Culture method for promoting secretion of extracellular vesicles and application

Abstract

The invention discloses a culture method for promoting secretion of extracellular vesicles and application of the culture method, and mesenchymal stem cells are cultured for 6-72 hours under a centrifugal condition (0.6-20 * g) by building a programmable control centrifugal culture platform. According to the scheme, exogenous molecules are not introduced, and the influence on cell activity is very small. Compared with stimulation methods such as flow shear force, ultrasonic waves, magnetic force and cell pulling, centrifugal stimulation equipment is low in cost, short in consumed time, easy and convenient to operate and capable of being expanded in a large-scale mode, the yield of extracellular vesicles secreted after centrifugal stimulation is remarkably increased compared with conventional culture, and meanwhile the yield of the extracellular vesicles secreted after centrifugal stimulation is remarkably increased. The produced extracellular vesicles have enhanced ability to promote cell migration and wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical applications, and particularly relates to a culture method for promoting the secretion of extracellular vesicles and its application. Technical Background

[0002] Extracellular vesicles (EVs) refer to subcellular structures formed by lipid bilayer encapsulation. EVs contain components similar to those of the parent cells, such as proteins, lipids, mRNAs, and regulatory miRNAs. As a key mediator of intercellular communication and material exchange, EVs play an important role in physiological regulation and disease processes due to their advantages such as high stability, low immunogenicity, high drug-loading capacity, and blood-brain barrier penetrability. EVs can deliver a variety of bioactive components (including easily degradable / inactivated drugs), participate in tissue repair, immune regulation, etc. by targeting and regulating cell functions, and are expected to become a new strategy to replace cell therapy. As a natural drug delivery system, EVs show great potential in regenerative medicine. For example, in stem cell therapy, EVs can act as signaling molecules to regulate the proliferation and differentiation of stem cells; in tissue engineering, EVs can be used as functional components of scaffolds to promote tissue repair and regeneration by releasing bioactive molecules. Improving the production and preparation efficiency of EVs with regenerative bioactivity is the key to promoting the clinical transformation of EVs.

[0003] The yield of extracellular vesicles obtained by traditional static culture methods is low, and the efficacy of bioactive molecules is limited. Existing studies have shown that the depolymerization or inhibition of polymerization of cellular actin can be induced by RGB short peptides or actin depolymerizing agents, which can increase the yield of extracellular vesicles of stem cells. However, the introduction of exogenous chemical drugs may affect the composition and purity of extracellular vesicles, leading to potential clinical biosafety problems. In addition, previous reports have shown that existing culture methods (such as stimulation means like magnetic field, ultrasound, fluid shear force, or mechanical stretching) can promote the increase in the yield and bioactivity of extracellular vesicles, but there are various problems such as the need for precise control instruments, affecting cell viability, inducing cell thermal effects, degrading thermosensitive components of EVs, and disturbing the EV membrane structure, which affect the purity and quality consistency of EVs.

[0004] Therefore, this patent is committed to developing a low-cost and high-efficiency production platform that can increase the yield of EVs with regenerative bioactivity, providing strong support for the clinical transformation of EVs. Summary of the Invention

[0005] An object of the present invention is to provide a culture method for promoting cells to secrete extracellular vesicles.

[0006] The present invention provides a culture method for promoting the secretion of extracellular vesicles. When the cell density in the culture flask reaches 80% to 90%, the culture medium is aspirated and replaced with serum-free medium, and the angle between the culture flask and the horizontal plane is adjusted to 0° to 90°, and cultured for 6 to 72 h under centrifugation conditions of 0.6 to 20×g.

[0007] Preferably, the angle between the culture flask and the horizontal plane is 30° to 90°.

[0008] Preferably, the centrifugation conditions are 5 to 15×g.

[0009] Preferably, the two centrifugal accelerations of the centrifugation conditions alternate with each other, the centrifugation time is 2 to 30 seconds, the centrifugation conditions for the first time period are 7.5 to 15×g, and the centrifugation conditions for the second time period are 3.5 to 11×g.

[0010] Preferably, the mesenchymal stem cells are one of human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells, and human dental pulp mesenchymal stem cells.

[0011] Preferably, the mesenchymal stem cells are cultured in a large-scale amplification manner using complete medium. When the cell growth density reaches 50 to 90%, serum-free medium is added and cultured for 6 to 72 h.

[0012] Preferably, the mesenchymal stem cells are cultured in a large-scale amplification manner using complete medium. When the cell growth density reaches 50 to 90%, after gently washing with PBS, serum-free medium is added, the angle between the culture flask and the horizontal plane is adjusted to 30° to 90°, and cultured for 6 to 72 h under centrifugation conditions of 0.6 to 20×g.

[0013] Preferably, the mesenchymal stem cells are cultured in a large-scale amplification manner using complete medium. When the cell growth density reaches 80 to 90%, after gently washing with PBS, serum-free medium is added, the angle between the culture flask and the horizontal plane is adjusted to 30° to 90°, and cultured for 6 to 72 h under centrifugation conditions of 0.6 to 20×g.

[0014] Preferably, it further includes the step of collecting extracellular vesicles: after collecting the culture medium, dead cells and cell debris are removed by centrifugation, then the liquid is concentrated by an ultrafiltration tube, and finally extracellular vesicles are obtained by ultracentrifugation.

[0015] Preferably, the steps for collecting extracellular vesicles include: subjecting the collected culture medium to centrifugation at 500×g for 10 min, then at 3000×g for 10 min, filtering through a 0.22-μm filter to remove dead cells and cell debris, concentrating with an ultrafiltration tube, and further removing cell debris by centrifugation at 10000×g for 30 min; centrifuging the culture medium at 120000×g for 120 min using an ultra-high-speed refrigerated centrifuge, discarding the upper liquid, and resuspending the recovered extracellular vesicles with PBS.

[0016] Another object of the present invention is to provide extracellular vesicles prepared by the above culture method.

[0017] Another object of the present invention is to provide an extracellular vesicle preparation for promoting wound healing, comprising the above extracellular vesicles.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Compared with fluid shear force, centrifugal stimulation enhances cell adhesion to the bottom of the culture medium and the stimulation is more uniform; compared with ultrasonic waves, centrifugal stimulation does not cause a significant thermal effect and avoids thermal damage; compared with magnetic fields, centrifugal stimulation does not require the introduction of magnetic materials that may interfere with cell viability; compared with tensile force, centrifugal stimulation does not require the preparation and pretreatment of materials loaded with cells, the cells are evenly stressed and the cell input can be expanded (the attachment area size of the stretching material is limited).

[0020] Compared with static culture, centrifugal stimulation increases the yield of extracellular vesicles and also enhances cell adhesion; compared with culture without centrifugation, the extracellular vesicles after centrifugal stimulation are enriched with more proteins related to cell proliferation and wound healing, and these proteins are mainly involved in biological processes such as the cell cycle and complement and coagulation cascades. Description of the Drawings

[0021] Figure 1 The figure shows the theoretical calculation results of cell surface pressure and surface velocity for the uniform speed group and variable speed group;

[0022] Figure 2 The figure shows the cell survival rate results under different culture conditions;

[0023] Figure 3 The figure shows the yield of extracellular vesicles under different culture conditions;

[0024] Figure 4 Figure A shows the migration results of extracellular vesicles from the control group and experimental group of this application acting on corneal epithelial cells; Figure 4 Figure B shows the statistical chart of the migration area results of extracellular vesicles from the control group and experimental group of this application acting on corneal epithelial cells;

[0025] Figure 5Figure of the proteomic analysis results of extracellular vesicles stimulated by Example 5 of the present application; Figure 5 Figure of the log2-fold change in protein abundances related to cell cycle, cell senescence, complement, and coagulation cascade in Comparative Example 2 and Example 5 of the present application; Figure 5 Figure of the subcellular localization analysis of differentially expressed proteins in extracellular vesicles in Comparative Example 2 and Example 5 of the present application. Detailed implementation manners

[0026] The content of the present invention will be further described below in conjunction with the specification drawings and specific embodiments, but it should not be construed as a limitation to the present invention; modifications or substitutions made to the methods, steps, and conditions of the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention. Unless otherwise specified, the experimental methods used in the examples are all conventional methods and techniques well-known to those skilled in the art, and the reagents or materials are all obtained through commercial channels.

[0027] Example 1

[0028] The present invention provides a culture platform for promoting the secretion of extracellular vesicles. A number of adjustable culture flask placement seats are evenly distributed along the circumferential direction on this culture platform, and each placement seat is connected to the culture platform through a hinge. The angle is adjusted through the linkage of a gear set and a hinge shaft or a multi-stage adjustable support rod, and the culture flask is fixed using an elastic clamping structure or a customized card slot. The central shaft coupling of the stepping motor is connected to the culture platform to form a coaxial drive to realize the rotation of the culture platform.

[0029] Example 2

[0030] The present embodiment provides a culture method for promoting the secretion of extracellular vesicles, and the culture method is as follows:

[0031] Step 1: Uniformly spread umbilical cord mesenchymal stem cells in a culture flask and add 4 mL of complete medium;

[0032] Step 2: Wait until it grows to 80% to 90% of the culture flask, gently wash it 2 to 3 times with PBS, and then add 50 mL of serum-free medium;

[0033] Step 3: Place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed through the angle adjustment component, and centrifuge and culture it for 12 h under the condition of 0.6×g (RCF);

[0034] Step 4: The collected culture medium is successively passed through 500×g for 10 min; 3000×g for 10 min, and a 0.22 μm filter membrane to remove dead cells and cell debris. After concentration with an ultrafiltration tube, it is further centrifuged at 10000×g for 30 min to remove cell debris;

[0035] Step 5: Centrifuge the culture medium at 120,000×g for 120 min using an ultra-high-speed refrigerated centrifuge, discard the upper liquid, and resuspend the recovered extracellular vesicles with PBS.

[0036] Example 3

[0037] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it horizontally placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of 15×g (RCF).

[0038] Example 4

[0039] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of 7.5×g (RCF).

[0040] Example 5

[0041] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of 15×g (RCF).

[0042] Example 6

[0043] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of 20×g (RCF);

[0044] Example 7

[0045] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of alternating between 7.5×g (RCF) and 3.5×g (RCF) every 5 seconds.

[0046] Example 8

[0047] Different from Example 2, in Step 3, place the culture flask on the culture flask placement seat on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, and centrifuge and culture it for 12 h under the condition of alternating between 15×g (RCF) and 11×g (RCF) every 5 seconds.

[0048] Comparative Example 1

[0049] Different from Example 2, in Step 2, when it grows to 80 to 90% of the culture flask, gently wash it 2 to 3 times with PBS, and add 4 mL of serum-free medium; in Step 3, place the culture flask on the culture flask holder on the culture platform in Example 1, and make it horizontally placed by adjusting the angle component, without applying centrifugal culture for 12 h;

[0050] Comparative Example 2

[0051] Different from Example 2, in Step 3, place the culture flask on the culture flask holder on the culture platform in Example 1, and make it vertically placed by adjusting the angle component, without applying centrifugal culture for 12 h.

[0052] According to Figure 1 the results, the uniform positive pressure on the cells mainly occurs during the uniform centrifugation process.

[0053] According to Figure 2 the results, compared with Comparative Example 1, under the centrifugation conditions of 7.5 to 15×g (RCF), the cell survival rate decreased slightly. While at 20×g (RCF), the cell survival rate decreased significantly. In contrast, the cell survival rate of the variable speed group (Examples 7 and 8) increased.

[0054] According to Figure 3 the results, centrifugal stimulation can significantly increase the extracellular vesicle yield: within the centrifugal force range of 0.6 to 15×g, the EVs yield shows an upward trend with the increase of centrifugal force, reaching 2 to 5 times that of conventional culture; but when the centrifugal force increases to 20×g, the yield decreases. In addition, the extracellular vesicle yield of the variable speed group (Examples 7 and 8) is higher than that of the uniform speed group.

[0055] Verification Example:

[0056] Human corneal epithelial cells were seeded into a 96-well culture plate at a density of 3x10 4 cells per well. After adherent for 24 h, culture overnight in serum-free medium until 90% confluence. Then, use a 200 μL pipette tip to scrape a straight line along the center line of each well. Next, add PBS (negative control group), 0°-0×g (conventional culture group), and EVs treated with 90°-15×g-11×g, 90°-0×g, and 90°-15×g to the 96-well plate at the same particle concentration respectively. Use an inverted fluorescence microscope to observe and obtain cell images, and calculate the migration rate through image J (migration area = initial area - area at the current time point). The results are as Figure 4 shown, the extracellular vesicles produced by the 90°-15×g experimental group have a better wound healing effect.

[0057] As Figure 5As shown in A-B, EVs stimulated by 90°-15×g have more proteins related to the extracellular matrix, cell proliferation, and innate immunity. The complement system is a component of innate immunity and jointly maintains physiological homeostasis through synergistic action with the hemostasis pathway. Proteolytic enzymes and their regulatory factors in the coagulation cascade not only participate in the dynamic balance of hemostasis and thrombosis but also play important roles in key physiological processes such as cell proliferation regulation and tissue repair. In addition, as Figure 5 shown in C, centrifugation stimulation increased the extracellular matrix components of extracellular vesicles compared to 90°-0×g, while the untreated group enriched more proteins related to cytoplasmic / nuclear localization.

[0058] Generally speaking, the yield of extracellular vesicles and the therapeutic effect on wound healing in the 90°-15×g experimental group are better than other culture conditions.

[0059] Finally, it is necessary to state here that the above embodiments are only used to further illustrate the technical solutions of the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention.

Claims

1. A culture method for promoting extracellular vesicle secretion, characterized in that: When the cell density in the culture flask reaches 80% to 90%, the culture medium is removed and replaced with serum-free medium, the angle between the culture flask and the horizontal plane is adjusted to 0° to 90°, and culture is carried out under centrifugation conditions of 0.6 to 20×g for 6 to 72 hours.

2. The method for promoting extracellular vesicle secretion according to claim 1, characterized in that: The angle between the culture bottle and the horizontal plane is 30° to 90°.

3. A culture method for promoting extracellular vesicle secretion according to claim 1 or 2, characterized in that: The centrifugation condition is 5 to 15×g.

4. A culture method for promoting extracellular vesicle secretion according to claim 1 or 2, characterized in that: The centrifugal conditions are as follows: two centrifugal speeds are alternated with each other, the centrifugal time is 2 to 30 seconds, the first centrifugal acceleration is 7.5 to 15×g, and the second centrifugal acceleration is 3.5 to 11×g.

5. The method for promoting extracellular vesicle secretion according to claim 2, characterized in that: The mesenchymal stem cells are cultured in a large-scale expansion manner using a complete culture medium. When the cell growth density reaches 50 to 90%, serum-free culture medium is added and cultured for 6 to 72 hours.

6. The method for promoting extracellular vesicle secretion according to claim 5, characterized in that: Mesenchymal stem cells were cultured in a large-scale expansion manner using complete culture medium. When the cell growth density reached 80 to 90%, serum-free culture medium was added after gentle washing with PBS. The angle between the culture flask and the horizontal plane was adjusted to 30° to 90°, and cultured under centrifugation conditions of 0.6 to 15×g for 6 to 72 hours.

7. The method for promoting extracellular vesicle secretion according to claim 6, characterized in that: The steps of collecting the extracellular vesicles include: collecting the culture medium, removing dead cells and cell debris by centrifugation, concentrating the liquid by ultrafiltration tube, and obtaining the extracellular vesicles by ultracentrifugation.

8. The method for promoting extracellular vesicle secretion according to claim 7, characterized in that: The steps for collecting extracellular vesicles include: passing the collected culture medium through 500×g, 10 min; 3000×g, 10 min; 0.22μm filter membrane to remove large vesicles, dead cells and cell debris, then concentrating the liquid with an ultrafiltration tube and further removing cell debris through 10000×g, 30 min; finally, centrifuging the culture medium with an ultrahigh-speed refrigerated centrifuge at 120000×g, 120 min, discarding the upper liquid, and resuspending with PBS to recover the extracellular vesicles.

9. Extracellular vesicles prepared by the culture method for promoting extracellular vesicle secretion according to any one of claims 1-8.

10. An extracellular vesicle preparation for promoting wound healing, characterized in that: Comprising the extracellular vesicles described in claim 9.

Images