Preparation method and application of mesenchymal stem cell exosome composition

Abstract

The invention relates to a preparation method and application of a mesenchymal stem cell exosome composition, and belongs to the technical field of mesenchymal stem cell medicines.The preparation method comprises the following steps that S1, a conditioned medium is prepared; s2, conditioned medium collection; s3, separating the exosome; s4, purifying the exosome; s5, preparing the composition; and S6, quality control and detection. According to the preparation method and the application of the mesenchymal stem cell exosome composition, the mesenchymal stem cell exosome composition prepared by adopting the preparation method can promote cell proliferation and migration capacity, can remarkably accelerate a tissue repair process, can play an important role in inhibiting tumor growth, can delay a skin aging process, and has a good application prospect. The composition can improve the safety of cell therapy, can reduce the risk of cell therapy, and has the beneficial effects of promoting tissue repair and regeneration, inhibiting tumor growth, resisting aging, and being high in safety and low in risk.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cell drugs, and specifically to a preparation method and application of a mesenchymal stem cell exosome composition. Background Art

[0002] Mesenchymal stem cells are a type of precursor cell with self-renewal ability and multiple differentiation potentials, which are widely present in various tissues and organs. In recent years, with the help of regenerative medicine technology, mesenchymal stem cells have shown great potential in tissue repair and regeneration. Among them, exosomes, as an important way of paracrine activity of mesenchymal stem cells, have also received widespread attention.

[0003] Exosomes are tiny vesicles secreted by cells, with a diameter of 40-100 nanometers. They are coated with a double lipid membrane structure of cells and contain biologically active lipids, proteins, receptors, mRNA, microRNA (miRNA), long non-coding RNA, and complete organelles such as mitochondria. Four transmembrane proteins CD9, CD63, CD81, and CD82 are commonly used molecular markers of exosomes. Exosomes can be released and endocytosed by most types of cells and are an important way for cells to communicate with each other.

[0004] At present, the methods for isolating exosomes mainly include ultra-high-speed centrifugation, density gradient centrifugation, size exclusion chromatography, filtration, polymer precipitation, immunoseparation, and isolation screening, etc. Among them, ultra-high-speed centrifugation is widely used due to its stable separation effect and simple operation.

[0005] Mesenchymal stem cell exosomes contain multiple proteins and RNAs, which have higher safety and greater tissue regeneration potential compared with a single cytokine. Therefore, they have great advantages in tissue regeneration. However, the extraction method of exosomes and their complex classification system still hinder their application.

[0006] In summary, mesenchymal stem cell exosomes, as a new type of intercellular communication medium, show great potential in tissue repair and regeneration. Therefore, a preparation method and application of a mesenchymal stem cell exosome composition are proposed to solve the above-mentioned problems. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a method for preparing a mesenchymal stem cell exosome composition and its application, which has the advantages of promoting tissue repair and regeneration, inhibiting tumor growth and having good anti-aging effects, and solves the problems of low purity, low yield and poor activity of the stem cell exosome composition prepared by the existing preparation method.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing a mesenchymal stem cell exosome composition, comprising the following steps:

[0009] S1. Conditioned medium preparation: select umbilical cord mesenchymal stem cells with good growth status and no contamination for culture; remove complete medium, wash with PBS 3 times to remove residual medium and impurities, and replace with serum-free medium;

[0010] S2. Collection of conditioned medium: After continuing to culture stem cells in the serum-free culture system for 48 hours, collect the culture supernatant as the conditioned medium. Use a pipette or a sterile pipette to collect the conditioned medium into a sterile 50 ml centrifuge tube; centrifuge at 300×g for 10 minutes at 4°C and retain the supernatant.

[0011] S3. Exosome isolation: The conditioned medium was transferred to a 50 ml centrifuge tube and centrifuged at 2000 × g, 4 ° C, for 20 min and 10000 × g, 4 ° C, for 30 min to remove cells, dead cells and cell debris; ultra-high speed centrifuge was used for centrifugation at a centrifugal force of 100000 × g at 4 ° C for 70 min, and the precipitate was retained;

[0012] S4. Exosome purification: Add pre-cooled PBS to wash the precipitate, centrifuge again at 100,000 × g at 4°C for 70 minutes, retain the precipitate, and obtain purer exosomes after precipitation; then use a small volume of 50-100 μl PBS or TBS to resuspend the precipitate to obtain an exosome suspension;

[0013] S5. Preparation of composition: mixing the separated exosomes with other drugs or bioactive molecules to form a composition with specific functions;

[0014] S6. Quality control and testing: During the preparation process, quality control should be carried out at each step, including monitoring of cell culture conditions, control of pretreatment and storage conditions of conditioned medium, and testing of the efficiency and purity of exosome separation and purification. At the same time, the morphological structure of exosomes can be observed using an electron microscope, and the expression of exosome marker proteins can be detected using methods such as protein blotting to ensure that the prepared exosome composition meets the quality requirements.

[0015] Furthermore, the lower the generation number of the umbilical cord mesenchymal stem cells in S1, the better the secretion ability. During the culture process in S1, complete culture medium needs to be used to culture the cells until the coverage rate reaches 70%-80%.

[0016] Furthermore, the mesenchymal stem cell culture equipment in S1 is a bioreactor and an oscillating incubator. The bioreactor can control the culture parameters of CO2, O2, and pH value in real time. The oscillating incubator is used for oscillating culture of cells, which is conducive to cell growth and division.

[0017] Furthermore, the serum-free culture medium in S1 can be a commercial serum-free culture medium dedicated to mesenchymal stem cells or a DMEM / F12 basic culture medium.

[0018] Furthermore, the conditioned medium in S2 can be stored at 4°C for one week, or stored at -80°C for a long time, but it should be noted that storage at -80°C may cause changes in the morphology of exosomes, which is not conducive to subsequent identification.

[0019] Furthermore, the time for separation and precipitation of exosomes in S3 is 30 minutes.

[0020] Furthermore, the time for purification and precipitation of exosomes in S4 is 40 minutes.

[0021] Furthermore, the exosome suspension in S4 can be stored at -80°C for up to one year.

[0022] Another technical problem to be solved by the present invention is to provide an application of a mesenchymal stem cell exosome composition. The mesenchymal stem cell exosome composition prepared by the method for preparing the mesenchymal stem cell exosome composition according to claim 1 is applied to disease treatment, tissue repair and drug screening.

[0023] Compared with the prior art, the present invention provides a method for preparing a mesenchymal stem cell exosome composition and its application, which has the following beneficial effects:

[0024] 1. The preparation method and application of the mesenchymal stem cell exosome composition. The mesenchymal stem cell exosome composition prepared by the above preparation method can promote the ability of cell proliferation and migration, can significantly accelerate the tissue repair process, can play an important role in inhibiting tumor growth, can delay the skin aging process, can improve the safety of cell therapy, can reduce the risk of cell therapy, and has the beneficial effects of promoting tissue repair and regeneration, inhibiting tumor growth, anti-aging effect, high safety and low risk.

[0025] 2. The preparation method and application of the mesenchymal stem cell exosome composition can significantly improve the purity and yield of exosomes by optimizing culture conditions, centrifugation parameters, ultrasonic treatment conditions and other steps, providing strong support for the widespread application of exosomes, and achieving the beneficial effects of high safety and low risk as well as standardized preparation and increased yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is a process flow chart of a method for preparing a mesenchymal stem cell exosome composition and its application proposed by the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] See also Figure 1 In this embodiment, a method for preparing a mesenchymal stem cell exosome composition comprises the following steps:

[0030] S1. Conditioned medium preparation: select umbilical cord mesenchymal stem cells with good growth status and no contamination for culture; remove complete medium, wash with PBS 3 times to remove residual medium and impurities, and replace with serum-free medium;

[0031] S2. Collection of conditioned medium: After continuing to culture stem cells in the serum-free culture system for 48 hours, collect the culture supernatant as the conditioned medium. Use a pipette or a sterile pipette to collect the conditioned medium into a sterile 50 ml centrifuge tube; centrifuge at 300×g for 10 minutes at 4°C and retain the supernatant.

[0032] S3. Exosome isolation: The conditioned medium was transferred to a 50 ml centrifuge tube and centrifuged at 2000 × g, 4 ° C, for 20 min and 10000 × g, 4 ° C, for 30 min to remove cells, dead cells and cell debris; ultra-high speed centrifuge was used for centrifugation at a centrifugal force of 100000 × g at 4 ° C for 70 min, and the precipitate was retained;

[0033] S4. Exosome purification: Add pre-cooled PBS to wash the precipitate, centrifuge again at 100,000 × g at 4°C for 70 minutes, retain the precipitate, and obtain purer exosomes after precipitation; then use a small volume of 50-100 μl PBS or TBS to resuspend the precipitate to obtain an exosome suspension;

[0034] S5. Preparation of composition: mixing the separated exosomes with other drugs or bioactive molecules to form a composition with specific functions;

[0035] S6. Quality control and testing: During the preparation process, quality control should be carried out at each step, including monitoring of cell culture conditions, control of pretreatment and storage conditions of conditioned medium, and testing of the efficiency and purity of exosome separation and purification. At the same time, the morphology of exosomes can be observed using an electron microscope, and the expression of exosome marker proteins can be detected using methods such as protein blotting to ensure that the prepared exosome composition meets the quality requirements.

[0036] Specifically, the lower the generation number of umbilical cord mesenchymal stem cells in S1, the better the secretion ability. During the culture process in S1, complete culture medium needs to be used to culture the cells until the coverage rate reaches 70%-80%.

[0037] Specifically, the mesenchymal stem cell culture equipment in S1 is a bioreactor and an oscillating incubator. The bioreactor can control the culture parameters of CO2, O2, and pH value in real time, and the oscillating incubator is used for oscillating culture of cells, which is conducive to cell growth and division.

[0038] Specifically, the serum-free culture medium in S1 can be a commercial serum-free culture medium dedicated to mesenchymal stem cells or a DMEM / F12 basic culture medium.

[0039] Specifically, the conditioned medium in S2 can be stored at 4°C for one week, or at -80°C for a long time, but it should be noted that storage at -80°C may cause changes in the morphology of exosomes, which is not conducive to subsequent identification.

[0040] Specifically, the time for exosome separation and precipitation in S3 was 30 min.

[0041] Specifically, the time for exosome purification precipitation in S4 was 40 min.

[0042] Specifically, the exosome suspension in S4 can be stored at -80°C for up to one year.

[0043] Embodiment 2:

[0044] In this embodiment, an application of a mesenchymal stem cell exosome composition is provided. The mesenchymal stem cell exosome composition prepared by the method for preparing a mesenchymal stem cell exosome composition according to claim 1 is applied to disease treatment, tissue repair and drug screening.

[0045] The beneficial effects of the above embodiments are:

[0046] The preparation method and application of the mesenchymal stem cell exosome composition, the mesenchymal stem cell exosome composition prepared by the above preparation method can promote the ability of cell proliferation and migration, can significantly accelerate the tissue repair process, can play an important role in inhibiting tumor growth, can delay the skin aging process, can improve the safety of cell therapy, can reduce the risk of cell therapy, and has the beneficial effects of promoting tissue repair and regeneration, inhibiting tumor growth, anti-aging effect, high safety and low risk.

[0047] The preparation method and application of the mesenchymal stem cell exosome composition can significantly improve the purity and yield of exosomes by optimizing steps such as culture conditions, centrifugation parameters and ultrasonic treatment conditions, providing strong support for the wide application of exosomes, and achieving the beneficial effects of high safety and low risk as well as standardized preparation and increased yield.

[0048] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0049] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a mesenchymal stem cell exosome composition, characterized in that: The following steps are involved: S1. Conditioned medium preparation: select umbilical cord mesenchymal stem cells with good growth status and no contamination for culture; remove complete medium, wash with PBS 3 times to remove residual medium and impurities, and replace with serum-free medium; S2. Conditioned medium collection: After continuing to culture stem cells in the serum-free culture system for 48 hours, collect the culture supernatant as the conditioned medium. Use a pipette or a sterile pipette to collect the conditioned medium into a sterile 50 ml centrifuge tube; centrifuge at 300 × g for 10 minutes at 4°C and retain the supernatant. S3. Exosome isolation: The conditioned medium was transferred to a 50 ml centrifuge tube and centrifuged at 2000 × g, 4 ° C, for 20 min and 10000 × g, 4 ° C, for 30 min to remove cells, dead cells and cell debris; ultra-high speed centrifuge was used for centrifugation at a centrifugal force of 100000 × g at 4 ° C for 70 min, and the precipitate was retained; S4. Exosome purification: Add pre-cooled PBS to wash the precipitate, centrifuge again at 100,000 × g at 4°C for 70 minutes, retain the precipitate, and obtain purer exosomes after precipitation; then use a small volume of 50-100 μl PBS or TBS to resuspend the precipitate to obtain an exosome suspension; S5. Preparation of composition: mixing the separated exosomes with other drugs or bioactive molecules to form a composition with specific functions; S6. Quality control and testing: During the preparation process, quality control should be carried out at each step, including monitoring of cell culture conditions, control of pretreatment and storage conditions of conditioned medium, and testing of the efficiency and purity of exosome separation and purification. At the same time, the morphological structure of exosomes can be observed using an electron microscope, and the expression of exosome marker proteins can be detected using methods such as protein blotting to ensure that the prepared exosome composition meets the quality requirements.

2. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The lower the generation number of umbilical cord mesenchymal stem cells in S1, the better the secretion ability. During the culture process in S1, complete culture medium needs to be used to culture the cells until the coverage rate reaches 70%-80%.

3. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The mesenchymal stem cell culture equipment in S1 is a bioreactor and an oscillating incubator. The bioreactor can control the culture parameters of CO2, O2, and pH value in real time. The oscillating incubator is used for oscillating culture of cells, which is conducive to cell growth and division.

4. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The serum-free culture medium in S1 can be a commercial serum-free culture medium dedicated to mesenchymal stem cells or a DMEM / F12 basic culture medium.

5. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The conditioned medium in S2 can be stored at 4°C for one week, or stored at -80°C for a long time, but it should be noted that storage at -80°C may cause changes in the morphology of exosomes, which is not conducive to subsequent identification.

6. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The time for separation and precipitation of exosomes in S3 is 30 minutes.

7. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The time for exosome purification and precipitation in S4 is 40 minutes.

8. The method for preparing a mesenchymal stem cell exosome composition according to claim 1, characterized in that: The exosome suspension in S4 can be stored at -80°C for up to one year.

9. An application of a mesenchymal stem cell exosome composition, characterized in that: The mesenchymal stem cell exosome composition prepared by the method for preparing the mesenchymal stem cell exosome composition according to claim 1 is used for disease treatment, tissue repair and drug screening.

Images