Stem cell freeze-dried powder for promoting skin healing and preparation method thereof

Abstract

The invention discloses stem cell freeze-dried powder for promoting skin healing and a preparation method of the stem cell freeze-dried powder. The freeze-dried powder comprises an effective component, a freeze-drying protective agent group and a polymer protective agent group, the effective component is umbilical cord mesenchymal stem cell conditioned medium freeze-dried supernatant, the protective agent group is selected from one or more of cane sugar, trehalose, lactose and mannitol, and the polymer protective agent group is selected from one or more of polyvinylpyrrolidone (PVP) and glucan. The preparation method comprises the following steps: collection of a conditioned culture medium, preparation of a freeze-drying protective agent solution, mixing treatment, low-temperature pre-freezing and freeze-drying, collection and packaging of freeze-dried powder and the like. The method can effectively retain stem cell-derived active factors and enhance the stability and redissolvability of the freeze-dried powder, and is suitable for skin wound repair, regenerative medicine and other scenes.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine and biopharmaceutical preparation technology, specifically to a freeze-dried stem cell powder that promotes skin healing and its preparation method. Background Technology

[0002] The skin is the largest organ in the human body, playing vital roles in barrier function, defense, and regulation. Once injured, such as by burns, postoperative wounds, chronic ulcers, or diabetic foot, if healing is delayed or improperly performed, it can easily lead to infection, scarring, or tissue necrosis. Traditional treatments mainly rely on topical ointments, sterile dressings, and growth factor injections. However, these methods suffer from problems such as short shelf life of active ingredients, low local retention rate, and incomplete reconstitution, making it difficult to achieve continuous and efficient tissue repair.

[0003] In recent years, mesenchymal stem cells (MSCs) have become a hot topic in tissue engineering and regenerative medicine research due to their low immunogenicity, multi-lineage differentiation potential, and paracrine effects. Among them, umbilical cord-derived MSCs have advantages such as convenient collection, low ethical burden, and abundant secretory factors. Studies have shown that their conditioned medium is rich in various repair-promoting factors such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and exosomes, which can effectively regulate the wound microenvironment and promote angiogenesis, cell migration, and epithelial remodeling.

[0004] However, existing stem cell culture media products generally suffer from practical problems such as unstable active ingredients, strong reliance on cold chain preservation, and difficulty in large-scale storage. While freeze-drying technology can significantly extend the storage period and improve transportation convenience, it is prone to denaturation and structural damage of active proteins in the absence of a scientific protective agent system, seriously affecting the efficacy of the products. Therefore, developing a stem cell-derived freeze-dried powder with high activity retention, good resolubility, and biocompatibility is a technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a freeze-dried stem cell powder that promotes skin healing and its preparation method. It has the advantages of stable components, long shelf life, convenient reconstitution, and strong applicability, and solves the problems of difficult preservation, low activity, and complicated preparation of existing stem cell derivative products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a freeze-dried stem cell powder that promotes skin healing, comprising the following components:

[0007] Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells;

[0008] Freeze-drying protectants: selected from one or more of sucrose, trehalose, lactose, and mannitol;

[0009] Polymer protective agents: selected from one or more of polyvinylpyrrolidone (PVP) and dextran.

[0010] Preferably, it includes the following components:

[0011] Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells;

[0012] Lyophilization protectant group: Trehalose;

[0013] Polymer protectant group: Polyvinylpyrrolidone (PVP).

[0014] Preferably, the mass percentage range of each component is as follows:

[0015] Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells, 5%–20%;

[0016] Lyophilization protectant: Trehalose, 40%–70%;

[0017] Polymer protectant: Polyvinylpyrrolidone (PVP40), 20%–40%.

[0018] Preferably, the mass percentages of each component are as follows:

[0019] Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells, 12%;

[0020] Lyophilization protectant: Trehalose, 58%;

[0021] Polymer protectant: Polyvinylpyrrolidone (PVP40), 30%.

[0022] A method for preparing a freeze-dried stem cell powder that promotes skin healing includes the following steps:

[0023] S1) Collection of conditioned medium: Umbilical cord mesenchymal stem cells are cultured. After the cells reach the preset degree of confluence, the culture medium is collected and subjected to multi-stage centrifugation and filtration to obtain cell-free conditioned medium.

[0024] S2) Preparation of freeze-drying protectant solution: Prepare sugar protectant and polymer protectant solutions separately. The sugar protectant is trehalose and the polymer protectant is polyvinylpyrrolidone (PVP40). Mix them according to a preset ratio to form a two-component freeze-drying protectant mixture.

[0025] S3) Mixing treatment of the cryoprotectant and culture medium: Mix the freeze-drying cryoprotectant mixture with an equal volume of conditioned culture medium and mix by vortexing to form a solution to be freeze-dried;

[0026] S4) Low-temperature pre-freezing and freeze-drying: After the mixture is rapidly frozen at -80℃, it is transferred to a freeze dryer to perform a sublimation drying process, forming a freeze-dried powder.

[0027] S5) Collection and packaging of lyophilized powder: The dried lyophilized powder is sampled, tested and vacuum-sealed to finally obtain the lyophilized powder product of umbilical cord mesenchymal stem cell conditioned culture medium.

[0028] Preferably, step S1) includes the following sub-steps:

[0029] S1.1) Stem cell culture: Umbilical cord mesenchymal stem cells were cultured in serum-free conditioned medium. The culture was terminated when the cell confluence reached 80% to 90%. The culture supernatant of the third to fifth generation cells with a confluence of 85% was collected.

[0030] S1.2) Primary centrifugation: The obtained culture supernatant was centrifuged for the first time at 4°C with a centrifugal force of 200-800×g for 5-10 minutes. The specific centrifugation parameters were 500×g and 5 minutes to remove dead cells and large granular cell debris.

[0031] S1.3) Secondary centrifugation treatment: The supernatant after the first centrifugation is centrifuged again at 4°C with a centrifugal force of 1000-3000×g for 10-15 minutes. The specific centrifugation parameters are 3000×g and 10 minutes to further remove small fragments and residual precipitates.

[0032] S1.4) Aseptic filtration: The supernatant after the second centrifugation was filtered and sterilized using a sterile filter membrane with a pore size of 0.22 μm to obtain a clear, transparent umbilical cord mesenchymal stem cell conditioned medium free of cell impurities, providing a basic liquid raw material for subsequent freeze-drying.

[0033] Preferably, step S2) includes the following sub-steps:

[0034] S2.1) Preparation of trehalose solution: Dissolve trehalose in a solvent obtained by mixing ultrapure water and PBS at a volume ratio of 65:35 to prepare a trehalose solution with a concentration range of 50-200mM, specifically 100mM. Stir with a magnetic stirrer until fully dissolved. The resulting solution is a non-reducing sugar protective agent solution.

[0035] S2.2) Preparation of PVP40 solution: Polyvinylpyrrolidone (PVP40) is directly added to the above trehalose solution to prepare a polymer protectant solution with a mass concentration of 1% to 5%, specifically 2%. Stir thoroughly until the solution is uniform and clear to form a synergistic composite protectant system.

[0036] S2.3) Sterilization and light-protected storage: The trehalose solution and PVP40 mixed solution were sterilized by autoclaving at 121℃ for 30 minutes. After cooling to room temperature, they were filtered through a 0.22μm filter membrane for sterilization. Finally, they were stored at 4℃ under light-protected conditions for later use.

[0037] Preferably, step S3) includes the following sub-steps:

[0038] S3.1) Mixing treatment: Mix the umbilical cord mesenchymal stem cell conditioned culture medium obtained in step S1) with the composite protectant solution obtained in step S2) at a volume ratio of 1:1, and mix by vortexing for 30-60 seconds to form a homogeneous pre-frozen mixture.

[0039] S3.2) Sample pretreatment: The mixture is temporarily stored at low temperature, and its appearance, pH and turbidity are tested to confirm that it meets the physicochemical stability requirements before freeze-drying and is ready to enter the freeze-drying process.

[0040] Preferably, step S4) includes the following sub-steps:

[0041] S4.1) Low-temperature rapid freezing: The pre-frozen mixture obtained in step S3) is rapidly placed in an environment of -80°C for freezing treatment for 4 to 6 hours, so as to pass through the nucleation critical temperature zone and form a structurally stable freeze-dried sample;

[0042] S4.2) Freeze-drying: Place the pre-frozen sample into a freeze dryer and execute the following procedure:

[0043] The temperature for the initial drying stage was set to -40℃, the vacuum degree to 10Pa, and the drying time to 12–16 hours.

[0044] During the secondary drying stage, the temperature is raised to 20°C, and the vacuum conditions are kept constant for 6 to 8 hours.

[0045] The residual moisture content is controlled to be below 3% throughout the entire process.

[0046] Preferably, step S5) includes the following sub-steps:

[0047] S5.1) Collection of freeze-dried powder: After freeze-drying, the freeze-dried powder is recovered from the desiccator in a sterile environment. The powder should be loose and free of lumps.

[0048] S5.2) Packaging and storage: The lyophilized powder is packaged in a light-proof glass bottle using a nitrogen-filling process, sealed with a cap and labeled with a batch number, and finally stored at 4°C to ensure the biological activity and physicochemical stability of the finished product.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] 1. This invention uses umbilical cord mesenchymal stem cell culture supernatant as the sole source of activity, avoiding the introduction of intact cells and exogenous additives, ensuring low immunogenicity and good applicability of the finished product, and meeting the needs of a wide range of users. By scientifically setting the pre-freezing rate and freeze-drying curve (temperature-time-vacuum degree) parameters, combined with a reasonable composite protective agent system, the structural damage rate of active factors during freeze-drying is effectively reduced, and the freeze-dried powder can still maintain good biological function after reconstitution.

[0051] 2. The product is a powder formulation that can be stably stored at 4°C, eliminating the need for cold chain transportation and significantly improving its feasibility for use in primary healthcare, emergency care, and remote areas. The entire process is controlled by standardized parameters, including key process parameters such as cell culture fusion, dual centrifugation conditions, protectant concentration and ratio, and freeze-drying curve, all of which can be quantified and tracked, providing a foundation for industrial production.

[0052] 3. After being reconstituted with PBS, the lyophilized powder forms a homogeneous and transparent liquid, which can be easily applied to the wound surface by various methods such as dripping, spraying, and dressing. It has good skin adhesion and the ability to maintain a moist environment, which helps to accelerate the tissue repair process.

[0053] 4. A synergistic protection system is formed by combining trehalose and PVP40, which takes into account vitrification protection, osmotic pressure regulation and protein stability regulation, and is significantly better than the protection effect of a single type of protectant in the freeze-drying process. Attached Figure Description

[0054] Figure 1 The present invention uses nanoparticle tracking analysis (NTA) technology to test the nanoparticle size of lyophilized powders with different protective agents;

[0055] Figure 2 The components contained in the analytical conditions culture medium of the ELISA kit of this invention;

[0056] Figure 3 Cell viability experiments of lyophilized powder of conditioned medium based on human fibroblast (HDF cell line) according to this invention: a) cytotoxicity test; b) cell proliferation test;

[0057] Figure 4 The results of cell migration experiments using the transwell chamber of this invention;

[0058] Figure 5 This invention uses an ELISA kit to test the anti-inflammatory effect of lyophilized conditioned medium powder on a macrophage inflammation model constructed with LPS.

[0059] Figure 6 This invention provides an in vivo experimental test to evaluate the effect of lyophilized culture medium powder on skin wound healing.

[0060] Figure 7 This is a graph showing the SA-β-gal staining experiment and statistical results of this invention;

[0061] Figure 8 This is a graph showing the results of the DNA damage marker γ-H2AX staining experiment and statistical analysis of the present invention.

[0062] Figure 9 This invention uses Western blotting to detect the relative expression levels of aging-related proteins p16, p53, and Lamin B1.

[0063] Figure 10 This is a diagram showing the formulation and structural composition of the freeze-dried powder of the present invention;

[0064] Figure 11 This is a flowchart of the freeze-dried powder preparation method of the present invention. Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0066] Please see Figures 1 to 11 This invention proposes a freeze-dried stem cell powder that promotes skin healing and its preparation method. It uses conditioned medium derived from umbilical cord mesenchymal stem cells as the active core component, supplemented by a sugar freeze-drying protectant and a polymer synergistic protectant to construct a stable embedding system. The stable powder form is obtained through low-temperature rapid freezing and freeze-drying processes. It has comprehensive effects such as promoting wound healing, inhibiting inflammatory response and antioxidant repair, and is suitable for skin wound care and regenerative repair applications.

[0067] The stem cell freeze-dried powder formulation of this invention has a clearly defined component combination and a clear functional division, including:

[0068] Active ingredient: Umbilical cord mesenchymal stem cell conditioned medium supernatant (CM). This component is derived from cytokines and exosomes secreted during the culture of P3-P5 generation umbilical cord mesenchymal stem cells. Through multi-stage centrifugation and processing with a 0.22μm sterile filter membrane, all cellular impurities are removed, yielding a supernatant rich in bioactive proteins such as EGF, TGF-β, VEGF, and HGF, as well as exosome structures. This substance is considered a key mediator for promoting tissue repair, accelerating epidermal regeneration, and regulating immune responses.

[0069] The freeze-drying protectant group: Trehalose, a non-reducing sugar, is selected. Trehalose is a recognized freeze-drying protectant that can form a glassy structure outside cells, reducing the risk of ice crystal damage to protein structures, while also possessing water-retention and antioxidant properties. Customer information indicates that trehalose in this invention is used to maintain the spatial conformational integrity of protein factors and exosomes in the supernatant, which helps restore functional activity upon reconstitution after freeze-drying.

[0070] Polymer protectant group: Polyvinylpyrrolidone (PVP40) is selected. PVP40 is a hydrophilic polymer with good film-forming properties and stability. In the lyophilization system, it can enhance the stability of proteins and prevent component aggregation and precipitation. It forms a synergistic protective network with trehalose, ensuring that the reconstituted liquid has a clear appearance and uniform structure. Original customer data indicates that this combination has good thermal stability and physicochemical compatibility during long-term storage and transportation of dry powder.

[0071] Furthermore, the process of this invention employs equal-volume mixing + standard pre-freezing + two-stage vacuum freeze-drying to form a powder formulation with low dryness, intact active substances, and good resolubility, which significantly improves ease of use and functional stability.

[0072] Through the combined design of the above components and processes, this invention constructs a cell-free, low-immunogenic, freeze-dried, and highly efficient reconstituted topical skin formulation, providing an innovative stem cell-derived solution for clinical wound management, postoperative care, and repair of aging skin.

[0073] This step aims to obtain a bioactive conditioned medium supernatant from umbilical cord mesenchymal stem cell culture, which will serve as the main active ingredient in the lyophilized powder. Specifically, it includes the following sub-steps:

[0074] S1.1) Stem cell culture

[0075] Umbilical cord tissue from healthy, full-term newborns, sourced legally and ethically reviewed, was used to isolate umbilical cord mesenchymal stem cells using a combined collagenase and trypsin enzymatic digestion method. Cells were seeded in serum-free conditioned medium and cultured under controlled conditions: 37°C, 95% humidity, and 5% CO2. Culture was terminated when cell confluence reached 80%–90%, preferably at 85% confluence. Cells from passages 3–5 (P3–P5) were selected to ensure high cell viability and stable secretory function.

[0076] This culture process does not require the addition of animal serum or exogenous stimulants, ensuring that the obtained supernatant contains only endogenous secretory factors, thus exhibiting good biosafety and compositional consistency.

[0077] S1.2) Primary centrifugation treatment

[0078] The collected supernatant was immediately refrigerated at 4°C and then centrifuged for the first time using a benchtop centrifuge. Centrifugation conditions were: 200–800 × g for 5–10 minutes, preferably 500 × g for 5 minutes. This treatment was used to remove dead cells, shed cell membranes, and large debris, and to preliminarily clarify the culture medium.

[0079] S1.3) Secondary centrifugal treatment

[0080] The supernatant obtained from the first centrifugation is further placed in a centrifuge tube and subjected to a second low-temperature centrifugation. The centrifugation conditions are: 1000-3000×g for 10-15 minutes, preferably 3000×g for 10 minutes, to remove residual cell debris, exogenous precipitates and tiny suspended impurities, and to improve the clarity and filterability of the subsequent supernatant.

[0081] S1.4) Aseptic filtration treatment

[0082] The supernatant obtained after double centrifugation was sterilized by filtration through a 0.22 μm pore size polyethersulfone (PES) sterile filter membrane. During filtration, air bubbles and shear forces should be avoided to protect the sensitive protein factors and exosome structures in the supernatant from damage.

[0083] The resulting clear, transparent liquid is the conditioned medium (CM) for umbilical cord mesenchymal stem cells, containing various cell-secreted bioactive substances, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and transforming growth factor β (TGF-β). These components possess multiple functions during skin repair, including regulating inflammation, stimulating fibroblast proliferation, and promoting angiogenesis.

[0084] The CM stock solution obtained in this step will serve as the base liquid raw material for subsequent lyophilization protectant mixing and will also directly participate in the construction of lyophilized powder as an active ingredient.

[0085] Preparation steps for the lyophilization protectant (S2)

[0086] To ensure the structural integrity and bioactivity of stem cell conditioned medium during freeze-drying, a complex protective agent consisting of sugars and polymers is added to reduce damage to sensitive components such as protein structures and exosome membranes caused by low-temperature freezing and drying. This step includes the following sub-steps:

[0087] S2.1) Preparation of trehalose solution: Weigh an appropriate amount of trehalose powder (high purity, low endotoxin level) and dissolve it in a mixture of ultrapure water and sterile PBS buffer at a volume ratio of 65:35. Stir thoroughly to form a trehalose solution ranging from 50 to 200 mM, with a specific concentration of 100 mM. Trehalose is a non-reducing disaccharide with good hydration and vitrification protection capabilities. At low temperatures, it can form a glassy structure to embed active ingredients, effectively preventing protein denaturation and exosome membrane rupture.

[0088] Use a magnetic stirrer to continuously stir at room temperature for 10-15 minutes until the sugar powder is completely dissolved and there are no visible suspended matter in the solution, forming a clear and transparent sugar protective agent solution.

[0089] S2.2) Preparation of PVP40 solution: Based on the trehalose solution, polyvinylpyrrolidone (PVP40) powder is further added at a mass concentration of 1% to 5%, with a specific ratio of 2%. This polymer is a hydrophilic polymer material that can help maintain the viscosity of the system, reduce the growth rate of ice crystals during freezing, and provide a physical protective barrier for the membrane structure during freeze-drying.

[0090] Continue stirring after mixing until a uniform and clear composite solution is formed, ensuring that the sugars and polymers are fully dissolved to form a synergistic freeze-drying protectant system.

[0091] S2.3) Sterilization and Light Protection: The prepared trehalose solution and PVP composite protective agent solution must be sterilized separately. Autoclave at 121℃ for 30 minutes to ensure the aseptic safety of the protective agent system during subsequent mixing and freeze-drying.

[0092] After sterilization and cooling, further sterilization filtration is performed using a 0.22μm polyethersulfone membrane. Filtration should be carried out under aseptic conditions to prevent secondary contamination.

[0093] Finally, the sterile composite cryoprotectant solution was stored in a 4°C refrigerator away from light, and the shelf life was controlled to be used within 7 days to ensure the stability and freezing protection capability of the cryoprotectant system.

[0094] This composite protective agent system possesses a dual protection mechanism:

[0095] 1) Trehalose inhibits ice crystal formation during the freezing stage and forms bioactive molecules such as glassy protective proteins during the drying stage;

[0096] 2) As a polymeric protective agent, PVP has a synergistic effect in stabilizing membrane structures and encapsulating exosomes.

[0097] Freeze-drying mixing and pretreatment step (S3): After obtaining clear and impurity-free umbilical cord mesenchymal stem cell conditioned medium (S1) and prepared compound freeze-drying protectant solution (S2), mixing and pre-freezing solution stability assessment are required to form a pre-frozen sample that can enter the freeze-drying process.

[0098] The specific steps are as follows:

[0099] S3.1) Mixing treatment: Mix the conditioned medium obtained in step S1) with the lyophilization protectant composite solution obtained in step S2) at a 1:1 volume ratio, place in a sterile centrifuge tube or lyophilization sample dish, and mix by vortexing for 30-60 seconds. This mixing ratio aims to balance the protectant concentration and the content of active ingredients in the supernatant, avoiding excessive concentration that may affect lyophilization efficiency or cause membrane protein aggregation and precipitation.

[0100] In this step, the mixing container and shaking operation should be completed in a clean bench to avoid the risk of contamination during subsequent freeze-drying. The resulting mixture should be a clear or microemulsion liquid, free of visible suspended impurities or sediment.

[0101] S3.2) Sample Pretreatment and Physicochemical Parameter Evaluation: To ensure the stability of the solution system before lyophilization, the mixture needs to be pretreated and observed, and its physicochemical parameters initially tested, including but not limited to the following:

[0102] Appearance inspection: Observe its color, transparency, and presence of flocculent sediment;

[0103] pH testing: Use a miniature pH meter to measure the pH value of the mixture. It should be controlled between 7.0 and 7.4 to accommodate the stable range of most exosomes and protein active components.

[0104] Turbidity detection: Detected using a turbidimeter or visible light absorption method. If the turbidity exceeds the standard, it can be treated by supplementing filtration.

[0105] If all the above parameters meet the requirements, the process can proceed to the low-temperature quick-freezing and freeze-drying process. If pH drift or abnormal turbidity occurs, the protective agent solution should be prepared again or the mixing ratio adjusted.

[0106] Low-temperature pre-freezing and freeze-drying steps (S4)

[0107] The quality of the finished freeze-dried powder is highly dependent on the temperature control and drying strategy design during the freeze-drying process. The freeze-drying process can be divided into three continuous processes: pre-freezing, primary drying, and secondary drying. To reduce the damage of ice crystals to the structure of active substances while improving drying efficiency, this embodiment adopts the following standardized processing path:

[0108] S4.1) Low-temperature quick-freezing

[0109] The pre-frozen mixture obtained in step S3) was rapidly transferred to an ultra-low temperature freezer at -80°C for quick freezing, with the freezing time controlled between 4 and 6 hours. By rapidly traversing the nucleation critical temperature zone, the water in the system was encouraged to form uniform and fine ice crystals, thereby minimizing the shear damage to proteins and peptides caused by ice crystal growth. During the freezing process, the sample was kept static to avoid disturbance that could cause uneven ice crystal arrangement.

[0110] S4.2) Freeze-drying (sublimation drying)

[0111] The quick-frozen solid mixture sample is transferred to a freeze-drying system (such as the LGJ-10 or a similar vacuum freeze dryer) and subjected to primary and secondary drying processes in sequence:

[0112] Primary drying stage:

[0113] Temperature setting: -40℃;

[0114] Vacuum control: 10 Pa;

[0115] Drying time: 12–16 hours;

[0116] Explanation of principle: This stage uses a sublimation mechanism to directly convert ice crystals in the sample into water vapor, removing most of the moisture.

[0117] Secondary drying stage:

[0118] Temperature setting: Increase to +20℃;

[0119] The vacuum level remains constant;

[0120] Drying time: 6-8 hours;

[0121] Explanation of principle: This stage mainly removes bound water from the sample to reduce residual moisture and prevent microbial contamination or protein denaturation during subsequent storage.

[0122] After drying, the system automatically returns to atmospheric pressure. According to testing standards, the total residual moisture content of the resulting lyophilized powder should be controlled below 3% to meet the stability requirements of pharmacopoeia and biological agents. Low-temperature rapid freezing and vacuum freeze-drying are the core processes in lyophilized powder preparation, and their temperature control profile design must balance component protection and energy efficiency. Rapid pre-freezing can improve structural stability and reduce the risk of active ingredient inactivation; during the sublimation drying stage, excessively rapid heating should be avoided to prevent sample collapse or structural damage.

[0123] The freeze-dried powder collection and packaging process is described in step S5.

[0124] After freeze-drying, the freeze-dried powder must be collected and packaged rapidly under sterile conditions to ensure the biosafety and physicochemical stability of the finished product. This embodiment uses the following process flow:

[0125] S5.1) Collection of freeze-dried powder

[0126] Once the freeze-drying process is complete, the system automatically returns to normal pressure and maintains room temperature. Operators, wearing sterile gloves, open the drying chamber door in a Class 100,000 cleanroom environment to sample and recover the powder from the freeze-drying dish or glass container.

[0127] The freeze-dried powder should be loose, uniform, and free of lumps or agglomerations. If the powder shows obvious signs of moisture absorption and clumping, abnormal color, or adhesion, the batch must be rejected. After visual inspection and initial screening based on physical flowability, a portion of the samples will be randomly tested for moisture retention, pH, and activity retention to ensure that the finished product meets quality control standards.

[0128] S5.2) Encapsulation and Storage

[0129] The collected lyophilized powder should be immediately sealed under an inert gas (such as nitrogen) protective atmosphere to prevent oxidation and moisture absorption. The preferred packaging containers are light-proof brown glass bottles or aluminum-plastic composite bags, with volumes customized according to the dosage specifications, typically ranging from 1 mL to 10 mL.

[0130] The packaging process includes the following key steps:

[0131] Filling: Fill the lyophilized powder into pre-sterilized containers according to the set dosage;

[0132] Vacuum evacuation and nitrogen filling: High-purity nitrogen is filled in after evacuation using a vacuum sealing device;

[0133] Sealing: Use a sterile cap to press the seal tightly to ensure that it is airtight and does not leak powder;

[0134] Labels: Affix batch number, production date, storage conditions, and product name, etc.

[0135] Refrigerated storage: Finally, place the sealed bottle in a refrigerated environment at 2-8°C for long-term storage.

[0136] The collection and packaging of lyophilized powder products not only affects the usability of the final product but also directly impacts subsequent reconstitution performance and application effectiveness. The nitrogen-filled sealing method used in this embodiment effectively delays oxidation and moisture adsorption, maintaining the structural integrity and activity stability of the powder, making it suitable for hospital, pharmacy, and research packaging applications.

[0137] In addition, the containers and packaging materials used for packaging must comply with the "Regulations on the Administration of Drug Packaging Materials and Containers" and have medical-grade certification.

[0138] To better understand the technical solution and effects of the present invention, the present invention will be further described below with reference to specific embodiments. However, it should be understood that the embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Any equivalent substitutions or improvements that can be made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

[0139] Example 1: Preparation and particle size integrity verification of lyophilized powder of umbilical cord mesenchymal stem cell conditioned medium based on a dual lyophilization protectant system

[0140] 1. Umbilical cord mesenchymal stem cells were cultured in serum-free conditioned medium. When the cell confluence was 80%–90%, the cell culture supernatant from the third to fifth passages was collected.

[0141] 2. Perform stepwise centrifugation on the collected supernatant to remove dead cells and cell debris. The first centrifugation parameters are 200–800 × g, 4°C, for 5–10 minutes, preferably 500 × g for 5 minutes at 4°C. The second centrifugation parameters are 1000–3000 × g, 4°C, for 5–15 minutes, preferably 3000 × g for 10 minutes at 4°C.

[0142] 3. The cell supernatant after centrifugation was filtered through a 0.22 μm filter membrane to obtain the conditioned medium for umbilical cord mesenchymal stem cells.

[0143] 4. The preparation process of lyophilized culture medium powder is as follows: First, two types of lyophilization protectant solutions are prepared. The sugar lyophilization protectant is a disaccharide, including sucrose, trehalose, lactose, and mannitol. The polymer lyophilization protectant is a high molecular weight polymer, including polyvinylpyrrolidone (PVP) and dextran. Trehalose is preferred as a non-reducing sugar protectant, and PVP40 is preferred as a polymer lyophilization protectant. The concentration range of trehalose is 50–200 mM, and the concentration range of PVP40 is 1%–5% (w / v). The solvent for the trehalose solution is a mixture of ultrapure water and PBS, with a preferred ratio of ddH2O:PBS = 65:35. The PVP40 preparation step involves adding the corresponding mass fraction of PVP40 to the above trehalose solution and mixing with a magnetic stirrer.

[0144] 5. All obtained solutions were sterilized by autoclaving, filtered through a 0.22 μm filter membrane, and stored away from light.

[0145] 6. Vortex mix equal volumes of the above-mentioned lyophilization protectant mixture and conditioned medium solution at a 1:1 ratio. Then place the mixture in a -80℃ freezer for rapid freezing. After rapid freezing, place it in a freeze dryer for lyophilization to obtain mesenchymal stem cell conditioned medium lyophilized powder (Pro-Lyo-MSC-CM).

[0146] The lyophilized medium powder obtained using two lyophilization protectants showed significantly higher particle size preservation than lyophilized medium powders obtained using only one protectant or without any protectant. For example... Figure 1 As shown: The lyophilized powder particle concentration with added trehalose and PVP40 lyophilization protectant is 2×10⁻⁶. 9 The particle size concentration was 10⁻⁶ / mL, while the particle size concentrations of the unprotected and the protected particles were 10⁻⁶ / mL. 8 On the order of magnitude of cells / mL. Figure 2 The analysis of the components of various conditioned media using ELISA shows that the conditioned media contain abundant growth factors, cytokines, and inflammation-related factors, which can play an important role in tissue repair, immune response, and other processes.

[0147] Figure 1 Nanoparticle tracking analysis (NTA) was used to test the nanoparticle size of lyophilized powders with different protectants. a) Particle size test of lyophilized powder of conditioned medium without protectant; b) Particle size test of lyophilized powder of conditioned medium with only trehalose protectant; c) Particle size test of lyophilized powder of conditioned medium with both trehalose and PVP40 protectants.

[0148] Example 2: Experimental study on cell proliferation and anti-inflammatory effects of lyophilized powder with dual protective agents

[0149] In a preferred embodiment of the present invention, a lyophilized powder of conditioned medium for umbilical cord mesenchymal stem cells is provided. The preparation method of the lyophilized powder utilizes trehalose as a non-reducing protectant, preferably at a concentration of 100 mM. At this concentration, the osmotic pressure of the prepared solution is consistent with that of the conditioned medium, minimizing the damage to the active components of the medium caused by the addition of the lyophilization protectant. PVP40 is used as a non-permeability protectant, preferably at a concentration of 2%. The lyophilized powder without a lyophilization protectant is named CM0, the powder using trehalose as a lyophilization protectant is named CM1, and the powder using both lyophilization protectants is named CM2. Cell viability experiments show that… Figure 3 CM2, when diluted two-fold, showed the most significant effect in promoting cell proliferation without significant cytotoxicity. Cell migration assays demonstrated that CM2 significantly promoted HDF cell migration, with a much better effect than CM1. Figure 4 An inflammation-induced macrophage model was constructed using LPS. Figure 5 The study found that CM2 effectively reduced the levels of IL-1β, IL-6, and TNF-α. In particular, the 2-fold diluted CM2 showed an anti-inflammatory effect comparable to that of the classic anti-inflammatory drug dexamethasone, indicating that CM2 has a good anti-inflammatory effect. Finally, a rat model of skin defects was constructed. Figure 6The lyophilized conditioned medium powder was reconstituted with PBS and applied daily to animal wounds. Daily application of CM2 effectively accelerated wound healing and increased the levels of growth factors VEGF and FGF, as well as the anti-inflammatory factor IL-10, at the wound site, thus promoting wound healing. In summary, the lyophilized conditioned medium powder protected by the two protective agents obtained in this invention has a significant effect on promoting skin wound healing. Compared with the traditional method of injecting conditioned medium into the wound, the application method is simpler, does not create new wounds, and is more practical.

[0150] Figure 3 Cell viability assays using lyophilized powder of conditioned medium based on human fibroblast (HDF) cell line. a) Cytotoxicity test; b) Cell proliferation test.

[0151] Figure 4 Results of cell migration experiments using transwell chambers;

[0152] Figure 5 The anti-inflammatory effects of lyophilized medium in a macrophage inflammation model constructed using LPS were tested using an ELISA kit. a) Effect on IL-1β; b) Effect on IL-6; c) Effect on TNF-α.

[0153] Figure 6 In vivo experiments were conducted to test the effect of lyophilized culture medium on skin wound healing. a. Left: Photographs of rat skin wound repair and healing using lyophilized culture medium under different conditions. a. Right: Analysis of wound healing area. b. ELISA kits were used to analyze the levels of growth factors FGF, VEGF, and anti-inflammatory factor IL-10 at the wound site on days 7 and 14 of wound healing.

[0154] Example 3: Validation experiment on the anti-aging mechanism of cells based on lyophilized powder:

[0155] The lyophilized conditioned medium powder prepared in Example 2 was used for anti-aging related functional tests. An exogenous aging model was constructed by irradiating HDF cells with X-rays. SA-β-gal staining experiments were then performed. Figure 7 DNA damage marker detection Figure 8 The relative expression levels of aging-related proteins were characterized using Western blotting (WB). Figure 9 This study aimed to assess the mitigation of cell senescence after using lyophilized conditioned medium powder. Senescent cells possess highly active β-galactosidase. In situ staining with X-Gal as a substrate, β-galactosidase catalyzes the formation of a deep blue product, which can be observed under an optical microscope. SA-β-gal staining experiments showed that… Figure 7Using CM2 diluted two times significantly reduced the proportion of senescent cells after radiation exposure compared to using CM1 alone as a trehalose lyophilization protectant. γ-H2AX is formed by the rapid phosphorylation of histone H2AX after DNA double-strand breaks (DSBs), and γ-H2AX immunofluorescence staining is an important method for detecting DNA damage. γ-H2AX staining results ( Figure 8 Consistent with SA-β-gal staining results, CM2 diluted two-fold showed the best effect in reducing DNA damage in senescent cells. Finally, Western blotting was used to detect the relative expression levels of senescence-related proteins p53, p16, and Lamin B1 in HDF senescent cells treated with lyophilized medium. Both p53 and p16 led to cell cycle arrest, SASP secretion (such as IL-6 and MMPs), and cell morphological changes. The p53 / p21 pathway dominated in replicative senescence (telomere shortening), while p16 was more significant in stress-induced senescence (such as oxidative damage and oncogene activation). Both were highly expressed in senescent cells. Lamin B1, a nuclear laminin, is another important characteristic of cellular senescence. Its loss is associated with epigenetic characteristics and senescence-related chromatin structure; therefore, lower Lamin B1 levels indicate more severe senescence. Western blotting results showed that compared with the X-ray model group, using CM2 diluted two-fold significantly downregulated the relative expression levels of p53 and p16 and upregulated the relative expression level of Lamin B1. The above results show that the lyophilized conditioned medium powder obtained by the present invention has anti-aging effects.

[0156] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A freeze-dried stem cell powder that promotes skin healing, characterized in that, It includes the following components: Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells; Freeze-drying protectants: selected from one or more of sucrose, trehalose, lactose, and mannitol; Polymer protective agents: selected from one or more of polyvinylpyrrolidone (PVP) and dextran.

2. The freeze-dried stem cell powder for promoting skin healing according to claim 1, characterized in that, It includes the following components: Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells; Lyophilization protectant group: Trehalose; Polymer protectant group: Polyvinylpyrrolidone (PVP).

3. The freeze-dried stem cell powder for promoting skin healing according to claim 2, characterized in that, The mass percentage ranges for each component are as follows: Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells, 5%–20%; Lyophilization protectant: Trehalose, 40%–70%; Polymer protectant: Polyvinylpyrrolidone (PVP40), 20%–40%.

4. The freeze-dried stem cell powder for promoting skin healing according to claim 3, characterized in that, The mass percentages of each component are as follows: Active ingredient: Lyophilized supernatant of conditioned medium for umbilical cord mesenchymal stem cells, 12%; Lyophilization protectant: Trehalose, 58%; Polymer protectant: Polyvinylpyrrolidone (PVP40), 30%.

5. A method for preparing a freeze-dried stem cell powder that promotes skin healing, applied to the freeze-dried stem cell powder for promoting skin healing as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1) Collection of conditioned medium: Umbilical cord mesenchymal stem cells are cultured. After the cells reach the preset degree of confluence, the culture medium is collected and subjected to multi-stage centrifugation and filtration to obtain cell-free conditioned medium. S2) Preparation of freeze-drying protectant solution: Prepare sugar protectant and polymer protectant solutions separately. The sugar protectant is trehalose and the polymer protectant is polyvinylpyrrolidone (PVP40). Mix them according to a preset ratio to form a two-component freeze-drying protectant mixture. S3) Mixing treatment of the cryoprotectant and culture medium: Mix the freeze-drying cryoprotectant mixture with an equal volume of conditioned culture medium and mix by vortexing to form a solution to be freeze-dried; S4) Low-temperature pre-freezing and freeze-drying: After the mixture is rapidly frozen at -80℃, it is transferred to a freeze dryer to perform a sublimation drying process, forming a freeze-dried powder. S5) Collection and packaging of lyophilized powder: The dried lyophilized powder is sampled, tested and vacuum-sealed to finally obtain the lyophilized powder product of umbilical cord mesenchymal stem cell conditioned culture medium.

6. The method for preparing a freeze-dried stem cell powder for promoting skin healing according to claim 5, characterized in that, Step S1) includes the following sub-steps: S1.1) Stem cell culture: Umbilical cord mesenchymal stem cells were cultured in serum-free conditioned medium. The culture was terminated when the cell confluence reached 80% to 90%. The culture supernatant of the third to fifth generation cells with a confluence of 85% was collected. S1.2) Primary centrifugation: The obtained culture supernatant was centrifuged for the first time at 4°C with a centrifugal force of 200-800×g for 5-10 minutes. The specific centrifugation parameters were 500×g and 5 minutes to remove dead cells and large granular cell debris. S1.3) Secondary centrifugation treatment: The supernatant after the first centrifugation is centrifuged again at 4°C with a centrifugal force of 1000-3000×g for 10-15 minutes. The specific centrifugation parameters are 3000×g and 10 minutes to further remove small fragments and residual precipitates. S1.4) Aseptic filtration: The supernatant after the second centrifugation was filtered and sterilized using a sterile filter membrane with a pore size of 0.22 μm to obtain a clear, transparent umbilical cord mesenchymal stem cell conditioned medium free of cell impurities, providing a basic liquid raw material for subsequent freeze-drying.

7. The method for preparing a freeze-dried stem cell powder for promoting skin healing according to claim 5, characterized in that, Step S2) includes the following sub-steps: S2.1) Preparation of trehalose solution: Dissolve trehalose in a solvent obtained by mixing ultrapure water and PBS at a volume ratio of 65:35 to prepare a trehalose solution with a concentration range of 50-200mM, specifically 100mM. Stir with a magnetic stirrer until fully dissolved. The resulting solution is a non-reducing sugar protective agent solution. S2.2) Preparation of PVP40 solution: Polyvinylpyrrolidone (PVP40) is directly added to the above trehalose solution to prepare a polymer protectant solution with a mass concentration of 1% to 5%, specifically 2%. Stir thoroughly until the solution is uniform and clear to form a synergistic composite protectant system. S2.3) Sterilization and light-protected storage: The trehalose solution and PVP40 mixed solution were sterilized by autoclaving at 121℃ for 30 minutes. After cooling to room temperature, they were filtered through a 0.22μm filter membrane for sterilization. Finally, they were stored at 4℃ under light-protected conditions for later use.

8. The method for preparing a freeze-dried stem cell powder for promoting skin healing according to claim 5, characterized in that, Step S3) includes the following sub-steps: S3.1) Mixing treatment: Mix the umbilical cord mesenchymal stem cell conditioned culture medium obtained in step S1) with the composite protectant solution obtained in step S2) at a volume ratio of 1:1, and mix by vortexing for 30-60 seconds to form a homogeneous pre-frozen mixture. S3.2) Sample pretreatment: The mixture is temporarily stored at low temperature, and its appearance, pH and turbidity are tested to confirm that it meets the physicochemical stability requirements before freeze-drying and is ready to enter the freeze-drying process.

9. The method for preparing a freeze-dried stem cell powder for promoting skin healing according to claim 5, characterized in that, Step S4) includes the following sub-steps: S4.1) Low-temperature rapid freezing: The pre-frozen mixture obtained in step S3) is rapidly placed in an environment of -80°C for freezing treatment for 4 to 6 hours, so as to pass through the nucleation critical temperature zone and form a structurally stable freeze-dried sample; S4.2) Freeze-drying: Place the pre-frozen sample into a freeze dryer and execute the following procedure: The temperature for the initial drying stage was set to -40℃, the vacuum degree to 10Pa, and the drying time to 12–16 hours. During the secondary drying stage, the temperature is raised to 20°C, and the vacuum conditions are kept constant for 6 to 8 hours. The residual moisture content is controlled to be below 3% throughout the entire process.

10. The method for preparing a freeze-dried stem cell powder for promoting skin healing according to claim 5, characterized in that, Step S5) includes the following sub-steps: S5.1) Collection of freeze-dried powder: After freeze-drying, the freeze-dried powder is recovered from the desiccator in a sterile environment. The powder should be loose and free of lumps. S5.2) Packaging and storage: The lyophilized powder is packaged in a light-proof glass bottle using a nitrogen-filling process, sealed with a cap and labeled with a batch number, and finally stored at 4°C to ensure the biological activity and physicochemical stability of the finished product.

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