Stem cell-based microvesicle as well as preparation method and application thereof

Abstract

The invention discloses a stem cell-based microvesicle as well as a preparation method and application thereof, belongs to the technical field of biological tissue engineering, and particularly relates to a preparation method of the stem cell-based microvesicle, which comprises the following steps: culturing stem cells until the cell fusion degree is 80-90%, performing hypoxia treatment, and cleaning for 2-3 times; standing and culturing the cleaned stem cells, then adding a calcium ion carrier, and carrying out ultrasonic treatment; the serum-free basic culture medium containing the calcium ion carrier is immediately absorbed and removed, the cells are cleaned through PBS, culture is conducted for 24 h, and cell supernatant is collected and merged respectively; performing first centrifugation to remove cell debris; performing second centrifugation to remove dead cells and large cell debris; carrying out third centrifugation to remove organelles; fourth centrifugation is carried out, large vesicles are removed, and a final supernatant is obtained; and filtering and centrifuging to obtain a microvesicle precipitate. The preparation method of the microvesicles aims at systematically improving the yield and biological activity of the stem cell microvesicles through low-oxygen pre-stimulation, ultrasonic-calcium ion carrier synergistic induction and differential centrifugation.

Description

Technical Field

[0001] This invention belongs to the field of bio-tissue engineering technology, and particularly relates to a stem cell-based microvesicle, its preparation method, and its application. Background Technology

[0002] Stem cell therapy has shown great potential in tissue repair, regenerative medicine, and disease treatment. However, direct application of stem cells faces numerous challenges, including immune rejection, tumorigenesis risks, ethical constraints, low in vivo survival rates, and low colonization efficiency. Recent research on the paracrine mechanisms of stem cells has revealed that one of the key mechanisms by which stem cells exert their therapeutic effects is through the secretion of various active factors, with microvesicles (such as exosomes and microvesicles) serving as crucial signal transduction carriers. These nanoscale or microscale membrane vesicles carry bioactive substances such as proteins, nucleic acids (e.g., mRNA, miRNA), and lipids from the source cells, mediating intercellular communication and regulating the biological behavior of recipient cells. They exhibit significant efficacy in promoting angiogenesis, inhibiting apoptosis, regulating immune responses, and stimulating in situ tissue regeneration.

[0003] Currently, the acquisition of stem cell microvesicles mainly relies on the isolation and purification of stem cells from conditioned culture media after in vitro culture. Conventional preparation methods often employ ultracentrifugation combined with ultrafiltration, size exclusion chromatography, and polymer precipitation for purification. However, existing technologies have several significant bottlenecks that severely restrict the clinical translation and industrial application of microvesicles. The natural secretion of microvesicles by stem cells under conventional conditions is limited, resulting in only a very small number of microvesicles being harvested from large amounts of culture medium, leading to extremely high production costs and making it difficult to meet the requirements for dosage consistency and sufficiency in large-scale treatments or commercial products. Most existing methods passively collect naturally secreted microvesicles from stem cells, lacking effective means to actively and purposefully intervene in stem cells to "promote" their microvesicle generation. The state of stem cells, the culture microenvironment, and specific inducing factors have a decisive influence on the yield and composition of microvesicles, but existing conventional culture systems have not systematically optimized these factors to achieve "high-yield" microvesicles.

[0004] Therefore, developing a method for efficiently generating microvesicles and significantly increasing their yield, while simultaneously ensuring or even enhancing the bioactivity and functional uniformity of the resulting microvesicles, is of paramount importance for advancing basic research, clinical therapeutic applications, and related product development of stem cell microvesicles. This will not only address the core pain points of current preparation technologies—low yield and high cost—but also lay a solid foundation for obtaining microvesicle therapeutic products with controllable quality and clearly defined efficacy. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a stem cell-based microvesicle, its preparation method, and its application. The microvesicle preparation method of this invention aims to systematically improve the yield and bioactivity of stem cell microvesicles through a process of hypoxia pre-stimulation, ultrasound-calcium ion carrier synergistic induction, and differential centrifugation for refined collection.

[0006] To achieve the above objectives, this invention provides a method for preparing stem cell-based microvesicles, comprising the following steps: 1) culturing stem cells in complete culture medium until cell confluence reaches 80-90%, subjecting them to hypoxia, discarding the culture medium, and washing 2-3 times; 2) statically culturing the washed stem cells from step 1) in serum-free basal culture medium, then adding calcium ion carrier to the serum-free basal culture medium and performing sonication; 3) immediately after the sonication in step 2), removing the serum-free basal culture medium containing calcium ion carrier, and washing the cells with PBS containing 1% bovine serum albumin. 1-3 times, add fresh serum-free basal medium and culture for 24h. Collect and combine cell supernatants at 6h, 12h and 24h of culture respectively; 4) First centrifugation to remove cell debris; second centrifugation to remove dead cells and large cell debris; third centrifugation to remove organelles; fourth centrifugation to remove large vesicles and obtain the final supernatant; 5) Filter the final supernatant obtained in step 4) with a filter membrane, centrifuge at 100,000-120,000g for 70-90min at 4℃, discard the supernatant to obtain the microvesicle precipitate; 6) Resuspend the microvesicle precipitate obtained in step 5), aliquot and store in a -80℃ refrigerator.

[0007] Preferably, the stem cells mentioned in step 1) are mesenchymal stem cells, which are one of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose mesenchymal stem cells. The culture environment conditions mentioned in step 1) are: 37°C, CO2 volume fraction 5%; the hypoxia treatment environment conditions mentioned in step 1) are: 37°C, CO2 volume fraction 5%, O2 volume fraction 2%, and the hypoxia treatment time is 10~14h; the washing in step 1) uses sterile PBS.

[0008] Preferably, the static culture time in step 2) is 0.5~1h, and the environmental conditions for static culture are: 37℃, CO2 volume fraction 5%; the final concentration of the calcium ion carrier in the serum-free basal medium in step 2) is 1μM.

[0009] Preferably, in step 2), the ultrasonic treatment uses degassed, preheated sterile saline solution to 37°C as the ultrasonic coupling agent, the ultrasonic treatment frequency is 200 kHz, and the ultrasonic treatment power is 0.5~1.5 W / cm². 2The ultrasonic treatment uses pulsed waves, with a working time of 0.5 seconds and a pause of 0.5 seconds, and the ultrasonic treatment time is 2 to 4 minutes.

[0010] Preferably, the environmental conditions for cultivation in step 3) are: 37°C and CO2 volume fraction of 5%.

[0011] Preferably, in step 4), the temperature of the first centrifugation is 4°C, the centrifugal force is 300g, and the time is 10min; in step 4), the temperature of the second centrifugation is 4°C, the centrifugal force is 2000g, and the time is 20min; in step 4), the temperature of the third centrifugation is 4°C, the centrifugal force is 10000g, and the time is 30min; and in step 4), the temperature of the fourth centrifugation is 4°C, the centrifugal force is 20000g, and the time is 45min.

[0012] Preferably, the filter membrane used in step 5) is a 0.22 μm filter membrane.

[0013] Preferably, the resuspension in step 6) is performed using sterile PBS or sterile saline.

[0014] The present invention also provides microvesicles prepared by the aforementioned preparation method.

[0015] The present invention also provides the application of the microvesicles in the preparation of products for repairing tissue damage.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a stem cell-based microvesicle, its preparation method, and its applications. The microvesicle preparation method aims to systematically improve the yield and bioactivity of stem cell microvesicles through a process involving hypoxia pre-stimulation, synergistic induction by ultrasound and calcium ion carriers, and refined collection via differential centrifugation. Hypoxia is a physiological stressor for stem cells, activating pathways such as HIF-1α and initiating stem cells into a state of high paracrine activity. Under serum-free conditions, a large amount of microvesicles are released through synergistic physical and chemical stimulation. Adding calcium ion carriers can rapidly increase intracellular calcium levels, activating microvesicle secretion. The use of low-intensity pulsed waves and ultrasound effectively reduces thermal damage and improves cell tolerance. The synergistic action of physical and chemical stimulation through different signaling pathways significantly increases yield. This invention maximizes the recovery of microvesicles continuously secreted by cells after stimulation and purifies them efficiently. Microvesicles are collected at 6h, 12h, and 24h of culture time, preventing reabsorption or degradation by cells in the culture environment. By gradually increasing centrifugation, cell debris, large organelles, and other impurities are removed, ultimately obtaining microvesicles of relatively uniform size, ensuring maximum recovery of cell-secreted microvesicles. This invention improves microvesicle yield from three aspects: secretion source, induction intensity, and recovery efficiency. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results of NTA assays for the cell microvesicles prepared in Example 1 and Comparative Example 1; Figure 2 A comparison of the secretion numbers of cell microvesicles prepared in Example 1 and Comparative Example 1; Figure 3 The results of CCK-8 assay for the cell microvesicles prepared in Example 1 and Comparative Example 1; Figure 4 The images show the appearance of the knee joints of rats in each group. A represents the sham-operated group, B represents the blank control group, C represents experimental group 1, and D represents experimental group 2. Detailed Implementation

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The description and embodiments of this invention are exemplary only. Unless otherwise specified, all materials used in this invention are commercially available.

[0020] Example 1 1) Umbilical cord mesenchymal stem cells were cultured in complete culture medium (37℃, CO2 volume fraction 5%) until the cell confluence was 85%. The cells were then treated with low oxygen for 12 hours in an environment of 37℃, CO2 volume fraction 5%, and O2 volume fraction 2%. The culture medium was then discarded and the cells were washed three times with sterile PBS.

[0021] 2) Stem cells were statically cultured in serum-free basal medium (37℃, 5% CO2) for 1 hour. Then, calcium ionophore (A23187) was added to the serum-free basal medium to a final concentration of 1 μM, followed by sonication. Degassed, preheated sterile saline solution at 37℃ was used as the sonication coupling agent at a frequency of 200 kHz and a power of 1 W / cm². 2 The process involves pulsed waves, with a 0.5s working cycle followed by a 0.5s pause, and ultrasonic treatment lasting 3 minutes.

[0022] 3) After sonication, the serum-free basal medium containing calcium ion carrier was immediately aspirated. The cells were washed twice with PBS containing 1% bovine serum albumin. Fresh serum-free basal medium was added and the cells were cultured (37°C, 5% CO2) for 24 hours. The cell supernatants were collected and combined at 6, 12 and 24 hours of culture.

[0023] 4) Centrifuge at 300g for 10 minutes at 4℃ to remove cell debris; centrifuge at 2000g for 20 minutes at 4℃ to remove dead cells and large cell debris; centrifuge at 10000g for 30 minutes at 4℃ to remove organelles; centrifuge at 20000g for 45 minutes at 4℃ to remove large vesicles and obtain the final supernatant.

[0024] 5) Filter the final supernatant through a 0.22μm filter membrane, centrifuge at 110000g for 80min at 4℃, discard the supernatant, and obtain the microvesicle precipitate.

[0025] 6) Resuspend the microvesicle precipitate in sterile physiological saline, aliquot, and store at -80°C. Figure 2 As shown, the number of microvesicles secreted was 7.891 × 10⁻⁶. 10 / mL.

[0026] Example 2 1) Adipose-derived mesenchymal stem cells were cultured in complete culture medium (37℃, CO2 volume fraction 5%) until the cell confluence was 85%. The cells were then subjected to hypoxia in an environment of 37℃, CO2 volume fraction 5%, and O2 volume fraction 2% for 10 hours. The culture medium was then discarded and the cells were washed three times with sterile PBS.

[0027] 2) Stem cells were statically cultured in serum-free basal medium (37℃, 5% CO2) for 0.5 h. Then, calcium ionophore (A23187) was added to the serum-free basal medium to a final concentration of 1 μM, followed by sonication. Degassed, preheated sterile saline solution at 37℃ was used as the sonic coupling agent at a frequency of 200 kHz and a power of 0.5 W / cm². 2 The process involves pulse waves, with a 0.5s working time followed by a 0.5s pause, and a total ultrasonic treatment time of 4 minutes.

[0028] 3) After sonication, the serum-free basal medium containing calcium ion carrier was immediately aspirated. The cells were washed twice with PBS containing 1% bovine serum albumin. Fresh serum-free basal medium was added and the cells were cultured (37°C, 5% CO2) for 24 hours. The cell supernatants were collected and combined at 6, 12 and 24 hours of culture.

[0029] 4) Centrifuge at 300g for 10 minutes at 4℃ to remove cell debris; centrifuge at 2000g for 20 minutes at 4℃ to remove dead cells and large cell debris; centrifuge at 10000g for 30 minutes at 4℃ to remove organelles; centrifuge at 20000g for 45 minutes at 4℃ to remove large vesicles and obtain the final supernatant.

[0030] 5) Filter the final supernatant through a 0.22μm filter membrane, centrifuge at 100000g for 90min at 4℃, discard the supernatant, and obtain the microvesicle precipitate.

[0031] 6) Resuspend the microvesicle precipitate in sterile physiological saline, aliquot, and store in a -80℃ refrigerator.

[0032] Example 3 1) Bone marrow mesenchymal stem cells were cultured in complete culture medium (37℃, CO2 volume fraction 5%) until the cell confluence was 85%. The cells were then treated with hypoxia in an environment of 37℃, CO2 volume fraction 5%, and O2 volume fraction 2% for 14 hours. The culture medium was then discarded and the cells were washed three times with sterile PBS.

[0033] 2) Stem cells were statically cultured in serum-free basal medium (37℃, 5% CO2) for 0.5 h. Then, calcium ionophore (A23187) was added to the serum-free basal medium to a final concentration of 1 μM, followed by sonication. Degassed, preheated sterile saline solution at 37℃ was used as the sonic coupling agent at a frequency of 200 kHz and a power of 1.5 W / cm². 2 The process involves pulsed waves, with a 0.5s working time, a 0.5s pause, and a 2-minute ultrasonic treatment.

[0034] 3) After sonication, the serum-free basal medium containing calcium ion carrier was immediately aspirated. The cells were washed twice with PBS containing 1% bovine serum albumin. Fresh serum-free basal medium was added and the cells were cultured (37°C, 5% CO2) for 24 hours. The cell supernatants were collected and combined at 6, 12 and 24 hours of culture.

[0035] 4) Centrifuge at 300g for 10 minutes at 4℃ to remove cell debris; centrifuge at 2000g for 20 minutes at 4℃ to remove dead cells and large cell debris; centrifuge at 10000g for 30 minutes at 4℃ to remove organelles; centrifuge at 20000g for 45 minutes at 4℃ to remove large vesicles and obtain the final supernatant.

[0036] 5) The final supernatant was filtered through a 0.22 μm filter membrane, centrifuged at 120,000 g for 70 min at 4 °C, and the supernatant was discarded to obtain the microvesicle precipitate.

[0037] 6) Resuspend the microvesicle precipitate in sterile PBS, aliquot, and store at -80°C.

[0038] Comparative Example 1 1) Umbilical cord mesenchymal stem cells were cultured in complete culture medium (37℃, CO2 volume fraction 5%) until the cell confluence was 85%. The cells were then treated with low oxygen for 12 hours in an environment of 37℃, CO2 volume fraction 5%, and O2 volume fraction 2%. The culture medium was then discarded and the cells were washed three times with sterile PBS.

[0039] 2) Stem cells were statically cultured in serum-free basal medium (37℃, 5% CO2) for 1 hour. Then, calcium ionophore (A23187) was added to the serum-free basal medium to a final concentration of 1 μM, followed by sonication. Degassed, preheated sterile saline solution at 37℃ was used as the sonic coupling agent at a frequency of 1 MHz and a power of 2 W / cm². 2 The process involves pulsed waves, with a 0.5s working cycle followed by a 0.5s pause, and ultrasonic treatment lasting 3 minutes.

[0040] 3) After sonication, the serum-free basal medium containing calcium ion carrier was immediately aspirated. The cells were washed twice with PBS containing 1% bovine serum albumin. Fresh serum-free basal medium was added and the cells were cultured (37°C, 5% CO2) for 24 hours. The cell supernatants were collected and combined at 6, 12 and 24 hours of culture.

[0041] 4) Centrifuge at 300g for 10 minutes at 4℃ to remove cell debris; centrifuge at 2000g for 20 minutes at 4℃ to remove dead cells and large cell debris; centrifuge at 10000g for 30 minutes at 4℃ to remove organelles; centrifuge at 20000g for 45 minutes at 4℃ to remove large vesicles and obtain the final supernatant.

[0042] 5) Filter the final supernatant through a 0.22μm filter membrane, centrifuge at 110000g for 80min at 4℃, discard the supernatant, and obtain the microvesicle precipitate.

[0043] 6) Resuspend the microvesicle precipitate in sterile physiological saline, aliquot, and store at -80°C. Figure 2 As shown, the number of microvesicles secreted was 2.688 × 10⁻⁶. 10 / mL.

[0044] Experimental Example 1 Nanoparticle tracking analysis (NTA) was employed using a NanoSight instrument to directly image and observe microvesicles with diameters ranging from 50 to 1000 nm in the suspensions prepared in Example 1 and Comparative Example 1 in real time. This allowed for precise determination of the number of microvesicles at different sizes, and the number of obtained microvesicles was also measured. Following step 3) of cultivation in Example 1 and Comparative Example 1, proliferation was detected using a CCK-8 assay.

[0045] like Figure 1 The results of NTA assay are shown. Compared with the preparation method of Comparative Example 1, the preparation method of Example 1 resulted in increased secretion of cell microvesicles, and the NTA assay results showed an increase in the number of cell microvesicles. Figure 2 The image shows a comparison of the number of microvesicles secreted in Example 1 and Comparative Example 1. The preparation method in Example 1 significantly increases the number of microvesicles secreted. Figure 3 As shown in the figure, the CCK-8 detection results show that the proliferation activity is unchanged compared to Comparative Example 1.

[0046] Experiment Example 2 Rat bone marrow mesenchymal stem cells (purchased from Shanghai Yaji) were used to prepare experimental group 1 microvesicles and experimental group 2 microvesicles using the preparation methods described in Example 1 and Comparative Example 1, respectively.

[0047] Eighteen 2-month-old SD rats underwent hair removal at the surgical site and were anesthetized intraperitoneally with 0.3 mL / 100 g of 10% chloral hydrate. A 1.5 cm arc-shaped incision was made bilaterally on the medial side of the knee joint to expose the non-weight-bearing surface of the femoral trochlea. One × 0.2 mm of cartilage tissue was scraped away to construct an articular cartilage defect model. Postoperatively, 100,000 units of penicillin were injected intramuscularly once daily for three consecutive days. Model rats were obtained two weeks after modeling. Six 2-month-old SD rats were included in the sham-operated group; only the knee joint was incised.

[0048] The rats were divided into three groups of six each. The blank control group was injected with 60 μL of physiological saline once a week for 8 weeks. Experimental group 1 was injected with 60 μL of experimental group microvesicles once a week for 8 weeks. Experimental group 2 was injected with 60 μL of experimental group microvesicles once a week. The sham-operated group was injected with 60 μL of physiological saline once a week for 8 weeks.

[0049] After the injection, the rats were sacrificed, and the femoral trochlea of ​​the experimental rats was exposed to observe the degree of tissue repair in the defect area, the smoothness of the repaired tissue, and the connection between the repaired tissue and the undamaged area.

[0050] like Figure 4 China A Figure 4 B, Figure 4 C and Figure 4As shown in Figure D, the knee joints of rats in the sham-operated group, blank control group, experimental group 1, and experimental group 2 are respectively observed. It can be seen that the joint surface of the rats in the sham-operated group is smooth and shiny, with no obvious damage to the articular cartilage. The joint surface of the blank control group is uneven, and the base of the defect area is partially filled. The joint surface of experimental group 1 is smoother than that of the blank control group, and the base of the defect area is mostly filled and shiny. The joint surface of experimental group 2 is similar in smoothness to that of the blank control group, but the base of the defect area is mostly filled and shiny.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing stem cell-based microvesicles, characterized in that, Includes the following steps: 1) Stem cells are cultured in complete culture medium until the cell confluence reaches 80-90%, subjected to hypoxia, the culture medium is discarded, and the cells are washed 2-3 times. 2) Stem cells washed in step 1) were statically cultured in serum-free basal medium, and then calcium ion carriers were added to the serum-free basal medium and subjected to sonication. 3) After the ultrasonic treatment described in step 2), the serum-free basal culture medium containing calcium ion carrier was immediately aspirated. The cells were washed 1-3 times with PBS containing 1% bovine serum albumin. Fresh serum-free basal culture medium was added and the cells were cultured for 24 hours. The cell supernatants were collected and combined at 6, 12 and 24 hours of culture, respectively. 4) First centrifugation to remove cell debris; The second centrifugation removes dead cells and large cell debris; the third centrifugation removes organelles. The fourth step is centrifugation to remove large vesicles and obtain the final supernatant; 5) The final supernatant obtained in step 4) is filtered through a filter membrane, centrifuged at 100,000~120,000g for 70~90min at 4℃, and the supernatant is discarded to obtain the microvesicle precipitate. 6) Resuspend the microvesicle precipitate obtained in step 5), aliquot it, and store it in a refrigerator at -80°C.

2. The preparation method according to claim 1, characterized in that, The stem cells mentioned in step 1) are mesenchymal stem cells, which are one of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose mesenchymal stem cells. The culture environment conditions mentioned in step 1) are: 37℃, CO2 volume fraction 5%; the hypoxia treatment environment conditions mentioned in step 1) are: 37℃, CO2 volume fraction 5%, O2 volume fraction 2%, and the hypoxia treatment time is 10~14h; the washing in step 1) uses sterile PBS.

3. The preparation method according to claim 1, characterized in that, The static culture time in step 2) is 0.5~1h, and the environmental conditions for static culture are: 37℃, CO2 volume fraction 5%; the final concentration of the calcium ion carrier in the serum-free basal medium in step 2) is 1μM.

4. The preparation method according to claim 1, characterized in that, In step 2), the ultrasonic treatment uses degassed, preheated sterile saline solution at 37°C as the ultrasonic coupling agent. The frequency of the ultrasonic treatment is 200 kHz, and the power of the ultrasonic treatment is 0.5~1.5 W / cm². 2 The ultrasonic treatment uses pulsed waves, with a working time of 0.5 seconds and a pause of 0.5 seconds, and the ultrasonic treatment time is 2 to 4 minutes.

5. The preparation method according to claim 1, characterized in that, The environmental conditions for cultivation described in step 3) are: 37℃, CO2 volume fraction 5%.

6. The preparation method according to claim 1, characterized in that, In step 4), the temperature of the first centrifugation is 4°C, the centrifugal force is 300g, and the time is 10min; in step 4), the temperature of the second centrifugation is 4°C, the centrifugal force is 2000g, and the time is 20min; in step 4), the temperature of the third centrifugation is 4°C, the centrifugal force is 10000g, and the time is 30min; in step 4), the temperature of the fourth centrifugation is 4°C, the centrifugal force is 20000g, and the time is 45min.

7. The preparation method according to claim 1, characterized in that, The filter membrane used in step 5) is a 0.22 μm filter membrane.

8. The preparation method according to claim 1, characterized in that, The resuspension described in step 6) uses sterile PBS or sterile saline.

9. Microvesicles prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the microvesicles as described in claim 9 in the preparation of products for repairing tissue damage.

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