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Preparation and application of exosome secreted by human derived blood or mesenchymal stem cell

A stem cell and blood technology, applied in the field of exosome and application, can solve the problems of restricting exosome research and application, small size of exosome, complicated extraction process, etc., and achieve the effect of easy storage and transportation, easy absorption and high biological stability

Inactive Publication Date: 2014-05-07
珠海霍普金斯医药研究院股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exosomes are small in size, low in density, complicated in extraction process, and low in yield, which restricts further research and application of exosomes.

Method used

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  • Preparation and application of exosome secreted by human derived blood or mesenchymal stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, the extraction of exosome in serum. Human blood comes from a wide range of sources and contains a large amount of exosomes. Serum is obtained by separating and removing blood cells and fibrinogen from the blood of healthy people. The cell debris and impurities in the serum were removed by differential centrifugation technology, and then the serum was ultracentrifuged at low temperature to remove the supernatant, and the resulting precipitate was exosome, which was resuspended in PBS and stored at -20°C. The specific process is as follows:

[0036] Collect healthy human blood, centrifuge to remove blood cells and fibrinogen in the blood, and obtain serum, which should be light yellow liquid.

[0037] 1. Dilute the serum with an equal volume of phosphate buffered saline (PBS).

[0038] 2. Centrifuge the serum at 800g for 10 minutes, retain the supernatant, discard the pellet, and remove the cells in the serum

[0039] 3. Centrifuge at 2000g for 10 minutes...

Embodiment 2

[0047] Example 2, the extraction of exosome in cell culture supernatant. Taking human mesenchymal stem cells (MSC) as an example, human mesenchymal stem cells are derived from the bone marrow, placenta and umbilical cord tissue of healthy people, and can be stably cultured under in vitro conditions, with strong cell activity and strong secretion. Contains a large amount of exosome. At the same time, the source of exosome secreted by mesenchymal stem cells is single and stable, and modern biotechnology can be used to transfect and express the required protein, which is highly controllable.

[0048] 1. Isolate mesenchymal stem cells from the bone marrow, placenta or umbilical cord blood of healthy people, and culture them in a synthetic medium without animal-derived components.

[0049] 2. Collect the replaced culture supernatant during the cell culture process and store it in a freezer at -20°C.

[0050] 3. Thaw the supernatant collected during cell culture, mix, centrifuge a...

Embodiment 3

[0059] Example 3, Purification of exosome in cell culture supernatant by immunoadsorption method. This method uses the antigen-antibody reaction to achieve the purpose of purification by adsorbing specific antigens on the surface of the exosome. It has the advantages of fast method, high purity and easy subsequent biochemical analysis, but the cost is relatively high.

[0060] 1. with the method in embodiment 2, obtain cell culture supernatant

[0061] 2. Centrifuge at 2000g for 10 minutes at 4°C, retain the supernatant, and remove the cell debris in the pellet.

[0062] 3. Wash the antibody-coated magnetic beads (antibody-coated Dynabeads) with PBS

[0063] 4. Precipitate the washed beads with a magnet and discard all supernatant

[0064] 5. Mix the obtained magnetic beads and the supernatant obtained in step 2 evenly, and incubate at 4°C for 24 hours

[0065] 6. Use a magnet to collect the magnetic beads that have adsorbed the exosome

[0066] 7. Wash the obtained magnet...

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Abstract

The invention discloses preparation and application of exosome secreted by human derived blood or mesenchymal stem cell. The preparation process of exosome comprises: extracting mesenchymal stem cells from health-human body blood or a stem cell culture supernatant, performing in-vitro culture and amplification, and utilizing means such as low-temperature separation, purification and the like to prepare high-purity high-bioactivity exosome. The blood or mesenchymal stem cell secreted exosome is added into medicines, health-care products and cosmetics according to ratios for developing of products with bioactivity, is capable of effectively regulating and controlling body cell signal transduction, activating cell regeneration, enhancing health cell proliferation and differentiation capacity, further regulating physiologic restoration or removing damaged, pathologic and aged cells of a body, fundamentally changing body physiologic functions, and helping to generate anti-tumor treatment effect by adjusting cellular immunity. The blood or mesenchymal stem cell secreted exosome is applicable to fields of medical science, health care and beauty treatment, and has wide application prospect.

Description

[0001] technical field [0002] The invention belongs to the field of biotechnology, and specifically relates to extracting mesenchymal stem cells from blood or stem cell culture supernatant of healthy human beings, and extracting exosome with biological activity from serum or culture supernatant through in vitro separation and application. Background technique [0003] Exosome is a nanoscale membranous vesicle secreted by cells. It was first discovered to be secreted by sheep reticulocytes 50 years ago, and it has been confirmed that it can be secreted by a variety of cells, such as: B cells, tree Steroid cells, mast cells, T cells, platelet cells, Schwann cells, tumor cells, sperm cells, etc. Exosomes are also found in various body fluids such as urine and plasma. [0004] Exosomes have specific biophysical and biochemical properties. Its diameter is 40-100 nanometers, its sucrose density is 1.13-1.19 g / ml, and it can be precipitated under a centrifugal force of 100,000 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K8/98A61K35/14A61K35/28A61K35/50A61K35/44A61P39/00A61P39/06A61P37/02A61P35/00A61P27/02A61Q19/08A61Q19/10
Inventor 王义刚赵柏松
Owner 珠海霍普金斯医药研究院股份有限公司
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