Preparation method of fibroblast-derived exosome, exosome and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of a fibroblast-derived exosome, the exosome and application of the exosome. According to the preparation method of the exosome, the exosome does not depend on high-cost exogenous growth factors, the overall cost is lower, the sustainability is higher, the traction force belongs to physical controllable stimulation, dependence on recombinant proteins such as VEGF / bFGF can be reduced, and the problems that the exosome is high in cost, prone to inactivation, different in batch, difficult to deliver and the like are solved. The yield and activity of the prepared exosome are synchronously enhanced, standardized quality control is easier, secretion increase of the exosome is achieved through YAP mediation, and the exosome has more stable and traceable functional characteristics along with selective enrichment of key angiogenic protein, so that establishment of amplified production and release standards is facilitated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing exosomes derived from fibroblasts, the exosomes themselves, and their applications. Background Technology

[0002] Skin wound repair is a common and complex medical problem in clinical practice. Acute trauma and chronic, refractory wounds often have limited repair due to insufficient blood supply in the early stages. Current treatment methods mainly include surgical skin grafting, wound dressings, and biological agents, but all have significant limitations: surgical skin grafting is limited by insufficient autologous skin sources and the risk of donor site damage; allogeneic or tissue-engineered skin is costly and lacks stability; traditional dressings are mainly protective and have limited healing-promoting capabilities; biological agents such as growth factors are expensive, have poor in vivo stability, and are difficult to maintain efficacy in the long term.

[0003] In recent years, tissue-engineered skin and exosome biopharmaceuticals have become important research directions for promoting wound repair and angiogenesis. Exosomes, due to their low immunogenicity and strong signal transduction capabilities, have become a key research area for promoting angiogenesis and tissue repair. However, existing exosome strategies still face problems such as insufficient yield, unstable activity, and lack of controllable functional regulation, especially in three-dimensional tissue engineering systems, where in vitro vascularization efficiency is low and there is a lack of simulation of the physiological and mechanical microenvironment. Furthermore, in the field of 3D bioprinted skin, researchers often promote vascularization by adding growth factors or optimizing scaffold materials, but the simulation of the dynamic mechanical microenvironment that actually exists in vivo remains relatively limited.

[0004] Although existing studies suggest that mechanical stimulation may enhance the pro-angiogenic effect of fibroblast exosomes, current technologies largely remain at the observational level, lacking a systematic and standardized system of mechanical parameters and a stable and reproducible method for exosome enhancement, hindering large-scale application. Furthermore, in tissue-engineered skin culture systems, current strategies primarily rely on exogenous growth factor supplementation, resulting in high costs, poor stability, and significant batch-to-batch variability. They also lack a systematic integration of the complete regulatory chain of "stretching—YAP signaling—exosome secretion and functional reprogramming." Therefore, a controllable process based on mechanical microenvironment regulation that can stably increase exosome production and enhance its pro-angiogenic capacity is still lacking. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing exosomes derived from fibroblasts, the exosomes and their applications.

[0006] This invention provides a method for preparing exosomes derived from fibroblasts, comprising the following steps:

[0007] Step 1: Take fibroblasts and seed them on a deformable culture medium substrate for culture;

[0008] Step 2: After the fibroblasts adhere to the culture wall, apply tensile stimulation to the deformable culture medium substrate, and obtain the culture supernatant after culturing.

[0009] Step 3: Take the culture supernatant and centrifuge it to obtain exosomes.

[0010] In some embodiments, in step 1, the fibroblasts are human skin fibroblasts.

[0011] In some embodiments, in step 1, the deformable culture substrate includes an elastic culture plate or a PCL fixation frame system.

[0012] In some embodiments, in step 2, the strain of the tension stimulation is 5% to 10%, and the duration of the tension stimulation is 5 to 10 days.

[0013] In some embodiments, the duration is 7 days.

[0014] In some embodiments, step 3, the separation includes: taking the culture supernatant and sequentially centrifuging it at differential speed to remove cells and debris, and then centrifuging the resulting supernatant at high speed to obtain exosomes.

[0015] This invention provides exosomes prepared by the aforementioned method.

[0016] This invention provides the use of the aforementioned exosomes in the preparation of products for promoting angiogenesis.

[0017] This invention provides the use of the aforementioned exosomes in the preparation of products for promoting tissue repair.

[0018] This invention provides a product comprising the aforementioned exosomes and pharmaceutically acceptable excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The method for preparing exosomes in this invention does not rely on high-cost exogenous growth factors, resulting in lower overall cost and stronger sustainability. The tension is a physically controllable stimulus, which can reduce dependence on recombinant proteins such as VEGF / bFGF and avoid their high cost, easy inactivation, batch differences and delivery difficulties.

[0021] (2) The yield and activity of the prepared exosomes are enhanced simultaneously and are easier to standardize and control. This invention provides a method to increase the secretion of exosomes through YAP-mediated synthesis, accompanied by selective enrichment of key pro-angiogenic proteins, so that the exosomes have more stable and traceable functional characteristics, which facilitates the establishment of scale-up production and release standards.

[0022] (3) The method for preparing exosomes in this invention is closer to the real microenvironment in vivo, which improves the convertibility of tissue-engineered skin culture in vitro. In vivo wounds and skin tissues are in a tensile and stretching environment for a long time, while conventional static culture ignores this factor. This invention incorporates the mechanical microenvironment into the culture strategy, which can "pre-train" the function of cells and exosomes in the in vitro stage, thereby improving the prevascularization efficiency and maturity of 3D printed dermis.

[0023] (4) This invention provides a clear mechanochemical transduction mechanism and key effector protein loading logic, which can be further used for parameter optimization (stretch amplitude / frequency / duration), coupling with material systems, and scale-up of exosome preparation processes, forming a platform-based technology path for sustainable iteration. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the technical process of the present invention;

[0025] Figure 2 The results of evaluating the length of vascular-like structures by CD31 immunofluorescence staining or histological analysis are shown. Figure A shows the results of vascular immunofluorescence staining, and Figure B shows the statistical results of vascular length.

[0026] Figure 3 The NTA assay was used to demonstrate the secretion-promoting effect of exosomes, where Exo-1 was the exosome in the non-tension group (control group) and Exo-2 was the exosome in the tension group.

[0027] Figure 4 The results of morphological identification of exosomes in the non-tension and tension groups are shown. Figure A shows the transmission electron microscopy results, Figure B shows the particle size statistics, and Figure C shows the Western blot detection results.

[0028] Figure 5This study demonstrates how tension-regulated printed tissue fibroblast-derived exosomes promote HUVEC proliferation, migration, and tube formation via BMP2-mediated pathways. Figure A shows the effect of different concentrations of BMP2 neutralizing antibody on the promotion of HUVEC proliferation by 20 μg / mL exosomes (Exo) using the MTT assay. Figures B and C show the effect of 1 μg / mL BMP2 neutralizing antibody on the promotion of HUVEC migration by 20 μg / mL exosomes using a scratch assay (Figure B) and the quantitative analysis of scratch wound area (Figure C). The scale bar is 100 μm. Figures D and E show the effect of 1 μg / mL BMP2 neutralizing antibody on the promotion of HUVEC tube formation by 20 μg / mL exosomes using a tube formation assay (Figure D) and the analysis of vascular-like structure length (Figure E). The scale bar is 100 μm. Data are presented as mean ± standard deviation, n=3, **P < 0.01, ***P < 0.001, compared with the PBS group at the same time point (Figures A and C), #P < 0.05, ##P < 0.01, ###P < 0.001, Comparison of the Exo + BMP2 neutralizing antibody group with the Exo group at the same time point;

[0029] Figure 6 The results show that fibroblast-derived exosomes regulated by tension promote the proliferation, migration, and tube formation of HUVECs via CXCL8. Figure A shows the effect of different concentrations of CXCL8 neutralizing antibody on the promotion of HUVEC proliferation by 20 μg / mL exosomes (Exo) using the MTT assay. Figures B and C show the effect of 2 μg / mL CXCL8 neutralizing antibody on the promotion of HUVEC migration by 20 μg / mL exosomes using the scratch assay (Figure B) and the quantitative analysis of the scratch wound area (Figure C). The scale bar is 100 μm. Figures D and E show the effect of 2 μg / mL CXCL8 neutralizing antibody on the promotion of HUVEC tube formation by 20 μg / mL exosomes using the tube formation assay (Figure D) and the analysis of the length of the vascular-like structure (Figure E). The scale bar is 100 μm. Data are mean ± standard deviation, n=3. **P < 0.01, ***P < 0.001, compared with the PBS group at the same time point (Figures A and C); ##P < 0.01, ###P < 0.001, compared with the Exo + CXCL8 neutralizing antibody group at the same time point;

[0030] Figure 7This study investigated how exosomes secreted by fibroblasts in printed tissues promote the migration of HUVECs. The effects of different concentrations of fibroblast-derived exosomes (Exo-1 and Exo-2) at 20 μg / mL on HUVEC migration were examined using a scratch assay in non-tension and tension-tension printed tissues (Figure C). Figure A shows a representative image with a scale bar of 100 μm; Figure B shows the quantitative analysis of scratch wound area; and Figure C shows the quantitative analysis of scratch wound area at an exosome concentration of 20 μg / mL. Data are presented as mean ± standard deviation, n=3. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the PBS control group at the same time point, #P < 0.05, ##P < 0.01, ###P < 0.001. Comparison between Exo-1 and Exo-2 groups at the same time point was also performed.

[0031] Figure 8 This study demonstrates how exosomes secreted by fibroblasts in 3D-printed tissue promote the formation of vascular-like structures in HUVECs. The effect of different concentrations of exosomes derived from fibroblasts (Exo-1 and Exo-2) in the non-tension and tension-tensioned groups on the formation of vascular-like structures in HUVECs was investigated using a tube formation assay. Figure A shows representative images with a scale bar of 100 μm; Figure B shows the quantitative analysis of vascular-like structure length; and Figure C shows the quantitative analysis of vascular-like structure length at an exosome concentration of 20 μg / mL. Data are presented as mean ± standard deviation, n=3. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the PBS control group at the same time point, ### P < 0.001. Comparison between the Exo-1 group and the Exo-2 group at the same time point.

[0032] Figure 9 The effect of continuous stretch stimulation for 4 days on exosome production in the stretch group and the non-stretch group was shown.

[0033] Figure 10 The study showed the effect of continuous stretch stimulation for 5 days on exosome production in the stretching group and the non-stretching group. Detailed Implementation

[0034] This invention provides a method for preparing exosomes derived from fibroblasts, the exosomes themselves, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0035] This invention, for the first time, constructs a complete mechanistic chain of "tension—YAP signaling pathway—enhanced exosome secretion from fibroblasts—selective loading of specific pro-angiogenic proteins (BMP2, CXCL8)—promotion of angiogenesis," clarifying that tension not only increases exosome secretion but also significantly enhances exosome biological function by activating the YAP signaling pathway to enrich and load key pro-angiogenic proteins. Unlike existing technologies that only report phenomenological studies of how mechanical stimulation affects cell behavior or exosome function, this invention systematically elucidates specific molecular pathways and key effector proteins, revealing a selective encapsulation mechanism by which BMP2 expression is not significantly increased intracellularly but is significantly enriched in exosomes. Furthermore, this invention establishes a controllable tension culture method based on a three-dimensional bioprinted tissue system, making mechanical stimulation a standardized and engineerable exosome function enhancement technology, rather than simply an experimental treatment condition.

[0036] The distinction lies in whether it is a common technique in existing technologies: The aforementioned distinctions are not common techniques in existing technologies. Although existing research involves the role of mechanical stimulation or exosomes in angiogenesis, most are single-factor studies or empirical observations, and have not formed a systematic technical solution of "mechanical stimulation—signaling pathway—exosome loading—functional enhancement". In particular, the process of regulating the selective loading of exosome proteins by activating YAP signaling and establishing a stable and reproducible process in 3D printed tissue models is not a conventional technical route in this field.

[0037] The reasons for using unconventional methods are as follows: existing research typically treats mechanical stimulation as a background factor regulating cell arrangement or gene expression, rather than as a core engineering means for precisely controlling exosome secretion and functional reprogramming. Furthermore, research on the selective encapsulation mechanism of proteins within exosomes is relatively limited, lacking a systematic design that combines molecular mechanisms with process parameters. In addition, 3D printing tissue culture and mechanical microenvironment regulation have long been relatively independent research paths, and those skilled in the art lack technical inspiration to integrate the two into a scalable exosome enhancement method. Therefore, it is difficult to naturally derive the technical solution of this invention from existing technologies.

[0038] Conventional solutions: For the problem of "insufficient vascularization of 3D-printed / tissue-engineered skin and unstable exosome production and angiogenesis activity", conventional solutions commonly used by those skilled in the art include: First, adding recombinant growth factors or small molecules (such as VEGF, bFGF, PDGF, etc.) exogenously during the in vitro culture stage to directly stimulate endothelial cell proliferation, migration and tube formation, or improving the vascularization microenvironment by optimizing biomaterials (increasing porosity, adjusting stiffness, introducing adhesion peptides, covalently binding growth factors); Second, to improve the efficacy of exosome-related treatments, more potent exosomes are often obtained by "changing cell source / cell pretreatment (hypoxia, inflammatory factors, drug stimulation) / directly concentrating exosome dosage", but there is often a lack of scalable and standardized activity enhancement processes; Third, at the mechanism verification level, inhibitors such as GW4869 are often used to prove that a certain phenomenon is "dependent on exosomes", but this cannot be translated into a production method that stably improves exosome production and function. Overall, conventional approaches tend to favor "biochemical factor-driven" or "material / dose stacking" methods, and rarely use the mechanical microenvironment as an engineerable means to systematically enhance exosome secretion and loading of specific functional proteins.

[0039] The differences between this invention and conventional methods are as follows: At the mechanism level, conventional approaches often rely on exogenous factors to promote angiogenesis or empirical pretreatment of exosomes, lacking a systematic explanation of how mechanical stimulation reprograms exosome secretion and loading. This invention, however, uses tension as the core regulatory mechanism, establishing a closed-loop pathway of "tension—YAP signal—enhanced fibroblast exosome secretion—selective loading of pro-angiogenic proteins (such as BMP2 and CXCL8)—enhanced angiogenesis," achieving simultaneous and controllable improvement in exosome yield and function. At the technical route level, conventional methods rely on exogenous growth factor supplementation or material modification. This invention introduces controllable tension into a 3D-printed dermal culture system, making mechanical stimulation a standardized and reproducible "process parameter," improving exosome quality attributes from the source. At the product characteristic level, conventional exosome enhancement methods are often non-specific (increased overall quantity or enhanced effect but unclear key products). This invention emphasizes the enrichment and loading of key pro-angiogenic proteins and verifiable molecular mechanisms, providing a clear approach and quality control basis for exosome function enhancement.

[0040] To address the problems existing in tissue-engineered skin and exosome-based angiogenesis strategies, such as unclear mechanisms, uncontrollable functional enhancement pathways, reliance on high-cost exogenous growth factors, and lack of physiological and mechanical microenvironment simulation in in vitro culture systems, this invention focuses on the core technical challenge of "mechanical stimulation regulating exosome functional enhancement." It systematically conducts in vitro and in vivo experimental studies and establishes a complete mechanism chain for tension-regulated exosome secretion and protein loading in fibroblasts.

[0041] The research and development objectives of this invention are: ① To clarify the molecular mechanism by which tensile stress regulates the secretion of fibroblast exosomes and the selective loading of pro-angiogenic proteins (such as BMP2 and CXCL8) through activation of the YAP signaling pathway, filling the research gap in the integration of mechanisms between mechanical stimulation and exosome functional reprogramming; ② To construct a standardized 3D printed tissue tensile culture system, establish a stable and reproducible mechanical parameter window, and provide an engineered preparation path for enhancing exosome function; ③ To improve the pro-angiogenic capacity of exosomes, reduce production costs, and enhance bioactivity stability without relying on high-cost recombinant growth factors; ④ To apply this mechanical regulation strategy to the in vitro pre-vascularization culture of 3D printed dermal tissue, improving the early blood supply reconstruction capacity after tissue transplantation; ⑤ To provide new technical ideas for exosome preparation technology based on physical microenvironment regulation, and promote the transformation and industrial application of exosome biological agents and tissue-engineered skin in the biomedical field.

[0042] The technical solution provided by this invention includes a method for introducing tension stimulation to promote angiogenesis during the 3D bioprinted dermal tissue culture process (e.g., using a PCL framework to fix and form a continuously tensile microenvironment, causing HUVECs to align along the tension direction and form longer vascular-like structures). Because tension provides a mechanical microenvironment for the printed tissue that more closely resembles the tension of in vivo skin, it induces endothelial cells to align in a specific direction and enhances their ability to form vascular-like structures. Therefore, it can significantly improve the in vitro prevascularization efficiency of 3D printed dermal tissue, reduce the risk of hypoxic necrosis caused by insufficient early perfusion after transplantation, and thus enhance tissue survival and integration potential.

[0043] The technical solution provided by this invention clearly demonstrates that the effect of tension-induced angiogenesis is mediated by exosomes, and this is verified through exosome secretion inhibition strategies (such as GW4869). Since inhibiting exosome synthesis or secretion can significantly weaken the growth of vascular-like structures induced by tension, it proves that this effect is mainly mediated by exosomes. Therefore, the focus of tension-induced angiogenesis is further narrowed from a macroscopic phenomenon to a key, operable, and amplifiable medium, giving the technical solution a clearer mechanism for action and facilitating subsequent process optimization, quality control, and product commercialization.

[0044] The technical solution provided by this invention includes a method for enhancing the secretion of exosomes from fibroblasts and improving their pro-angiogenic function through tension stimulation (the amount of exosomes in the tension group increased significantly, and the promoting effect on HUVEC proliferation, migration, and tubule formation was stronger than that in the non-tension group). Since tension increases the overall release level of exosomes and enhances the driving effect of exosomes on key endothelial cell behaviors (proliferation, migration, and tubule formation), it is possible to simultaneously increase exosome production and activity without relying on the continuous addition of high-cost exogenous growth factors, significantly improving the application efficiency and stability of exosomes as a pro-angiogenic biological agent.

[0045] The technical solution provided by this invention elucidates how tension promotes exosome secretion by activating the YAP signaling pathway in fibroblasts and drives the enrichment and loading of key pro-angiogenic proteins (such as BMP2 and CXCL8) within exosomes; this is verified by the decrease in exosome secretion and the reduction in BMP2 and CXCL8 expression within exosomes after YAP inhibition. By introducing the clearly defined upstream mechanochemical transduction node YAP as a regulatory switch and identifying key proteins exerting effects within exosomes as core effector molecules, this solution not only explains the mechanism by which tension enhances exosome function but also achieves controllable regulation: by regulating YAP activity, exosome secretion levels and protein loading characteristics can be adjusted, thereby stably and reproducibly obtaining exosomes with stronger pro-angiogenic capabilities, improving batch consistency and the feasibility of industrial applications.

[0046] Some alternatives to the present invention include tension-enhanced exosome spray formulations.

[0047] Alternative solutions preparation and use:

[0048] Preparation: The exocrine cells of fibroblasts obtained by tension stimulation were resuspended in sterile PBS or isotonic buffer, and the concentration was adjusted to a suitable range (e.g., 50–200 μg / mL). An appropriate amount of pharmaceutical stabilizer or protectant was added, mixed well, and then filled into sterile spray containers and stored at low temperature.

[0049] Usage: After routine disinfection and cleaning of the wound, aim the sprayer at the wound and spray the exosome solution evenly to cover the wound surface. Use once daily or every other day until the wound heals.

[0050] Advantages: The administration method is more convenient, especially suitable for irregular or large-area wounds, and can achieve rapid and uniform coverage; the mist-like particles help increase the contact area with the wound and improve the uniformity of distribution; the packaging has good sealing properties, which is convenient for clinical bedside use and home care; it does not change the biological activity of exosomes and is suitable for rapid treatment of acute wounds.

[0051] Disadvantages: Some drug may be lost during the spraying process; the adhesion is still limited, and it is easily washed away by wound exudate, resulting in a short retention time; compared with sustained-release formulations, more frequent administration is required; it has high requirements for storage conditions, and its long-term stability needs further verification.

[0052] Existing spray formulations are mostly complex solutions of various natural ingredients, often lacking clear targets and molecular mechanisms of action, and failing to systematically define precise effective concentration ranges. In contrast, the spray formulation of this invention is based on a single, clearly defined active ingredient, possessing a clear molecular mechanism and a defined concentration window, making it more precise and controllable in terms of technical logic. However, this existing spray technology is not adopted because, while spray formulations improve the convenience of drug delivery, their core problems remain poor adhesion, easy loss from the wound surface, and low bioavailability. Compared to solution formulations, they only optimize the form and do not fundamentally solve the problem of short drug retention time. Furthermore, drug loss or evaporation occurs during spraying, increasing raw material waste. For small wounds, spraying is less precise than direct dripping. Therefore, considering the stability of therapeutic efficacy and resource utilization, this existing technology is not the preferred choice.

[0053] Some alternatives to the present invention include tension-enhanced exosome sustained-release film agents.

[0054] Alternative solutions preparation and use:

[0055] Preparation: Dissolve chitosan or other biodegradable biopolymers in a suitable solvent, add exosomes obtained by tensile stress enhancement, mix gently, and then add an appropriate amount of plasticizer; cast the mixed solution into a film, dry at room temperature or low temperature, peel off and shape, sterilize, and seal for storage.

[0056] Usage: After routine disinfection and cleaning of the wound, cut the film to a shape suitable for the size of the wound, apply it to the wound surface, ensuring a tight fit, and change it every 3-4 days until the wound heals.

[0057] Advantages: The film adheres well, effectively preventing exosome loss and isolating external contamination; it enables continuous sustained release, prolonging the action time of exosomes and reducing the frequency of dressing changes; it combines the functions of a physical barrier for wounds and drug release; it is relatively convenient to store at room temperature and facilitates transportation.

[0058] Disadvantages: The preparation process is relatively complex, requiring control of membrane thickness and uniform distribution of exosomes; it has poor adaptability to wounds with a lot of exudate, which may affect the stability of adhesion; the cost is higher than that of simple solution formulations; the long-term activity and stability of exosomes in the membrane need to be rigorously verified.

[0059] In general, spray formulations are more suitable for scenarios requiring rapid and convenient drug administration, while sustained-release films are better suited for wound types requiring long-term, continuous treatment. The two alternatives each have their own characteristics in terms of administration method, drug retention time, and applicable scenarios, and can be selected based on wound type and clinical needs.

[0060] Existing membrane formulations are mostly single-carrier membranes or preparations with added antibiotics or growth factors, and do not use the core active ingredient of this invention as the functional basis. In contrast, the sustained-release system of this invention focuses on an active ingredient with a clearly defined target and mechanism, emphasizing mechanism-driven formulation design. However, this existing membrane technology is not adopted because: the membrane has limited water absorption, and for chronic or deep wounds with significant exudate, the membrane is prone to detachment due to exudate accumulation, affecting the sustained-release effect; secondly, membrane preparation involves steps such as casting and thickness control, which are complex processes requiring advanced equipment and operation, thus increasing production costs; furthermore, membrane replacement after application may pull on newly formed tissue, increasing the risk of secondary damage. Therefore, considering the stability of therapeutic efficacy, the scope of application, and industrialization feasibility, this existing technological approach is not prioritized.

[0061] The alternative to the tension application device of the present invention can be: in the implementation scheme, a PCL frame or elastic culture plate is used to form a continuous tension microenvironment, or a commercial cell tension system (such as the Flexcell tension loading system), a programmable mechanical stretching platform or a bioreactor loading system can be used instead.

[0062] Different stretching devices can all achieve mechanical stimulation of cells by controlling the strain amplitude, frequency, and duration. Commercial tension systems offer advantages such as precisely adjustable strain parameters, high repeatability, and suitability for standardized experiments and large-scale production; bioreactor systems enable simultaneous processing of multiple samples, improving production efficiency; and simple elastic frame structures are less expensive and more suitable for early-stage research and development. Although these devices differ in structure, as long as they can provide stable and controllable stretching stimulation, they can all achieve the technical effect of activating the YAP signaling pathway and enhancing exosome secretion and function. This invention does not further limit their application.

[0063] The exosome extraction technology of this invention can be replaced by: ultracentrifugation to extract exosomes in the implementation scheme, or size exclusion chromatography, ultrafiltration concentration, immunomagnetic bead method or commercial exosome extraction kit.

[0064] Size exclusion chromatography yields high purity and minimal protein contamination, making it suitable for subsequent mechanistic studies; ultrafiltration is simple to operate and suitable for large-scale production; immunomagnetic bead extraction can improve specificity, but it is more expensive. Different extraction methods vary in purity, yield, and cost, but as long as particles conforming to exosome characteristics are obtained and their biological activity is maintained, they can all promote the proliferation, migration, and tube formation of HUVECs. This invention does not impose further limitations on these methods.

[0065] The alternative materials for the bioprinting scaffold of the present invention can be: in the implementation scheme, GelMA, HAMA and fibrinogen are used as bio-inks, or collagen, gelatin, sodium alginate, PEG-based hydrogel or composite hydrogel system can be used as alternatives.

[0066] Collagen exhibits excellent cell adhesion, more closely resembling the natural extracellular matrix environment; sodium alginate demonstrates good moldability, making it suitable for rapid printing; PEG-based hydrogels possess stable mechanical properties, facilitating parameter control. While different materials vary in mechanical properties and degradation rates, as long as they can form a stable three-dimensional structure and allow for cell survival and mechanical transfer, they can all achieve the effect of enhanced exosome secretion induced by stretch stimulation. This invention does not impose further limitations in this regard.

[0067] The alternative to the YAP signaling regulation method of the present invention can be: in the implementation scheme, drug inhibitors are used to verify YAP dependence, or siRNA interference, CRISPR gene editing or other YAP / TAZ regulation methods can be used as alternatives.

[0068] siRNA interference has the advantages of being simple to operate and highly specific; gene editing methods can achieve stable knockdown or deletion, making them more suitable for long-term mechanism studies; drug inhibitors are simple to operate and highly reversible, making them suitable for functional validation. Different regulatory methods differ in stability and cost, but all can be used to verify the mechanism by which stretch stimulation regulates exosome secretion and protein loading through YAP signaling. This invention does not further limit these aspects.

[0069] The cell source alternatives of the present invention can be: in the implementation scheme, human skin fibroblasts and HUVECs are used, or dermal fibroblast cell lines, microvascular endothelial cells or other sources of endothelial cells can be used as alternatives.

[0070] Microvascular endothelial cells more closely resemble the local vascular environment of the skin, potentially enhancing physiological relevance; their cell line source is stable, facilitating large-scale production. Different cell sources vary in proliferative and angiogenic capabilities, but as long as they possess exosome secretion and angiogenesis potential, similar pro-angiogenic effects can be achieved. This invention does not impose further limitations in this regard.

[0071] In summary, the stretching device, exosome extraction method, bioprinting material, signal modulation method, and cell source in the above-described embodiments can all be replaced by functionally equivalent technical products. As long as the core technical effects of stretch stimulation activating the YAP signaling pathway, enhancing exosome secretion, and promoting angiogenesis protein enrichment can be achieved, they all fall under the category of alternative embodiments of the present invention.

[0072] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0073] Example 1: Method and application of tension stimulation for promoting angiogenesis in 3D-printed dermal tissue.

[0074] Preparation steps: Prepare a bio-ink solution by mixing GelMA, HAMA, and fibrinogen in a predetermined ratio and incubating at 37°C. Human skin fibroblasts and HUVECs are mixed into the bio-ink in a predetermined ratio, then loaded into the bioprinting nozzle. A dermal construct is printed using a 3D bioprinting device according to the predetermined structure, and UV cross-linked and cured to form a stable scaffold. The printed construct is fixed within a PCL frame, kept under continuous tension (e.g., 5%-10% continuous static tension or periodic tension). It is then incubated at 37°C in a 5% CO2 incubator for 7 days, with the culture medium (4:1 volume ratio of EGM2 medium to complete DMEM medium) changed periodically. The technical flow chart is shown below. Figure 1 As shown. The control group construct was not subjected to tension treatment, while the inhibitor group was subjected to tension treatment and GW4869 inhibitor was added.

[0075] Application: Mature, cultured, stretched dermal tissue is printed and used for in vitro vascularization assessment or transplantation into animal skin defect models. The length, number of branches, and orientation of the vascular-like structures are evaluated using CD31 immunofluorescence staining or histological analysis. Results are as follows: Figure 2 As shown.

[0076] Working principle: Stretch tension mimics the in vivo skin tension microenvironment, inducing endothelial cells to align in the direction of stretch and enhancing their ability to form angiogenic structures. Simultaneously, stretch stimulation activates the YAP signaling pathway within fibroblasts, causing YAP nuclear translocation and regulating downstream gene expression, thereby enhancing exosome secretion. Increased exosome secretion further acts on HUVECs, promoting their proliferation, migration, and tube formation, ultimately significantly improving the in vitro prevascularization efficiency of 3D-printed dermis and reducing the risk of early hypoxic necrosis after transplantation. Further validation using a GW4869 inhibitor to suppress exosome secretion showed that inhibiting exosome synthesis or secretion significantly weakens the stretch-induced angiogenic structure growth, proving that this effect is mainly mediated by exosomes. Therefore, stretch-induced angiogenesis is further narrowed down from a macroscopic phenomenon to a key, operable, and amplifiable medium.

[0077] as follows Figure 3 The results show that the secretion-promoting effect of exosomes was demonstrated by NTA detection. Exo-1 was the non-stretch group exosome (control group), and Exo-2 was the stretch group exosome. The results showed that the peak value of Exo-2 group was more than 3 times that of Exo-1 group.

[0078] Example 2: Method for preparing tension-enhanced fibroblast exosomes and their angiogenesis-promoting applications

[0079] Preparation steps: Human skin fibroblasts were seeded into elastic culture plates or PCL fixation frames. After cell adhesion, continuous stretching stimulation (e.g., 5%-10% strain for 7 days) was applied. The culture supernatant was collected, and cells and debris were removed by differential centrifugation. Exosomes were extracted by ultracentrifugation and resuspended in PBS for storage. Exosomes were identified using nanoparticle tracking analysis, transmission electron microscopy, and Western blot. The results are as follows: Figure 4 As shown.

[0080] Application method: Add the stretching group exosomes (e.g., 20 μg / mL) to the HUVEC culture system for proliferation, scratch, and tube formation experiments. Alternatively, add the exosomes to a 3D-printed dermal system to evaluate angiogenesis.

[0081] Working principle: Stretch stimulation activates the YAP signaling pathway in fibroblasts, enhancing YAP nuclear translocation and promoting the expression of exosome biogenetic proteins, thereby increasing exosome secretion. Simultaneously, stretch stimulation drives the selective enrichment of pro-angiogenic proteins (such as BMP2 and CXCL8) in exosomes. Exosomes rich in BMP2 and CXCL8, after being taken up by HUVECs, can activate downstream signaling pathways such as PI3K-AKT and MAPK, enhancing endothelial cell proliferation, migration, and lumen formation, achieving a dual effect of increased production and enhanced function.

[0082] Example 3: Verification of YAP-dependent regulation and application of exosome function enhancement mechanism

[0083] Preparation steps: Fibroblasts were cultured under stretch stimulation conditions, with a YAP inhibitor treatment group included. The culture supernatant from both the stretch group and the stretch plus YAP inhibition group was collected, and exosomes were extracted. Western blot was used to detect the expression levels of proteins such as BMP2 and CXCL8 in the exosomes. The effects of exosomes from different treatment groups on HUVEC proliferation, migration, and tube formation ability were compared. The results are as follows: Figure 5 and Figure 6 As shown.

[0084] Usage: The differences in the pro-angiogenic effects of exosomes are evaluated through in vitro functional experiments or animal models.

[0085] Working principle: Stretch stimulation activates the YAP signaling pathway, where YAP, as a key node in mechanochemical transduction, regulates exosome secretion and protein loading. When YAP is inhibited, exosome secretion levels and the expression of BMP2 and CXCL8 within exosomes significantly decrease, weakening their pro-angiogenic capacity. This result demonstrates that stretch-enhanced exosome function depends on YAP-mediated signal transduction. By regulating stretch parameters or YAP activity, controllable optimization of exosome secretion levels and functional characteristics can be achieved, improving preparation stability and batch-to-batch consistency.

[0086] Example 4

[0087] Further scratch assays were used to observe the effects of exosomes in the tension group and the non-tension group on HUVEC migration. The results showed that compared with the PBS control group, the concentration of exosomes in the non-tension group had no significant effect on HUVEC migration at 10 μg / mL, but significantly promoted HUVEC migration at 20-30 μg / mL; the concentration of exosomes in the tension group significantly promoted HUVEC migration at 10-30 μg / mL. Figure 7 A, 7B). At an exosome concentration of 20 μg / mL, the exosomes in the tension-controlled group had a significantly greater effect on promoting the migration of HUVECs than those in the non-tension-controlled group. Figure 7 (A, 7C). These results indicate that exosomes secreted by fibroblasts in tension-stimulated 3D-printed tissues have a stronger pro-migration effect on HUVECs.

[0088] Finally, the effect of exosomes in the tension group and the non-tension group on the formation of vascular-like structures by HUVECs was examined using an in vitro angiogenesis experiment. The results showed that at exosome concentrations of 20 and 30 μg / mL, the length of the vascular-like structures formed by HUVECs in both groups was significantly greater than that in the PBS control group. Figure 8 A, 8B); at a concentration of 20 μg / mL, the tubular structures formed by HUVECs under the action of the tension-controlled exosomes were longer than those of the non-tension-controlled exosomes. Figure 8 (A, 8C). These results indicate that exosomes from the tension group have a stronger effect on promoting HUVEC tube formation than exosomes from the non-tension group.

[0089] Example 5

[0090] Based on Example 2, the generation and effects of exosomes in the stretching group and the control group were investigated by setting different durations of continuous stretching stimulation. The results are as follows: Figure 9 and Figure 10 As shown.

[0091] The results showed that exosome production was low after 5 days of continuous stretch stimulation, and the exosomes also had a poor driving effect on endothelial cells. Fluorescence imaging revealed that this was due to insufficient culture time, resulting in incomplete cell expansion within the material.

[0092] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes, characterized in that, Includes the following steps: Step 1: Take fibroblasts and seed them on a deformable culture medium substrate for culture; Step 2: After the fibroblasts adhere to the culture wall, apply tensile stimulation to the deformable culture medium substrate, and obtain the culture supernatant after culturing. Step 3: Take the culture supernatant and centrifuge it to obtain exosomes.

2. The preparation method according to claim 1, characterized in that, In step 1, the fibroblasts are human skin fibroblasts.

3. The preparation method according to claim 1, characterized in that, In step 1, the deformable culture medium substrate includes an elastic culture plate or a PCL fixation frame system.

4. The method according to claim 1, characterized in that, In step 2, the strain of the tension stimulation is 5% to 10%, and the duration of the tension stimulation is 5 to 10 days.

5. The method according to claim 4, characterized in that, The duration is 7 days.

6. The method according to claim 1, characterized in that, In step 3, the separation includes: taking the culture supernatant and centrifuging it sequentially to remove cells and debris, and then centrifuging the resulting supernatant at high speed to obtain exosomes.

7. Exosomes prepared by the method according to any one of claims 1 to 6.

8. The use of the exosomes of claim 7 in the preparation of products for promoting angiogenesis.

9. The use of the exosomes of claim 7 in the preparation of products for promoting tissue repair.

10. The product, characterized in that, It includes the exosomes as described in claim 7 and pharmaceutically acceptable excipients.

Images