Method for efficiently separating and preparing exosome

Abstract

The invention relates to the technical field of biomedicine, in particular to a method for efficiently separating and preparing exosomes, which comprises the following steps: S10, providing human umbilical cord mesenchymal stem cells; s20, culturing the human umbilical cord mesenchymal stem cells by using an exosome induction culture medium; the exosome induction culture medium contains a first inflammatory factor; a second inflammatory factor is added in the culture process; the first inflammatory factor comprises at least one selected from the following groups: IL-6 and TNF-alpha; the second inflammatory factor comprises CCL2; and S30, collecting the supernatant of the culture medium in the step S20, and centrifugally collecting to obtain the exosome. The method can improve the yield of the exosomes, has the advantage of simple operation, is suitable for extracting a large amount of exosomes, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a method for efficiently separating and preparing exosomes. Background Art

[0002] Exosomes are membranous vesicles with a diameter of 30 - 150 nm and a density of 1.0 - 1.18 g / mL released extracellularly after the fusion of endosomes (also known as multivesicular bodies) in the cell nucleus and the cell membrane. The membrane of exosomes contains abundant substances such as cholesterol, glycerophospholipids, sphingolipids, and ceramides, as well as luminal and transmembrane proteins similar to the cell membrane structure. Exosomes can alter the gene regulatory network of target cells by transporting various bioactive molecules such as proteins, mRNAs, and miRNAs to the target cells.

[0003] Mesenchymal stem cells are a type of pluripotent stem cells, derived from the mesoderm and ectoderm in the early stage of development, and can be isolated from various tissues such as bone marrow, adipose tissue, synovium, bone, muscle, and umbilical cord, and can be differentiated into various tissues after induction. Exosomes derived from mesenchymal stem cells can transfer between cells through their protein and RNA components, and play roles similar to those of mesenchymal stem cells in tissue repair, immunosuppression, and regulation of immune function. And compared with cell therapy, exosomes have the advantages of more stable properties, convenient storage and transportation, and no risk of immune rejection reaction and tumor formation brought by transplanted cells.

[0004] Existing exosomes derived from mesenchymal stem cells can be obtained by collecting the supernatant of mesenchymal stem cells. The main extraction methods include ultracentrifugation, gel chromatography, ultrafiltration, immunomagnetic bead method, and kit extraction method. However, the current exosome preparation methods have problems such as low yield and low extraction efficiency, and it is difficult to meet the actual application requirements. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for efficiently separating and preparing exosomes, which can improve the yield of exosomes, has the advantage of simple operation, is suitable for large-scale extraction of exosomes, and has good clinical application prospects.

[0006] To this end, in the first aspect, the present invention provides a method for efficiently separating and preparing exosomes, which includes:

[0007] S10. Provide human umbilical cord mesenchymal stem cells;

[0008] S20. Culture the human umbilical cord mesenchymal stem cells with an exosome induction medium; the exosome induction medium contains a first inflammatory factor; and a second inflammatory factor is added during the culture process; the first inflammatory factor includes at least one selected from the group consisting of IL-6 and TNF-α; the second inflammatory factor includes CCL2;

[0009] S30. Collect the culture medium supernatant in step S20, and collect exosomes by centrifugation.

[0010] By using a culture medium containing a first inflammatory factor and adding a second inflammatory factor during the culture process, it is possible to simulate and stimulate cells to produce an inflammatory response, thereby inducing a large amount of exosomes to be produced by mesenchymal stem cells (MSCs) and increasing the yield of exosomes.

[0011] In some embodiments, the first inflammatory factor is IL-6, and the second inflammatory factor is CCL2.

[0012] In some embodiments, the conditions for the culture include:

[0013] S21. First culture: Culture the human umbilical cord mesenchymal stem cells with the exosome induction medium for 12 - 14 h;

[0014] S22. Second culture: After the first culture, add the second inflammatory factor and culture for 8 - 12 h;

[0015] S23. Third culture: After the second culture, replace the culture medium with DMEM / F12 medium prepared with exosome-free fetal bovine serum (FBS) and culture for 48 - 72 h.

[0016] By the above method, the first inflammatory factor and the second inflammatory factor stimulate MSCs step by step, and their effect on inducing the production of exosomes is better than adding the above two inflammatory factors simultaneously.

[0017] In some embodiments, the second culture includes: first continue to culture under low temperature and low oxygen conditions for 2 - 4 h, and then continue to culture under normal temperature and low oxygen conditions for 6 - 8 h.

[0018] In some embodiments, the low oxygen condition includes: the volume percentage of oxygen is 0.5% - 1.5%.

[0019] In some embodiments, the low oxygen condition culture includes: placing it in an incubator and continuously introducing a mixed gas of 93.5% - 94.5% N2: 5% CO 2 : 0.5% - 1.5% O 2 to maintain saturated humidity.

[0020] It is found through experiments that culturing under low oxygen and low temperature conditions after adding the second inflammatory factor has a positive effect on the yield of exosomes.

[0021] In some embodiments, the low temperature condition includes: 18 - 23 °C.

[0022] In some embodiments, in the exosome induction medium, the content of the first inflammatory factor is 5-20 ng / mL.

[0023] In some embodiments, the final concentration of the second inflammatory factor added is 10-20 ng / mL.

[0024] In some embodiments, step S10 includes: taking a healthy neonatal umbilical cord specimen, washing the umbilical cord with sterile PBS solution to remove blood stains and other impurities, stripping the umbilical artery and umbilical vein, separating the Wharton's jelly tissue of the umbilical cord, thoroughly washing and then cutting it into tissue blocks about 1-3 mm 3 in size, adding 0.2% type II collagenase and hyaluronidase and digesting at 37°C for 1-2 h, centrifuging at 500 g for 5 min to collect cells; resuspending the cell pellet with α-MEM medium containing 10% FBS + 1% penicillin-streptomycin, inoculating into a culture flask, and placing it in a 37°C, 5% CO 2 incubator for culture. According to the cell growth situation, replace the fresh culture medium every 2-3 days until the cell confluence reaches more than 80%. The obtained human umbilical cord mesenchymal stem cells are used for step S20.

[0025] Compared with the prior art, the beneficial effects of the present invention include:

[0026] By adding inflammatory factors and optimizing the culture conditions, the present invention promotes the expression level of exosomes, thereby increasing the extraction yield of exosomes. This method is simple to operate, suitable for mass production, and has good application prospects. Moreover, the source of the mesenchymal stem cells of the present invention can be the umbilical cord. The collection of the umbilical cord is a non-invasive operation, does not involve legal and ethical issues, and has the advantages of wide source and scalable application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to limit the present invention. In the drawings:

[0028] Figure 1 : TEM imaging diagram of exosomes prepared according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be fully conveyed to those skilled in the art.

[0030] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form new technical solutions; all the technical features and preferred features mentioned herein can be combined with each other to form new technical solutions; the various components involved or their preferred components can be combined with each other to form new technical solutions.

[0031] In the present invention, unless otherwise stated, the numerical range "a~b" represents an abbreviated representation of any real number combination between a and b; the "range" disclosed in the present invention can be in the form of one or more lower limits and one or more upper limits respectively.

[0032] Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0033] Exosomes are membranous vesicles with a diameter of 30~150nm and a density of 1.0~1.18g / mL released extracellularly after the fusion of endosomes (also known as multivesicular bodies) in the cell nucleus with the cell membrane. Exosomes derived from mesenchymal stem cells can play roles such as tissue repair, immunosuppression, and regulation of immune function. However, existing exosome preparation methods have problems such as low yield and low extraction efficiency, and it is difficult to meet the actual application requirements.

[0034] Therefore, the present invention mainly provides a method for efficiently isolating and preparing exosomes by optimizing the culture of mesenchymal stem cells.

[0035] In some embodiments, a method for isolating and preparing exosomes is provided, which includes:

[0036] S10. Provide human umbilical cord mesenchymal stem cells;

[0037] S20. Culture the human umbilical cord mesenchymal stem cells with an exosome induction medium; the exosome induction medium contains a first inflammatory factor; and a second inflammatory factor is added during the culture process; the first inflammatory factor includes at least one selected from the group consisting of IL-6 and TNF-α; the second inflammatory factor includes CCL2;

[0038] S30. Collect the culture supernatant in step S20, and centrifuge to collect exosomes.

[0039] By using a medium containing a first inflammatory factor and adding a second inflammatory factor during the culture process, it is possible to simulate and stimulate the cells to produce an inflammatory response, thereby inducing mesenchymal stem cells (MSCs) to produce a large amount of exosomes and improving the yield of exosomes.

[0040] In some embodiments, the first inflammatory factor is IL-6, and the second inflammatory factor is CCL2. In some embodiments, the first inflammatory factor is a combination of IL-6 and TNF-α, and the second inflammatory factor is CCL2.

[0041] In some embodiments, the exosome induction medium can be obtained by adding the above-mentioned inflammatory factors to a commonly used stem cell medium. In some examples, the stem cell medium can be a basal medium supplemented with serum, such as DMEM medium containing 10% FBS, α-MEM medium containing 10% FBS, etc. In addition, the stem cell medium can also contain antibiotics, such as penicillin and streptomycin, which can be added according to the required usage concentration. In some examples, the exosome medium includes α-MEM medium containing 10% FBS, the first inflammatory factor, and the second inflammatory factor is added during the culture process.

[0042] In some embodiments, the culture conditions include:

[0043] S21. First culture: The human umbilical cord mesenchymal stem cells are cultured with the exosome induction medium for 12-14 h. For example, the culture time can be about 12 h, 13 h, 14 h, etc.;

[0044] S22. Second culture: After the first culture, the second inflammatory factor is added and cultured for 8-12 h. For example, the culture time can be about 8 h, 9 h, 10 h, 11 h, 12 h, etc.;

[0045] S23. Third culture: After the second culture, the medium is replaced with DMEM / F12 medium prepared with exosome-free fetal bovine serum (FBS) and cultured for 48-72 h. For example, the culture time can be about 48 h, 52 h, 56 h, 60 h, 62 h, 64 h, 66 h, 68 h, 70 h, 72 h, etc.

[0046] By the above method, the first inflammatory factor and the second inflammatory factor stimulate the MSC step by step, and their effect on the induction of exosomes is better than adding the above two inflammatory factors simultaneously.

[0047] In some embodiments, the conditions for the first culture and the third culture are 37 °C, 95% air: 5% CO 2 .

[0048] In some embodiments, the second culture includes: first continuing to culture under low temperature and low oxygen conditions for 2-4 h. For example, it can be about 2 h, 3 h, 4 h, etc.; then continuing to culture under normal temperature and low oxygen conditions for 6-8 h. For example, it can be about 6 h, 7 h, 8 h, etc.

[0049] In some embodiments, the hypoxic condition includes: the volume percentage of oxygen is 0.5% to 1.5%, for example, it can be about 0.5%, 1.0%, 1.5%, etc.

[0050] In some embodiments, the hypoxic condition culture includes: placing in an incubator, continuously introducing 93.5% to 94.5% N 2 : 5% CO 2 : 0.5% to 1.5% O 2 of the mixed gas, and maintaining saturated humidity.

[0051] It is found through experiments that culturing under hypoxic and low-temperature conditions after adding the second inflammatory factor has a positive effect on the yield of exosomes.

[0052] In some embodiments, the low-temperature condition includes: 18 to 23 °C, for example, it can be about 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, etc.

[0053] In some embodiments, the normal temperature refers to the common temperature used for culturing cells, such as 37 °C.

[0054] In some embodiments, in the exosome induction medium, the content of the first inflammatory factor is 5 to 20 ng / mL, for example, it can be about 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, etc.

[0055] In some embodiments, the final concentration of the added second inflammatory factor is 10 to 20 ng / mL, for example, it can be about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, etc.

[0056] In some embodiments, the step S10 includes: taking a healthy neonatal umbilical cord specimen, washing the umbilical cord with sterile PBS solution to remove blood stains and other impurities, stripping the umbilical artery and umbilical vein, separating the Wharton's jelly tissue of the umbilical cord, thoroughly washing and then cutting it into tissue blocks of about 1 to 3 mm 3 tissue blocks, adding 0.2% type II collagenase and hyaluronidase, digesting at 37 °C for 1 to 2 h, centrifuging at 500 g for 5 min to collect cells; adding α-MEM medium containing 10% FBS + 1% penicillin-streptomycin to resuspend the cell pellet, inoculating into a culture flask, and placing it at 37 °C, 5% CO 2Cultivate in an incubator. According to the cell growth situation, replace the fresh culture medium every 2 - 3 days until the cell confluence reaches more than 80%, and use the obtained human umbilical cord mesenchymal stem cells for step S20.

[0057] In some embodiments, step S20 includes:

[0058] S21. First cultivation: Cultivate the human umbilical cord mesenchymal stem cells with the exosome induction medium for 12 - 14 h. The exosome induction medium includes α-MEM containing 10% FBS and 5 - 20 ng / mL of a first inflammatory factor, and the first inflammatory factor includes one or both of IL-6 and TNF-α;

[0059] S22. Second cultivation: After the first cultivation, add the second inflammatory factor, which includes CCL2; cultivate under low-temperature and low-oxygen conditions for 2 - 4 h, and then continue to cultivate under normal-temperature and low-oxygen conditions for 6 - 8 h; the low-temperature and low-oxygen conditions include 18 - 23°C, and a mixed gas of 93.5% - 94.5% N 2 : 5% CO 2 : 0.5% - 1.5% O 2 is introduced; the normal-temperature and low-oxygen conditions include 37°C, and a mixed gas of 93.5% - 94.5% N 2 : 5% CO 2 : 0.5% - 1.5% O 2 is introduced;

[0060] S23. Third cultivation: After the second cultivation, replace the culture medium with DMEM / F12 medium prepared with FBS without exosomes, and cultivate for 48 - 72 h.

[0061] In some embodiments, step S30 includes:

[0062] Collect the culture medium supernatant obtained from the third cultivation, and centrifuge to collect the exosomes.

[0063] The technical solutions and beneficial effects of the present invention will be further described below in conjunction with embodiments.

[0064] Example 1

[0065] This example provides a method for preparing exosomes, which specifically includes:

[0066] S10. Take a healthy neonatal umbilical cord specimen, wash the umbilical cord with sterile PBS solution to remove blood stains and other impurities, strip the umbilical artery and umbilical vein, isolate the Wharton's jelly tissue of the umbilical cord, and after thorough washing, cut it into pieces about 1 - 3 mm 3Tissue blocks: Weigh a certain amount of tissue blocks, add 0.2% type II collagenase and hyaluronidase, and digest at 37°C for 2 h. Centrifuge at 500 g for 5 min to collect the cells. Resuspend the cell pellet in α-MEM medium containing 10% FBS + 1% penicillin-streptomycin, inoculate into a culture flask, and place in an incubator at 37°C with 95% air: 5% CO 2 2. Incubate in an incubator. According to the cell growth situation, replace the fresh medium every 2 days until the cell confluence reaches more than 80%. The obtained human umbilical cord mesenchymal stem cells are used in step S21.

[0067] S21. First culture: Replace the fresh medium, add 10 ng / mL of IL-6 (the first inflammatory factor) to the medium, and continue to culture human umbilical cord mesenchymal stem cells. The culture conditions are 37°C, 95% air: 5% CO 2 , and the culture time is 12 h.

[0068] S22. Second culture: After the first culture, add 15 ng / mL of CCL2 (the second inflammatory factor) to the medium, and culture according to the following method: First, culture under low-temperature and low-oxygen conditions for 4 h. The low-temperature and low-oxygen conditions are 18°C, continuously introduce a mixed gas of 94% N 2 : 5% CO 2 : 1% O 2 to maintain saturated humidity; then culture under normal-temperature and low-oxygen conditions for 6 h. The normal-temperature and low-oxygen conditions are 37°C, continuously introduce a mixed gas of 94% N 2 : 5% CO 2 : 1% O 2 to maintain saturated humidity.

[0069] S23. Third culture: After the second culture, replace the medium with DMEM / F12 medium prepared with exosome-free FBS and culture for 48 h.

[0070] S30. Collect the supernatant of the medium obtained from the third culture and collect the exosomes by ultracentrifugation.

[0071] The above experiment for preparing exosomes is performed in triplicate. The morphology of the prepared exosomes is identified by transmission electron microscopy (TEM); the particle size distribution of the exosomes is detected by nanoparticle tracking analysis (NTA); calculated based on the protein concentration in the exosomes, the average content of the obtained exosomes is 1.65 mg / mL.

[0072] Examples 2 - 6

[0073] Except for the following differences in the second culture, exosomes are prepared according to the same method as in Example 1, and the exosome content is detected. Among them, the normal temperature is 37°C, and the normal oxygen condition is 95% air: 5% CO 2, the low temperature condition is 18°C, and the low oxygen condition is 94% N 2 : 5% CO 2 : 1% O 2 .

[0074] Example 2: Normal temperature and normal oxygen conditions were used throughout the process.

[0075] Example 3: Low temperature and low oxygen conditions were used throughout the process.

[0076] Example 4: First, culture under normal temperature and low oxygen conditions for 4 h, and then culture under low temperature and low oxygen conditions for 6 h.

[0077] Example 5: First, culture under normal temperature and normal oxygen conditions for 4 h, and then culture under normal temperature and low oxygen conditions for 6 h.

[0078] Example 6: First, culture under low temperature and low oxygen conditions for 4 h, and then culture under normal temperature and normal oxygen conditions for 6 h.

[0079] Comparative Example 1

[0080] Exosomes were prepared in the same manner as in Example 1, except that the first culture time was 22 h and the second culture was omitted, and the exosome content was detected.

[0081] Comparative Example 2

[0082] Exosomes were prepared in the same manner as in Example 1, except that the first inflammatory factor was CCL2 and the second inflammatory factor was IL-6, and the exosome content was detected.

[0083] Comparative Example 3

[0084] Exosomes were prepared in the same manner as in Example 1, except that the first culture and the second culture were omitted, and the exosome content was detected.

[0085] The particle size distribution and content of the exosomes prepared in the above Examples 1-6 and Comparative Examples 1-3 are shown in Table 1.

[0086] Table 1

[0087] Average particle size (nm) Exosome content (mg / mL) Example 1 112 1.65 Example 2 116 1.24 Example 3 115 1.47 Example 4 112 1.34 Example 5 114 1.25 Example 6 113 1.21 Comparative Example 1 115 0.77 Comparative Example 2 114 1.04 Comparative Example 3 113 0.62

[0088] It can be seen from the above results that by using the preparation method provided by the present invention, the yield of exosomes is significantly improved.

[0089] Examples 7-10

[0090] Exosomes were prepared in the same manner as in Example 1, except that the low temperature conditions in the second culture were as shown in Table 2, and the exosome content was detected. The results are shown in Table 2.

[0091] Table 2

[0092] Temperature of low-temperature condition (°C) Exosome content (mg / mL) Example 1 18 1.65 Example 7 15 1.36 Example 8 20 1.74 Example 9 23 1.69 Example 10 25 1.41

[0093] Examples 11 to 14

[0094] Except that the oxygen content in the hypoxic condition in the second culture is as shown in Table 3 (when adjusting the oxygen concentration, the N 2 concentration is adjusted accordingly, and the CO 2 concentration remains unchanged), exosomes were prepared in the same manner as in Example 1, and the exosome content was detected. The results are shown in Table 3.

[0095] Table 3

[0096] Oxygen content (%) Exosome content (mg / mL) Example 1 1 1.65 Example 11 0.5 1.42 Example 12 0.3 1.26 Example 13 1.5 1.66 Example 14 2 1.37

[0097] Examples 15 to 17

[0098] Except that the inflammatory factors and their contents in the culture medium are as shown in Table 4, exosomes were prepared in the same manner as in Example 1, and the exosome content was detected. The results are shown in Table 4.

[0099] Table 4

[0100]

[0101] As mentioned above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for efficiently separating and preparing exosomes, characterized in that: The following steps are involved: S10, providing human umbilical cord mesenchymal stem cells; S20, culturing the human umbilical cord mesenchymal stem cells with an exosome induction medium; the exosome induction medium contains a first inflammatory factor; and a second inflammatory factor is added during the culturing process; the first inflammatory factor comprises at least one selected from the group consisting of IL-6 and TNF-α; the second inflammatory factor comprises CCL2; S30, collecting the culture medium supernatant in step S20, and centrifuging to obtain exosomes.

2. The method for efficiently separating and preparing exosomes according to claim 1, characterized in that: The first inflammatory factor is IL-6, and the second inflammatory factor is CCL2.

3. The method for efficiently separating and preparing exosomes according to claim 1, characterized in that: The culture conditions include: S21, first culture: culturing the human umbilical cord mesenchymal stem cells with the exosome induction medium for 12 to 14 hours; S22, second culture: after the first culture, adding the second inflammatory factor and culturing for 8 to 12 hours; S23, third culture: After the second culture, the culture medium was replaced with DMEM / F12 medium containing FBS without exosomes, and cultured for 48 to 72 hours.

4. The method for efficiently separating and preparing exosomes according to claim 1, characterized in that: The second culturing comprises: firstly continuing culturing for 2 to 4 hours under low temperature and low oxygen conditions, and then continuing culturing for 6 to 8 hours under normal temperature and low oxygen conditions.

5. The method for efficiently separating and preparing exosomes according to claim 4, characterized in that: The hypoxic condition includes: the volume percentage of oxygen is 0.5% to 1.5%.

6. The method for efficiently separating and preparing exosomes according to claim 4, characterized in that: The hypoxic condition culture comprises: placing in a constant temperature box, continuously introducing a mixed gas of 93.5% to 94.5% N2: 5% CO2: 0.5% to 1.5% O2, and maintaining saturated humidity.

7. The method for efficiently separating and preparing exosomes according to claim 4, characterized in that: The low temperature conditions include: 18-23°C.

8. The method for efficiently separating and preparing exosomes according to claim 1, characterized in that: In the exosome induction medium, the content of the first inflammatory factor is 5 to 20 ng / mL.

9. The method for efficiently separating and preparing exosomes according to claim 1, characterized in that: The final concentration of the second inflammatory factor added is 10-20 ng / mL.

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