A high-mechanical-strength cell membrane sheet rich in extracellular matrix and a preparation method and application thereof

Abstract

The application discloses a kind of high mechanical strength cell membrane pieces rich in extracellular matrix and its preparation method and application, belong to the technical field of biomaterials.The preparation method of the present application comprises: using special serum-free cell membrane piece culture medium to culture dental mesenchymal stem cells, until forming complete cell membrane piece;The special serum-free cell membrane piece culture medium is mixed by vitamin C injection, insulin-like growth factor-1, serum-free cell culture additive and MEM alpha culture medium.The application also discloses the cell membrane piece prepared by the above method and application.The method of the present application can effectively improve the extracellular matrix content of cell membrane piece, improve the mechanical strength of membrane piece, meet the mechanical use requirements of clinical operation and in vivo transplantation;And process is simple, inoculation density is low, culture cycle is short, production cost is low, suitable for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a cell membrane sheet rich in extracellular matrix with high mechanical strength, its preparation method, and its application. Background Technology

[0002] Since its initial report by Okano et al. in 1993, cell sheet technology has gradually developed into an important cell transplantation method in the fields of tissue engineering and regenerative medicine. This technology can completely preserve intercellular connections, the extracellular matrix (ECM), and endogenous bioactive factors, which is beneficial for post-transplant cell survival, functional integration, and tissue reconstruction. Currently, cell sheets have shown promising applications in various fields, including corneal, myocardial, liver, bone, and periodontal regeneration.

[0003] Currently, commonly used methods for preparing cell sheets mainly fall into two categories. One category utilizes culture surfaces modified with thermosensitive materials (such as PNIPAAm) to achieve non-enzymatic desorption of the cell layer through cooling. The other category involves long-term addition of high doses of vitamin C (e.g., 50 μg / mL) to induce cells to synthesize and deposit large amounts of ECM, thereby forming a self-supporting cell sheet. Furthermore, in bone tissue engineering, mechanical dissection methods have been used to obtain bone marrow mesenchymal stem cell sheets for implant surface modification. It is worth noting that existing technologies have reported the combined use of vitamin C and serum-free culture systems, such as adding vitamin C to passage media to maintain cell proliferation activity. However, this method serves the cell expansion stage and has not been specifically optimized for the large-scale deposition and disseminated properties of ECM required for sheet induction.

[0004] While the aforementioned methods have advanced cell sheet technology, several technical bottlenecks remain in its translation to clinical applications. First, temperature-sensitive culture dishes rely on imported materials, resulting in high costs, and harvesting the sheets requires prolonged room temperature treatment, a complex process prone to cell stress. Traditional vitamin C induction methods have excessively long culture cycles (e.g., 14–21 days for human dental follicle stem cells), failing to meet clinical timeliness requirements, and high-density seeding and high-concentration vitamin C further increase costs. Second, and more critically, existing methods generally result in insufficient ECM content in cell sheets. ECM not only provides structural support and mechanical integrity for cells but also stores and regulates various growth factors, mediating cell-microenvironment interactions. A lack of ECM directly affects the stability of transplanted sheets, the vascularization process, and the recruitment and differentiation capacity of host cells, thus limiting regeneration efficacy.

[0005] The scarcity of ECMs directly leads to another core performance deficiency in cell sheet membranes—insufficient mechanical strength. Mechanical strength (including tensile strength, tear resistance, elastic modulus, and suture retention capacity) is crucial for the successful transplantation of membranes and their adaptation to the in vivo mechanical environment. In clinical procedures, membranes must withstand mechanical forces such as forceps handling, rolling, suturing, or attachment; after transplantation, they must resist the loads from tissue fluid flow, muscle contraction, or bone defect areas. However, membranes prepared using existing technologies are fragile. For example, the ultimate tensile strength of conventional vitamin C-induced mesenchymal stem cell membranes is only tens to hundreds of kilopascals, far lower than that of natural soft tissues (such as the cornea and myocardium), making them prone to rupture, retraction, or deformation during procedures. Currently, a few studies have attempted to enhance the mechanical properties of membranes through chemical cross-linking agents (such as genipin) or nanoscaffold composites, but cross-linking agents have residual cytotoxicity, and composite scaffolds damage the natural ECM structure. Other methods employ dynamic mechanical loading to train membranes, but these methods involve complex equipment, high costs, and long cycles, making widespread adoption difficult. In summary, existing technologies lack a simple and safe method to significantly improve the mechanical strength of membranes while maintaining cell viability and natural ECM structure.

[0006] Therefore, a method for preparing cell sheets with high ECM content and excellent mechanical strength based on a serum-free culture system with well-defined components and low-dose vitamin C induction is needed. This method is characterized by low cost, short cycle, simple operation, and no dependence on temperature-sensitive materials. It solves the problems of high preparation cost, long culture cycle, insufficient ECM deposition, low mechanical strength of the membrane, and the failure of existing serum-free / vitamin C methods to be used for efficient membrane preparation in the prior art. This has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for preparing cell membranes rich in extracellular matrix and with high mechanical strength, which can effectively increase the extracellular matrix content of the cell membranes, improve the mechanical strength of the membranes, and meet the mechanical requirements of clinical surgical operations and in vivo transplantation; moreover, the process is simple, with low seeding density, short culture cycle, and low production cost, making it suitable for clinical application.

[0008] A second objective of this invention is to provide a cell membrane sheet with high mechanical strength and rich in extracellular matrix prepared by the above method.

[0009] A third objective of this invention is to provide applications for this high-mechanical-strength cell membrane sheet rich in extracellular matrix.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a method for preparing a cell membrane sheet with high mechanical strength and rich in extracellular matrix, characterized by comprising the following steps: seeding dental follicle mesenchymal stem cells in a culture container and culturing them in a special serum-free cell membrane sheet culture medium until a complete cell membrane sheet is formed on the surface of the culture container; gently rinsing the cell membrane sheet with sterile physiological saline, causing the cell membrane sheet to detach and obtaining a cell membrane sheet with an intact surface; The dedicated serum-free cell sheet culture medium is composed of vitamin C injection, insulin-like growth factor-1, serum-free cell culture additive, and MEMα culture medium, wherein the volume ratio of serum-free cell culture additive to MEMα culture medium is 1:10~40; the vitamin C content is 40~60 μg / ml; and the insulin-like growth factor-1 content is 20~40 ng / mL.

[0011] In some embodiments of the present invention, dental follicle mesenchymal stem cells are human dental follicle mesenchymal stem cells (MSCs), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2024384.

[0012] In some embodiments of the present invention, P2-P4 generation dental follicle mesenchymal stem cells are used for seeding.

[0013] In some embodiments of the present invention, the cell seeding density is 2000-3000 cells / cm². 2 After inoculation, the culture container was placed in an incubator at 37°C and 5% CO2 for static culture.

[0014] In some embodiments of the present invention, the cell seeding density is 2500 cells / cm². 2 .

[0015] In some embodiments of the present invention, during culture, the culture medium is replaced for the first time after the cells adhere to the culture vessel, and fresh dedicated serum-free cell membrane culture medium is used to continue culturing; fresh dedicated serum-free cell membrane culture medium is replaced every 2-4 days until a complete cell membrane is formed on the surface of the culture vessel.

[0016] In some embodiments of the present invention, the volume ratio of serum-free cell culture additive to MEMα medium in the dedicated serum-free cell sheet culture medium is 1:20; the vitamin C content is 50 μg / ml, and the insulin-like growth factor-1 content is 20~40 ng / mL.

[0017] In some embodiments of the present invention, slight wrinkles are observed at the edges of the cell membrane during culture, indicating that the cell membrane has formed; The training period is 10 months and 1 day.

[0018] The second aspect of the present invention discloses a cell membrane sheet with high mechanical strength rich in extracellular matrix prepared by the above method.

[0019] The third aspect of this invention discloses the use of the high mechanical strength cell membrane sheet rich in extracellular matrix in the preparation of medicaments for the prevention and / or treatment of periodontal diseases. Compared with the prior art, the present invention has the following beneficial effects: The preparation method of this invention can effectively improve the extracellular matrix content and mechanical properties of cell membranes, endowing them with the tensile, tear-resistant, and suture-compatible properties required for clinical operation, without impairing cell viability and the natural ECM structure. It also fills the application gap in existing serum-free and vitamin C combined regimens. Cell membranes prepared using this method possess excellent biological properties, clinical applicability, and safety, providing reliable technical support for the clinical translation of high-quality cell membranes.

[0020] This invention utilizes the synergistic effect of vitamin C and insulin-like growth factor-1 in the culture medium to significantly increase the content of glycosaminoglycans and collagen in cell membranes, reduce inoculation density, and effectively improve the mechanical properties of the membranes.

[0021] The cell membrane sheets prepared by this invention have high cell viability, and their sterility and endotoxin indicators meet the relevant standards for human implants. They also exhibit good stability and can meet the needs of clinical application and logistics. Furthermore, in vivo experiments have confirmed that they have excellent bone regeneration promoting capabilities.

[0022] In addition, in vivo safety tests showed that the cell sheet had no systemic toxicity or tumorigenic potential, and the relevant indicators were stable and the tissue morphology was normal, providing a reliable safety guarantee for its clinical translation. Attached Figure Description

[0023] Appendix Figure 1 This is a graph showing the detection results of glycosaminoglycans (sGAG).

[0024] Appendix Figure 2 This is a graph showing the detection results of hydroxyproline (HYP).

[0025] Appendix Figure 3 This is a diagram showing the test results of the mechanical properties of the cell membrane sheet of the present invention.

[0026] Appendix Figure 4 This is a graph showing the cell viability and transport stability test results of the cell membrane sheet of the present invention, wherein... Figure 4 Figure A shows the results of the cell viability test. Figure 4 B is a graph showing the results of the transportation stability test.

[0027] Appendix Figure 5 The images shown are three-dimensional reconstructions and tomographic CT images of Experiment Example 4 of this invention; where Control represents the control group and CS represents the cell sheet group.

[0028] Appendix Figure 6 The graph shows the results of quantitative analysis of bone morphometrics in Experiment 4; where Control represents the control group and CS represents the cell sheet group.

[0029] Appendix Figure 7 The images show HE and Masson trichrome stained sections from Experiment 4 of this invention; Control represents the control group, and CS represents the cell sheet group.

[0030] Appendix Figure 8 This is a graph showing the quantitative analysis results of the regeneration region in Experiment Example 4 of the present invention; Control represents the control group, and CS represents the cell membrane group.

[0031] Appendix Figure 9 This is a graph showing the results of the hematological and biochemical index examination in Experiment Example 4 of this invention; wherein... Figure 9 Image A shows the white blood cell count results. Figure 9 B shows the results of ALT measurement, a liver function indicator. Figure 9 C represents the results of UREA (renal function indicator) measurement. Figure 9 D is the electrolyte Ca. 2+ Measurement results diagram Figure 9 E is the electrolyte Mg 2+ The measurement results are shown in the figure; Control represents the control group, and CS represents the cell sheet group.

[0032] Appendix Figure 10 HE staining images of the main organs (heart, liver, spleen, lung, and kidney) in Experiment Example 4 of this invention; where Control represents the control group and CS represents the cell sheet group.

[0033] Appendix Figure 11 This is a gross observation diagram of Experimental Example 5 of the present invention; where Positive control represents the positive control group; Negative control represents the negative control group; CS represents the cell sheet group.

[0034] Appendix Figure 12 This is a graph showing the tumor volume quantification results of Experiment 5 of the present invention; where Positive control represents the positive control group; Negative control represents the negative control group; and CS represents the cell sheet group.

[0035] Appendix Figure 13 This is an HE-stained section image of Experimental Example 5 of the present invention; where Positive control represents the positive control group; Negative control represents the negative control group; CS represents the cell sheet group. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this application can be purchased on the market or prepared by existing methods.

[0037] The dental follicle mesenchymal stem cells of this invention are human dental follicle mesenchymal stem cells (MSCs), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2024384, deposited on December 6, 2024, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0038] First, second-generation (P2) dental follicle mesenchymal stem cells were used at a rate of 5 × 10⁻⁶. 6 Dental follicle mesenchymal stem cells were cryopreserved at a density of 10 cells / mL to construct a master cell bank (MCB). After thawing, the batch of dental follicle mesenchymal stem cells was passaged to the fourth generation (P4) and cryopreserved again to establish a working cell bank (WCB). When preparing cell sheets, dental follicle mesenchymal stem cells were retrieved from the master cell bank or working cell bank for seeding.

[0039] Example 1 This embodiment discloses a method for preparing the cell membrane sheet of the present invention, as detailed below: 1 . Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Take 0.105 mL of vitamin C injection solution (2 mL: 0.5 g), 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and mix them thoroughly with 25 mL of serum-free cell culture additive to obtain a mixed solution; then mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this embodiment. The working concentration of vitamin C is 50 μg / mL, and the working concentration of insulin-like growth factor-1 is 30 ng / mL.

[0040] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0041] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0042] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0043] 2.3 Culture Medium Replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for this embodiment. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0044] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. Around day 10, when slight wrinkles appeared at the edges of the cell membrane, it indicated that a complete cell membrane had formed and its adhesion to the bottom of the culture dish began to decrease.

[0045] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0046] Example 2 This embodiment discloses a method for preparing the cell membrane sheet of the present invention, as detailed below: 1 . Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Take 0.084 mL of vitamin C injection solution (2 mL: 0.5 g), 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and mix them thoroughly with 25 mL of serum-free cell culture additive to obtain a mixed solution; then mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this embodiment. The working concentration of vitamin C is 40 μg / mL, and the working concentration of insulin-like growth factor-1 is 30 ng / mL.

[0047] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0048] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0049] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0050] 2.3 Culture Medium Replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for this embodiment. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0051] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. Around day 10, when slight wrinkles appeared at the edges of the cell membrane, it indicated that a complete cell membrane had formed and its adhesion to the bottom of the culture dish began to decrease.

[0052] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0053] Example 3 This embodiment discloses a method for preparing the cell membrane sheet of the present invention, as detailed below: 1. Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Take 0.126 mL of vitamin C injection solution (2 mL: 0.5 g), 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and mix them thoroughly with 25 mL of serum-free cell culture additive to obtain a mixed solution; then mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this embodiment. The working concentration of vitamin C is 60 μg / mL, and the working concentration of insulin-like growth factor-1 is 30 ng / mL.

[0054] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0055] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0056] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0057] 2.3 Culture Medium Replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for this embodiment. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0058] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. Around day 10, when slight wrinkles appeared at the edges of the cell membrane, it indicated that a complete cell membrane had formed and its adhesion to the bottom of the culture dish began to decrease.

[0059] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0060] Example 4 This embodiment discloses a method for preparing the cell membrane sheet of the present invention, as detailed below: 1. Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Take 0.105 mL of vitamin C injection solution (2 mL: 0.5 g), 0.105 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and mix them thoroughly with 25 mL of serum-free cell culture additive to obtain a mixed solution; then mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this embodiment. The working concentration of vitamin C is 50 μg / mL, and the working concentration of insulin-like growth factor-1 is 20 ng / mL.

[0061] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0062] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0063] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0064] 2.3 Culture Medium Replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for this embodiment. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0065] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. Around day 10, when slight wrinkles appeared at the edges of the cell membrane, it indicated that a complete cell membrane had formed and its adhesion to the bottom of the culture dish began to decrease.

[0066] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0067] Example 5 This embodiment discloses a method for preparing the cell membrane sheet of the present invention, as detailed below: 1 . Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Take 0.105 mL of vitamin C injection solution (2 mL: 0.5 g), 0.21 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and mix them thoroughly with 25 mL of serum-free cell culture additive to obtain a mixed solution; then mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this embodiment. The working concentration of vitamin C is 50 μg / mL, and the working concentration of insulin-like growth factor-1 is 40 ng / mL.

[0068] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0069] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0070] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0071] 2.3 Culture Medium Replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for this embodiment. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0072] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. Around day 10, when slight wrinkles appeared at the edges of the cell membrane, it indicated that a complete cell membrane had formed and its adhesion to the bottom of the culture dish began to decrease.

[0073] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0074] Comparative Example 1 Compared with Example 1, the working concentration of vitamin C in the culture medium used for the cell sheet in Comparative Example 1 was 15 μg / mL, and it did not contain insulin-like growth factor-1; the cell seeding and culture conditions were the same. The specific method for preparing the cell sheet in Comparative Example 1 is as follows: 1 . Cell Culture Preparation Mix 0.0315 mL of vitamin C injection solution (2 mL: 0.5 g) with 25 mL of serum-free cell culture additive to obtain a mixed solution; mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet culture medium for this comparative example; the working concentration of vitamin C is 15 μg / mL.

[0075] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of this comparative example 1 to prepare a cell suspension.

[0076] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0077] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture.

[0078] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for Comparative Example 1. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0079] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: Cell growth status and membrane formation process were observed daily during culture. In Comparative Example 1, the cell membrane formation time was similar to that in Example 1, approximately 10 days, but the membrane wrinkling was milder and less noticeable.

[0080] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to allow them to detach intact from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0081] Comparative Example 2 Compared with Example 1, the working concentration of vitamin C in the culture medium used for the cell sheet in Comparative Example 2 was 34 μg / mL, and it did not contain insulin-like growth factor-1; the cell seeding and culture conditions were the same. The specific method for preparing the cell sheet in Comparative Example 2 is as follows: 1 . Cell Culture Preparation Take 0.0714 mL of vitamin C injection solution (2 mL: 0.5 g) and mix it with 25 mL of serum-free cell culture additive to obtain a mixed solution; mix 25 mL of this mixed solution with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this comparative example 2; the working concentration of vitamin C is 34 μg / mL.

[0082] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of this comparative example 2 to prepare a cell suspension for later use.

[0083] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 3000 cells / cm². 2 .

[0084] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0085] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for Comparative Example 2. Thereafter, this specific culture medium was replaced every 3 days to ensure a sufficiently nutrient-rich environment for cell growth and to maintain a continuous induction effect.

[0086] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During the culture period, cell growth status and membrane formation process were observed daily. In Comparative Example 2, the cell membrane formation time was similar to that of Example 1, approximately 10 days, but the membrane wrinkling phenomenon was milder and less noticeable.

[0087] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0088] Comparative Example 3 Compared with Example 1, Comparative Example 3 did not contain insulin-like growth factor-1; the working concentration of vitamin C, cell seeding, and culture conditions were the same. The specific method for preparing the cell membrane sheet in Comparative Example 3 is as follows: 1 . Cell Culture Preparation Take 0.105 mL of vitamin C injection solution (2 mL: 0.5 g) and mix it evenly with 25 mL of serum-free cell culture additive to obtain a mixed solution; mix 25 mL of this mixed solution evenly with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of this comparative example 3; the working concentration of vitamin C is 50 μg / mL.

[0089] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of this comparative example 3 to prepare a cell suspension.

[0090] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0091] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0092] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for Comparative Example 3. Thereafter, this specific culture medium was replaced every 3 days to ensure a sufficiently nutrient-rich environment for cell growth and to maintain a continuous induction effect.

[0093] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During the culture period, the cell growth status and membrane formation process were observed daily. In Comparative Example 3, the cell membrane formation time was similar to that of Example 1, approximately 10 days; however, the membrane wrinkles in Comparative Example 3 were slightly increased, and the opaque area of ​​the membrane appearance was less.

[0094] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0095] Comparative Example 4 Compared with Example 1, the working concentration of vitamin C in the culture medium used for the cell sheet in Comparative Example 4 was 15 μg / mL; the concentration of insulin-like growth factor-1, cell seeding, and culture conditions were all the same. The specific method for preparing the cell sheet in Comparative Example 4 is as follows: 1 . Cell Culture Preparation Take 0.0315 mL of vitamin C injection solution (2 mL: 0.5 g), 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL), and 25 mL of serum-free cell culture additive and mix them evenly to obtain a mixed solution; mix 25 mL of this mixed solution with 500 mL of MEMα medium evenly to obtain the cell sheet culture medium for this comparative example; the working concentration of vitamin C is 15 μg / mL, and the working concentration of insulin-like growth factor-1 is 30 ng / mL.

[0096] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of Comparative Example 4 to prepare a cell suspension.

[0097] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0098] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0099] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh, specially formulated cell membrane sheet culture medium for this comparative example. Thereafter, the culture medium was replaced every 3 days to ensure a sufficiently nutrient-rich environment for cell growth and to maintain a continuous induction effect.

[0100] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During the culture period, the cell growth status and membrane formation process were systematically observed daily. In Comparative Example 4, the cell membrane formation time was similar to that of Example 1, approximately 10 days; however, the cell membrane formed in Comparative Example 4 exhibited edge shrinkage and curling, and the overall transparency of the membrane was higher, with relatively fewer opaque areas.

[0101] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0102] Comparative Example 5 Compared with Example 1, the working concentration of vitamin C in the culture medium used for the cell sheet in Comparative Example 5 was 34 μg / mL; the concentration of insulin-like growth factor-1, cell seeding, and culture conditions were all the same. The specific method for preparing the cell sheet in Comparative Example 4 is as follows: 1 . Cell Culture Preparation 0.0714 mL of vitamin C injection solution (2 mL: 0.5 g), 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL) and 25 mL of serum-free cell culture additive were mixed evenly to obtain a mixed solution; 25 mL of this mixed solution was mixed evenly with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of Comparative Example 5; wherein the working concentration of vitamin C was 34 μg / mL and the working concentration of insulin-like growth factor-1 was 30 ng / mL.

[0103] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of this comparative example 5 to prepare a cell suspension.

[0104] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes, adjusting the cell density to a controlled seeding density of 2500 cells / cm². 2 .

[0105] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0106] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh Comparative Example 5 cell sheet culture medium. Thereafter, the culture medium was replaced every 3 days to ensure a nutrient-rich environment for cell growth and to maintain continuous induction.

[0107] 3. Cell sheet formation and harvesting 3.1 Membrane Formation Monitoring: During the culture period, the cell growth status and membrane formation process were systematically observed daily. In Comparative Example 5, the cell membrane formation time was basically the same as in Example 1, approximately 10 days. However, the cell membrane formed in Comparative Example 5 exhibited edge shrinkage and curling, and the overall transparency of the membrane was higher, with relatively fewer opaque areas.

[0108] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0109] Comparative Example 6 Compared with Example 1, Comparative Example 6 used a culture medium that did not contain vitamin C; the concentration of insulin-like growth factor-1, cell seeding, and culture conditions were the same. The specific method for preparing the cell membrane in Comparative Example 6 is as follows: 1 . Cell Culture Preparation 0.1575 mL of insulin-like growth factor-1 (concentration 0.1 mg / mL) was mixed with 25 mL of serum-free cell culture additive to obtain a mixed solution; 25 mL of this mixed solution was mixed with 500 mL of MEMα medium to obtain the cell sheet-specific culture medium of Comparative Example 6; wherein the working concentration of insulin-like growth factor-1 was 30 ng / mL.

[0110] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of Comparative Example 6 to prepare a cell suspension.

[0111] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes, adjusting the cell density to a controlled seeding density of 2500 cells / cm². 2 .

[0112] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0113] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for Comparative Example 6. Thereafter, this specific culture medium was replaced every 3 days to ensure a sufficiently nutrient-rich environment for cell growth and to maintain a continuous induction effect.

[0114] 3. Cell sheet formation and harvesting 3.1 Membrane Formation Monitoring: During the culture period, the cell growth status and membrane formation process were systematically observed daily. In Comparative Example 6, the cell membrane formation time was basically the same as in Example 1, approximately 10 days. However, the cell membrane formed in Comparative Example 6 exhibited edge shrinkage and curling, and the overall transparency of the membrane was relatively high, with relatively few opaque areas.

[0115] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0116] Comparative Example 7 Comparative Example 7 discloses a method for preparing cell membrane sheets based on the conventional vitamin C method. Compared with Example 1, it uses a serum-containing culture medium with only vitamin C added and no insulin-like growth factor-1 added. The specific culture method of this comparative example is as follows: 1 . Cell Culture Preparation Take 50 mL of Gibco brand fetal bovine serum and add it to 450 mL of MEMα medium to prepare a conventional serum-containing mesenchymal stem cell culture medium. Add 0.105 mL of vitamin C injection solution (2 mL: 0.5 g) to the conventional serum-containing mesenchymal stem cell culture medium to make the working concentration of vitamin C 50 μg / mL, thus obtaining the cell sheet-specific culture medium of Comparative Example 7.

[0117] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of Comparative Example 7 to prepare a cell suspension.

[0118] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes, adjusting the cell density to a seeding density of 5000 cells / cm². 2 .

[0119] 2.2 Culture conditions: The inoculated culture dishes were placed in an incubator at 37℃ and 5% CO2 for static culture.

[0120] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh cell membrane sheet culture medium specifically designed for Comparative Example 7. Thereafter, this specific culture medium was replaced every 3 days to ensure a sufficiently nutrient-rich environment for cell growth and to maintain a continuous induction effect.

[0121] 3. Cell sheet formation and harvesting 3.1 Membrane formation monitoring: During culture, cell growth and membrane formation were observed daily. After 4 weeks of continuous culture, slight wrinkles appeared at the edges of the cell membranes, indicating that complete cell membranes had formed and their adhesion to the bottom of the culture dish began to decrease.

[0122] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0123] Comparative Example 8 Comparative Example 8 discloses a method for preparing cell membrane sheets based on the conventional vitamin C method with added insulin-like growth factor-1. Compared with Example 1, the culture medium used is a serum-containing culture medium. The specific method of this comparative example is as follows: 1. Cell Culture Preparation Using MEMα medium supplemented with 10% Gibco fetal bovine serum as the basal culture medium, 0.105 mL of vitamin C injection (2 mL: 0.5 g) was added to bring the working final concentration of vitamin C to 50 μg / mL; at the same time, 0.1575 mL of insulin-like growth factor-1 (0.1 mg / mL) was added to bring the working concentration of insulin-like growth factor-1 to 30 ng / mL.

[0124] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the special culture medium of Comparative Example 8 to prepare a cell suspension.

[0125] 2. Cell seeding and culture The cell suspension was seeded into standard cell culture dishes, and the cell density was adjusted to an inoculation density of 5000 cells / cm². 2 The cells were then incubated statically in a 37°C, 5% CO2 incubator. The culture medium was replaced with fresh vitamin C-containing medium every 3 days to ensure adequate nutrition for cell growth and to maintain a sustained induction effect.

[0126] 3. Harvesting of cell membrane sheets 3.1 Membrane formation monitoring: During culture, cell growth status and membrane formation process were observed daily. After 14 days of continuous culture, slight wrinkles were observed at the edges of the cell membranes, indicating that complete cell membranes had formed and their adhesion to the bottom of the culture dish began to decrease.

[0127] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0128] Comparative Example 9 The culture medium in this comparative example differs from that in Example 1. This comparative example uses a commercially available serum-free culture medium system to prepare cell membranes; the specific procedure is as follows: 1. Cell Culture Preparation First, prepare the cell sheet-specific culture medium: Use standard cell culture dishes and select commercially available premixed serum-free cell culture medium (RP02010, Zhong Sheng Su Yuan) as the basic culture system. Before use, add 0.105 mL of vitamin C injection solution (2 mL: 0.5 g) to the serum-free culture medium to achieve a working concentration of 50 μg / mL; simultaneously add 0.1575 mL of insulin-like growth factor-1 (0.1 mg / mL), achieving a working concentration of 30 ng / mL for insulin-like growth factor-1.

[0129] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0130] 2. Cell seeding and culture 2.1 Cell Seeding: Seed the above cell suspension into standard cell culture dishes and adjust the cell density to 2500 cells / cm². 2 .

[0131] 2.2 Culture conditions: Place the inoculated culture dishes in an incubator at 37℃ and 5% CO2 for static culture for 10 days.

[0132] 2.3 Culture medium replacement: The first culture medium replacement was performed 24 hours after inoculation, using fresh, specially formulated cell membrane sheet culture medium for this comparative example. Thereafter, the culture medium was replaced every 3 days to ensure sufficient nutrients for cell growth and maintain a sustained induction effect.

[0133] 3. Harvesting of cell membrane sheets 3.1 Membrane formation monitoring: During culture, cell growth status and membrane formation process were observed daily. After 14 days of continuous culture, slight wrinkles were observed at the edges of the cell membranes, indicating that complete cell membranes had formed and their adhesion to the bottom of the culture dish began to decrease.

[0134] 3.2 Membrane harvesting: Gently rinse the cell membranes with sterile physiological saline to encourage complete detachment from the culture dish surface. Then, transfer the harvested cell membranes to a new culture dish or designated preservation solution for later use.

[0135] Comparative Example 10 This comparative example discloses a method for preparing cell membrane sheets based on temperature-responsive culture dishes.

[0136] 1. Cell Culture Preparation Using commercially available temperature-responsive cell culture dishes (SuperCell) TM The series of temperature-sensitive intelligent cell culture consumables, DCTP012, Dachuan Heyi Biotechnology, has a poly(N-isopropylacrylamide) (PIPAAm) polymer layer covalently bonded to its culture surface.

[0137] Cell culture medium: MEMα medium supplemented with 10% Gibco fetal bovine serum.

[0138] The revived P4 generation dental follicle mesenchymal stem cells were resuspended in the above-mentioned special culture medium to prepare a cell suspension for later use.

[0139] 2. Cell seeding and culture The cell suspension was seeded into standard cell culture dishes, and the cell density was adjusted to 7 × 10⁶ cells / mL. 5 cells / cm 2After inoculation, the culture dishes were placed in an incubator at 37°C and 5% CO2 and incubated statically for about 24 hours.

[0140] 3. Harvesting of cell membrane sheets Once the cells have merged, remove the culture dish from the 37°C incubator and transfer it to an experimental environment with a preset temperature of 20°C, where it will be incubated for 30 minutes.

[0141] After continuous incubation at room temperature for 10 days (similar to the time in Example 1), the edges of the cell layer were observed to gradually curl and shrink from the culture surface, eventually detaching completely to form a complete cell membrane sheet. Subsequently, the culture medium containing the free cell membrane sheet was carefully transferred to a new collection tube using a pipette to obtain the cell membrane sheet of this comparative example.

[0142] Experimental Example 1 This experiment systematically investigated the extracellular matrix content of cell membrane sheets obtained under different preparation conditions using glycosaminoglycans (GAG) and collagen (calculated as hydroxyproline) as detection indicators.

[0143] 1. Glycosaminoglycan (GAG) determination Cell membrane fragments were disrupted, and 1 mL of papain extraction reagent was added. The mixture was heated at 65°C for 3 hours until no visible tissue fragments remained. The cells were then centrifuged at 10000 g for 10 minutes. The sGAG content was determined using a sulfated glycosaminoglycan (sGAG) assay kit (Biocolor, UK). The results are attached. Figure 1 As shown, the specific analysis is as follows: (1) By Figure 1 As can be seen from A, Example 1 (vitamin C 50 μg / mL, insulin-like growth factor-1 30 ng / mL) had the highest sGAG content, while the sGAG content of Examples 2-5 decreased slightly, but there was no statistically significant difference among the groups. This result indicates that the formulation of the present invention can stably maintain a high level of glycosaminoglycan synthesis within the above concentration range, with the parameter combination of Example 1 representing the optimal conditions.

[0144] (2) By Figure 1 As shown in Figure B, compared with Example 1, the sGAG content of Comparative Examples 1 to 3, which do not contain insulin-like growth factor-1, is significantly reduced (p<0.0001); even though the vitamin C concentration of Comparative Example 3 is the same as that of Example 1, its sGAG content is still significantly lower than that of Example 1. This indicates that insulin-like growth factor-1 is a key component for the synthesis of high-glycosaminoglycans, and it has a synergistic promoting effect with vitamin C.

[0145] (3) By Figure 1As can be seen from Example C, compared with Example 1, the sGAG content of Comparative Examples 4 to 6, which contained different concentrations of vitamin C, showed a gradient decreasing trend with decreasing vitamin C concentration, and were all significantly lower than those of Example 1 (p<0.001 or p<0.0001), with Comparative Example 6, which did not contain vitamin C, having the lowest sGAG content. This result indicates that vitamin C concentration is positively correlated with glycosaminoglycan synthesis capacity, 50 μg / mL is the optimal working concentration of vitamin C, and that vitamin C has a synergistic effect with insulin-like growth factor-1.

[0146] (4) By Figure 1 As can be seen from Example D, compared with Example 1, the sGAG content of the conventional vitamin C method (Comparative Example 7), the conventional vitamin C method with added insulin-like growth factor-1 (Comparative Example 8), and the commercial serum-free culture medium system (Comparative Example 9) were all significantly reduced (p<0.0001). This result indicates that the vitamin C, insulin-like growth factor-1, and serum-free culture system of the present invention have a synergistic promoting effect at low inoculation densities (2500 cells / cm²). 2 This method can achieve a higher efficiency in extracellular matrix synthesis than existing conventional methods.

[0147] (5) By Figure 1 As can be seen from Example E, compared with Example 1, the sGAG content of Comparative Example 10 prepared using the temperature-responsive culture dish method was significantly reduced (p<0.01). Under the same film-forming time (10 days), its GAG synthesis ability was significantly inferior to that of the method of this application, proving that the culture medium formulation of this application has a significantly better promoting effect on extracellular matrix synthesis than the temperature-responsive culture dish preparation method.

[0148] In summary, this invention significantly enhances the content of cell membrane glycosaminoglycans (sGAGs) at low inoculation densities by synergistically adding specific concentrations of vitamin C and insulin-like growth factor-1 to serum-free culture medium, resulting in superior preparation efficiency and product quality compared to existing conventional methods.

[0149] 2. Collagen content determination (calculated as hydroxyproline) After disrupting the cell membrane fragments, 1 mL of 6 mol / L hydrochloric acid was added, and the mixture was digested at 110°C for 2 hours. The pH was then adjusted to 6-8 with 10 mol / L sodium hydroxide solution, followed by centrifugation at 25°C and 16000g for 20 minutes. The supernatant was collected. The hydroxyproline (HYP) content was determined using a hydroxyproline assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The hydroxyproline detection results are attached. Figure 2 As shown, the specific analysis is as follows: Depend on Figure 2As can be seen from A, when the working concentration of vitamin C is 40~60 μg / mL and the working concentration of insulin-like growth factor-1 is 20~40 ng / mL, the hydroxyproline content of the cell membrane sheets prepared in each example is not significantly different and is maintained at a high level; among them, the hydroxyproline content of Example 1 (vitamin C 50 μg / mL, insulin-like growth factor-1 30 ng / mL) is the optimal level.

[0150] Depend on Figure 2 As can be seen from B, the hydroxyproline content of the cell membrane sheet prepared in Example 1 was significantly higher than that of Comparative Examples 1 to 3, which did not contain insulin-like growth factor-1 (p<0.0001). Even if the vitamin C concentration was increased to the same level as in Example 1 (Comparative Example 3), the hydroxyproline content could not be restored to the level of Example 1, which proves that insulin-like growth factor-1 is a key component for maintaining the high extracellular matrix synthesis capacity of the cell membrane sheet.

[0151] Depend on Figure 2 As can be seen from Example C, the hydroxyproline content of the cell membrane sheets prepared in Example 1 was significantly higher than that of Comparative Examples 4 to 6, where vitamin C concentration was reduced or absent (p<0.0001 / p<0.001). The hydroxyproline content of the cell membrane sheets decreased with decreasing vitamin C concentration, reaching its lowest level when vitamin C was completely absent (Comparative Example 6). This result indicates that vitamin C and insulin-like growth factor-1 have a synergistic effect, and both are essential for maintaining high extracellular matrix synthesis capacity.

[0152] Depend on Figure 2 As can be seen from D, the hydroxyproline content of the cell membrane sheets prepared in Example 1 was significantly higher than that of Comparative Example 7 based on the conventional vitamin C method, Comparative Example 8 based on the conventional vitamin C method with added insulin-like growth factor-1, and Comparative Example 9 based on the commercial serum-free culture medium system (p<0.0001). This demonstrates that the vitamin C, insulin-like growth factor-1, and serum-free culture system of the present invention work synergistically at low inoculation densities (2500 cells / cm²). 2 Under these conditions, compared to existing culture medium systems, it can still significantly enhance the extracellular matrix synthesis capacity of cell membranes.

[0153] Depend on Figure 2 As can be seen from E, the hydroxyproline content of the cell membrane sheets prepared in Example 1 was significantly higher than that of Comparative Example 10 based on temperature-responsive culture dishes (p<0.0001), and the cell seeding density of Example 1 (2500 cells / cm²) was also significantly higher. 2 ) is far lower than the comparative example 10 (7×10 5 pcs / cm 2 This indicates that the preparation method of the present invention can still achieve a higher level of collagen synthesis under extremely low seeding density conditions, significantly reducing the dependence on the initial seeding amount of cells.

[0154] The above results indicate that the present invention, through the synergistic effect of vitamin C, insulin-like growth factor-1 and serum-free culture system, can significantly increase the hydroxyproline content of cell membranes at optimized concentrations, and has significant advantages over existing conventional preparation methods in terms of collagen synthesis capacity and tolerance to inoculation density.

[0155] 3. Experimental Conclusions This experiment systematically measured and analyzed two core extracellular matrix indicators, glycosaminoglycans (sGAG) and collagen (hydroxyproline), clarifying the technical advantages and key influencing factors of this invention. The experimental results confirmed that the synergistic effect of vitamin C (optimal concentration 50 μg / mL) and insulin-like growth factor-1 (optimal concentration 30 ng / mL) is crucial for enhancing the extracellular matrix synthesis capacity of cell membranes; both are indispensable, and their concentrations must be controlled within a specific range. Compared to existing conventional culture media systems and traditional physical preparation methods, the synergistic formulation of this invention significantly increases the sGAG and hydroxyproline content of cell membranes, and requires a lower inoculation density.

[0156] The cell membrane preparation method of the present invention can effectively improve the biological quality of the membrane, solve the problem of insufficient extracellular matrix synthesis in the prior art, and provide a reliable technical solution for the preparation of high-quality cell membranes.

[0157] Experimental Example 2 To verify the mechanical properties of the cell membrane sheets prepared in this invention, and to simulate the mechanical environment during surgical clamping, suturing, and transplantation, a universal testing machine was used to measure the tensile strength of the cell membrane sheets prepared in Examples 1-5 and Comparative Examples 1-10. The test results are as follows: Figure 3 As shown. The specific analysis is as follows: (1) By Figure 3 As can be seen from A, the maximum tensile force of the cell membrane sheets prepared in Examples 1 to 5 was maintained in the range of 0.007 to 0.0085 N, and there was no statistically significant difference among the groups. This proves that the preparation process of the present invention has good stability within the range of vitamin C concentration of 40-60 μg / mL and insulin-like growth factor-1 concentration of 20-40 ng / mL, and the mechanical properties of the cell membrane sheets are uniform and reliable.

[0158] (2) By Figure 3As can be seen from B, the maximum tensile force of the cell membrane sheet prepared in Example 1 was significantly higher than that of Comparative Examples 1 to 3 (p<0.0001), and there was no significant difference among Comparative Examples 1 to 3. This result indicates that adjusting the vitamin C concentration alone or using vitamin C alone cannot achieve mechanical strength comparable to that of the present invention, and the synergistic effect of vitamin C and insulin-like growth factor-1 is a necessary condition for improving the mechanical properties of the cell membrane sheet.

[0159] (3) By Figure 3 As can be seen from Example C, the maximum tensile force of the cell membrane sheet prepared in Example 1 was significantly higher than that of Comparative Examples 4 to 6 (p<0.01 or p<0.001), and there was no significant difference among Comparative Examples 4 to 6. This result indicates that under the condition of a fixed insulin-like growth factor-1 concentration, a vitamin C concentration below the threshold defined in this invention or its complete absence will lead to a significant decrease in the mechanical strength of the cell membrane sheet. The vitamin C concentration threshold is a necessary condition to ensure mechanical properties; below this threshold, insulin-like growth factor-1 cannot compensate for the performance defects on its own.

[0160] (4) By Figure 3 D shows that The maximum tensile force of the cell membrane sheet in Example 1 was significantly higher than that of the membrane sheet prepared by the conventional vitamin C method (Comparative Example 7), the conventional vitamin C method with added insulin-like growth factor-1 (Comparative Example 8), and the commercial serum-free system (Comparative Example 9). This proves that while significantly reducing cell seeding density and shortening the membrane formation cycle, the mechanical properties of the membrane sheet prepared by the present invention are still significantly better than those of the existing conventional preparation system.

[0161] (5) By Figure 3 As can be seen from E, the maximum tensile force of the cell sheet prepared in Example 1 is significantly higher than that in Comparative Example 10 (p<0.01), and the maximum tensile force of the cell sheet prepared in Comparative Example 10 is close to 0, indicating almost no mechanical strength. This result shows that cell sheets prepared by the traditional temperature-responsive culture dish method cannot meet the mechanical requirements of surgical operations, while cell sheets prepared by the method of this invention can meet the mechanical environment requirements during clinical transplantation.

[0162] In summary, this invention, through the synergistic effect of vitamin C, insulin-like growth factor-1, and a serum-free system, can stably prepare cell sheets with significantly superior mechanical properties compared to existing conventional, commercial, and temperature-responsive systems. Furthermore, it exhibits a wide process parameter window and good stability, effectively overcoming the shortcomings of existing cell sheet preparation technologies, such as poor mechanical properties, high seeding density, and long film formation cycles. Example 3 This experiment systematically conducted three core performance tests on the cell membrane prepared in Example 1: cell viability, aseptic safety, and storage and transport stability, in order to verify its biological function and feasibility for clinical application.

[0163] 1. Cell viability test Cell viability within cell sheets was determined using Calcein-AM / PI staining (live / dead cell dual-color fluorescence staining) combined with microscopic observation and quantitative analysis. The specific procedures are as follows: Freshly prepared cell membrane sheets were prepared, and the surface culture medium was discarded. The sheets were gently rinsed with phosphate-buffered saline (PBS) for 10 seconds to thoroughly remove any residual culture medium. To achieve cell viability staining, the cell membrane sheets were first incubated with a 1:1000 dilution of calcein-AM solution (Beyotime Biotechnology Co., Ltd., Shanghai, China) in PBS at 37°C in the dark for 30 minutes. After removing the calcein-AM solution, the cell membrane sheets were then incubated with a 1:1000 dilution of PI solution (Beyotime Biotechnology Co., Ltd., Shanghai, China) in PBS at 37°C in the dark for 10 minutes. After staining, the PI solution was removed, and the cell membrane sheets were rinsed again with PBS for approximately 10 seconds to remove any unbound free dye. Fluorescence images were captured using a fluorescence microscope, and the stained cells were quantitatively analyzed using ImageJ software to calculate cell viability.

[0164] Test results: Live / dead cell staining showed that the cell viability within the cell sheet was ≥95%. Figure 4 A). This indicates that the cell membrane prepared by this invention can still maintain extremely high cell viability after preparation and can perform its expected biological functions normally.

[0165] 2. Sterility and endotoxin testing Test methods: In accordance with the relevant provisions of the Chinese Pharmacopoeia, the cell membrane smears were subjected to aseptic examination by direct inoculation, and the bacterial endotoxin content was detected by the Limulus Amebocyte Lysate (LAL) method.

[0166] Test results: The sterility test results showed that no microbial growth was observed in any of the submitted cell membrane samples during the 14-day culture period; the endotoxin content was all below 0.5 EU / mL, fully meeting the relevant standards for human implants, ensuring that the cell membrane can meet the sterility and safety requirements for clinical applications.

[0167] 3. Storage and Transportation Stability Test Test Method: The packaged cell membrane sheets were placed in a preservation solution (physiological saline) at 4°C and then placed on a simulated transport vibration table (reciprocating shaker, Jiangsu Changzhou Dedu Precision Instruments Co., Ltd.) at an oscillation frequency of 75 times / minute for 24 hours to simulate the logistics and transportation conditions from actual production to clinical use. After the treatment, the cell viability of the cell membrane sheets was retested according to the specific operating procedure in "1. Cell Viability Test" of this test case to evaluate their preservation and transportation stability.

[0168] Test results: After treatment under the above conditions, the cell viability of the cell sheet remained above 90%. Figure 4 B). This demonstrates that the cell membrane sheet of the present invention has good stability, can meet the logistical requirements from the production end to the clinical end, and ensures the effectiveness of its clinical application.

[0169] Test Example 4 This study used a rat in situ periodontal defect model to systematically evaluate the in vivo efficacy and safety of the cell membrane sheet of the present invention in promoting periodontal tissue regeneration.

[0170] 1. Experimental Materials and Grouping All animal experiments strictly followed the requirements of the "Guidelines for the Feeding and Use of Laboratory Animals" (8th edition, National Academy of Sciences Press, 2011) and the ARRIVE 2.0 guidelines (full name: Animal Research: Reporting of In Vivo Experiments Guidelines 2.0), and were approved by the Ethics Committee of the State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, with approval number WCHSIRB-AT-2025-534.

[0171] Twenty-four healthy, 8-week-old male Sprague-Dawley rats were purchased from Chengdu Duosai Experimental Animal Co., Ltd. The rats were housed in an SPF-grade environment with a controlled temperature of 25℃ and humidity of 50%, using a 12-hour light / 12-hour dark cycle. They had free access to food and water and underwent one week of acclimatization before the start of the experiment.

[0172] The experimental animals were randomly divided into two groups of eight animals each, as follows: (1) Control group: only periodontal defects were prepared, and no materials were implanted in the defect area.

[0173] (2) Cell membrane patch group (CS group): Freshly prepared cell membrane patches were implanted at the defect site according to the method of Example 1.

[0174] 2. Experimental Operation 2.1 Establishment of a periodontal defect model Following the methods described in relevant literature, a periodontal defect model was constructed in rats under general anesthesia and aseptic conditions. A mucoperiosteal flap was raised from the buccal side of the right mandibular first molar in rats, and impurities in the alveolar bone, periodontal ligament, and root surface were thoroughly cleaned. A periodontal defect measuring 3mm (length) × 2mm (width) × 1mm (depth) was prepared using a round dental drill. According to the grouping requirements, the defect area in the control group remained empty, while freshly prepared cell membrane sheets were implanted into the defect area in the CS group. After repositioning the tissue flap, interrupted sutures were performed using 6-0 Vicichal sutures (Ethicon, Netherlands) to complete the model construction.

[0175] The following literature was referenced for establishing the periodontal defect model: Wei X, Guo S, Liu Q, Liu L, Huo F, Wu Y, Tian W. Dental Follicle StemCells Promote Periodontal Regeneration through Periodontal-Mediated MacrophageInfiltration and Reprogramming in an Inflammatory Microenvironment. Int J MolSci 2023; 24: 6353 [PMID: 37047322 DOI: 10.3390 / ijms24076353] Yu JL, Yang C, Liu L, Lin A, Guo SJ, Tian WD. Optimal goodmanufacturing practice-compliant production of dental follicle stem cellsheet and its application in Sprague-Dawley rat periodontitis. World J StemCells. 2025 May 26;17(5):104116. doi: 10.4252 / wjsc.v17.i5.104116. PMID: 40503360; PMCID: PMC12149792. 2.2 Postoperative management and sample collection The rats were dynamically monitored for 5 consecutive weeks after surgery to observe their general condition. Five weeks after surgery, the rats were euthanized by intravenous injection of 150 mg / kg sodium pentobarbital, and samples were collected afterwards.

[0176] The collected samples were divided into two categories: (1) Target tissue samples: rat mandibular bone specimens were collected and immediately fixed in 4% paraformaldehyde solution for subsequent Micro-CT scanning and histological testing; (2) Safety assessment samples: organ tissue specimens such as heart, liver, spleen, lung, and kidney were collected simultaneously for histopathological analysis; peripheral blood samples were collected via tail vein at baseline (preoperative), 3 days postoperatively, 1 week postoperatively, and 1 month postoperatively for hematological and biochemical index testing.

[0177] After the study was completed, all transplant materials were recovered and the animal carcasses were disposed of in accordance with the institution’s biosafety and humane treatment protocols.

[0178] 3. Evaluation Methods and Quality Control: 3.1 Three-blind design To minimize bias, a triple-blind design was adopted, with three independent researchers responsible for the surgical procedure, sample collection and storage, and statistical analysis of the experimental data, respectively, to ensure the objectivity of the experimental results.

[0179] 3.2 Micro-computed tomography (micro-CT) evaluation The fixed mandibular bone specimen was fixed in 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 2 days and scanned using a SkyScan 1176 desktop X-ray micro-CT system (SkyScan, Belgium). The scan data were reconstructed and the images were observed. The new bone formation area in the images was located and analyzed, and the bone volume / tissue volume (BV / TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were quantitatively measured.

[0180] 3.3 Histological assessment The fixed mandibular bone specimens were sequentially decalcified, dehydrated with gradient ethanol, and embedded in paraffin. Continuous horizontal sections of the defect area with a thickness of 8 μm were prepared using a microtome (SP1600, Leica, Germany). Hematoxylin-eosin (HE) staining (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and Masson's trichrome staining (Shanghai Baomet Biotechnology Co., Ltd., Shanghai, China) were performed on the stained sections to observe the formation of new bone (NB), collagen deposition, and histological characteristics in the defect area.

[0181] 4. Test Results 4.1 Periodontal regeneration efficacy Three-dimensional (3D) and computed tomographic micro-CT images are attached. Figure 5 As shown: Compared with the control group, the CS group showed a large amount of new bone formation in the periodontal defect area, and the new bone filling effect was significantly better than that of the control group.

[0182] The quantitative analysis results are attached. Figure 6As shown, the CS group had significantly higher bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) than the control group, while the trabecular bone separation (Tb.Sp) was significantly lower in the CS group than in the control group. All of the above indicators were statistically significant compared with the control group (p<0.05).

[0183] HE-stained and Masson's trichrome stained sections are attached. Figure 7 As shown: In the defect area of ​​the CS group, a large amount of new bone (NB) was formed, and the collagen fibers were arranged in a neat and orderly manner. The area of ​​the regenerated area was significantly larger than that of the control group (p<0.0001).

[0184] The quantitative analysis results of the regeneration area are attached. Figure 8 As shown.

[0185] 4.2 Security Results The results of hematological and biochemical indicators are attached. Figure 9 The HE-stained section image is attached. Figure 10 As shown in the figure. The results indicated that no significant systemic toxicity was observed in the CS group rats, and all hematological and biochemical indicators remained stable, providing a reliable safety basis for the further clinical translation and application of this cell sheet.

[0186] Experimental Example 5 This study aims to comprehensively evaluate the in vivo tumorigenic potential and systemic toxicity of the cell membrane of the present invention, further verify its safety for clinical application, and provide reliable experimental evidence for subsequent clinical translation.

[0187] 1. Experimental Materials and Grouping 1.1 Laboratory Animals Using 5-week-old female BALB / c nude mice, whose immunodeficiency characteristics can eliminate the interference of the body's immune response on the experimental results, ensuring the accuracy of the experimental results.

[0188] 1.2 Grouping and Transplantation Materials: The experimental animals were randomly divided into 3 groups. Mice in each group were subcutaneously injected with the corresponding experimental material in their backs. The specific grouping and injection materials are as follows: Positive control: HeLa cells were injected, which are known tumorigenic cells and served as a positive reference for tumorigenicity. Negative control: Injected with sterile saline; served as a negative reference for non-tumorigenicity and non-toxicity. Cell sheet assembly (CS): Injection of fresh cell sheets prepared according to the method of Example 1.

[0189] 2. Experimental Operation All animal experiments were conducted in strict accordance with the Guidelines for the Housing and Use of Laboratory Animals (8th Edition) and the ARRIVE 2.0 guidelines, and were approved by the Ethics Committee of the State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, approval number WCHSIRB-AT-2025-534. Animals were housed in a specific pathogen-free (SPF) environment with strictly controlled environmental conditions: temperature 25°C, humidity 50%, and a 12-hour light / 12-hour dark cycle. Daily care was provided by professionally trained personnel.

[0190] 2.1 Subcutaneous transplantation All procedures were performed under sterile conditions, with designated group materials injected subcutaneously into the back of each mouse.

[0191] 2.2 Observation and Monitoring From week 1 to week 6, the formation of subcutaneous nodules at the injection site was monitored twice a week; after week 6, monitoring was conducted once a week.

[0192] Due to the strong tumorigenicity and rapid growth of HeLa cells, mice in this group were euthanized at week 4; the remaining groups were observed until week 16 for further evaluation of long-term safety before euthanasia. Euthanasia was performed using cervical dislocation under deep anesthesia, with the entire process adhering to the principle of minimizing animal suffering.

[0193] 2.3 Sample Collection and Pathological Analysis Gross examination: After euthanizing the mice, a comprehensive gross examination was performed on the injection site and major organs (including the heart, lungs, liver, spleen, kidneys, brain and local lymph nodes) to assess for the presence of abnormal cell proliferation or metastatic lesions.

[0194] Tissue processing and staining: The collected tissue specimens were immediately fixed with 4% paraformaldehyde, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

[0195] Histopathological analysis: Through microscopic observation, systematically assess whether the transplanted cells induced tumor formation or abnormal tissue proliferation locally or systemically.

[0196] Throughout the experiment, all procedures were performed in accordance with the principle of minimizing animal suffering. After the experiment, the recovery of the transplanted materials and the disposal of the animal carcasses complied with biosafety and ethical standards.

[0197] 3. Experimental Results General observation diagram is attached. Figure 11 As shown in the attached figure, the quantitative results of tumor volume are as follows. Figure 12 As shown: Throughout the observation period, only the positive control group mice developed visible tumors at the injection site. No tumor formation was observed at any time point in either the negative control group or the cell sheet group.

[0198] HE-stained slide images are attached. Figure 13 As shown: The tissue structure at the injection site of the cell sheet group mice was intact and the morphology was normal. No abnormal cell proliferation or infiltration was found. HE staining sections of all major organs (brain, heart, liver, spleen, lungs, and kidneys) showed normal tissue morphology, without any pathological changes or metastatic lesions.

[0199] In summary, the results of this experiment show that the cell membrane of the present invention has no tumorigenic potential in vivo and does not produce any systemic toxicity in experimental animals, and its in vivo application has good safety.

[0200] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a cell membrane sheet rich in extracellular matrix with high mechanical strength, characterized in that, The process includes the following steps: seeding dental follicle mesenchymal stem cells into a culture container and culturing them in a special serum-free cell sheet culture medium until a complete cell sheet forms on the surface of the culture container; gently rinsing the cell sheet with sterile physiological saline, causing the cell sheet to detach and obtaining a cell sheet with an intact surface; The dedicated serum-free cell sheet culture medium is composed of vitamin C injection, insulin-like growth factor-1, serum-free cell culture additive, and MEMα culture medium, wherein the volume ratio of serum-free cell culture additive to MEMα culture medium is 1:10~40; the vitamin C content is 40~60 μg / ml; and the insulin-like growth factor-1 content is 20~40 ng / mL.

2. The method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1, characterized in that, Dental sac mesenchymal stem cells (MSCs) are deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2024384.

3. The method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1 or 2, characterized in that, P2-P4 generation dental follicle mesenchymal stem cells were used for inoculation.

4. The method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1, characterized in that, The cell seeding density is 2000-3000 cells / cm2; after seeding, the culture container is placed in an incubator at 37℃ and 5% CO2 for static culture.

5. A method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1 or 4, characterized in that, The cell seeding density was 2500 cells / cm2.

6. The method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1, characterized in that, During culture, the culture medium is replaced for the first time after the cells adhere to the culture vessel, and fresh serum-free cell membrane medium is used to continue culturing. Fresh serum-free cell membrane medium is replaced every 2-4 days until a complete cell membrane is formed on the surface of the culture vessel.

7. A method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1, characterized in that, The serum-free cell membrane culture medium contains serum-free cell culture additives and MEMα medium in a volume ratio of 1:20; vitamin C content is 50 μg / ml; and insulin-like growth factor-1 content is 20~40 ng / mL.

8. The method for preparing a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 1, characterized in that, During culture, slight wrinkles were observed at the edges of the cell membrane, indicating that the cell membrane had formed. The training period is 10 months and 1 day.

9. A cell membrane sheet rich in extracellular matrix with high mechanical strength, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The application of a high-mechanical-strength cell membrane sheet rich in extracellular matrix according to claim 9, characterized in that, Application in the preparation of medicines for the prevention and / or treatment of periodontal diseases.

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