubes, and storing the exosome precipitate for later use. According to the method, in-vitro experiments prove that co-culture of exosome secreted by early-generation bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells can significantly promote the proliferative activity and related functions of the mesenchymal stem cells; and the methodis an effective strategy for potentially improving cell functions in a large-scale cell culture process.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of bioengineering, in particular to a method for preparing exosomes and a stem cell proliferation reagent containing exosomes. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a type of pluripotent stem cells with self-renewal and multiple differentiation potentials, which can differentiate into various tissues and cells after induction. It is an ideal seed cell in regenerative medicine. [0003] Umbilical cord mesenchymal stem cells (Human Umbilical Mesenchymal Stem Cells, hUC-MSCs), as a kind of tissue-derived mesenchymal stem cells, have stronger differentiation potential and proliferation ability. It is worth noting that after a large number of subcultures, some cells can still maintain their pluripotency, while some cells differentiate in a specific direction or begin to age, that is, the growth rate gradually slows down, and the cell sha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775G01N33/68
CPCC12N5/0663C12N2502/1358C12N5/0665G01N33/6872C12N2509/00G01N2333/70596
Inventor 不公告发明人
Owner 广东佰鸿干细胞再生医学有限公司
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