Function-enhanced mesenchymal stem cell preparation obtained by utilizing arthritis effusion and hypoxia pretreatment of mesenchymal stem cells as well as preparation method and application of function-enhanced mesenchymal stem cell preparation

Abstract

The invention discloses a function-enhanced mesenchymal stem cell preparation obtained by utilizing arthritis effusion and hypoxia pretreatment of mesenchymal stem cells as well as a preparation method and application of the function-enhanced mesenchymal stem cell preparation, and relates to the technical field of biological medicines. The preparation method of the function-enhanced mesenchymal stem cell preparation obtained by utilizing arthritis effusion and hypoxia pretreatment of mesenchymal stem cells comprises the following steps: S1, obtaining mesenchymal stem cells and performing hypoxia amplification passage; s2, pretreatment of arthritis effusion; S3, pre-stimulation of the arthritis effusion of the mesenchymal stem cells to obtain function-enhanced mesenchymal stem cells; s4, index detection and screening; s5, preparing a function-enhanced mesenchymal stem cell preparation; optimized low-oxygen pretreatment is adopted, so that the cell activity is improved; the mesenchymal stem cells subjected to joint pretreatment of arthritis effusion and hypoxia enhance expression and secretion of anti-inflammatory factors and immunomodulatory molecules in the cells; the pre-stimulated mesenchymal stem cells show a stronger immunosuppression function in vitro.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a functional enhanced mesenchymal stem cell preparation obtained by using arthritis effusion combined with hypoxic preconditioning of mesenchymal stem cells, and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal Stem Cell (MSC) has made some progress in the field of cell therapy drugs for arthritis, and some products have entered the clinical and market. However, there are still some limiting factors in the existing MSC related products and preparation process, including: 1. Insufficient stability of clinical efficacy of MSC cell therapy: Most of the commercially available osteoarthritis MSC cell products directly use MSC cultured and expanded in vitro, without specific functional enhancement treatment, resulting in unstable cell therapy effect. Literature and clinical practice show that MSC without specific pretreatment is easily affected by the local inflammatory microenvironment of the joint after transplantation, resulting in unstable function and significant individual differences in efficacy, and some patients have poor efficacy, limiting the predictability and reliability of clinical application. 2. Existing products are not optimized for the local inflammatory microenvironment of the joint: The existing method of MSC treating osteoarthritis generally ignores the negative impact of inflammatory factors (such as IL-1β, TNF-α, IFN-γ) on MSC function. Studies have shown that MSC without pre-adaptation may rapidly lose function, reduce viability, and even accelerate death after entering the inflammatory joint, and cannot effectively inhibit the inflammatory response and repair cartilage damage in the joint for a long time. 3. Not effectively using the patient's own arthritic effusion resources: Patients with knee arthritis in the III or IV stage often have a large amount of arthritic effusion, and the traditional treatment method is mostly direct disposal or simple drainage, without using the potential enabling substances in the effusion. 4. Lack of hypoxic environment pretreatment leading to decreased MSC cell activity: The current MSC preparation process generally uses 21% O2 normoxic culture conditions, which is significantly different from the natural hypoxic microenvironment (2%-8% O2) in vivo. Studies have shown that normoxic culture can accelerate MSC aging, reduce their proliferation capacity, differentiation potential and therapeutic activity, making it difficult to meet the demand for high-quality and high-activity MSC in clinical practice. 5. The existing method lacks optimized effusion stimulation concentration and processing conditions: Some studies suggest that moderate inflammatory stimulation can enhance MSC function. However, for the complex biological system of patient's arthritic effusion, there is still a lack of systematic optimization research on stimulation concentration and action time. Direct use of high concentration or long time stimulation in clinical or experimental practice may cause MSC overactivation or induce cytotoxicity, thereby weakening its therapeutic benefit. 6. The preparation process lacks standardization and stability control: The existing MSC preparation methods are diverse, but lack clear and standardized preparation processes and quality control standards. Especially in terms of pre-stimulation and hypoxic culture, the related process parameters are not clear and standardized, resulting in uneven quality of MSC products, making it difficult to repeat and guarantee the clinical efficacy. Therefore, based on the current situation, it is urgent to develop a functional enhanced mesenchymal stem cell preparation obtained by using arthritic effusion combined with hypoxic pretreatment of mesenchymal stem cells, as well as its preparation method and application, to meet the actual application. SUMMARY

[0003] In view of the deficiencies of the prior art described above, the main purpose of the present application is to provide a functional enhanced mesenchymal stem cell preparation using arthritis effusion combined with hypoxic pretreatment and a preparation method and application thereof. The method significantly improves the biological activity and therapeutic efficacy of MSCs by introducing an optimized hypoxic pretreatment strategy. Specifically, the present application uses a hypoxic condition during MSC culture, allowing the cells to be in a near-physiological hypoxic state in vivo, thereby maintaining their high proliferation capacity and original biological properties. Hypoxic culture can effectively increase the proliferation rate of MSCs, delay cell aging, enhance differentiation potential and therapeutic activity. On this basis, the combination of arthritis effusion and hypoxic pretreatment can further induce MSCs to up-regulate the expression and secretion of anti-inflammatory factors such as IL-10, IDO-1, PGE2 and immunomodulatory molecules. MSCs pretreated by this combination show stronger immunosuppressive ability in vitro and can effectively inhibit inflammatory response and protect joint cartilage tissue. To achieve the above purpose, the present application adopts the following technical solutions:

[0004] A preparation method for obtaining a functional enhanced mesenchymal stem cell preparation by pretreating mesenchymal stem cells with arthritis effusion combined with hypoxia, comprising the following steps:

[0005] S1, obtaining mesenchymal stem cells and hypoxic expansion and passage;

[0006] S2, pretreatment of arthritis effusion:

[0007] S3, obtaining functional enhanced mesenchymal stem cells by arthritis effusion pre-stimulation of mesenchymal stem cells;

[0008] S4, index detection and screening;

[0009] S5, preparation of functional enhanced mesenchymal stem cell preparation.

[0010] As a preferred scheme: the oxygen concentration of hypoxic expansion and passage in step S1 is 2%-8%.

[0011] As a preferred scheme: the arthritis effusion pre-stimulation concentration in step S3 is 5%-60%, and the pre-stimulation treatment time is 12-72 hours.

[0012] As a preferred scheme: the arthritis effusion pre-stimulation concentration in step S3 is 30%, and the pre-stimulation treatment time is 48 hours.

[0013] As a preferred scheme: step S1 is specifically: obtaining mesenchymal stem cells, expanding in a hypoxic incubator from P0 generation, using serum-free complete culture medium, and passing mesenchymal stem cells to P3 generation after culturing to 80%-90% confluence for subsequent pre-stimulation.

[0014] As a preferred solution: the step S2 is specifically: collecting the arthritis effusion, centrifuging to remove the cell and tissue block part, filtering the supernatant to remove bacteria, pre-freezing the supernatant and then vacuum freeze-drying to obtain the effusion freeze-dried powder, which is sealed and stored for later use.

[0015] As a preferred solution: the step S3 is specifically: redissolving the treated arthritis effusion using mesenchymal stem cell complete culture medium to prepare a solution; adding the mesenchymal stem cells passed to the P3 generation to the solution for pre-stimulation, after the pre-stimulation is completed, removing the culture medium added with the arthritis effusion solution, washing the mesenchymal stem cells with PBS, and then adding no arthritis effusion culture medium for continuous culture to the P4 generation to obtain the functional enhanced mesenchymal stem cells.

[0016] As a preferred solution: the index detection screening in the step S4 includes phenotype detection screening, function detection screening and safety detection screening, the phenotype detection screening is that the expression rate of CD73 + , CD90 + , CD105 + of the functional enhanced mesenchymal stem cells is greater than or equal to 95%, and the expression rate of CD34 - , CD45 - is less than or equal to 2%; the function detection screening is that the T cell proliferation inhibition rate of the functional enhanced mesenchymal stem cells is greater than or equal to 50%, and the secretion levels of IL-10 and PGE2 are at least 2 times higher than those of the untreated mesenchymal stem cells; and the safety detection screening is that the functional enhanced mesenchymal stem cells are free of bacterial, fungal and mycoplasma contamination, and the chromosome karyotype is normal.

[0017] As a preferred solution: the step S5 is specifically: adjusting the concentration of the functional enhanced mesenchymal stem cells, the concentration is 0.5-5x10 6 cells / joint / time, and resuspending the cells in a carrier to prepare a functional enhanced mesenchymal stem cell preparation capable of injection.

[0018] A functional enhanced mesenchymal stem cell preparation prepared by the preparation method.

[0019] The functional enhanced mesenchymal stem cell preparation is used for the preparation of an arthritis drug.

[0020] As a preferred solution: the administration mode of the arthritis drug includes intra-articular injection, arthroscopic injection and intravenous administration.

[0021] Compared with the prior art, the present application has obvious advantages and beneficial effects, specifically:

[0022] First, by adopting an optimized low-oxygen pretreatment technology, the cell activity is improved: the mesenchymal stem cells are cultured in a low-oxygen condition in the present application, so that the mesenchymal stem cells maintain the proliferation ability and original state in a physiological low-oxygen environment close to the body; the low-oxygen culture can significantly improve the mesenchymal stem cell proliferation rate, delay cell aging, improve cell differentiation potential and therapeutic activity;

[0023] Second, the functional enhanced mesenchymal stem cells with stronger immune regulation function are obtained: the mesenchymal stem cells pretreated by the arthritis effusion and the low oxygen are significantly enhanced in the expression and secretion of anti-inflammatory factors and immune regulation molecules such as IL-10, IDO-1 and PGE2 in the cells; compared with the mesenchymal stem cells without pretreatment, the pretreated mesenchymal stem cells exhibit stronger immune inhibition function in vitro, which can effectively inhibit the inflammatory reaction and protect the cartilage tissue;

[0024] Third, the concentration and treatment time of the effusion pretreatment are optimized: the optimal stimulation concentration and stimulation time are determined in the present application; the optimization condition effectively avoids the problems of excessive activation or insufficient stimulation of cell function, and ensures the stability and safety of the MSC treatment function;

[0025] Fourth, the patient's own arthritis effusion resources are innovatively utilized: the arthritis effusion of the patient with late-stage knee osteoarthritis is ingeniously utilized in the present application, which is freeze-dried and prepared into a standardized powder preparation, and then used for MSC pretreatment; this method fully utilizes the inflammatory factors in the effusion for pretreatment, avoids resource waste, reduces the treatment cost, and improves the functional characteristics of MSCs;

[0026] Fifth, the animal model verifies that the therapeutic effect is significantly enhanced: the knee osteoarthritis rat animal model is used for pharmacodynamic verification in the present application, and the results show that the pretreated mesenchymal stem cells exhibit obvious drug advantages in the animal body; the experiments show that the pretreated mesenchymal stem cells are obviously superior to the traditional mesenchymal stem cells in cartilage protection, inflammation inhibition and joint function improvement, which confirms that they have significant clinical treatment potential.

[0027] In order to more clearly illustrate the structural features and effects of the present application, specific embodiments will be described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The flow chart of the preparation method of the functional enhanced mesenchymal stem cell preparation of the present application by using arthritis effusion combined with low-oxygen pretreatment of mesenchymal stem cells;

[0029] Figure 2 The component analysis comparison chart of arthritis effusion of different patients with knee osteoarthritis in Example 2 of the present application;

[0030] Figure 3This is a schematic diagram illustrating the process of culturing and expanding MSCs to generation P3 under hypoxic conditions in Example 1 of the present invention.

[0031] Figure 4 This is an image showing the epimorphological morphology of P3 generation MSC cells in Example 1 of the present invention.

[0032] Figure 5 This is a schematic diagram showing the comparison of MSC viability in each group after pre-stimulation of fluid accumulation in Example 3 of the present invention;

[0033] Figure 6 These are morphological images of MSCs in each group after pre-stimulation of fluid accumulation in Example 3 of the present invention;

[0034] Figure 7 This is a graph showing the comparison of MSC proliferation capacity among different groups after pre-stimulation with fluid accumulation in Example 3 of the present invention.

[0035] Figure 8 This is a graph showing the statistical results of the inhibition rate of lymphocyte proliferation by MSCs in each group after pre-stimulation of effusion in Example 4 of the present invention;

[0036] Figure 9 This is a graph showing the changes in the levels of growth factors and inflammatory factors secreted by MSCs in each group after pre-stimulation of effusion in Example 5 of the present invention.

[0037] Figure 10 This is a schematic diagram of the experimental design for pre-stimulated MSC treatment of a rat knee osteoarthritis model in Example 6 of the present invention;

[0038] Figure 11 The above is a graph showing the OARSI cartilage degeneration scores of the knee joints of rats before and after treatment in each group in Example 6 of this invention.

[0039] Figure 12 This is a schematic diagram of the experimental design for pre-stimulated MSC treatment of a rat knee osteoarthritis model in Example 6 of the present invention;

[0040] Figure 13 These are the movement trajectories of rats in each group before and after treatment in the open field test in Example 6 of the present invention. Detailed Implementation

[0041] The present invention is as follows Figures 1 to 13 As shown, a method for preparing a functionally enhanced mesenchymal stem cell preparation by utilizing arthritis effusion combined with hypoxia pretreatment of mesenchymal stem cells includes the following steps:

[0042] S1. Acquisition and hypoxic expansion passage of mesenchymal stem cells.

[0043] S2. Pretreatment of arthritis effusion:

[0044] S3, arthritis effusion pre-stimulation of mesenchymal stem cells to obtain function-enhanced mesenchymal stem cells;

[0045] S4, index detection screening;

[0046] S5, preparation of function-enhanced mesenchymal stem cell preparation.

[0047] The oxygen concentration of the low-oxygen expansion passage in this step S1 is 2%-8%.

[0048] The arthritis effusion pre-stimulation concentration in this step S3 is 5%-60%, and the pre-stimulation treatment time is 12-72 hours.

[0049] The arthritis effusion pre-stimulation concentration in this step S3 is 30%, and the pre-stimulation treatment time is 48 hours.

[0050] System optimization of arthritis effusion pre-stimulation concentration and treatment time: the present application determines the experimental data of the influence of different concentrations of arthritis effusion on the function of mesenchymal stem cells, and determines the appropriate stimulation concentration. This optimization condition effectively avoids the problem of excessive activation or insufficient stimulation of cell function, and ensures the stability and safety of mesenchymal stem cells as drugs for treating arthritis.

[0051] This step S1 is specifically: obtaining mesenchymal stem cells, expanding in a low-oxygen incubator from P0 generation, using serum-free complete culture medium, and culturing mesenchymal stem cells to 80%-90% confluence before passing to P3 generation for subsequent pre-stimulation.

[0052] This step S2 is specifically: collecting arthritis effusion, centrifuging to remove cellular components, filtering and sterilizing the supernatant, vacuum freeze-drying the pre-frozen supernatant to obtain effusion freeze-dried powder, and sealing and storing for standby use.

[0053] Using arthritis effusion of arthritis patients, the arthritis effusion is preserved by freeze-drying and prepared into a standardized powder preparation, which is then used for MSC pre-stimulation. This method fully utilizes the inflammatory factors in the effusion for pre-stimulation, avoids waste of resources, reduces treatment costs, and improves the functional properties of MSCs.

[0054] This step S3 is specifically: resolubilizing the treated arthritis effusion using mesenchymal stem cell complete culture medium to prepare a solution; adding the mesenchymal stem cells passed to P3 generation to the above-mentioned solution for pre-stimulation, after pre-stimulation, removing the arthritis effusion solution, washing the mesenchymal stem cells with PBS, and then adding arthritis effusion-free medium for further culture to P4 generation to obtain function-enhanced mesenchymal stem cells.

[0055] The index detection screening in this step S4 includes phenotype detection screening, function detection screening and safety detection screening. The phenotype detection screening is: CD73+ CD90 + CD105 + Expression rate ≥95%, CD34 - CD45 - Expression rate ≤2%; the functional screening criteria are: T cell proliferation inhibition rate of functionally enhanced mesenchymal stem cells ≥50%, and IL-10 and PGE2 secretion levels at least 2 times higher than those of untreated mesenchymal stem cells; the safety screening criteria are: functionally enhanced mesenchymal stem cells free from bacterial, fungal and mycoplasma contamination, and with normal chromosome karyotype.

[0056] Step S5 specifically involves adjusting the concentration of functionally enhanced mesenchymal stem cells to 0.5-5 × 10⁻⁵. 6 Cells / joints / subunits are resuspended in a carrier to create an injectable, functionally enhanced mesenchymal stem cell formulation. This carrier may contain hyaluronic acid, chitosan, PRP, alginate microbeads, decellularized matrix gel, or other thermosensitive gels, providing long-lasting sustained-release properties.

[0057] A functionally enhanced mesenchymal stem cell preparation obtained by a preparation method.

[0058] Application of a functionally enhanced mesenchymal stem cell preparation in the preparation of drugs for treating arthritis.

[0059] This arthritis medication can be administered via intra-articular injection, arthroscopic injection, and intravenous administration. Intravenous injection (MSCs can reach the affected joint area via homing mechanisms), or local tissue perfusion (such as periarticular injection); low-dose single injection (e.g., 1×10⁻⁶) 5 Although the cell count is low, it can still partially play an immunomodulatory role; although PBS or regular saline is not a sustained-release carrier, it can still achieve basic cell delivery.

[0060] Mesenchymal stem cells (MSCs): MSCs derived from bone marrow, adipose tissue, or umbilical cord, as well as dental pulp MSCs, synovial MSCs, amniotic membrane MSCs, menstrual blood MSCs, and periosteal MSCs, can be used. Although these cells have slightly lower activity, they still exhibit certain immunomodulatory and cartilage repair capabilities, making them suitable as alternative cell sources for the technical route of this invention. Bone marrow MSCs are preferred, and after strict selection and passage control, they are expanded to the P3–P4 generation. The final formulation contains 0.5–1 × 10⁻⁶ cells per dose. 6 MSCs serve as seed cells for treating arthritis. MSCs are the "primary active ingredient" in drugs that directly repair cartilage and inhibit inflammation.

[0061] The arthritis effusion used for pre-stimulation can use knee osteoarthritis effusion, rheumatoid arthritis, psoriatic arthritis and other joint disease effusion of patients, which is rich in inflammatory factors and can effectively pre-stimulate MSC; or use serum or plasma of knee osteoarthritis patients, and obtain inflammatory factor-rich liquid by centrifugation, ultrafiltration, dialysis or chromatography method, as a substitute for effusion pre-stimulation agent; or artificial arthritis effusion can be used, which contains hyaluronic acid, IL-1β, TNF-α, IFN-γ, IL-6, MMP-3 / 13 key inflammatory factors and matrix degradation products, which can replace natural effusion to achieve pre-stimulation effect.

[0062] After the priority hypoxia pre-culture, the arthritis effusion stimulation is carried out again, or the hypoxia culture and the effusion pre-stimulation are carried out synchronously (that is, the MSC is added into the arthritis effusion under the hypoxia environment).

[0063] In view of the deficiencies and defects of the existing mesenchymal stem cell treatment method for arthritis, the present application focuses on solving the following technical problems:

[0064] 1) The problem of unstable drug efficacy of MSC for treating osteoarthritis: The conventional MSC is not effectively pretreated, and is prone to functional degradation or cell activity reduction after entering the inflammatory microenvironment of the patient, so that the treatment effect is uncertain.

[0065] 2) The problem of not fully utilizing the patient's own arthritis effusion resources in the prior art: The existing therapy ignores the specific inflammatory microenvironment in the joint cavity of the patient, and does not effectively use the biological active substances of the effusion for cell function regulation, resulting in waste of resources.

[0066] 3) The problem of weakening of cell function caused by conventional normoxic environment culture of MSC: The conventional 21% O2 culture environment is not conducive to maintaining the original active state of MSC in vivo, which leads to premature aging of cells, reduction of differentiation potential and proliferation activity.

[0067] 4) The problem of lack of optimization and standardization of effusion stimulation concentration and stimulation conditions: The existing technology is not clear about the specific concentration and time parameters of the effusion stimulation of MSC, which is prone to the problems of insufficient stimulation or excessive stimulation.

[0068] 5) The problem of lack of standardization of existing MSC preparation method, which is difficult to stably control the quality of cells: Lack of unified process parameters and quality control standards leads to inconsistent product quality.

[0069] Example 1: Hypoxic culture expansion and morphological observation of MSC

[0070] Experimental purpose: To prepare high-quality mesenchymal stem cells (MSCs) for subsequent arthritis effusion pre-stimulation. By culturing MSCs under hypoxic conditions, their proliferative activity and stemness are maintained to avoid cell aging during conventional in vitro expansion. The hypoxic environment is closer to the bone marrow microenvironment, which can improve the proliferation and differentiation potential of MSCs.

[0071] Methods and steps:

[0072] 1. MSC isolation and culture: Commercial umbilical cords were used to obtain P0 seed cells, which were then expanded and cultured in a hypoxic incubator with 2-8% O2 in serum-free complete medium. The cells were inoculated in appropriate density in adherent bottles in a 37°C, 5% CO2 environment, and the medium was changed every 2-3 days.

[0073] 2. Subculture expansion to P3 generation: Cells were subcultured when they reached 80-90% confluence, and were continuously subcultured to the 3rd generation (P3), with the morphology and proliferation of cells at each generation being recorded.

[0074] 3. Morphological observation: Phase contrast microscopy was used to observe the morphology of P3 MSCs, and cell uniformity and signs of aging and apoptosis were evaluated.

[0075] Hypoxic culture environment: The cells were maintained at an oxygen concentration of 2-8% O2 during the expansion phase to simulate the bone marrow microenvironment, maintain MSC stemness, and enhance their proliferative capacity.

[0076] Culture medium and auxiliary components: A full-component MSC medium was used as the basis (mesenchymal stem cell serum-free basal medium + 5% platelet lysate + appropriate growth factors), which was used for both hypoxic expansion and recovery culture after effusion pre-stimulation.

[0077] Hypoxic expansion: MSCs were subcultured from P0 to P3 under 2-8% O2, with a single subculture density of 4x10 3 -6x10 3 cells / cm 2 , subcultured every 3-4 days. Experimental data showed that the doubling time of MSCs under hypoxic conditions was shortened from 50 hours under normoxic conditions to 36 hours, with a cumulative expansion of more than 200 times (normoxic control 80 times), ensuring an adequate cell source for subsequent experiments and treatment.

[0078] Results and analysis: MSCs were successfully expanded to P3 under hypoxic conditions (2-8% O2). Figure 3 The flowchart shows the subculture expansion of MSCs in a hypoxic culture environment, including the hypoxic culture device and schematic diagram of the state of cells at each generation. Hypoxic culture helps to inhibit premature aging of MSCs and improve their proliferative capacity. Figure 4Phase contrast micrographs (4x, 10x) of P3 MSCs under hypoxic condition: cells show uniform spindle shape and good adhesion, without obvious signs of cell deformation or aging. The key step of this example is to use hypoxic pre-culture of MSCs, which helps to keep the cells in a "young" state and improve the quality of cells in subsequent experiments.

[0079] Example 2: Composition analysis of knee osteoarthritis synovial fluid

[0080] Objective: To analyze the composition of knee osteoarthritis synovial fluid, and evaluate the content of inflammatory factors and cartilage degradation products. By understanding the biochemical characteristics of the synovial fluid, we can provide a basis for setting the concentration gradient of the synovial fluid for MSC pre-stimulation and judging the pre-stimulation effect.

[0081] Methods and procedures:

[0082] Collection of synovial fluid samples: Collect synovial fluid from several patients with knee osteoarthritis, and record the basic information of the patients and the Kellgren-Lawrence classification.

[0083] Composition detection: Use multi-factor analysis and biochemical methods to detect the levels of cytokines and matrix molecules in the synovial fluid.

[0084] Patient's synovial fluid lyophilized powder: Synovial fluid from patients with knee osteoarthritis in stages III-IV is collected, centrifuged to remove cells and impurities, then pre-frozen at -80°C and vacuum freeze-dried into powder, which is re-dissolved in proportion before use. As natural factors for cell pre-stimulation, it is rich in inflammatory factors such as IL-1β, TNF-α, IL-6 and some cartilage matrix degradation products, which can trigger the immune regulation function of MSCs.

[0085] Results comparison: Compare and analyze the composition of synovial fluid from different patients, and summarize the common characteristics and differences.

[0086] Results and analysis: Figure 2 The composition analysis of synovial fluid from different patients with knee osteoarthritis is shown. Elevated levels of inflammatory factors (such as IL-6, IL-8, TNF-α, IL-1β, etc.) were detected in the synovial fluid of each patient. These pro-inflammatory factors are present in high concentrations in the synovial fluid, reflecting the chronic inflammatory microenvironment of the OA joint. In addition, the content of cartilage degradation products (such as sulfated glycosaminoglycan sGAG, proteoglycan fragments) is also high, indicating that the cartilage matrix is continuously being broken down. There are differences in the levels of certain components among different patients, but the overall trend shows that OA synovial fluid is an environment rich in inflammatory mediators and matrix fragments. These findings provide a basis for subsequent MSC pre-stimulation: using synovial fluid containing these inflammatory factors can "activate" the immune regulation function of MSCs, and the concentration of synovial fluid needs to be controlled to avoid cytotoxicity caused by high concentration.

[0087] Example 3: Effect of arthritic effusion pre-stimulation on MSC survival and proliferation (concentration gradient experiment)

[0088] Objective of the experiment: To explore the effect of different concentrations of arthritic effusion pre-stimulation on MSC survival rate and proliferative capacity, and to determine the appropriate pre-stimulation concentration range. It is hypothesized that moderate concentration of effusion stimulation can promote MSC activity, while too high concentration may have inhibitory effect on cells.

[0089] Methods and procedures:

[0090] Pre-stimulation treatment: P3 MSCs were divided into groups: the hypoxia control group was added with medium without effusion, and the experimental groups were added with low concentration (e.g. 10%), moderate concentration (e.g. 30%) and high concentration (e.g. 50%) of patient arthritic effusion for pre-stimulation (the rest was medium), i.e. the pre-stimulation experimental groups were divided into hypoxia + 10% effusion, hypoxia + 30% effusion, and hypoxia + 50% effusion groups, and cultured for another 48 hours under hypoxic conditions.

[0091] Cell survival / viability assay: After 48 hours of pre-stimulation, CCK-8 or MTT method was used to detect the survival rate / metabolic activity of MSCs in each group, and the percentage change was calculated relative to the unstimulated control group.

[0092] Cell morphology and number observation: Record the morphological changes of cells in each group, and count the total number of cells to compare the increase or decrease. Pay special attention to whether high concentration of effusion causes abnormal cell morphology or reduction in cell number.

[0093] Subculture proliferation assay: MSCs in each group after pre-stimulation were subcultured to P4, inoculated with equal amount of cells, and cultured for several days (e.g. 120 hours), and the proliferation rate and doubling time of P4 cells in each group were evaluated by cell counting or growth curve.

[0094] Results and analysis: Different concentrations of arthritic effusion pre-stimulation showed concentration-dependent effect on MSC survival and proliferation. Figure 5 The comparison results of MSC survival rate / viability after 48 hours of pre-stimulation were summarized: there was no significant difference in survival rate between the hypoxia + 10% effusion group and the hypoxia control group, the hypoxia + 30% effusion group had a slight increase, while the MSC survival rate in the hypoxia + 50% effusion group decreased relatively, showing that high-intensity inflammatory stimulation had a certain inhibitory effect on cells. Figure 6 The morphology and proliferation of MSCs before and after pre-stimulation were compared: microscopic photographs showed that the morphology of MSCs stimulated by hypoxia + 30% effusion was similar to that of the hypoxia control group, and the cells still grew uniformly adherent; while the cell morphology in the hypoxia + 50% effusion group showed slight stress changes, and the total number of cells had a slight decrease within 48 hours, suggesting that over-stimulation might cause some cells to stop proliferating or die. Further, Figure 7The proliferation ability of P4 MSCs after different pre-stimulation treatments is shown: after equal inoculation of P4 cells in each group, the cell growth curve of the low oxygen + 30% effusion group is steep, and the cell number is multiplied more than the control group and the high concentration group within 120 hours; on the contrary, the proliferation rate of the low oxygen + 50% effusion group is the slowest. As can be seen, moderate arthritis effusion stimulation (such as 30% concentration) can promote the proliferation activity of MSCs, while too low stimulation has no obvious effect, and too high stimulation may be harmful. This example highlights the importance of the pre-stimulation concentration gradient: selecting an appropriate inflammatory stimulation intensity can optimize MSC function, and avoid cell damage caused by excessive stimulation.

[0095] Arthritis effusion pre-stimulation: P3 MSCs were used as seed cells, and the reconstituted arthritis effusion was added at a volume fraction of 10%, 30%, and 50% for gradient experiments, and 30% was finally determined as the optimal stimulation concentration. The effusion was incubated with MSCs for 48 hours, after which the effusion was removed and fresh culture medium was used for continuous culture to P4. The experiment showed that the MSCs in the low oxygen + 30% effusion group had a proliferation rate of 4.5 times within 72 hours, which was significantly higher than that of the low oxygen control group (3.5 times); the low oxygen + 50% effusion group showed a slight decrease in activity.

[0096] Example 4: Detection of the immunosuppressive function of pre-stimulated MSCs (mixed lymphocyte reaction)

[0097] Purpose of the experiment: To verify whether arthritis effusion pre-stimulation enhances the immunosuppressive function of MSCs. The mixed lymphocyte reaction (MLR) was used to evaluate the inhibitory ability of pre-stimulated MSCs on T lymphocyte proliferation, and compared with unstimulated MSCs. Inflammatory factor pre-activation is expected to enhance the immunosuppressive effect of MSCs.

[0098] Methods and steps:

[0099] MSC preparation: P4 MSCs treated by different methods (low oxygen control group vs. low oxygen + appropriate concentration effusion pre-stimulation group, such as 30%) were selected and counted after washing with PBS.

[0100] Establishment of MLR system: Allogeneic donor peripheral blood mononuclear cells (PBMCs) were extracted and stimulated with phytohemagglutinin to proliferate T cells, which were co-cultured with MSCs of different groups (such as MSC:T cell ratio = 1:5 or 1:10). The culture with only T cells without MSCs was set as the positive proliferation control.

[0101] Proliferation inhibition determination: After several days of culture, 3 H-thymine incorporation method or CFSE staining flow cytometry was used to determine the level of T cell proliferation. The inhibition rate of MSCs on T cell proliferation was calculated = [(positive control proliferation value - co-culture group proliferation value) / positive control proliferation value] x 100%.

[0102] Data comparison: compare the difference in inhibition rate between unstimulated MSCs and effusion pre-stimulated MSCs, and test for significance using statistical methods.

[0103] Results and analysis: effusion pre-stimulated MSCs exhibit stronger immunosuppressive activity. Figure 8 A column chart showing the inhibition rate of T cell proliferation in each group of MSCs in the mixed lymphocyte reaction: unstimulated MSCs produced a certain degree of inhibition on T cell proliferation, while arthritis effusion pre-stimulated MSCs significantly enhanced the inhibitory effect. Among them, the low oxygen + 30% effusion group had the highest inhibition rate, which was several percentage points higher than the low oxygen control group in inhibiting T cell proliferation, and the difference was statistically significant. After MSCs were "pre-stimulated" in an inflammatory environment, a series of immune suppressor molecules were up-regulated, such as IDO enzyme and PGE2, thereby more effectively inhibiting the proliferation of T cells. The key to this example is immune function detection: the results prove that moderate concentration of arthritis effusion pre-stimulation can "activate" the immunosuppressive potential of MSCs, making them more effective in inhibiting T cell proliferation in MLR, providing a more powerful cellular functional basis for MSC treatment of inflammatory diseases.

[0104] Example 5: Pre-stimulation of MSC immunomodulatory factor secretion analysis

[0105] Purpose of the experiment: to explore how arthritis effusion pre-stimulation changes the level of immune modulatory factors secreted by MSCs, in order to reveal the mechanism of pre-stimulation enhancing the immune function of MSCs. Detect the key anti-inflammatory factors (such as IL-10, PGE2) in the MSC culture supernatant and the IDO enzyme activity or expression in the MSC cells, and compare the differences between pre-stimulation and unstimulation.

[0106] Methods and steps:

[0107] Supernatant collection: re-add fresh culture medium to unstimulated MSCs and MSCs pre-stimulated with different concentrations of effusion for 48 hours, culture for a period of time (such as 48 hours), collect the culture supernatant of each group and centrifuge to remove cells.

[0108] Cell lysis: collect the corresponding MSC cell samples in parallel, use lysis method to obtain intracellular proteins or extract RNA for subsequent IDO detection.

[0109] Factor determination: use ELISA kit to determine the concentration of interleukin-10 (IL-10) and prostaglandin E2 (PGE2) in each group of supernatant. For IDO, colorimetric method is used to detect its enzyme activity or qPCR to detect the mRNA expression level of IDO gene.

[0110] Result analysis: compare the relative changes of IL-10, PGE2 concentration and IDO activity / expression between pre-stimulation group and hypoxia control group, and perform statistical analysis.

[0111] Results and analysis: pre-stimulation of arthritis effusion significantly increased the level of MSC secretion of immunomodulatory molecules. Figure 9 The levels of major anti-inflammatory factors secreted by pre-stimulated MSCs were summarized: it can be seen that the contents of IL-10 and PGE2 in the supernatant of the pre-stimulation group (especially the low oxygen + 30% effusion group) were significantly higher than those of the low oxygen control group, both of which were several times higher than those of the control group, and the difference was statistically significant. In addition, the IDO enzyme activity and / or mRNA expression in the MSCs of the pre-stimulation group were also significantly higher than those of the low oxygen control group. For example, the IDO activity of the MSCs in the low oxygen + 30% effusion group was several times higher than that of the low oxygen control group, which means that the pre-stimulated MSCs have stronger tryptophan metabolism and immunosuppressive capacity. These results are consistent with the literature reports that "a series of immunomodulatory molecules (such as PGE2, IL-10, NO, etc.) are up-regulated in MSCs after being stimulated by inflammatory factors". Therefore, this embodiment confirms the mechanism of enhanced immune function of pre-stimulated MSCs at the molecular level: the moderate inflammatory environment stimulates the up-regulation of MSC secretion of anti-inflammatory factors and immunity, so that the MSCs have stronger immunomodulatory effect in the inflammatory environment.

[0112] Example 6: Effect verification of pre-stimulated MSCs in treating knee arthritis animal models

[0113] Purpose of the experiment: to evaluate the effect of MSCs pre-stimulated by arthritis effusion in treating osteoarthritis (OA) in vivo. By comparing the treatment results of pre-stimulated MSCs (including normoxia + effusion group, hypoxia + effusion group), unstimulated MSCs (normoxia group, hypoxia group) and placebo control (PBS group) in rat knee arthritis model, the effect of pre-stimulation strategy on cartilage protection and joint function is verified from the aspects of histology and functionality.

[0114] Methods and steps:

[0115] Establishment of OA animal model: monosodium iodoacetate (MIA) intra-articular injection was used to induce rat knee osteoarthritis model. Specifically, a certain dose of MIA (such as 2.5 mg) was injected into the right knee joint cavity of the rat to cause cartilage cell damage and joint degeneration. The model was observed for 1-2 weeks to ensure the formation of cartilage lesions.

[0116] Grouping and treatment: the model rats were randomly divided into 6 groups (n≥10 for each group):

[0117] ① Sham group: only joint puncture and injection of PBS of the same volume were performed, without injection of MIA, as a normal control;

[0118] ②PBS group: After MIA modeling, an equal volume of PBS was injected into the joint as a placebo control;

[0119] ③ Normoacid group: After MIA modeling, an equal volume of conventionally cultured MSC suspension was injected into the joint;

[0120] ④ Hypoxia group: After MIA modeling, an equal volume of hypoxic cultured MSC suspension was injected into the joint.

[0121] ⑤ Normoxy + Effusion Group: After MIA modeling, MSC suspension cultured in normoxic conditions and pre-stimulated with 30% effusion was injected into the joint;

[0122] ⑥ Hypoxia + Effusion Group: After MIA modeling, MSC suspension cultured under hypoxia and pre-stimulated with 30% effusion was injected into the joint.

[0123] The first cell / control drug is administered one week after modeling, and subsequent injections can be given multiple times according to the protocol (e.g., once every 2 weeks, for a total of 2–3 times) to evaluate the therapeutic effect of MSCs under different treatment conditions.

[0124] Animal treatment dose: 0.5–5 × 10⁻⁵ after adjusting the final concentration of P4 generation MSCs. 6 One cell / joint is used for intra-articular injection.

[0125] Histological evaluation: 4–6 weeks after the final treatment, rats in each group were sacrificed, and right knee joint specimens were collected for macroscopic and microscopic morphological evaluation. First, macroscopic observation was performed, recording the smoothness, continuity, and defects of the cartilage surface. The degree of cartilage degeneration was quantitatively scored according to the OARSI cartilage damage scoring system. The results of macroscopic and microscopic evaluations were combined to determine the differences in cartilage protection effects among the treatment groups.

[0126] Functional assessment: Behavioral tests (such as the open field test) were performed on rats during and at the end of the experiment. The spontaneous movement trajectories and total distances of movement were recorded for each group of rats to assess the impact of joint pain on mobility. Animals with less cartilage damage and lower pain levels were expected to have a higher range of movement trajectories and activity levels.

[0127] Results and Analysis: Pre-stimulated MSCs showed better cartilage protection and functional improvement effects in vivo than unstimulated MSCs. Figure 10 This paper outlines the experimental design of a rat knee osteoarthritis model, including the modeling method, treatment protocols for each group, and sampling time points. Figure 11The results of the morphological observation of the knee joint cartilage of each group of rats (after sacrifice) are compared: the figure shows the knee joint cartilage morphology of the sham operation group, the PBS group, the normoxia group, the normoxia + effusion group, the hypoxia group and the hypoxia + effusion group at the time of sampling after the last treatment. Macroscopic observation shows that the cartilage surface of the sham operation group is smooth, uniform in color, and the structure is complete; the cartilage of the PBS group is severely eroded, the defect is obvious, and the articular surface is rough; the erosion of the normoxia group is less than that of the PBS group, but the structure is still discontinuous; the continuity of the cartilage surface of the normoxia + effusion group is improved, and the defect area is reduced; the cartilage of the hypoxia group is relatively flat and less worn; the cartilage of the hypoxia + effusion group has the best appearance, the surface is smooth and complete, and is close to the normal state. The results of microscopic histological staining are consistent with macroscopic observation: the content of proteoglycan in the PBS group is significantly reduced, and the structure of the cartilage layer is damaged; the staining of the MSC treatment group is improved, and the staining intensity and structural integrity of the hypoxia + effusion group are similar to those of the sham operation group, indicating that it has the most significant effect in protecting cartilage and reducing joint degeneration. The results of the quantitative OARSI cartilage degeneration score are shown in Figure 12 : The figure shows the OARSI cartilage injury score results of the sham operation group, the PBS group, the normoxia group, the normoxia + effusion group, the hypoxia group and the hypoxia + effusion group 4-6 weeks after the last treatment. The PBS group has the highest score (4.2), indicating that the cartilage degeneration is the most serious; the scores of the normoxia group and the normoxia + effusion group are reduced to 3.5 and 2.8, respectively, and the cartilage injury is alleviated; the score of the hypoxia group is further reduced to 1.5; the score of the hypoxia + effusion group is the lowest (1.2), close to the level of the sham operation group (0.8). Statistical analysis shows that the hypoxia + effusion group has significant differences compared with the PBS group, the normoxia group and the hypoxia group (p<0.05 or p<0.01), indicating that it has the best effect in reducing cartilage degeneration. Functionally, Figure 13 The activity trajectory graph of the rat open field experiment is given: the activity range of the untreated OA rats is limited, the trajectory is scattered and limited, and the activity is reduced due to joint pain; the activity trajectory of the normoxia group is increased; while the movement trajectory range of the rats in the hypoxia + effusion group is the widest, and they walk and explore more actively in the open field, covering an area significantly larger than that of the other groups. This suggests that pre-stimulated MSCs alleviate joint pain and restore some joint function, allowing rats to move more freely. In summary, Example 6 fully verifies the superior efficacy of pre-stimulated MSCs through animal experiments: in the knee osteoarthritis model, pre-stimulated MSCs are more effective than unstimulated MSCs in protecting cartilage, reducing inflammation and improving joint function, providing strong experimental evidence for the application of this strategy in the clinical treatment of OA.

[0128] The cell function enhancement effect is significant: after MSCs are treated with hypoxia + 30% effusion, the T cell proliferation inhibition rate is increased to 80% (only 45% in the hypoxia control group); the secretion amount of immune regulatory factors IL-10 and PGE2 is increased by 2-3 times, and the intracellular IDO enzyme activity is significantly up-regulated.

[0129] Good safety and morphological stability: MSCs cultured under low oxygen were spindle-shaped and adherent with uniform morphology, and showed no signs of aging. After 48 hours of pre-stimulation with hydrops, the cells continued to proliferate to P4 without chromosomal abnormalities or acute necrosis.

[0130] Outstanding results in animal models: In the MIA-induced rat OA model, the OARSI cartilage damage score of the low oxygen + hydrops group was 1.8, which was significantly lower than that of the non-pre-stimulated normoxic group (3.5) and the PBS group (4.2), with a decrease of 49% and 57% (p<0.05), respectively. Histological and morphological observations showed that the cartilage surface of this group was smooth and continuous, and the matrix structure was close to that of the sham operation group, which was significantly better than that of the normoxic group and the PBS control group, and the latter two groups still showed different degrees of cartilage erosion. Functional behavior test results also support the above histological conclusions: the open field experiment showed that the low oxygen + hydrops group of rats had the widest range of spontaneous activity trajectory coverage, continuous and uniform distribution of movement path, and significantly higher total distance than the normoxic group and the PBS group, indicating that this treatment not only effectively reduced joint cartilage degeneration and inflammatory response, but also significantly improved joint function and motor ability.

[0131] By 2-8% low oxygen environment pretreatment and 30% patient arthritis hydrops pre-stimulation, the proliferation, immunomodulatory ability and in vivo efficacy of MSCs are simultaneously optimized; and all parameters (oxygen concentration, hydrops ratio, stimulation duration) have clear quantitative ranges.

[0132] The benefits of the present application over the prior art are:

[0133] I. Effectiveness

[0134] 1) Cell level: After low oxygen (2%-8% O2) expansion and 30% patient hydrops pre-stimulation, the doubling time of MSCs was shortened from 50h in normoxic conditions to 36h, and the proliferation rate was increased by 30% within 72h. The T cell inhibition rate increased from 45% to 80%, and the anti-inflammatory secretion levels of IL-10 and PGE2 increased by ≥2 times, indicating that the cells had a stronger "immune mediation" phenotype.

[0135] 2) Animal level: In the MIA-induced rat OA model, the OARSI cartilage damage score of the hypoxia+effusion group was 1.8, which was significantly lower than that of the normoxia group without pre-stimulation (3.5) and the PBS model control group (4.2), with a decrease of 49% and 57% respectively (p<0.05). Histomorphological observation results showed that the cartilage surface of this group was continuous and smooth, and the matrix structure integrity was close to the sham operation group, which was significantly better than the normoxia group and the PBS control group, and the latter two groups still showed different degrees of cartilage erosion. The functional behavior test results also supported the above histological conclusion: the open field experiment showed that the rats in the hypoxia+effusion group had the widest range of spontaneous activity trajectory coverage, continuous and uniform motion path, and significantly higher total distance than the normoxia group and the PBS group, indicating that this treatment not only effectively reduced joint cartilage degeneration and inflammatory response, but also significantly improved joint function and motor ability.

[0136] II. Safety

[0137] 1) Residual and contamination control

[0138] Arthritis effusion was freeze-dried, redissolved and filtered; if necessary, combined with gamma ray sterilization, both to remove pathogens and avoid traditional chemical inducer residues.

[0139] 2) Genetic and phenotypic stability: hypoxic environment delays MSC replication stress, karyotype remains normal; CD73 + / CD90 + / CD105 + expression rate ≥95%, CD34 - / CD45 - expression rate ≤2%, avoid mixing hematopoietic or immunogenic cells.

[0140] 3) Low risk of uncontrolled inflammation: no up-regulation of MMP-13 was observed during pre-stimulation; no abnormal serum inflammatory factor surge or synovial membrane proliferation was detected in animals, and no acute gout-like reaction or excessive bone hyperplasia occurred.

[0141] 4) Clinical manageability: the product is autologous or allogeneic low-immunogenic MSC, without the need for long-term immunosuppression; the injection path is the same as conventional intra-articular HA or PRP, without additional surgical risk.

[0142] III. Results landing (transformation and industrialization)

[0143] Process can be scaled up:

[0144] 1) Key parameters are clear (hypoxia 2-8%, effusion 30%, 48h pre-stimulation); closed bioreactor batch production and freeze-dried powder components can be modularly replicated;

[0145] 2) Resource conservation and cost optimization: using daily arthritic effusion from local hospitals as raw material, "waste to raw material", replacing expensive recombinant factors; low oxygen rapid expansion reduces the number of passages, the overall production cost can be reduced by 20-30%.

[0146] 3) Clear regulatory path: the cells themselves meet the definition of "in vitro expanded MSC" in the "Cell Therapy Product Management Method (Draft)"; arthrocentesis is a clinical path, and the single-arm exploratory trial can be carried out according to the "Good Clinical Practice" first, and then compared with randomization.

[0147] 4) Potential for expanded indications: the same pre-stimulation platform can be used for hip, shoulder, ankle OA and even post-traumatic arthritis after fine tuning; the cell-derived exosomes can also be developed as a cell-free injection, which can be used with HA, PRP or minimally invasive arthroscopic surgery, which is conducive to rapid cooperation with orthopedic hospitals and rehabilitation centers.

[0148] The application highlights the following four irreplaceable technical pillars:

[0149] 1. Dual pretreatment synergy window

[0150] Specific parameter closed loop - first expand to P3 under 2%-8% O2 hypoxia, then stimulate for 48 hours with 30% volume fraction of freeze-dried-resolved arthritic effusion; experiments have shown that the three indicators of "proliferation, IL-10 / IDO-1 expression up-regulation, and T cell inhibition ≥80%" can be simultaneously met; any single hypoxia or single inflammatory factor pulse cannot simultaneously meet the three functional thresholds in the same batch of cells.

[0151] 2. Patient's autologous (or allogeneic) effusion freeze-dried powder as a pre-stimulation inducer

[0152] Compared with commercially available recombinant IFN-γ / TNF-α, it has innovation:

[0153] a) Natural multi-factor synergy, no need for expensive recombinant proteins;

[0154] b) Freeze-drying-resolving process retains growth factor / chemokine activity and facilitates cross-batch standardization;

[0155] c) Resource reuse, converting traditional medical waste into functional raw materials, significantly reducing costs and reducing immunogenicity risk.

[0156] 3. In-situ long-term repair and animal-level evidence

[0157] MSCs prepared by the preparation method in the MIA rat knee OA model:

[0158] a) Joint swelling and pain are significantly relieved;

[0159] b) OARSI cartilage damage score reduced by 67%;

[0160] c) Animal activity trajectory significantly extended.

[0161] In summary, the double pretreatment of hypoxia and effusion, the self-effusion freeze-drying inducer, and the long-lasting repair effect verified by animals, all have irreplaceable advantages compared with any single pre-stimulation or conventional MSC culture scheme.

[0162] The design focus of the present application is:

[0163] First, improve cell activity by using optimized hypoxic pretreatment technology: the present application uses hypoxic conditions for the culture of mesenchymal stem cells, allowing mesenchymal stem cells to maintain proliferative capacity and original state in a physiological hypoxic environment close to the body; hypoxic culture can significantly improve the proliferation rate of mesenchymal stem cells, delay cell aging, and improve cell differentiation potential and therapeutic activity;

[0164] Second, obtain function-enhanced mesenchymal stem cells with stronger immune regulation function: mesenchymal stem cells pretreated with arthritis effusion and hypoxia significantly enhance the expression and secretion of anti-inflammatory factors and immune regulation molecules such as IL-10, IDO-1, and PGE2 in cells; compared with unstimulated mesenchymal stem cells, pre-stimulated mesenchymal stem cells exhibit stronger immune suppression function in vitro, which can effectively inhibit inflammatory response and protect cartilage tissue;

[0165] Third, optimize the concentration and processing time of effusion pre-stimulation: the present application determines the optimal stimulation concentration and stimulation time; this optimized condition effectively avoids the problem of excessive activation or insufficient stimulation of cell function, ensuring the stability and safety of MSC treatment function;

[0166] Fourth, innovative use of patients' own arthritis effusion resources: the present application ingeniously uses the arthritis effusion of patients with late-stage knee osteoarthritis, which is freeze-dried and prepared into a standardized powder preparation, and then used for MSC pre-stimulation; this method makes full use of the inflammatory factors in the effusion for pre-stimulation, avoiding resource waste, reducing treatment costs while improving the functional properties of MSCs;

[0167] Fifth, animal model verification shows significant enhancement of therapeutic effect: the present application uses a knee osteoarthritis rat animal model for pharmacodynamic verification, and the results show that pre-treated mesenchymal stem cells exhibit obvious drug advantages in vivo; experiments show that pre-stimulated mesenchymal stem cells are significantly superior to traditional mesenchymal stem cells in terms of cartilage protection, inflammation inhibition, and joint function improvement, confirming their significant clinical treatment potential.

[0168] The above merely describes preferred embodiments of the present application, and is not intended to limit the technical scope of the present application in any way. Any minor modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application shall still fall within the technical scope of the present application.

Claims

1. A preparation method for obtaining a functional enhanced mesenchymal stem cell preparation by using arthritic effusion combined with hypoxic preconditioning of mesenchymal stem cells, characterized by: It comprises the following steps: S1, obtaining mesenchymal stem cells and low-oxygen expansion and passage; S2, pretreatment of arthritis effusion: S3, arthritis effusion pre-stimulation of mesenchymal stem cells to obtain function-enhanced mesenchymal stem cells; S4, index detection screening; S5, preparation of function-enhanced mesenchymal stem cell preparation.

2. The method for preparing a functional enhanced mesenchymal stem cell preparation using arthritis effusion combined with hypoxic preconditioning of mesenchymal stem cells according to claim 1, characterized in that: The oxygen concentration of low-oxygen expansion and passage in step S1 is 2%-8%.

3. The method for preparing a functional enhanced mesenchymal stem cell preparation using arthritis effusion combined with hypoxic preconditioning of mesenchymal stem cells according to claim 1, characterized in that: The pre-stimulation concentration of arthritis effusion in step S3 is 5%-60%, and the pre-stimulation treatment time is 12-72 hours.

4. The method for preparing a functional enhanced mesenchymal stem cell preparation using arthritis effusion combined with hypoxic preconditioning of mesenchymal stem cells according to claim 3, characterized in that: The pre-stimulation concentration of arthritis effusion in step S3 is 30%, and the pre-stimulation treatment time is 48 hours.

5. The method for preparing a functionally enhanced mesenchymal stem cell preparation by utilizing arthritis effusion combined with hypoxia pretreatment of mesenchymal stem cells according to claim 1, characterized in that: The step S1 is: obtaining mesenchymal stem cells, expanding in a low-oxygen incubator from P0 generation, using serum-free complete culture medium, and culturing mesenchymal stem cells to 80%-90% confluence before passing to P3 generation for subsequent pre-stimulation. 6.The method of claim 1, wherein the method is characterized by: The step S2 is: collecting arthritis effusion, centrifuging to remove cellular components, filtering and sterilizing the supernatant, vacuum freeze-drying the supernatant after pre-freezing, obtaining effusion freeze-dried powder, and sealing and storing for standby use.

7. The method for preparing a functionally enhanced mesenchymal stem cell preparation by utilizing arthritis effusion combined with hypoxia pretreatment of mesenchymal stem cells according to claim 1, characterized in that: The step S3 is: resolubilizing the treated arthritis effusion using mesenchymal stem cell complete culture medium to prepare a solution; adding mesenchymal stem cells passed to P3 generation to the solution for pre-stimulation, removing the arthritis effusion solution after pre-stimulation, washing the mesenchymal stem cells with PBS, and adding arthritis effusion-free culture medium for further culture to P4 generation to obtain function-enhanced mesenchymal stem cells.

8. The method for preparing a functionally enhanced mesenchymal stem cell preparation by utilizing arthritis effusion combined with hypoxia pretreatment of mesenchymal stem cells according to claim 1, characterized in that: The index detection screening in the step S4 includes phenotype detection screening, function detection screening and safety detection screening. The phenotype detection screening is that the expression rate of CD73 + , CD90 + , CD105 + of the function-enhanced mesenchymal stem cells is ≥ 95%, and the expression rate of CD34 - , CD45 - is ≤ 2%; the function detection screening is that the T cell proliferation inhibition rate of the function-enhanced mesenchymal stem cells is ≥ 50%, and the secretion levels of IL-10 and PGE2 are at least 2 times higher than those of the untreated mesenchymal stem cells; and the safety detection screening is that the function-enhanced mesenchymal stem cells are not contaminated by bacteria, fungi and mycoplasma, and the chromosome karyotype is normal.

9. The method for preparing a functionally enhanced mesenchymal stem cell preparation by utilizing arthritis effusion combined with hypoxia pretreatment of mesenchymal stem cells according to claim 1, characterized in that: The step S5 is specifically: the functional enhanced mesenchymal stem cells adjust the concentration, the concentration is 0.5-5×10 6 Cells / joint / second, and resuspended in the carrier to make the functional enhanced mesenchymal stem cell preparation capable of injection.

10. A function-enhanced mesenchymal stem cell preparation prepared by the preparation method of any one of claims 1-9.

11. Use of the function-enhanced mesenchymal stem cell preparation of claim 10 in the preparation of an arthritis drug.

12. Use according to claim 11, characterized in that: The administration mode of the arthritis drug includes intra-articular injection, arthroscopic injection, and intravenous administration.

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