Cell microvesicles based on freezing extraction as well as extraction method and application of cell microvesicles

Abstract

The invention is suitable for the technical field of biological medicine, and provides a cell microvesicle based on freezing extraction and an extraction method and application thereof, ginsenoside Rh2 is used for pre-stimulating human umbilical cord mesenchymal stem cells (hUC-MSCs), a freezing extraction technology is used for extracting cell-derived frozen microvesicles (CDCs), and the novel engineered vesicles Rh2-CDCs are prepared. The method can effectively improve in-vivo blood coagulation and fibrinolytic system functions, reduce multi-organ injury, improve the treatment effect and improve the biological stability of nano-drug treatment, and the freezing extraction technology not only greatly shortens the extraction time and reduces the production cost, but also can obtain the microvesicles with higher concentration, so that the microvesicles have better application prospects. The technical bottleneck of the traditional exosome in application is expected to be overcome; besides, the freezing extraction technology is also suitable for vesicle extraction of other types of cells, and the extracted CDCs can be used as a nano-particle platform to deliver other drugs and active ingredients, so that the freezing extraction method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a cell microvesicle based on cryoextraction, its extraction method, and its application. Background Technology

[0002] In recent years, the use of mesenchymal stem cells (MSCs) as drug delivery platforms has attracted much attention, and exosomes (Exos), as a very important category of extracellular vesicles, have become a current research hotspot due to their high utilization value. Exos are considered ideal carriers due to their low immunogenicity and high bioactivity. However, the application and promotion of Exos are limited by several problems: 1) Exos extraction is difficult; their tiny size and complex separation requirements make the extraction process very challenging; 2) Existing purification techniques are complex and cumbersome, with low purification efficiency, making it difficult to meet the requirements of large-scale production; 3) Exos are prone to degradation during extraction, storage, and transportation, affecting their stability and bioactivity; 4) Although traditional differential ultracentrifugation is the most commonly used method for Exos extraction, it faces problems such as long processing time, high equipment costs, complex operation, and the potential mechanical damage to the Exos structure caused by high centrifugal force. Therefore, simplifying extracellular vesicle extraction techniques is necessary for the development and application of biomaterials. Summary of the Invention

[0003] The purpose of this invention is to provide a cell microvesicle based on cryoextraction, its extraction method, and its application, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: In the first aspect, a cell microvesicle based on cryo-extraction, the extraction method of which includes the following steps: hUC-MSCs pretreatment: hUC-MSCs were digested with trypsin and collected into centrifuge tubes, centrifuged, and the supernatant was discarded. Preparation of cell-derived frozen microvesicles (CDCs): Pretreated hUC-MSCs were collected and resuspended with sterile PBS, frozen at -80°C for 24 h, and then rapidly thawed in a 37°C water bath. Immediately after thawing, the cells were centrifuged at 4°C to remove cell debris, and the supernatant was filtered. The vesicle-like particles were then enriched by two ultracentrifugation cycles, which were then stored at -80°C. Preparation of Rh2-CDCs: hUC-MSCs were pre-stimulated with ginsenoside Rh2. hUC-MSCs and ginsenoside Rh2 were co-incubated at 37℃ for 24h. After trypsin digestion, the suspended hUC-MSCs were collected and centrifuged in centrifuge tubes. The supernatant was discarded, and the cells were resuspended in sterile PBS. The treated hUC-MSCs were frozen at -80℃ for 24h and then rapidly thawed in a 37℃ water bath. Immediately after thawing, the cells were centrifuged at 4℃ to remove cell debris and the supernatant was filtered. The cells were then enriched by two ultracentrifugation cycles to obtain vesicle-like particles, which were then Rh2-CDCs and stored at -80℃.

[0005] Secondly, the present invention also provides an application of cryo-extraction-based cell microvesicles in the preparation of drug delivery systems.

[0006] Thirdly, the present invention also provides an application of cell microvesicles based on cryoextraction in the preparation of DIC therapeutic drugs.

[0007] Compared with the prior art, the specific beneficial effects of the present invention are as follows: This invention utilizes cryoextraction technology and pre-stimulation with ginsenoside Rh2 to successfully extract engineered microvesicles Rh2-CDCs. This method has significant advantages in improving vesicle extraction concentration and efficiency, solving the problem of low extraction efficiency of Exos. CDCs have stronger membrane integrity and yield, which allows for the extraction of more vesicles with the same number of MSCs. Compared with traditional Exos extraction technology, the yield of microvesicles is 10 times higher than that of Exos, and the extraction yield after pre-stimulation with ginsenoside Rh2 is even 15 times higher. Furthermore, CDCs and Rh2-CDCs have the potential to become highly efficient nanocarrier platforms, which can be used as carriers in drug delivery systems. This system has a significant function in regulating coagulation and inflammatory responses under pathological conditions, and can effectively improve multiple coagulation indicators in animal models of disseminated intravascular coagulation (DIC), including shortening prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT), increasing fibrinogen (FIB) levels, and reducing fibrin degradation products (FDP) and D-dimer levels. At the same time, it significantly inhibits microthrombus formation in multiple organ tissues, revealing the important role of this carrier in regulating the body's microenvironment, and providing a general technical platform for developing novel nanomedicine therapy strategies. In summary, the extraction method provided by the embodiments of the present invention has good universality and scalability. It can not only be used for loading and delivery of various bioactive components, but also be widely applied to multiple therapeutic areas such as cardiovascular and cerebrovascular diseases, tumors, inflammatory diseases, and immune-related diseases. The cell-derived carrier technology involved provides a key foundation for integrating traditional drugs with advanced delivery systems and constructing a multifunctional nanobiomedicine platform, which has significant prospects for industrial application and broad clinical translational value. Attached Figure Description

[0008] Figure 1 Morphological characterization of hUC-MSCs provided in embodiments of the present invention; wherein, A is a schematic diagram of stem cell trilineage differentiation; B is a representative image of osteogenic differentiation of hUC-MSCs; C is a representative image of adipogenic differentiation of hUC-MSCs; and D is a representative image of chondrogenic differentiation of hUC-MSCs. Figure 2 The following are the biological characterization results of hUC-MSCs provided in the embodiments of the present invention; wherein, A is a flow cytometry isotype control image (negative control); B is a flow cytometry analysis image of CD105; C is a flow cytometry analysis image of CD73 and CD90; and D is a flow cytometry analysis image of CD11b, CD19, CD34, CD45 and HLA-DR. Figure 3 The following are the preparation methods, morphological characteristics, and particle size distribution results of Exos, CDCs, and Rh2-CDCs provided in the embodiments of the present invention; A is a schematic diagram of the extraction process of Exos, CDCs, and Rh2-CDCs; B is the diameter of Exos, CDCs, and Rh2-CDCs determined by nanoparticle tracking analysis; C is a representative TEM image of Exos, CDCs, and Rh2-CDCs. Figure 4 The following are the results of the expression and functional differences of biomarkers for Exos, CDCs, and Rh2-CDCs provided in the embodiments of the present invention: A shows the concentration determination of Exos, CDCs, and Rh2-CDCs; B shows the HPLC statistical analysis of cells and supernatants of Rh2-prestimulated hUC-MSCs; C and D show the HPLC results of cells and supernatants of Rh2-prestimulated hUC-MSCs (n=4); E shows the results of Western blot analysis of specific biomarkers (CD9, CD81, TSG101, Calnexin) for Exos, CDCs, and Rh2-CDCs. <0.001, <0.0001; Figure 5The diagram shows the changes in coagulation and fibrinolysis functions in each group provided in the embodiments of the present invention; where A is the time axis for animal model construction and drug administration; BG is the detection and statistical analysis graph of rat plasma coagulation function indicators (n = 6), namely: PT (prothrombin time); APTT (activated partial thromboplastin time); TT (thrombin time); Fib (fibrinogen); FDP (fibrin(ogen) degradation products); D-dimer (D-dimer); <0.05, <0.01, <0.001, <0.0001; Figure 6 The organ changes of the heart in each group of Wistar rats provided in the embodiments of the present invention are shown in the following images: gross changes of the heart (top) obtained by photographing under the same light conditions; HE staining analysis (middle) and PTAH staining analysis (bottom) observed under a microscope; scale bar = 100 μm. Figure 7 The organ changes of the kidneys of various Wistar rats provided in the embodiments of the present invention are shown in the gross changes of the kidneys (top) obtained by photographing under the same light conditions, and the HE staining analysis (middle) and PTAH staining analysis (bottom) obtained by microscopic observation. Scale bar = 50 μm. Figure 8 The organ changes of the liver of each Wistar rat group provided in the embodiments of the present invention are shown in the gross changes of the liver (top) obtained by photographing under the same light, and the HE staining analysis (middle) and PTAH staining analysis (bottom) obtained by microscopic observation. Scale bar = 100 μm. Figure 9 The organ changes of the spleen in each group of Wistar rats provided in the embodiments of the present invention are shown in the macroscopic changes of the spleen obtained under the same light conditions (top), HE staining analysis (middle) and PTAH staining analysis (bottom) observed under a microscope, scale bar = 100 μm; Figure 10 The organ changes in the lungs of various Wistar rats provided in the embodiments of the present invention are shown in the following images: gross changes in the lungs (top) obtained under the same lighting conditions; HE staining analysis (middle) and PTAH staining analysis (bottom) observed under a microscope; scale bar = 100 μm. Figure 11 The images show the organ changes in the brains of various Wistar rats provided in the embodiments of the present invention. The images show the gross changes in the brain (top) obtained by photographing under the same light conditions, and the HE staining analysis (middle) and PTAH staining analysis (bottom) obtained by microscopic observation. Scale bar = 100 μm. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0011] Example 1: A cell microvesicle based on cryo-extraction, the extraction method of which is as follows: Figure 3 As shown in Figure A, the specific steps include: (1) Processing of hUC-MSCs: Discard the culture medium in the hUC-MSCs culture dish, add 1.5 mL of trypsin to digest for 90 s, collect the suspended hUC-MSCs into a 15 mL centrifuge tube, centrifuge at 1000 g for 5 min, discard the supernatant, and resuspend in 3 mL of sterile PBS. (2) Preparation of CDCs: hUC-MSCs were frozen at -80℃ for 24h and then rapidly thawed in a 37℃ water bath. Immediately after thawing, they were placed at 4℃ and centrifuged at 5000g for 30min to remove cell debris. The supernatant was filtered through a 0.22μm filter membrane and then subjected to two cycles of 100000g and 60min ultracentrifugation to enrich vesicle-like particles. (3) Storage of CDCs: Store at -80℃; (4) Preparation of Rh2-CDCs: hUC-MSCs were pre-stimulated with ginsenoside Rh2. Ginsenoside Rh2 was added to the culture medium of hUC-MSCs culture dish to make the concentration of ginsenoside Rh2 reach 16 μM. The culture medium was incubated at 37℃ for 24 h. The culture medium was discarded, and 1.5 mL of trypsin was added for 90 s digestion. The suspended hUC-MSCs were collected into 15 mL centrifuge tubes and centrifuged at 1000 g for 5 min. The supernatant was discarded and the cells were resuspended with 3 mL of sterile PBS. The treated hUC-MSCs were frozen at -80℃ for 24 h and then rapidly thawed in a 37℃ water bath. After thawing, the cells were immediately placed at 4℃ and centrifuged at 5000 g for 30 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane. Then, the cells were enriched by two cycles of 100000 g and 60 min ultracentrifugation. (5) Storage of Rh2-CDCs: Store at -80℃.

[0012] Analysis was performed on hUC-MSCs, CDCs, Rh2-CDCs, etc. in Example 1: 1. Morphological and biological characteristics of hUC-MSCs: 1.1 In order to systematically and comprehensively characterize the morphological and biological characteristics of hUC-MSCs, experiments were conducted to analyze the characteristics, purity, and multi-lineage differentiation potential of mesenchymal stem cells, such as... Figure 1 As shown, the details are as follows: A schematic diagram of multi-directional differentiation potential is shown below. Figure 1 As shown in Figure A, the ability of hUC-MSCs to differentiate into osteoblasts, adipocytes and chondrocytes under specific induction conditions is summarized in a clear and organized manner, outlining their "stem cell" characteristics and multi-lineage differentiation potential. Results of osteogenic differentiation capacity assessment as follows Figure 1 As shown in Figure B, after hUC-MSCs were cultured in osteogenic induction medium, calcium nodule formation was observed by Alizarin Red staining. The figure shows obvious red mineralized nodules, indicating that the cells have a high osteogenic differentiation capacity (scale bar = 50 μm). The results of the adipogenic differentiation capacity assessment are as follows: Figure 1 As shown in Figure C, after adipogenic induction culture, the accumulation of intracellular lipid droplets was detected by Oil Red O staining. The results showed that typical orange-red lipid droplets appeared in the cytoplasm, confirming that hUC-MSCs have adipogenic differentiation potential (scale bar = 50 μm). Results of chondrogenic differentiation capacity assessment as follows Figure 1 As shown in Figure D, cell clusters (microspheres) were formed through chondrogenic induction culture and Alcian blue staining was performed. Glycosaminoglycans in the cartilage matrix showed a blue positive reaction, proving that they successfully differentiated into chondrocytes (scale bar = 50 μm). This indicates that hUC-MSCs possess multi-directional differentiation stemness. 1.2. Flow cytometry was used to systematically identify the surface antigens of hUC-MSCs, and the results are as follows: Figure 2 As shown, the quantitative results indicate that the cells highly express CD73, CD90, and CD105 (positive rate >95%), but do not express CD11b, CD19, CD34, CD45, and HLA-DR (negative rate >99%), which fully meets the phenotypic definition criteria of mesenchymal stem cells of the International Society for Cell Therapy (ISCT). In summary, both functional differentiation and molecular phenotype confirmed that the hUC-MSCs used possess typical MSC characteristics and meet purity standards, providing important biological evidence for their subsequent use as drug carriers or cell therapy products.

[0013] 2. Systematic physical characterization and molecular phenotypic identification were performed on Exos, CDCs, and Rh2-CDCs, clarifying their preparation methods, morphological characteristics, particle size distribution, biomarker expression, and functional differences, such as... Figure 3 , Figure 4 As shown, the details are as follows: Extraction and preparation strategies such as Figure 3 As shown in Figure A, Exos were isolated from the culture supernatant of hUC-MSCs by ultracentrifugation; CDCs were enriched by cryoextraction and ultracentrifugation cycle of frozen microvesicles; Rh2-CDCs were obtained by using the same CDCs extraction process after hUC-MSCs were prestimulated with ginsenoside Rh2. This strategy aims to enhance the anticoagulation and immunomodulatory functions of vesicles. Nanoparticle size tracking analysis (NTA) results are as follows Figure 3 As shown in Figure B, the NTA particle size distribution shows that the average particle size of Exos is 148 nm, while that of CDCs and Rh2-CDCs is 135 nm and 133 nm, respectively, which are consistent with typical particle size characteristics. Morphological observation by transmission electron microscopy (TEM) Figure 3 As shown in C, TEM images show that Exos, CDCs, and Rh2-CDCs all exhibit typical spherical structures of lipid bilayer membranes, with complete morphology, clear boundaries, and good structural integrity. The results of the vesicle concentration comparison are as follows Figure 4 As shown in Figure A, compared with Exos, CDCs and Rh2-CDCs have significantly higher extraction yields, demonstrating their potential advantages in large-scale preparation. The results of the verification of the mechanism of action of ginsenoside Rh2 pretreatment are as follows: Figure 4 As shown in Figure BD, high-performance liquid chromatography (HPLC) analysis revealed that no Rh2 peak was detected in hUC-MSCs pretreated with ginsenoside Rh2 (e.g., Figure 4 As shown in C), a distinct Rh2 peak is visible in the supernatant (as shown in Figure C). Figure 4 As shown in D), the possibility of Rh2 directly loading drugs is ruled out, indicating that its functional regulation is mainly achieved through cell pretreatment; Results of Western Blot analysis of protein biomarkers are as follows Figure 4 As shown in Figure E, Exos highly expresses CD9, TSG101, and CD81, but does not express Calnexin; while CDCs and Rh2-CDCs, in addition to being positive for CD9, CD81, and TSG101, also show positive Calnexin, suggesting that their origin and composition are significantly different from Exos, belonging to a new type of vesicle population with greater heterogeneity. In summary, multiple dimensions have systematically confirmed that CDCs and Rh2-CDCs are novel vesicles that are significantly different from Exos in terms of origin, yield, and protein composition, and possess the following advantages: In terms of extraction and preparation: CDCs have a simpler preparation process, higher yield, and better scalability and application potential; Physical and chemical properties: CDCs and Rh2-CDCs show significant differences from Exos in terms of physical properties, chemical composition and molecular phenotype, indicating that they are a separate type of vesicle entity. In terms of functional regulation: pre-stimulation with ginsenoside Rh2 can further regulate the functional properties of vesicles, enhancing their potential as drug carriers and their targeting ability. These results fully demonstrate that CDCs and Rh2-CDCs are novel nanoparticles with promising applications, providing a solid experimental foundation for their subsequent development and application in drug preparation.

[0014] 3. Effects of ginsenosides Rh2, Exos, CDCs and Rh2-CDCs on coagulation and fibrinolysis in DIC model rats: DIC model rat construction: Female Wister rats weighing 160-180 g and aged 5-6 weeks were injected via the tail vein with 10 mg / kg lipopolysaccharide (LPS) slowly over 1 hour to construct a DIC rat model. According to the diagnostic criteria of DIC diagnosis and management guidelines, the following conditions were met for successful model establishment: PT prolongation of more than 3 seconds / ATPP prolongation of more than 5 seconds; presence of D-dimer; FIB reduction of more than 25%; and microthrombus formation shown in pathological tissue sections. Grouping and treatment: Patients were randomly divided into 6 groups: control group, model group, ginsenoside Rh2 group, Exos group, CDCs group, and Rh2-CDCs group (n=6). The control group received 0.9% saline via tail vein injection. Eight hours after modeling, the ginsenoside Rh2 group received 5 mg / kg ginsenoside Rh2; the Exos group received 400 μg / kg Exos; the CDCs group received 400 μg / kg CDCs; and the Rh2-CDCs group received 400 μg / kg Rh2-CDCs. All interventions were administered via tail vein injection. The effects of different intervention strategies on improving systemic hypercoagulable state were evaluated by detecting changes in key coagulation and fibrinolysis markers in the plasma of rats in each group. The results are as follows Figure 5 As shown, compared with the model group, all treatment groups improved coagulation dysfunction to some extent, specifically with a significant increase in Fib levels, and shorter PT, APTT and TT compared with the model group. At the same time, the fibrinolytic markers D-dimer and FDP levels decreased significantly. Among them, the Rh2-CDCs treatment group showed the best reversal effect in all the above indicators: the highest Fib concentration, the closest recovery of PT, APTT, and TT to the normal range, and the lowest D-dimer and FDP levels; the CDCs group was second, but its efficacy was still better than the Rh2 monotherapy group and the Exos treatment group; the results indicate that Rh2-CDCs can most effectively alleviate the systemic hypercoagulable state and excessive fibrinolytic reaction caused by DIC, suggesting that it has significant potential as a novel treatment strategy in improving the balance of the coagulation-fibrinolytic system; The above results provide key experimental evidence for the application of Rh2-CDCs in the preparation of drugs for DIC and related coagulation disorders, suggesting their great potential and broad application prospects in regulating coagulation function and immune-targeted regulation.

[0015] 4. Analysis of the organ protection effects of Rh2-CDCs and CDCs on DIC rats: Disseminated intravascular coagulation (DIC) can cause microthrombus formation in organs throughout the body. A series of histopathological examinations are performed on the heart, liver, spleen, lungs, kidneys, and brain. Hematoxylin and eosin (H&E) staining is used to observe changes in tissue structure and inflammatory responses, while PTAH staining is used to detect fibrin thrombus formation. This aims to clarify the distribution of microthrombi, ischemic necrosis, and hemorrhage in various organs, providing a basis for diagnosis and treatment. The results are as follows: Figures 6 to 11 As shown, the details are as follows: Gross and pathological changes of the heart, such as Figure 6 As shown, the control group had intact myocardial structure and a normal, rosy appearance; while in the LPS model group, the heart volume increased, and severe edema and dark red color were observed, with a small amount of myocardial tissue necrosis, cell nucleus condensation and fragmentation, vacuolar degeneration, and microthrombi forming in the blood vessels of the myocardium; in the Rh2 and Exos treatment groups, the myocardial color began to improve and become rosy, hypertrophy recovered, necrosis decreased, swelling lessened, vacuolar degeneration decreased sequentially, and PTAH showed a significant increase in thrombus dissolution; furthermore, in the treatment of CDCs and Rh2-CDCs, even more obvious changes were observed, with almost no fibrous accumulation in the heart and no obvious thrombi, indicating that the Rh2-CDCs group had the best treatment effect; Gross and pathological changes of the kidneys, such as Figure 7As shown, the kidneys are target organs for microthrombosis and inflammation in DIC. In the control group, the kidneys were rosy and structurally intact, with normal glomeruli, tubules, and interstitium. In the LPS group, the kidneys were darker, slightly congested and blackish, with mild edema of the tubular epithelial cells and scattered inflammatory cell infiltration. Swelling of the tubular epithelial cells containing homogeneous, powdery protein-like fragments and forming casts was observed. Mild thrombosis was observed in PTAH. In the Rh2 and Exos treatment groups, the capillary congestion in the glomeruli and tubulointerstitium decreased sequentially, the degree of tubular dilation decreased sequentially, and the scattered inflammatory cell infiltration decreased. In PTAH, the accumulation of homogeneous fibrous material in the tubules decreased sequentially. It is evident that CDCs and Rh2-CDCs treatments had the best therapeutic effects and good renal structural recovery. Gross and pathological changes of the liver, such as Figure 8 As shown, the liver, as the main site of synthesis of most clotting factors and anticoagulant proteins, plays an indicative role in the pathology of DIC. The liver tissue in the control group was normal; the liver in the LPS model group was congested and enlarged, with slight damage to the lobular structure, severe vacuolar degeneration and significant swelling of a large number of hepatocytes, narrowing of the sinusoids, and extensive inflammatory cell infiltration and microthrombus formation. PTAH staining showed extensive fibrin microthrombi in the lobules. In Rh2 and Exos treatment, a small amount of vacuolar degeneration and inflammatory cell infiltration were observed in hepatocytes, and the symptoms were significantly relieved. In comparison, CDCs and Rh2-CDCs had better treatment effects, with a significant reduction in inflammatory cell infiltration. The latter had the best treatment effect, with intact lobular structure and good normal morphology, almost returning to normal, and PTAH showed a significant reduction in thrombi. Gross and pathological changes of the spleen, as follows: Figure 9 As shown, the spleen plays an important role in DIC by clearing coagulation products, regulating immunity, and maintaining blood homeostasis. The spleen structure was normal in the control group. In the LPS model group, obvious splenic congestion, enlarged splenic megakaryocytes, and lymphocyte proliferation were observed. PTAH showed that it was accompanied by the development of microvascular thrombosis and fibrin deposition. After Rh2 and Exos treatment, significant improvement was observed, with reduced lymphocyte proliferation. PTAH showed a reduction in splenic fibrin deposition. After CDCs and Rh2-CDCs treatment, there was almost no fibrin deposition, with the latter showing the best treatment effect. Gross and pathological changes of the lungs, as follows: Figure 10As shown, the lungs, as an important site of microcirculation, are the main target of DIC. In the control group, the lung structure was intact, and the alveolar wall structure was clear and complete. In the LPS group, pulmonary congestion was observed, and under the microscope, the alveolar walls of the lung tissue were thickened, the alveolar cavities were narrowed and there was neutrophil infiltration. PTAH showed local thrombus formation. After treatment, the Rh2 and Exos groups showed mild thickening of the alveolar walls of the lung tissue and scattered neutrophil infiltration, with the degree of thickening generally decreasing in that order. The CDCs and Rh2-CDCs treatments showed better results, with more complete recovery of the alveolar walls, and PTAH showed that microthrombi were almost invisible. Gross and pathological changes of the brain, such as Figure 11 As shown, the brain may be severely affected by DIC. Normal brain tissue is generally intact and has a clear structure. In the LPS group, the brain tissue shows slight congestion and is dark in color. Severe edema of brain cells and increased gaps are visible, as well as inflammatory cell infiltration. In PTAH, extensive fibrin thrombus formation is seen in the brain microvessels (especially capillaries and arterioles), obstructing blood flow. In the Rh2 and Exos treatment groups, the gaps between brain tissues are narrowed, and the infiltration of inflammatory cells is reduced. In the CDCs and Rh2-CDCs groups, near-normal brain tissue is visible, and thrombi are no longer visible in PTAH.

[0016] In summary, interventions with Rh2, Exos, CDCs, and Rh2-CDCs improved the pathological state of various organs to varying degrees. Among them, the CDCs and Rh2-CDCs groups showed particularly significant effects in reducing microthrombi, inhibiting inflammatory responses, and restoring tissue structure, with the Rh2-CDCs group exhibiting the best therapeutic effect. These results, from a histopathological perspective, confirm that Rh2-CDCs have a highly effective multi-organ protective capacity, providing solid experimental evidence for potential therapeutic drugs for DIC and various coagulation-related diseases.

[0017] This invention, for the first time, employs cryoextraction to extract Rh2-CDCs from hUC-MSCs pre-stimulated with ginsenoside Rh2, constructing a novel engineered microvesicle. Characterization of Rh2-CDCs and CDCs confirms their stable physicochemical properties and good biocompatibility. In an LPS-induced DIC rat model, Rh2-CDCs exhibit remarkable therapeutic potential, significantly improving systemic intravascular coagulation function, specifically manifested in improved coagulation indicators and reduced DIC marker levels, and effectively inhibiting microthrombus formation in multiple organs including the heart, liver, spleen, lungs, kidneys, and brain. This invention not only reveals the specific mechanisms of action of CDCs and Rh2-CDCs in regulating coagulation function and microenvironment homeostasis, providing a new strategy for the treatment of DIC, but also promotes the development and optimization of cell-derived vesicle extraction technology. The cryoextraction technology used in this invention is not only applicable to hUC-MSCs, but can also be widely extended to other cell types. In terms of active ingredient pretreatment, it is not limited to ginsenoside Rh2, but is also applicable to other drugs or bioactive molecules. Therefore, this invention not only brings breakthrough progress to the preparation of therapeutic drugs for coagulation disorders such as DIC, but also provides a therapeutic platform with broad application prospects for drugs for other diseases, and demonstrates important value in the development of novel nanocarriers and translational medicine.

[0018] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell microvesicle based on cryo-extraction, characterized in that, The extraction method includes the following steps: hUC-MSCs pretreatment: hUC-MSCs were digested with trypsin and collected into centrifuge tubes, centrifuged, and the supernatant was discarded. Preparation of CDCs: Pretreated hUC-MSCs were collected and resuspended with sterile PBS, frozen at -80°C for 24 h, and then rapidly thawed in a 37°C water bath. Immediately after thawing, the cells were centrifuged at 4°C to remove cell debris and the supernatant was filtered. Then, the vesicle-like particles were enriched by two ultracentrifugation cycles, which were then stored at -80°C. Preparation of Rh2-CDCs: hUC-MSCs were pre-stimulated with ginsenoside Rh2. hUC-MSCs and ginsenoside Rh2 were co-incubated at 37℃ for 24h. After trypsin digestion, the suspended hUC-MSCs were collected and centrifuged in centrifuge tubes. The supernatant was discarded, and the cells were resuspended in sterile PBS. The treated hUC-MSCs were frozen at -80℃ for 24h and then rapidly thawed in a 37℃ water bath. Immediately after thawing, the cells were centrifuged at 4℃ to remove cell debris and the supernatant was filtered. Then, two ultracentrifugation cycles were performed to enrich vesicle-like particles, which were then Rh2-CDCs and stored at -80℃.

2. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the pretreatment steps of hUC-MSCs, the centrifugation process specifically involves centrifuging at 1000g for 5 minutes.

3. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of CDCs preparation, the centrifugation treatment at 4°C specifically refers to centrifuging at 5000g for 30 minutes.

4. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of CDCs preparation, the ultracentrifugation specifically involves centrifuging at 100,000g for 60 min.

5. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of preparing Rh2-CDCs, the specific process of collecting suspended hUC-MSCs into centrifuge tubes and centrifuging them is as follows: centrifuge at 1000g for 5 minutes.

6. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of preparing Rh2-CDCs, the centrifugation treatment at 4°C specifically refers to centrifuging at 5000g for 30 minutes.

7. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of preparing Rh2-CDCs, the ultracentrifugation specifically refers to centrifugation at 100,000g for 60 min.

8. The cell microvesicles based on cryo-extraction according to claim 1, characterized in that, In the steps of CDCs preparation and Rh2-CDCs preparation, the supernatant filtration specifically involves filtering the supernatant through a 0.22 μm filter membrane.

9. The use of cryo-extraction-based cell microvesicles as described in any one of claims 1-8 in the preparation of a drug delivery system.

10. The use of cryo-extraction-based cell microvesicles as described in any one of claims 1-8 in the preparation of DIC therapeutic agents.

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