Preparation method of exosome and stem cell proliferation reagent containing exosome

A technology of stem cell proliferation and exosomes, applied in the field of bioengineering, can solve the problem of no large-scale cell expansion, easy to meet quality standards, easy to use, and harmless ingredients

Inactive Publication Date: 2018-10-12
广东芙金干细胞再生医学有限公司
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  • Summary
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  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to create new types of materials called extracellular vesicles (EVs) which contain various molecules such as proteins or lipids with biological functions like carrying genetic material into target tissues within them. These EV' s have potential applications in medicine but also usefulness outside this field due to its ability to stimulate regenerative processes inside somatic organs.

Problems solved by technology

The technical problem addressed in this patented text relates to developing novel compositions containing certain molecular elements such as proteins, lipids, nucleic acids, carbohydrates, peptides, polysaccharide fragments, nucleotides, amino acid sequences, antibodies, cytokines, chemokines, etc., which could potentially improve therapeutic effects when delivered directly onto affected areas like cancerous sites without causing harmful side effect(such as inflammations).

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] Example 2 Separation, Purification and Culture of Human 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 in 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 ...

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 mediu...

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Abstract

The invention discloses a preparation method of exosome and a stem cell proliferation reagent containing the exosome. The preparation method of the exosome comprises the steps that 1, a culture mediumwithout exosome serum is added bone marrow mesenchymal stem cells to dilute the bone marrow mesenchymal stem cells into a cell suspension, the cell suspension is inoculated into a cell culture bottleto be cultured, supernate obtained after culture is collected, and the exosome is extracted; 2, cell fragments, dead cells and impurities in the supernate are removed; the supernate is removed, thena phosphate buffer salt solution (PBS) is added to dissolve and collect exosome precipitate, the exosome precipitate is filtered through a sterile filter membrane, and the obtained exosome precipitateis packaged in a sterile centrifugal tube and stored for later use. According to the method, in-vitro experiments prove that the proliferation activity and related functions of the mesenchymal stem cells can be remarkably promoted by co-culturing the exosome secreted by the early-generation bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells, and the method is an effective strategy for potentially improving the function of cells in a large-scale cell culture process.

Description

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Claims

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Application Information

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Owner 广东芙金干细胞再生医学有限公司
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