Three-dimensional culture device for extracellular vesicles in microgravity environment and method for preparing target extracellular vesicles

Abstract

The invention discloses a three-dimensional culture device for extracellular vesicles in a microgravity environment and a method for preparing target extracellular vesicles. The device comprises a base and a three-dimensional culture mechanism mounted on the base, the target extracellular vesicles are prepared, three-dimensional dynamic culture is achieved by simulating a microgravity environment, the interaction between cells is improved, and meanwhile cell proliferation and efficient secretion of the extracellular vesicles are promoted; in the culture process, bubbles in a culture container are removed, so that the culture environment has the characteristics of low shear force, low turbulence, efficient nutrition transfer and the like, the yield of the extracellular vesicles is remarkably improved, the biological activity and functionality of the extracellular vesicles can be kept, and the treatment effect of the extracellular vesicles can be enhanced; the method has the advantages of stable production effect, high efficiency, simple and convenient operation steps, economy and the like, and is a good choice for realizing the yield and function improvement of the extracellular vesicles in vitro.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a three-dimensional extracellular vesicle culture device in a microgravity environment and a method for preparing target extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale membrane vesicles secreted by cells into the extracellular matrix. They carry a large number of bioactive molecules with therapeutic effects and have natural receptor cell targeting, good biosafety and stability. They show broad application prospects in the treatment of various human diseases. However, the clinical application of EVs still faces many challenges. Existing methods for preparing EVs mainly involve cell culture in two-dimensional culture dishes or culture flasks. However, the two-dimensional culture environment limits the cell growth surface area and the interaction between cells and the extracellular matrix, which changes the biological characteristics of the cells and further affects the quality and function of the secreted EVs. Based on this, how to effectively maintain the biological characteristics of cells so that they can secrete EVs of stable quality and function during the treatment process is a key issue that needs to be urgently addressed in the current clinical application of EVs. In recent years, three-dimensional cell culture technology represented by microgravity (μg) environment has gradually emerged. It not only provides a cell culture environment with extremely low shear force, but also facilitates the efficient transport of nutrients, thereby cultivating three-dimensional spherical cell populations. Related studies have reported that microgravity can promote intercellular signaling and improve extracellular matrix remodeling. However, the effects of microgravity on the production and function of extracellular vesicles remain understudied. Summary of the Invention

[0003] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a three-dimensional extracellular vesicle culture device in a microgravity environment and a method for preparing target extracellular vesicles. The microgravity environment simulated by the three-dimensional culture device is used to culture target cells and isolate target extracellular vesicles, and the culture process is precisely controlled to obtain extracellular vesicles with significantly improved yield and function. The culture method has the characteristics of low shear force, low turbulence and efficient nutrient transfer, which helps to improve cell-to-cell interactions and enhance secretion production, providing scheme selection and technical support for in vitro realization of influencing extracellular vesicle yield and function through a microgravity environment.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A three-dimensional extracellular vesicle culture device in a microgravity environment includes a base 1 and a three-dimensional culture mechanism 2 installed on the base 1, wherein the three-dimensional culture mechanism 2 includes a parallel support plate 201 consisting of a bottom plate 2011 and a top plate 2012, and a first motor 202 is installed on the bottom plate 2011, wherein the power output shaft of the first motor 202 is connected to one end of a first longitudinal rotating shaft 203 through a coupling, and the other end of the first longitudinal rotating shaft 203 is fixedly connected to a side plate 2041 on one side of a U-shaped mounting groove 204, and the side plate 2042 on the other side of the U-shaped mounting groove 204 is rotatably connected to the top plate 2012 through a second longitudinal rotating shaft 205; a second motor 206 is installed at the bottom of the U-shaped mounting groove 204, wherein the power output shaft of the second motor 206 is connected to one end of a transverse rotating shaft 207 through a coupling, and the other end of the transverse rotating shaft 207 is fixedly connected to a culture container mounting seat 208, and a culture container 3 is movably connected to the culture container mounting seat 208.

[0006] A gas mixing valve 4 is provided at the center of the top cover of the culture container 3 , and the gas mixing valve 4 is connected to an axially fixed gas exchanger 5 in the culture container 3 . A liquid inlet 6 and a plurality of Luer interface one-way valves 7 are provided on the side wall of the culture container 3 .

[0007] The first motor 202 and the second motor 206 are electrically connected to the controller 8 respectively to control the on / off of the circuit and the motor speed.

[0008] A method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment, specifically comprising the following steps:

[0009] Step 1: Digest the target cells in the logarithmic growth phase in the culture dish with 0.25% Trypsin-EDTA at 36-37°C for 1-2 minutes, centrifuge the digested target cells at 800-1000 rpm for 3-5 minutes, and collect the cell pellet; add fetal bovine serum medium without extracellular vesicles to the cell pellet to resuspend the cell pellet to obtain a cell suspension and adjust its final density to 3×10 5 ~5×10 5 / mL;

[0010] Step 2: Place the cell suspension prepared in step 1 in a microgravity extracellular vesicle three-dimensional culture device to simulate the microgravity environment for three-dimensional cell culture:

[0011] 2.1) Injecting the cell suspension prepared in step 1 into the liquid inlet of the sterile culture container 3 of the extracellular vesicle three-dimensional culture device in a microgravity environment, and filling the remaining space of the culture container 3 with fresh extracellular vesicle-free fetal bovine serum culture medium;

[0012] 2.2) After connecting the culture container 3 to the three-dimensional culture structure 2 of the microgravity extracellular vesicle three-dimensional culture device, the entire device is placed in a cell culture incubator at 37°C and 5% CO2. The initial rotation speed of the extracellular vesicle three-dimensional culture device is set to 8-12 rpm, and the rotation speed is increased by 1-2 rpm every 1-2 hours to offset the increased sedimentation rate. After rotation begins, the target cells form aggregates. As the rotation speed increases, the volume of the target cell aggregates increases. When the target cell aggregates maintain a state of continuous free fall without hitting the wall, the rotation speed is maintained unchanged and the culture is carried out for 48-72 hours.

[0013] 2.3) Remove the culture vessel 3 from the three-dimensional culture structure 2, and orient the culture vessel 3 with the liquid inlet 6 facing upward. Allow the culture vessel 3 to stand for 3-5 minutes to allow the target cell aggregates to settle at the bottom of the culture vessel 3. Open the liquid inlet 6, aspirate 2 / 3-4 / 5 of the cell supernatant above the culture vessel 3, and store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel 3 with fresh extracellular vesicle-free fetal bovine serum culture medium, close the liquid inlet 6, continue culturing under the same conditions as step 2.2) for 48-72 hours, and collect the cell supernatant again using the same method.

[0014] Step 3: The cell supernatant collected in step 2.3) was centrifuged at 200-400 rcf for 10-15 minutes at 0-4°C, and the precipitate was discarded to remove the cells. Then, the supernatant was centrifuged at 2000-3000 rcf for 10-15 minutes, and the precipitate was discarded to remove dead cells. Then, the supernatant was centrifuged at 10000-120000 rcf for 20-30 minutes, and the precipitate was discarded to remove cell debris and impurities. Finally, the supernatant was centrifuged at 100000-120000 rcf for 70-90 minutes. The resulting precipitate is the target extracellular vesicles, which is resuspended in PBS and stored at -80°C.

[0015] The target cells include any one or more of stem cells, epithelial cells, fibroblasts, tumor cells, and immune cells;

[0016] After the remaining space of the culture container is filled with fresh extracellular vesicle-free fetal bovine serum culture medium in steps 2.1) and 2.3), bubbles in the culture container 3 need to be removed to minimize turbulence.

[0017] The specific method for minimizing turbulence is as follows: first, 3 to 5 ml of extracellular vesicle-free fetal bovine serum culture medium is drawn into a sterile syringe and connected to the Luer interface one-way valve 7 of the culture container 3. Then, another empty sterile syringe is connected to the other Luer interface one-way valve 7. The two Luer interface one-way valves 7 are opened. The culture container 3 is placed horizontally with the empty syringe vertically on top to facilitate the floating of bubbles. The extracellular vesicle-free fetal bovine serum culture medium is injected into the culture container 3 from the port of the syringe containing the extracellular vesicle-free fetal bovine serum culture medium, and the liquid and bubbles are discharged from the port of the empty syringe at the same time. The above operation is repeated until all bubbles are removed from the culture container 3. Finally, the Luer interface one-way valve 7 is closed and the valve cover is covered.

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

[0019] 1. Traditional two-dimensional culture environments limit the cell growth surface area and interactions between cells and the extracellular matrix, making it difficult to truly simulate the in vivo microenvironment, thereby limiting the efficiency of cell secretion of extracellular vesicles. The present invention achieves three-dimensional culture by simulating a microgravity environment. The first motor 202 drives the first longitudinal shaft 203, and the second motor 206 drives the transverse shaft 207. Cells self-assemble into spherical structures, effectively improving cell-cell interactions, thereby promoting both cell proliferation and efficient secretion of extracellular vesicles.

[0020] 2. Under static culture conditions, uneven nutrient distribution and inadequate waste removal limit long-term cell growth and exosome secretion efficiency. The present invention achieves dynamic cell culture by simulating a microgravity environment. A gas mixing valve 4 on a culture vessel 3 is connected to an axially fixed gas exchanger 5 within the culture vessel 3. The sidewall of the culture vessel 3 is provided with a liquid inlet 6 and multiple Luer-connected one-way valves 7. During the culture process, bubbles are removed from the culture vessel, resulting in a culture environment with low shear force, low turbulence, and efficient nutrient transfer, thereby significantly increasing the yield of extracellular vesicles.

[0021] 3. The present invention utilizes programmable rotational speed adjustment. The first motor 202 and the second motor 206 are electrically connected to the controller 8, controlling the on / off circuit and motor speed. This can alter the direction of the cell gravity vector within the culture environment, thereby offsetting the effects of gravity and achieving a microgravity environment for extracellular vesicle production. This microgravity environment not only increases extracellular vesicle production but also maintains the biological activity and functionality of the extracellular vesicles, enhancing their therapeutic effects. The increased production of extracellular vesicles in the present invention promotes tissue repair and regeneration, and ameliorates disease.

[0022] In summary, the microgravity-based three-dimensional extracellular vesicle culture device and preparation method of the present invention have the advantages of stable and efficient production effects, simple and economical operation steps, and are a good choice for achieving extracellular vesicle production and function improvement in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structure of the three-dimensional extracellular vesicle culture device in a microgravity environment of the present invention.

[0024] Figure 2 This is the identification of extracellular vesicles in the examples of the present invention.

[0025] Figure 3 This is the effect of microgravity environment on the proliferation of periodontal ligament stem cells in an embodiment of the present invention.

[0026] Figure 4 In the embodiment of the present invention, the microgravity environment significantly improves the production of extracellular vesicles and protein cargo, wherein: Figure 4 (A) is the number of vesicles secreted by a single cell; Figure 4 (B) Protein content in a single vesicle.

[0027] Figure 5 The extracellular vesicles in the embodiment of the present invention significantly enhance the osteogenic differentiation potential of periodontal ligament stem cells, wherein: Figure 5 (A) is the expression level of the osteogenic gene OCN of periodontal ligament stem cells in the blank control group, two-dimensional culture control group and microgravity group. Figure 5 (B) is the expression level of the osteogenic gene ALP of periodontal ligament stem cells in the blank control group, two-dimensional culture control group and microgravity group. Figure 5 (C) The expression levels of the osteogenic gene CLO1A1 of periodontal ligament stem cells in the blank control group, two-dimensional culture control group, and microgravity group. DETAILED DESCRIPTION

[0028] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to embodiments.

[0029] A three-dimensional extracellular vesicle culture device in a microgravity environment includes a base 1 and a three-dimensional culture mechanism 2 installed on the base 1, wherein the three-dimensional culture mechanism 2 includes a parallel support plate 201 consisting of a bottom plate 2011 and a top plate 2012, and a first motor 202 is installed on the bottom plate 2011, wherein the power output shaft of the first motor 202 is connected to one end of a first longitudinal rotating shaft 203 through a coupling, and the other end of the first longitudinal rotating shaft 203 is fixedly connected to a side plate 2041 on one side of a U-shaped mounting groove 204, and the side plate 2042 on the other side of the U-shaped mounting groove 204 is rotatably connected to the top plate 2012 through a second longitudinal rotating shaft 205; a second motor 206 is installed at the bottom of the U-shaped mounting groove 204, wherein the power output shaft of the second motor 206 is connected to one end of a transverse rotating shaft 207 through a coupling, and the other end of the transverse rotating shaft 207 is fixedly connected to a culture container mounting seat 208, and a culture container 3 is movably connected to the culture container mounting seat 208.

[0030] A gas mixing valve 4 is provided at the center of the top cover of the culture container 3 , and the gas mixing valve 4 is connected to an axially fixed gas exchanger 5 in the culture container 3 . A liquid inlet 6 and a plurality of Luer interface one-way valves 7 are provided on the side wall of the culture container 3 .

[0031] The first motor 202 and the second motor 206 are electrically connected to the controller 8 respectively to control the on / off of the circuit and the motor speed.

[0032] A method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment, specifically comprising the following steps:

[0033] Step 1: Digest the target cells in the logarithmic growth phase in the culture dish with 0.25% Trypsin-EDTA at 36-37°C for 1-2 minutes, centrifuge the digested target cells at 800-1000 rpm for 3-5 minutes, and collect the cell pellet; add the cell pellet to the fetal bovine serum medium without extracellular vesicles to resuspend the cell pellet to obtain a cell suspension and adjust its final density to 3×10 5 ~5×10 5 / mL;

[0034] Step 2: Place the cell suspension prepared in step 1 in a microgravity extracellular vesicle three-dimensional culture device to simulate the microgravity environment for three-dimensional cell culture:

[0035] 2.1) Injecting the cell suspension prepared in step 1 into the liquid inlet (6) of the sterile culture container (3) of the extracellular vesicle three-dimensional culture device in a microgravity environment, and filling the remaining space of the culture container (3) with fresh extracellular vesicle-free fetal bovine serum culture medium;

[0036] 2.2) After connecting the culture container (3) to the three-dimensional culture structure (2) of the extracellular vesicle three-dimensional culture device in a microgravity environment, the entire device is placed in a cell culture incubator under culture conditions of 37°C and 5% CO2; the initial rotation speed of the extracellular vesicle three-dimensional culture device is set to 8-12 rpm, and the rotation speed is increased by 1-2 rpm every 1-2 hours to offset the increased sedimentation rate. After the rotation begins, the target cells form aggregates. As the rotation speed increases, the volume of the target cell aggregates increases. When the target cell aggregates maintain a state of continuous free fall without hitting the wall, the rotation speed is maintained unchanged and culture is carried out for 48-72 hours;

[0037] 2.3) Remove the culture container (3) from the three-dimensional culture structure (2), and place the liquid inlet (6) of the culture container (3) upward, and let it stand for 3 to 5 minutes to allow the target cell aggregates to settle on the bottom of the culture container (3); open the liquid inlet (6), aspirate 2 / 3 to 4 / 5 of the cell supernatant above the culture container (3), and store the collected cell supernatant at -80°C; inject fresh extracellular vesicle-free fetal bovine serum culture medium into the culture container (3) to fill the remaining space, close the liquid inlet (6), continue to culture under the same conditions as step 2.2) for 48 to 72 hours, and collect the cell supernatant again according to the same method;

[0038] Step 3: The cell supernatant collected in step 2.3) was centrifuged at 200-400 rcf for 10-15 minutes at 0-4°C, and the precipitate was discarded to remove the cells. Then, the supernatant was centrifuged at 2000-3000 rcf for 10-15 minutes, and the precipitate was discarded to remove dead cells. Then, the supernatant was centrifuged at 10000-120000 rcf for 20-30 minutes, and the precipitate was discarded to remove cell debris and impurities. Finally, the supernatant was centrifuged at 100000-120000 rcf for 70-90 minutes. The resulting precipitate is the target extracellular vesicles, which is resuspended in PBS and stored at -80°C.

[0039] The target cells include any one or more of stem cells, epithelial cells, fibroblasts, tumor cells, and immune cells;

[0040] After the remaining space of the culture container is filled with fresh extracellular vesicle-free fetal bovine serum culture medium in steps 2.1) and 2.3), the bubbles in the culture container (3) need to be removed to minimize turbulence.

[0041] The specific method of minimizing turbulence is as follows: first, use a sterile syringe to suck 3-5 ml of cell-free extracellular vesicle fetal bovine serum medium, connect it to the luer interface one-way valve (7) of the culture container (3), then connect another empty sterile syringe to another luer interface one-way valve (7), open both luer interface one-way valves (7), place the culture container (3) horizontally and the empty syringe vertically above it to facilitate bubble floating, inject the cell-free extracellular vesicle fetal bovine serum medium into the culture container (3) from the syringe port containing the cell-free extracellular vesicle fetal bovine serum medium, while discharging liquid and bubbles from the empty syringe port, repeat the above operation until all bubbles are removed from the culture container (3), and finally close the luer interface one-way valve (7) and cover the valve cover.

[0042] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0043] Example 1

[0044] Microgravity group: periodontal ligament stem cells are cultured in a microgravity environment; the culture process includes the following specific steps:

[0045] Step one: the periodontal ligament stem cells in the logarithmic growth phase in the 150 mm culture dish are digested with 2 mL 0.25% Trypsin-EDTA at 37°C for 1 minute, then centrifuged at 800 rpm for 3 minutes to collect the cell precipitate, resuspend the cell precipitate with cell-free extracellular vesicle fetal bovine serum medium to obtain a cell suspension and adjust the final density to 3.2 x 10 5 cells / mL;

[0046] Step two: the cell suspension prepared in step one is placed in the extracellular vesicle three-dimensional culture device based on the microgravity environment to simulate a microgravity environment for three-dimensional cell culture:

[0047] 2.1) inject 25 mL of the cell suspension obtained in step one into a sterile culture container through the inlet, and fill the remaining space in the culture container with cell-free extracellular vesicle fetal bovine serum medium;

[0048] 2.2) connect the culture container to the three-dimensional culture mechanism of the extracellular vesicle three-dimensional culture device in the microgravity environment, then place the entire device in a cell culture incubator with a culture condition setting of 37°C and 5% CO2; the initial rotation speed of the three-dimensional culture device is set to 8 rpm, and the rotation speed is increased by 1 rpm every 1 hour after the rotation starts to offset the increasing sedimentation rate, and the most appropriate rotation speed is when the periodontal ligament stem cell aggregates maintain a continuous free-fall state without touching the wall;

[0049] 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture vessel from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the culture vessel to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture vessel. Open the liquid inlet and aspirate the cell supernatant from the upper two-thirds of the culture vessel, minimizing the removal of cells. Store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel with fresh extracellular vesicle-free fetal bovine serum culture medium. Close the liquid inlet and continue culturing for 48 hours under the same conditions as in step 2.2). Collect the culture medium again according to the same procedure.

[0050] Step 3: Centrifuge the culture medium collected in step 2.3) at 200 rcf for 10 minutes at 4°C, discard the precipitate to remove cells, then centrifuge at 2000 rcf for 10 minutes, discard the precipitate to remove dead cells, then centrifuge at 10000 rcf for 20 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 100000 rcf for 70 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

[0051] Two-dimensional culture control group: Periodontal ligament stem cells were cultured in a 150 mm culture dish. The culture process included the following specific steps:

[0052] Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 1 minute, then centrifuge at 800 rpm for 3 minutes to collect the cell pellet, resuspend the cell pellet in extracellular vesicle-free fetal bovine serum medium to obtain a cell suspension and adjust its final density to 3.2 × 10 5 / mL;

[0053] Step 2: Take 25 mL of the cell suspension prepared in step 1 and inoculate it into a 150 mm culture dish. Add 5 mL of fetal bovine serum medium without extracellular vesicles. Set the culture conditions to 37°C and 5% CO2. Collect the cell supernatant every 48 hours and store at -80°C.

[0054] Step 3: Centrifuge the cell supernatant collected in step 2 at 200 rcf for 10 minutes at 4°C, discard the precipitate to remove cells, then centrifuge at 2000 rcf for 10 minutes, discard the precipitate to remove dead cells, then centrifuge at 10000 rcf for 20 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 100000 rcf for 70 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

[0055] It can be concluded that compared with the two-dimensional culture control group, the proliferation ability of periodontal ligament stem cells in the microgravity group was significantly enhanced (Appendix Figure 3 ); Under the same number of periodontal ligament stem cells, the secretion volume and protein content of extracellular vesicles in the microgravity group were significantly increased (Appendix Figure 4 ); and the microgravity group's extracellular vesicles significantly enhanced the osteogenic differentiation ability of periodontal ligament stem cells (Appendix Figure 5 In summary, the microgravity group achieved an improvement in the production and function of target extracellular vesicles.

[0056] Example 2:

[0057] Microgravity group: Periodontal ligament stem cells were cultured in a microgravity environment. The culture process included the following specific steps:

[0058] Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 1.5 minutes, then centrifuge at 900 rpm for 4 minutes to collect the cell pellet, resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 4 × 10 5 / mL;

[0059] Step 2: Place the cell suspension prepared in step 1 in a microgravity-based extracellular vesicle three-dimensional culture device to simulate a microgravity environment for three-dimensional cell culture:

[0060] 2.1) Inject 20 mL of the cell suspension obtained in step 1 into a sterile culture container through the liquid inlet, and fill the remaining space of the culture container with extracellular vesicle-free fetal bovine serum culture medium;

[0061] 2.2) After connecting the culture container to the 3D culture mechanism of the microgravity extracellular vesicle 3D culture device, the entire device was placed in a cell culture incubator with culture conditions set to 37°C and 5% CO2. The initial rotation speed of the 3D culture device was set to 10 rpm. After the rotation began, the periodontal ligament stem cells formed aggregates. The rotation speed was increased by 2 rpm every hour to offset the increased sedimentation rate. The optimal rotation speed was achieved when the periodontal ligament stem cell aggregates maintained a state of continuous free fall without hitting the wall.

[0062] 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture vessel from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the culture vessel to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture vessel. Open the liquid inlet and aspirate the cell supernatant from the upper 3 / 4 of the culture vessel, minimizing the removal of cells. Store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel with fresh extracellular vesicle-free fetal bovine serum culture medium. Close the liquid inlet and continue culturing for 48 hours under the same conditions as in step 2.2). Collect the culture medium again according to the same procedure.

[0063] Step 3: Centrifuge the culture medium collected in step 2.3) at 300 rcf for 12 minutes at 3°C, discard the precipitate to remove cells, then centrifuge at 2500 rcf for 12 minutes, discard the precipitate to remove dead cells, then centrifuge at 11000 rcf for 25 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 110000 rcf for 80 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

[0064] Two-dimensional culture control group: Periodontal ligament stem cells were cultured in a 150 mm culture dish. The culture process included the following specific steps:

[0065] Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 1.5 minutes, then centrifuge at 900 rpm for 4 minutes to collect the cell pellet, resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 4 × 10 5 / mL;

[0066] Step 2: Take 20 mL of the cell suspension prepared in step 1 and inoculate it into a 150 mm culture dish. Then add 10 mL of extracellular vesicle-free fetal bovine serum culture medium. The culture conditions are set to 37°C and 5% CO2. The cell supernatant is collected every 48 hours and stored at -80°C.

[0067] Step 3: Centrifuge the cell supernatant collected in step 2 at 300 rcf for 12 minutes at 3°C, discard the precipitate to remove cells, then centrifuge at 2500 rcf for 12 minutes, discard the precipitate to remove dead cells, then centrifuge at 11000 rcf for 25 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 110000 rcf for 80 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

[0068] It can be concluded that compared with the two-dimensional culture control group, the proliferation ability of periodontal ligament stem cells in the microgravity group was significantly enhanced (Appendix Figure 3 ); Under the same number of periodontal ligament stem cells, the secretion volume and protein content of extracellular vesicles in the microgravity group were significantly increased (Appendix Figure 4 ); and the microgravity group's extracellular vesicles significantly enhanced the osteogenic differentiation ability of periodontal ligament stem cells (Appendix Figure 5 In summary, the microgravity group achieved an improvement in the production and function of target extracellular vesicles.

[0069] Example 3:

[0070] Microgravity group: Periodontal ligament stem cells were cultured in a microgravity environment. The culture process included the following specific steps:

[0071] Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 2 minutes, then centrifuge at 1000 rpm for 5 minutes, collect the cell pellet, and resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 5×10 5 / mL;

[0072] Step 2: Place the cell suspension prepared in step 1 in a microgravity-based extracellular vesicle three-dimensional culture device to simulate a microgravity environment for three-dimensional cell culture:

[0073] 2.1) Inject 16 mL of the cell suspension obtained in step 1 into a sterile culture container through the liquid inlet, and fill the remaining space of the culture container with extracellular vesicle-free fetal bovine serum culture medium;

[0074] 2.2) After connecting the culture container to the 3D culture mechanism of the microgravity extracellular vesicle 3D culture device, the entire device was placed in a cell culture incubator with culture conditions set to 37°C and 5% CO2. The initial rotation speed of the 3D culture device was set to 12 rpm. After the rotation began, the periodontal ligament stem cells formed aggregates. The rotation speed was increased by 2 rpm every 2 hours to offset the increased sedimentation rate. The optimal rotation speed was achieved when the periodontal ligament stem cell aggregates maintained a state of continuous free fall without hitting the wall.

[0075] 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture vessel from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the culture vessel to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture vessel. Open the liquid inlet and aspirate the cell supernatant from the upper 4 / 5 of the culture vessel, minimizing the removal of cells. Store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel with fresh extracellular vesicle-free fetal bovine serum culture medium. Close the liquid inlet and continue culturing for 48 hours under the same conditions as in step 2.2). Collect the culture medium again according to the same procedure.

[0076] Step 3: Centrifuge the culture medium collected in step 2.3) at 400 rcf for 15 minutes at 2°C, discard the precipitate to remove cells, then centrifuge at 3000 rcf for 15 minutes, discard the precipitate to remove dead cells, then centrifuge at 12000 rcf for 30 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 120000 rcf for 90 minutes. The resulting precipitate is the periodontal ligament stem cell extracellular vesicles, which is resuspended in PBS and stored at -80°C.

[0077] Two-dimensional culture control group: Periodontal ligament stem cells were cultured in a 150 mm culture dish. The culture process included the following specific steps:

[0078] Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 2 minutes, then centrifuge at 1000 rpm for 5 minutes, collect the cell pellet, and resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 5×10 5 / mL;

[0079] Step 2: Take 16 mL of the cell suspension prepared in step 1 and inoculate it into a 150 mm culture dish. Then add 14 mL of extracellular vesicle-free fetal bovine serum culture medium. The culture conditions are set to 37°C and 5% CO2. The cell supernatant is collected every 48 hours and stored at -80°C.

[0080] Step 3: Centrifuge the cell supernatant collected in step 2 at 400 rcf for 15 minutes at 2°C, discard the precipitate to remove cells, then centrifuge at 3000 rcf for 15 minutes, discard the precipitate to remove dead cells, then centrifuge at 12000 rcf for 30 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 120000 rcf for 90 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

[0081] It can be concluded that compared with the two-dimensional culture control group, the proliferation ability of periodontal ligament stem cells in the microgravity group was significantly enhanced (Appendix Figure 3 ); Under the same number of periodontal ligament stem cells, the secretion volume and protein content of extracellular vesicles in the microgravity group were significantly increased (Appendix Figure 4 ); and the microgravity group's extracellular vesicles significantly enhanced the osteogenic differentiation ability of periodontal ligament stem cells (Appendix Figure 5 In summary, the microgravity group achieved an improvement in the production and function of target extracellular vesicles.

[0082] In the prior art, cells capable of producing target extracellular vesicles are one or more of stem cells, epithelial cells, fibroblasts, tumor cells, and immune cells. In the examples of the present invention, stem cells are used to obtain extracellular vesicles. Because activity decreases after six generations, the stem cells used in the present invention are preferably from passages 3 to 6.

[0083] (1) Identification of extracellular vesicles (Appendix Figure 2 ):

[0084] Extracellular vesicles from the microgravity group and the two-dimensional culture control group were collected and dropped onto a copper grid. They were fixed with 2.5% glutaraldehyde solution and then stained with 2% uranyl acetate solution. They were observed and photographed under a transmission electron microscope.

[0085] The results are as follows Figure 2 Shown: The extracted two-dimensional culture control group ( Figure 2 A) Microgravity group ( Figure 2 B) Extracellular vesicles have a typical lipid bilayer "cup-and-saucer" structure, which is consistent with the general characteristics of extracellular vesicles.

[0086] (2) Cell proliferation ability detection (Appendix Figure 3 ):

[0087] On the second and fourth days of culture, cells were collected from the microgravity and 2D control groups, respectively. The collected cells from each group were incubated with 0.25% Trypsin-EDTA in a 37°C incubator to digest them into single cells. A 1 mL cell suspension was then prepared, and 20 μL was placed on a cell counter plate for counting and recording.

[0088] The results are as follows Figure 3 As shown: On the second day of culture, the number of cells in the two-dimensional culture control group expanded by 2.5 times, while that in the microgravity group expanded by 6.2 times; on the fourth day of culture, the number of cells in the two-dimensional culture control group and the microgravity group increased by 4.8 times and 13.8 times, respectively, indicating that the microgravity culture environment significantly promoted the proliferation ability of periodontal ligament stem cells.

[0089] (3) Extracellular vesicle production and protein cargo analysis (Appendix Figure 4):

[0090] Extracellular vesicles (EVs) from each group were collected and diluted proportionally, and the amount of EV secretion was measured using nanoparticle tracking analysis. Protein standards were then fully dissolved and prepared into a 25 mg / ml protein standard solution, which was diluted to a final concentration of 0.5 mg / ml. 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard solution were added to the standard wells of a 96-well plate, and the standard diluent was added to make up to 20 μl, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. An appropriate volume of EV sample was transferred to a sample well of a 96-well plate, and the standard diluent was added to make up to 20 μl. 200 μl of BCA working solution was added to each well, and the cells were incubated at 37°C for 20–30 minutes. The absorbance at OD = 562 nm was measured using a microplate reader, and the protein concentration was calculated based on the standard curve and the volume of EV sample used. The results were further analyzed by combining BCA experiment with nanoparticle tracking analysis technology.

[0091] The results are as follows Figure 4 As shown: Under the same number of periodontal ligament stem cells, the secretion of extracellular vesicles in microgravity culture environment was significantly higher than that in the control group ( Figure 4 A), and the concentration of proteins contained in its vesicles was significantly higher than that in the control group ( Figure 4 B) The above results indicate that microgravity culture environment can significantly increase the secretion amount and protein content of extracellular vesicles of periodontal ligament stem cells.

[0092] (4) Analysis of the function of extracellular vesicles in promoting osteogenesis (Appendix Figure 5 ):

[0093] Periodontal ligament stem cells were seeded in 6-well plates and divided into blank control group, microgravity group, and two-dimensional culture control group. After overnight culture, the culture medium of each group was replaced with osteogenic induction medium. The osteogenic induction medium was replaced every 3 days and extracellular vesicles (100 μg / mL) of the microgravity group and two-dimensional culture control group were added to the medium. After 7 days of co-induction, samples were collected, RNA was extracted and reverse transcription experiments were performed, and finally real-time quantitative polymerase chain reaction was performed to detect the expression of osteogenic-related genes OCN ( Figure 5 A) ALP( Figure 5 B), COL1A1( Figure 5 C) The relative mRNA expression levels of the blank control group, microgravity group, and two-dimensional culture control group were analyzed to obtain the differences.

[0094] The results are as follows Figure 5As shown in the results: compared with the blank control group, both the microgravity group and the two-dimensional culture control group can promote the expression of osteoblast-related genes in periodontal ligament stem cells; at the same time, compared with the two-dimensional culture control group, the expression of osteoblast-related genes induced by the microgravity group showed a further upward trend. The above results indicate that extracellular vesicles in the microgravity culture environment significantly enhance the osteoblast differentiation potential of periodontal ligament stem cells.

Claims

1. A three-dimensional extracellular vesicle culture device in a microgravity environment, comprising a base (1) and a three-dimensional culture mechanism (2) mounted on the base (1), characterized in that: The three-dimensional culture mechanism (2) comprises a parallel support plate (201) consisting of a bottom plate (2011) and a top plate (2012); a first motor (202) is mounted on the bottom plate (2011); a power output shaft of the first motor (202) is connected to one end of a first longitudinal rotating shaft (203) via a coupling; the other end of the first longitudinal rotating shaft (203) is fixedly connected to a side plate (2041) on one side of a U-shaped mounting groove (204); and the U-shaped mounting groove (204) is fixedly connected to a side plate (2041) on one side of the U-shaped mounting groove (204). The side plate (2042) on the other side is rotatably connected to the top plate (2012) via a second longitudinal rotating shaft (205); a second motor (206) is installed at the bottom of the U-shaped mounting groove (204); a power output shaft of the second motor (206) is connected to one end of a transverse rotating shaft (207) via a coupling; the other end of the transverse rotating shaft (207) is fixedly connected to a culture container mounting seat (208), and a culture container (3) is movably connected to the culture container mounting seat (208).

2. The three-dimensional extracellular vesicle culture device in a microgravity environment according to claim 1, characterized in that: A gas mixing valve (4) is provided at the center of the top cover of the culture container (3), and the gas mixing valve (4) is connected to an axially fixed gas exchanger (5) in the culture container (3). A liquid inlet (6) and a plurality of Luer interface one-way valves (7) are provided on the side wall of the culture container (3).

3. The three-dimensional extracellular vesicle culture device in a microgravity environment according to claim 1, characterized in that: The first motor (202) and the second motor (206) are respectively electrically connected to a controller (8) to control the on / off of the circuit and the speed of the motors.

4. A method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment based on the device according to any one of claims 1 to 3, characterized in that: The specific steps include: Step 1: Digest the target cells in the logarithmic growth phase in the culture dish with 0.25% Trypsin-EDTA at 36-37°C for 1-2 minutes, centrifuge the digested target cells at 800-1000 rpm for 3-5 minutes, and collect the cell pellet; add fetal bovine serum medium without extracellular vesicles to the cell pellet to resuspend the cell pellet to obtain a cell suspension and adjust its final density to 3×10 5 ~5×10 5 / mL; Step 2: Place the cell suspension prepared in step 1 in a microgravity extracellular vesicle three-dimensional culture device to simulate the microgravity environment for three-dimensional cell culture: 2.1) Injecting the cell suspension prepared in step 1 into the liquid inlet (6) of the sterile culture container (3) of the extracellular vesicle three-dimensional culture device in a microgravity environment, and filling the remaining space of the culture container (3) with fresh extracellular vesicle-free fetal bovine serum culture medium; 2.2) After connecting the culture container (3) to the three-dimensional culture structure (2) of the extracellular vesicle three-dimensional culture device in a microgravity environment, the entire device is placed in a cell culture incubator under culture conditions of 37°C and 5% CO2; the initial rotation speed of the extracellular vesicle three-dimensional culture device is set to 8-12 rpm, and the rotation speed is increased by 1-2 rpm every 1-2 hours to offset the increased sedimentation rate. After the rotation begins, the target cells form aggregates. As the rotation speed increases, the volume of the target cell aggregates increases. When the target cell aggregates maintain a state of continuous free fall without hitting the wall, the rotation speed is maintained unchanged and culture is carried out for 48-72 hours; 2.3) Remove the culture container (3) from the three-dimensional culture structure (2), and place the liquid inlet (6) of the culture container (3) upward, and let it stand for 3 to 5 minutes to allow the target cell aggregates to settle on the bottom of the culture container (3); open the liquid inlet (6), aspirate 2 / 3 to 4 / 5 of the cell supernatant above the culture container (3), and store the collected cell supernatant at -80°C; inject fresh extracellular vesicle-free fetal bovine serum culture medium into the culture container (3) to fill the remaining space, close the liquid inlet (6), continue to culture under the same conditions as step 2.2) for 48 to 72 hours, and collect the cell supernatant again according to the same method; Step 3: The cell supernatant collected in step 2.3) was centrifuged at 200-400 rcf for 10-15 minutes at 0-4°C, and the precipitate was discarded to remove the cells. Then, the supernatant was centrifuged at 2000-3000 rcf for 10-15 minutes, and the precipitate was discarded to remove dead cells. Then, the supernatant was centrifuged at 10000-120000 rcf for 20-30 minutes, and the precipitate was discarded to remove cell debris and impurities. Finally, the supernatant was centrifuged at 100000-120000 rcf for 70-90 minutes. The resulting precipitate is the target extracellular vesicles, which is resuspended in PBS and stored at -80°C.

5. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 4, characterized in that: The target cells include any one or more of stem cells, epithelial cells, fibroblasts, tumor cells, and immune cells.

6. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 4, characterized in that: After filling the remaining space of the culture container with fresh extracellular vesicle-free fetal bovine serum culture medium in steps 2.1) and 2.3), bubbles in the culture container (3) must be removed to minimize turbulence.

7. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 6, characterized in that: The specific method for minimizing turbulence is as follows: first, use a sterile syringe to draw 3 to 5 ml of extracellular vesicle-free fetal bovine serum culture medium, connect it to the Luer interface one-way valve (7) of the culture container (3), then connect another empty sterile syringe to another Luer interface one-way valve (7), open the two Luer interface one-way valves (7), place the culture container (3) horizontally and place the empty syringe vertically on top to facilitate the floating of bubbles, inject the extracellular vesicle-free fetal bovine serum culture medium into the culture container (3) from the port of the syringe containing the extracellular vesicle-free fetal bovine serum culture medium, and at the same time discharge liquid and bubbles from the port of the empty syringe, repeat the above operation until all bubbles are removed from the culture container (3), and finally close the Luer interface one-way valve (7) and cover the valve cover.

8. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 4, characterized in that: The specific steps include: Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 1 minute, then centrifuge at 800 rpm for 3 minutes to collect the cell pellet, resuspend the cell pellet in extracellular vesicle-free fetal bovine serum medium to obtain a cell suspension and adjust its final density to 3.2 × 10 5 / mL; Step 2: Place the cell suspension prepared in step 1 in a microgravity-based extracellular vesicle three-dimensional culture device to simulate a microgravity environment for three-dimensional cell culture: 2.1) Inject 25 mL of the cell suspension obtained in step 1 into a sterile culture container through the liquid inlet, and fill the remaining space of the culture container with extracellular vesicle-free fetal bovine serum culture medium; 2.2) After connecting the culture container to the 3D culture mechanism of the microgravity extracellular vesicle 3D culture device, the entire device was placed in a cell culture incubator with culture conditions set to 37°C and 5% CO2. The initial rotation speed of the 3D culture device was set to 8 rpm. After the rotation began, the periodontal ligament stem cells formed aggregates. The rotation speed was increased by 1 rpm every hour to offset the increased sedimentation rate. The optimal rotation speed was achieved when the periodontal ligament stem cell aggregates maintained a state of continuous free fall without hitting the wall. 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture container from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the container to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture container. Open the liquid inlet, aspirate the cell supernatant from the upper 2 / 3 of the culture vessel, and store the collected cell supernatant at -80°C. Inject fresh extracellular vesicle-free fetal bovine serum medium into the culture vessel to fill the remaining space. Close the liquid inlet, continue culturing under the same conditions as step 2.2) for 48 hours, and collect the medium again according to the same steps. Step 3: Centrifuge the culture medium collected in step 2.3) at 200 rcf for 10 minutes at 4°C, discard the precipitate to remove cells, then centrifuge at 2000 rcf for 10 minutes, discard the precipitate to remove dead cells, then centrifuge at 10000 rcf for 20 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 100000 rcf for 70 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

9. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 4, characterized in that: The specific steps include: Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 1.5 minutes, then centrifuge at 900 rpm for 4 minutes to collect the cell pellet, resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 4 × 10 5 / mL; Step 2: Place the cell suspension prepared in step 1 in a microgravity-based extracellular vesicle three-dimensional culture device to simulate a microgravity environment for three-dimensional cell culture: 2.1) Inject 20 mL of the cell suspension obtained in step 1 into a sterile culture container through the liquid inlet, and fill the remaining space of the culture container with extracellular vesicle-free fetal bovine serum culture medium; 2.2) After connecting the culture container to the 3D culture mechanism of the microgravity extracellular vesicle 3D culture device, the entire device was placed in a cell culture incubator with culture conditions set to 37°C and 5% CO2. The initial rotation speed of the 3D culture device was set to 10 rpm. After the rotation began, the periodontal ligament stem cells formed aggregates. The rotation speed was increased by 2 rpm every hour to offset the increased sedimentation rate. The optimal rotation speed was achieved when the periodontal ligament stem cell aggregates maintained a state of continuous free fall without hitting the wall. 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture vessel from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the culture vessel to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture vessel. Open the liquid inlet and aspirate the cell supernatant from the upper 3 / 4 of the culture vessel. Store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel with fresh extracellular vesicle-free fetal bovine serum culture medium. Close the liquid inlet and continue culturing for 48 hours under the same conditions as in step 2.2). Collect the culture medium again according to the same procedure. Step 3: Centrifuge the culture medium collected in step 2.3) at 300 rcf for 12 minutes at 3°C, discard the precipitate to remove cells, then centrifuge at 2500 rcf for 12 minutes, discard the precipitate to remove dead cells, then centrifuge at 11000 rcf for 25 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 110000 rcf for 80 minutes. The resulting precipitate is the extracellular vesicles of periodontal ligament stem cells, which is resuspended in PBS and stored at -80°C.

10. The method for preparing target extracellular vesicles by three-dimensional culture of extracellular vesicles in a microgravity environment according to claim 4, characterized in that: The specific steps include: Step 1: Digest periodontal ligament stem cells in the logarithmic growth phase in a 150 mm culture dish with 2 mL of 0.25% Trypsin-EDTA at 37°C for 2 minutes, then centrifuge at 1000 rpm for 5 minutes, collect the cell pellet, and resuspend the cell pellet in fetal bovine serum medium without extracellular vesicles to obtain a cell suspension and adjust its final density to 5 × 10 5 / mL; Step 2: Place the cell suspension prepared in step 1 in a microgravity-based extracellular vesicle three-dimensional culture device to simulate a microgravity environment for three-dimensional cell culture: 2.1) Inject 16 mL of the cell suspension obtained in step 1 into a sterile culture container through the liquid inlet, and fill the remaining space of the culture container with extracellular vesicle-free fetal bovine serum culture medium; 2.2) After connecting the culture container to the 3D culture mechanism of the microgravity extracellular vesicle 3D culture device, the entire device was placed in a cell culture incubator with culture conditions set to 37°C and 5% CO2. The initial rotation speed of the 3D culture device was set to 12 rpm. After the rotation began, the periodontal ligament stem cells formed aggregates. The rotation speed was increased by 2 rpm every 2 hours to offset the increased sedimentation rate. The optimal rotation speed was achieved when the periodontal ligament stem cell aggregates maintained a state of continuous free fall without hitting the wall. 2.3) After culturing the periodontal ligament stem cells for 48 hours, remove the culture vessel from the three-dimensional culture structure and place it in a biosafety cabinet with the liquid inlet facing upward. Allow the culture vessel to stand for 3 minutes to allow the periodontal ligament stem cell aggregates to settle at the bottom of the culture vessel. Open the liquid inlet and aspirate the cell supernatant from the top 4 / 5 of the culture vessel. Store the collected cell supernatant at -80°C. Fill the remaining space in the culture vessel with fresh extracellular vesicle-free fetal bovine serum culture medium. Close the liquid inlet and continue culturing for 48 hours under the same conditions as in step 2.2). Collect the culture medium again according to the same procedure. Step 3: Centrifuge the culture medium collected in step 2.3) at 400 rcf for 15 minutes at 2°C, discard the precipitate to remove cells, then centrifuge at 3000 rcf for 15 minutes, discard the precipitate to remove dead cells, then centrifuge at 12000 rcf for 30 minutes, discard the precipitate to remove cell debris and impurities, and finally centrifuge the supernatant at 120000 rcf for 90 minutes. The resulting precipitate is the periodontal ligament stem cell extracellular vesicles, which is resuspended in PBS and stored at -80°C.

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