Mesenchymal stem cell aggregate co-activated by matrix and cytokine and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a kind of mesenchymal stem cell aggregate jointly enabled by matrix and cytokine and application, the aggregate is by mesenchymal stem cell in the medium added cell culture matrix and IGF-1 is cultivated and obtains pretreated mesenchymal stem cell, continue in 3D cell culture system and obtain by generation culture, the calcium adhesion cell culture matrix is prepared by 1-3 parts hE-cad-Fc and 1-3 parts hN-cad-Fc;The aggregate structure obtained is complete, boundary is clear, high expression intracellular endogenous calcium adhesion and phosphorylated IGF-1 receptor, the curative effect of complex etiology, refractory, recurrent immune disorder diseases is significantly improved, such as systemic sclerosis, xerophthalmia and systemic lupus erythematosus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mesenchymal stem cell aggregate and its application that is jointly empowered by matrix and cytokines. Background Technology

[0002] Stem cell therapy is one of the most promising treatment methods of the 21st century. It utilizes the self-renewal, differentiation, and paracrine properties of stem cells to regenerate damaged cells and tissues in the human body. Mesenchymal stem cell (MSC) therapy accounts for 46% of all current stem cell clinical trials, covering a variety of indications including degenerative diseases, autoimmune diseases, organ fibrosis, and cancer, and has become a hot topic in translational research for next-generation cell therapy. However, the therapeutic function of MSCs in most clinical stages is not as effective as demonstrated in preclinical studies, failing to achieve the expected therapeutic effects. Besides the heterogeneity of MSCs and the functional decline caused by their limited in vitro expansion capacity, another important reason is the altered cell survival and functional response caused by changes in the in vivo environment after in vivo infusion. The problems of MSC heterogeneity and functional decline caused by in vitro expansion have been largely solved by the cell empowerment culture system previously constructed in our laboratory. However, how to improve their environmental responsiveness, survival, and therapeutic potential after in vivo through pre-induction during the in vitro expansion stage is a problem that urgently needs to be solved in the clinical application of MSCs.

[0003] To address this issue, various empowerment culture techniques have been developed to enhance the tolerance, responsiveness, and therapeutic potential of MSCs entering the lesion microenvironment. Common MSC empowerment strategies include hypoxia treatment, 3D culture, and the addition of nutritional factors (growth factors, cytokines, or hormones) to promote in vitro expansion of MSCs and improve their therapeutic function. In recent years, with the development of tissue engineering, the use of tissue engineering scaffold materials (including hydrogels, porous scaffolds, and microcarriers) to load MSCs and implant them into lesion sites has demonstrated the function of engineered MSCs in promoting the repair of damaged tissues. Our research group has also developed a series of cadherin-modified biomaterial microspheres to mediate MSC aggregation using a "one-pot" method to prepare composite aggregates of microspheres and MSCs. Results show that this method can effectively regulate the differentiation and secretion characteristics of MSCs and has good application prospects in organoid construction and the repair of local tissue injuries such as cartilage, blood vessels, and colorectal tissue. However, the precise control of the biocompatibility and biodegradability of exogenous tissue engineering scaffold materials cannot yet fully meet the requirements of stem cell therapy technology and stem cell drug development.

[0004] Building upon previous research, the inventor's research group discovered that, compared to commonly used MSC-activating cytokines (such as IL1-β, IFN-γ, or TNF-α), the artificial extracellular matrix of cadherin fusion protein combining E-Cad-Fc and N-cad-Fc, in synergy with IGF-1, can more efficiently activate the biological activities of MSCs, such as proliferation and secretion. They innovatively developed a technology for MSC amplification using cadherin fusion protein artificial extracellular matrix combined with IGF-1, and further creatively achieved a "two-step" preparation of pure cell aggregates of MSCs (i.e., scaffold-free MSC aggregates) using 3D culture technology. This not only reduces MSC apoptosis but also significantly improves the expression of MSCs' therapeutic functions in immune and inflammatory diseases, such as tolerance to the lesion microenvironment, immune regulation, and anti-inflammation. This provides a revolutionary new technology for cell function regulation in the treatment of autoimmune and inflammatory diseases and the development of cell drugs. Summary of the Invention

[0005] The primary objective of this invention is to provide a mesenchymal stem cell aggregate jointly empowered by matrix and cytokines. The aggregate is obtained by culturing pretreated mesenchymal stem cells in a culture medium supplemented with cell culture substrate and IGF-1, and then passaged in a 3D cell culture system. The cell culture substrate is prepared from 1-3 parts hE-cad-Fc and 1-3 parts hN-cad-Fc.

[0006] Preferably, the mesenchymal stem cell aggregates jointly empowered by the matrix and cytokines are prepared by the following method: (1) 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc are diluted to a total protein concentration of 1-30 μg / mL, added to a cell culture plate treated with tissue culture, incubated, and washed to obtain the cell culture substrate;

[0007] (2) Mesenchymal stem cells are seeded into cell culture plates cultured in step (1) and continuously passaged to obtain matrix-empowered mesenchymal stem cells.

[0008] (3) The matrix-energized mesenchymal stem cells obtained in step (2) are cultured until the cells reach 50-60% confluence, 0-15 ng / mL of human recombinant IGF-1 is added, and the cells are cultured until 70-80% confluence is reached. The cells are then digested and collected.

[0009] (4) The cells obtained in step (3) are added to a 3D cell culture system for culture to obtain mesenchymal stem cell aggregates that are jointly empowered by matrix and cytokines.

[0010] Preferably, the incubation time in step (1) is 0.5-24hrs and the incubation temperature is 4-37℃.

[0011] Preferably, the culture medium used in step (3) is a complete culture medium for in vitro amplification of MSCs, with additional IGF-1 added.

[0012] The second objective of this invention is to provide a method for preparing mesenchymal stem cell aggregates pretreated with cadherin and IGF-1, wherein the mesenchymal stem cell aggregates jointly energized by the matrix and cytokines are prepared by the following method: (1) 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc are diluted to a total protein concentration of 1-30 μg / mL, added to a cell culture plate treated with tissue culture, incubated, and washed to obtain the cell culture substrate;

[0013] (2) The mesenchymal stem cells were seeded into the cell culture plate with cell culture material obtained in step (1) and passaged to obtain matrix-empowered mesenchymal stem cells.

[0014] (3) The matrix-energized mesenchymal stem cells obtained in step (2) are cultured until the cells reach 50-60% confluence, 0-15 ng / mL of human recombinant IGF-1 is added, and the cells are cultured until 70-80% confluence is reached. The cells are then digested and collected.

[0015] (4) The cells obtained in step (3) are added to a 3D cell culture system for culture to obtain mesenchymal stem cell aggregates that are jointly empowered by matrix and cytokines.

[0016] A third objective of this invention is to provide the application of the aforementioned matrix and cytokine-enhanced mesenchymal stem cell aggregates in the preparation of drugs for treating immune diseases.

[0017] Preferably, the immune diseases include autoimmune diseases and inflammatory immune diseases.

[0018] Preferably, the autoimmune diseases include one or more of systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, hyperthyroidism, juvenile diabetes, diabetic nephropathy, primary thrombocytopenic purpura, and autoimmune hemolytic anemia; and the inflammatory immune diseases include one or more of lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, and polyneuritis.

[0019] The beneficial effects of the present invention are as follows: (1) The present invention provides a mesenchymal stem cell aggregate (E / N-IGF-3D) jointly empowered by matrix and cytokines. The mesenchymal stem cell aggregate is obtained by culturing pretreated mesenchymal stem cells in a 3D cell culture system. The pretreated mesenchymal stem cells are obtained by culturing mesenchymal stem cells on cadherin cell culture medium and in a medium with additional IGF-1. The cadherin cell culture medium is prepared from 1-3 parts hE-cad-Fc and 1-3 parts hN-cad-Fc. The aggregate is pretreated with E / N-cad and 0-15 ng / mL IGF-1. The resulting aggregate has a complete cell structure and clear boundaries. The expression of endogenous cadherin, IGF-1 receptor and its phosphorylated receptor in E / N-IGF-3D cells is significantly increased, the anti-inflammatory function is significantly upregulated, and the cell metabolic pathway is more biomimetic to the metabolic form of MSCs in vivo, which is beneficial to improving the therapeutic function expression after cell reinfusion. In vitro cell experiments showed that E / N-IGF-3D aggregates can effectively inhibit the proliferation of activated T cells, efficiently reverse the fibrotic phenotype of skin fibroblasts, and promote the repair of damaged endothelial cells and improve the integrity of vascular intima.

[0020] (2) When the E / N-IGF-3D aggregate described in this invention is used to treat systemic sclerosis, the E / N-IGF-3D aggregate treatment group effectively inhibits the immune infiltration of lymphocytes and monocytes in the lesion site, reduces pro-inflammatory CD4+ lymphocytes, and increases anti-inflammatory Treg cells; at the same time, the E / N-IGF-3D aggregate has a significant effect in reversing dermal fibrosis and pulmonary fibrosis; in addition, the E / N-IGF-3D aggregate significantly improves the vascular integrity and permeability of fibrotic lung tissue.

[0021] (3) When the E / N-IGF-3D aggregate described in this invention is used to treat Sjögren's syndrome, the E / N-IGF-3D aggregate treatment group significantly upregulates salivary gland flow rate, increases salivary gland / body weight ratio, reduces inflammatory cell infiltration, reduces atrophy of acini and ducts, reduces salivary gland collagen deposition and fibrosis, and at the same time increases the expression of aquaporin AQP5 and ion transporter NKCC1 in damaged salivary glands, effectively improving salivary gland function in Sjögren's syndrome;

[0022] (4) When the E / N-IGF-3D aggregate described in this invention is used to treat systemic lupus erythematosus in MRL / Lpr mice, the E / N-IGF-3D aggregate treatment group is more effective in inhibiting perirenal lymph node and spleen enlargement and inhibiting lymphocyte proliferation; it significantly reduces the physiological and biochemical indicators of systemic lupus erythematosus mice, including the content of protein, creatinine, and urea nitrogen in urine, as well as various pathological parameters such as the level of anti-dsDNA antibody, TNF-α and IgG concentration in blood; moreover, the pathological features of thickening of glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation in systemic lupus erythematosus mice in the E / N-IGF-3D aggregate treatment group are significantly reduced; at the same time, the E / N-IGF-3D aggregate, by regulating immune cells, more effectively inhibits inflammation and further improves kidney damage in MRL / Lpr mice. Attached Figure Description

[0023] The accompanying drawings illustrate the disclosed embodiments and are used to explain the principles of the disclosed embodiments. However, it should be understood that the drawings are intended for illustrative purposes only and are not intended to define limitations of the invention.

[0024] Figure 1 Preparation of hENFc

[0025] Note: (A) Immobilization amount-protein concentration curves of hE-cad-Fc and hN-cad-Fc; (B) Immobilization amount-time curves of hE-cad-Fc and hN-cad-Fc; (C) Interaction curves of hE-cad-Fc and hN-cad-Fc; (D) Detection of hE-cad-Fc and hN-cad-Fc immobilization amounts on different matrix surfaces.

[0026] Figure 2 Characterization of hENFc

[0027] Note: (A) Elemental analysis of different matrix surfaces; (B) Stability testing of different matrices; (C) Distribution and structural analysis of hE-cad-Fc and hN-cad-Fc on different matrix surfaces; (D) Hydrophilicity / hydrophobicity testing of different matrix surfaces.

[0028] Figure 3 Detection of the proliferation capacity of different generations of hMSCs on different matrix surfaces

[0029] Note: (A) Cell doubling time; (B) Cell number

[0030] Figure 4 Statistical analysis of hMSCs volumes at different passages on different matrix surfaces

[0031] Figure 5 Detection of endogenous cadherin expression in different generations of hMSCs on different matrix surfaces

[0032] Note: (A) Real-time PCR detection; (B) Western blotting detection

[0033] Figure 6 OCT4 gene and protein expression in different generations of hMSCs on different matrix surfaces

[0034] Figure 7 Senescence detection of different generations of hMSCs on different matrix surfaces

[0035] Note: (A) Cell cycle detection; (B) β-galactosidase staining

[0036] Figure 8 Apoptosis detection of different generations of hMSCs on different matrix surfaces

[0037] Figure 9 Multiple differentiation capacity assays of different generations of hMSCs on different matrix surfaces

[0038] Note: (A) Oil Red O staining and detection of adipogenic gene expression; (B) Alizarin Red staining and detection of osteogenic gene expression; (C) Alcian Blue staining and detection of chondrogenic gene expression.

[0039] Figure 10 MSC EN Oncogene and tumor suppressor gene expression point map

[0040] Figure 11 MSC EN karyotype analysis

[0041] Figure 12 MSC EN Tumorigenicity detection

[0042] Note: (A) Light micrograph of mouse tumor size; (B) Mouse body weight; (C) Tumor volume

[0043] Figure 13 The effect of E / N-cad combined with different factor pretreatment on MSC function

[0044] Figure 14 Effects of different concentrations of IGF pretreatment on cell proliferation

[0045] Figure 15 Effects of different concentrations of IGF pretreatment on endogenous cadherin, stemness, and anti-inflammatory function in cells

[0046] Figure 16 The expression levels of cadherin, IGF-1 receptor, anti-inflammatory, chemotactic and regeneration-related genes after E / N-cadherin combined treatment were observed.

[0047] Figure 17E / N-IGF-3D aggregate morphology

[0048] Figure 18. Expression levels of endogenous cadherin, IGF-1 receptor, and stem gene in E / N-IGF-2D and E / N-IGF-3D aggregates.

[0049] Figure 19 E / N-IGF-3D aggregate IGF-1 receptor expression and phosphorylation activation level

[0050] Figure 20 E / N-IGF-3D aggregates and the expression levels of genes related to anti-inflammatory effects, glucose metabolism, and mitochondrial function.

[0051] Figure 21 Morphology and particle size distribution of E / N-IGF-3D aggregate secreted exosomes

[0052] Figure 22 E / N-IGF-3D aggregate secretory exosome marker protein expression levels

[0053] Figure 23 E / N-IGF-3D aggregate anti-inflammatory and anti-fibrotic miRNA expression levels

[0054] Figure 24 Survival of E / N-IGF-3D aggregates under inflammatory conditions

[0055] (A) Live / dead staining; (B) CCK8 detection

[0056] Figure 25. Anti-apoptotic ability of E / N-IGF-3D aggregates under inflammatory conditions.

[0057] Figure 26 E / N-IGF-3D aggregates inhibit the proliferation of activated PBMCs.

[0058] Figure 27. The ability of E / N-IGF-3D aggregates to promote the repair of damaged endothelium.

[0059] (A) Flow cytometry detection of apoptosis in damaged endothelial cells; (B) Statistical analysis of apoptosis in damaged endothelial cells; (C) Western blot detection of phenotype and apoptosis markers in damaged endothelial cells; (D) PCR detection of phenotype in damaged endothelial cells.

[0060] Figure 28 E / N-IGF-3D aggregates inhibit fibrosis

[0061] Figure 29 E / N-IGF-3D aggregates inhibit inflammatory infiltration at the lesion site of systemic sclerosis.

[0062] Figure 30E / N-IGF-3D aggregates inhibit skin fibrosis in systemic sclerosis lesions.

[0063] Figure 31 E / N-IGF-3D aggregates inhibit pulmonary fibrosis in systemic sclerosis lesions.

[0064] Figure 32 E / N-IGF-3D aggregates improve the integrity of damaged blood vessels in systemic sclerosis.

[0065] Figure 33 E / N-IGF-3D aggregates improve vascular permeability in systemic sclerosis lesions.

[0066] Figure 34 E / N-IGF-3D aggregates are effective in treating Sjögren's syndrome.

[0067] (A) Salivary gland flow velocity; (B) Salivary gland morphology diagram; (C) Salivary gland / body weight ratio; (D) Pathological evaluation of salivary glands (H&E staining, glycogen staining, and Masson staining); (E) Fluorescent staining of salivary glands (aquaporin AQP5 and ion transporter NKCC1).

[0068] Figure 35 Morphological study of E / N-IGF-3D aggregates in mice with systemic lupus erythematosus.

[0069] Note: (A) Hair observation; (B) Spleen morphology and index; (C) Perirenal lymph node morphology and index

[0070] Figure 36 Physiological and biochemical indicators of mice with systemic lupus erythematosus treated with E / N-IGF-3D aggregates

[0071] Note: (A) Urine protein level; (B) Creatinine level; (C) Blood urea nitrogen level; (D) Blood anti-dsDNA antibody level; (E) Blood TNF-α level; (F) Blood IgG level

[0072] Figure 37 Kidney pathological staining and scoring in mice with systemic lupus erythematosus treated with E / N-IGF-3D aggregates.

[0073] Note: (A) Kidney pathological staining; (B) Kidney score; (C) Immunofluorescence staining of immune complexes.

[0074] Figure 38 Analysis of immune cells in mice treated with E / N-IGF-3D aggregates for systemic lupus erythematosus

[0075] Note: (A) Proportion of Treg cells in the spleen; (B) Proportion of Treg cells in the perirenal lymph nodes; (C) CD4+ in the spleen. +T cell percentage; (D) Perirenal lymph node CD4 + T cell ratio; (E) CD11b+ cell effector T cell ratio Detailed Implementation

[0076] Non-limiting embodiments of the present invention will be described in more detail with reference to specific embodiments, which should not be construed as limiting the scope of the invention in any way.

[0077] Unless otherwise specified, all reagents used in the following examples are commercially available.

[0078] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0079] In the following embodiments, the hE-cad-Fc modified matrix is ​​referred to as hEFc, and the hN-cad-Fc modified matrix is ​​referred to as hNFc. hE-cad-Fc and hN-cad-Fc have been disclosed in the inventor's previous invention patents CN109337857B "Uses of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc" and CN115040695B "Application of an active interface for fusion proteins based on VE-cad-Fc / N-cad-Fc", respectively, and the corresponding sequences have been disclosed in detail. Therefore, they will not be listed again in this patent.

[0080] The matrix modified by the combined application of hE-cad-Fc and hN-cad-Fc is abbreviated as hENFc.

[0081] All data are presented as mean ± standard deviation. Statistical differences among groups were assessed using one-way ANOVA. p < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicated no significant difference. Unless otherwise stated, all in vitro experimental results were from three independent parallel experiments, and all animal experimental results were from five independent parallel experiments. All results were statistically analyzed using GraphPad Prism version 8.0 software.

[0082] Example 1: Preparation of hENFc matrix and novel mesenchymal stem cells

[0083] 1. Preparation of hENFc matrix

[0084] The purified hE-cad-Fc and / or hN-cad-Fc were diluted in PBS to a total protein concentration range of 1-30 μg / mL, and 150 μL of protein solution was added to tissue culture plates (TCPS) at a ratio of 150 μL / cm². After incubation at 4-37°C for 0.5-24 hours, the substrate was washed 5 times with PBS.

[0085] 2. Adsorption capacity-concentration and adsorption capacity-time determination of hE-cad-Fc and hN-cad-Fc

[0086] To assess the dose-concentration relationship of hE / N-cad-Fc immobilized on TCPS plates, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to achieve concentration ranges of 0, 1, 3, 5, 10, 20, and 30 μg / mL. 100 μL of each diluted hE-cad-Fc was added to each well of a 96-well plate, and the plates were incubated at 37°C for 120 min, followed by washing the plates five times with PBS. To assess the dose-time relationship of hE / N-cad-Fc immobilized on TCPS plates, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to a concentration of 5 μg / mL. 100 μL of each diluted hE-cad-Fc was added to each well of a 96-well plate, and the plates were incubated at 37°C for 0, 30, 60, 90, 120, 150, and 180 min, followed by washing the plates five times with PBS. Finally, the modified surface was measured by enzyme-linked immunosorbent assay (ELISA). In short, each matrix was incubated with 1.0% (w / v) bovine serum albumin (BSA) for 1 h to block nonspecific interactions, and then incubated with mouse anti-E-cadherin antibody (extracellular domain, 1:2000 dilution) or rabbit anti-N-cadherin antibody (extracellular domain, 1:2000 dilution) at 37°C for 2 hrs. After washing 5 times with PBS, the modified surface was incubated with HRP-labeled goat anti-rabbit / mouse IgG (H+ L) antibody (1:5000 dilution) for 2 hrs, then TMB solution was added and incubated in the dark at 37°C. After 15 min, stop buffer was added and the absorbance was measured at 450 nm using a microplate reader.

[0087] 3. Hydrophilicity / hydrophobicity detection, elemental analysis, and morphology detection of hENFc matrix.

[0088] The prepared TCPS, hEFc, hNFc, and hENFc matrix surfaces were washed five times with ultrapure water and then air-dried. The surface hydrophilicity of different surfaces was assessed using a water contact angle (WCA) meter. Elemental changes in the matrix surfaces before and after modification were evaluated using X-ray photoelectron spectroscopy (XPS) with a monochromatic MgKα radiation source (1253.6 eV). High-resolution spectra of C1s, N1s, O1s, and S2p were analyzed using Avantage software, with C1s (binding energy 284.8 eV) used as a charge-corrected reference. The morphology and roughness of the modified surfaces were characterized using atomic force microscopy (AFM).

[0089] 4. Stability testing of hENFc matrix

[0090] The purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 1-30 μg / mL, and 100 μL was added to each well of a 96-well plate. After incubation at 4-37°C for 0.5-24 hours, the plate was washed 5 times with PBS. Finally, the modified surface was measured by ELISA. In short, each matrix was incubated with 1.0% (w / v) BSA for 1 hour to block non-specific interactions, and then incubated with goat anti-human IgG (H+L) antibody (1:5000 dilution) for 2 hours. TMB solution was then added and incubated in the dark at 37°C. After 15 minutes, stop buffer was added and the absorbance was measured at 450 nm using a microplate reader.

[0091] 6. Results

[0092] 6.1 Identification of hN-cad-Fc and hE-cad-Fc and preparation of hENFc

[0093] like Figure 1 As shown in Figure A, the hE-cad-Fc and hN-cad-Fc immobilized on the TCPS surface are related to the original solution concentration. When the Cad-Fc concentration is below 5 μg / mL, the amount of surface protein immobilized is directly proportional to the concentration, and saturation is reached when the Cad-Fc concentration is 10 μg / mL. Figure 1 B demonstrated that the amount of surface protein immobilization is also affected by the immobilization time, and the maximum amount of protein immobilization is reached when incubation is 120 min. Figure 1C shows that fusion protein matrices with different hE-cad-Fc and hN-cad-Fc ratios can be prepared by controlling the Cad-Fc concentration and fixation time. To avoid the interaction between the two Cad-Fcs in solution, I first observed the minimum concentration of interaction between the two Cad-Fcs using microthermophoresis, which was 400 μg / mL, much higher than the concentration of the protein solution at saturation fixation (10 μg / mL). To construct cadherin fusion protein matrices with different isoforms and ratios, we kept the total concentration of hE-cad-Fc and hN-cad-Fc constant at 5 μg / mL, while changing their ratio (hE-cad-Fc:hN-cad-Fc = 2:1, 1:1, and 1:2) to prepare hEFc, hNFc, and hENFc matrices with different Cad-Fc ratios. Figure 1 D).

[0094] 6.2 Physicochemical characterization of hENFc matrix

[0095] XPS analysis showed that the N1s element content increased significantly after Cad-Fc surface modification, indicating that Cad-Fc was successfully immobilized on the TCPS surface. Figure 2 A). Figure 2 B shows that these Cad-Fc matrices can be stably maintained in the culture medium for 5 days. Morphological analysis of these matrices by AFM showed that nanorod-like structures of approximately 5-7 nm could be clearly observed on the surface of the TCPS plate. Figure 2 C). For example Figure 2 As shown in Figure D, the hydrophilicity of these Cad-Fc matrices is significantly higher than that of TCPS. Cad-Fc is effectively immobilized on the TCPS plate and enhances the hydrophilicity of the culture plate surface.

[0096] The inventors investigated the effect of hENFc matrix on the adhesion and proliferation of hMSCs. They found that as the proportion of hE-cad-Fc in the cadherin fusion protein matrix increased, OCT4 expression in hMSCs also increased. Specifically, when the proportion of hE-cad-Fc was greater than or equal to that of hN-cad-Fc, hMSC expression... OCT4 The gene levels were significantly higher than those of hMSCs cultured on TCPS surfaces, and their expression levels no longer increased with the increase of hE-cad-Fc ratio. Therefore, the hENFc composite matrix (hE-cad-Fc:hN-cad-Fc = 1:1) was selected as the preferred matrix for subsequent studies.

[0097] Example 2: Preparation and Function of hENFc Matrix-Enabled hMSCs

[0098] 1. Preparation of hENFc matrix-empowered hMSCs

[0099] First, hMSCs were cultured on TCPS to passage 2; then, hMSCs were cultured at a rate of 1 × 10⁶ cells per bottle. 6 One cell was seeded into a T-75 cell culture flask containing hEFc, hNFc, or hENFc matrix, and cultured continuously for 15 passages under standard expansion conditions to obtain empowered hMSCs of different passages.

[0100] The following experiments used unmodified TCPS matrix to prepare hMSCs of various passages as controls. hMSCs enabled on hEFc matrix are referred to as MSCs. E hMSCs empowered on an hNFc matrix are abbreviated as MSCs N hENFc-enabled hMSCs are abbreviated as MSCs. EN hMSCs cultured normally on TCPS substrate are abbreviated as MSCs T .

[0101] 2. Detection of the proliferation capacity of hENFc matrix-empowered hMSCs

[0102] At the end of each passage, all cells on different substrate surfaces were collected and counted after removing dead cells using trypan blue. Alternatively, MSCs were seeded into TCPS, hEFc, hNFc, or hENFc substrates, incubated for 24 and 48 h, and then cell viability was measured using CCK-8. The CCK-8 viability assay was performed as follows: the supernatant was discarded, and the cells were washed 5 times with PBS. 100 μL of CCK-8 (1:10 dilution) was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance was measured at 450 nm using a microplate reader. To compare cell proliferation rates at different passages, doubling time (DT) and population doubling level (PDL) were calculated using the following formulas:

[0103]

[0104]

[0105] Where N(t) is the number of cells at passage, N(to) is the number of cells inoculated at the previous passage, A(48) is the cell absorbance value at 48 h, and A(24) is the cell absorbance value at 24 h.

[0106] 3. Detection of the dryness of hENFc matrix-empowered hMSCs

[0107] MSCs were harvested at the 6th, 11th, and 15th passages, respectively. T and MSC ENTotal RNA was extracted using the following method: all culture medium was discarded, cells were washed three times with PBS, and 1 mL of Trizol solution was added to each well to lyse the cells. The cells were then transferred to 1.5 mL centrifuge tubes, 200 μL of chloroform was added, and the tubes were shaken vigorously for 15 seconds. After incubation at room temperature for 5 min, the tubes were centrifuged at 12000 g for 15 min at 4°C. The supernatant was transferred to a new centrifuge tube, 500 μL of isopropanol was added, and the tubes were incubated at room temperature for 10 min. After centrifugation at 12000 g for 10 min at 4°C, the supernatant was discarded. 1 mL of 75% ethanol was added to each tube, and the tubes were mixed and centrifuged at 7500 g for 5 min at 4°C. The supernatant was discarded, and the tubes were air-dried at room temperature. Finally, 20 μL of sterile, enzyme-free ddH2O was added to each tube to dissolve the RNA. The tubes were incubated at 55°C for 10 min, aliquoted, and stored at -80°C. Total protein was extracted using the following method. All culture medium was discarded, and cells were washed with PBS. 100 μL of strong lysis buffer was added to each well and incubated on ice for 10 min. All liquid was scraped off using a cell scraper and transferred to centrifuge tubes. The tubes were centrifuged at 13000 g for 15 min at 4°C. The supernatant was collected, and the protein concentration in each sample was determined according to the BCA kit instructions. Finally, loading buffer (1:5 dilution) was added to the supernatant of each tube, and the tubes were boiled for 10 min. After aliquoting, the tubes were stored at -80°C.

[0108] E-cadherin, N-cadherin, and [other compounds] were detected by quantitative real-time PCR and Western blotting. OCT4 Gene and protein expression status. The quantitative real-time PCR method was as follows: Following the Vazyme instructions, 2 μL of cDNA from each group was added to each tube as a template, along with 1 μL of primers. Additionally, 10 μL of 5×ChamQ Universal SYBR qPCR MasterMix and 7 μL of RNase-free water were added to each group. After sample addition, the tubes were briefly centrifuged for 5 seconds using a handheld centrifuge, then placed in a quantitative real-time PCR instrument and amplified according to the following program: 95°C 30 sec, 95°C 3 sec, 60°C 10 sec (40 replicates), 95°C 15 sec, 60°C 60 sec, 95°C 15 sec. The obtained data used β-actin expression as an internal control. Three replicates were set up for each sample, and 2... -ΔΔCtStatistical calculations were performed using the following Western blotting method: Samples were added to a 10% SDS-polyacrylamide gel for electrophoresis. Electrophoresis was stopped when the sample front migrated to a point near the bottom of the glass plate. The PAGE gel was removed, and the absorbent sponge, PAGE gel, PVDF membrane, and absorbent sponge were clamped together in sequence using a transfer clamp. The membrane was placed in an electroblotting apparatus, and electroblotting buffer was added. Electroblotting was performed at 100 V for 90 min. After transfer, the PVDF membrane was removed and blocked with 5% BSA at room temperature for 2 hours. It was then incubated with primary antibody at 37°C for 2 hours or overnight at 4°C. The membrane was then washed with TBST solution at room temperature for 5 min, repeated three times. HRP-labeled secondary antibody was added and incubated at room temperature for 2 hours. The membrane was then washed with TBST solution at room temperature for 5 min, repeated five times. Finally, an appropriate amount of chromogenic solution was added to the PVDF membrane, and the images were observed and photographed using a Western blotting instrument. The graphs were statistically analyzed using ImageJ.

[0109] The primer sequences used in the implementation are as follows:

[0110]

[0111] 4. Periodicity detection of hENFc matrix-empowered hMSCs

[0112] At the 6th, 11th, and 15th generations, 1 × 10⁻⁶ were harvested respectively. 6 MSC T and MSC EN Cells were fixed in 70% cold ethanol for 2 hours to overnight; after washing 3 times with PBS, the cells were centrifuged at 1000 rpm for 10 min at room temperature to obtain cell pellets; 100 μL of RNase A solution was added to each sample, the cells were resuspended and incubated at 37°C for 30 min; 400 μL of PI staining solution was added and mixed well, and the cells were incubated at 4°C in the dark for 30 min; the red fluorescence at the excitation wavelength of 488 nm was recorded by flow cytometry.

[0113] 5. Senescence detection of hENFc matrix-empowered hMSCs

[0114] For aging-related β-galactosidase assays, MSCs were detected using an SA-β-gal staining kit at passages 6, 11, and 15. T and MSC ENβ-galactosidase activity was measured. In short, cells were cultured in modified or unmodified 6-well plates to 80% confluence, washed with PBS, and fixed with 4% formaldehyde for 15 min. Subsequently, the cells were incubated overnight at 37°C with freshly prepared β-galactosidase staining solution in a CO2-free oven. After washing five times with PBS, images were taken using an optical microscope, and the number of β-galactosidase-positive cells was counted randomly per 200 cells in the field of view.

[0115] 6. Apoptosis detection of hENFc matrix-empowered hMSCs

[0116] At the 6th, 11th, and 15th passages, 1 × 10⁻⁶ mol / L trypsin without EDTA was harvested. 6 MSC T and MSC EN Cells were resuspended in Binding Buffer (1:9 dilution) diluted with 1 ml of deionized water. 5 µL of Annexin V / FITC was added to every 100 µL of cell suspension, and the mixture was incubated at room temperature in the dark for 5 min. 5 µL of propidium iodide solution and 400 µL of PBS were added, and the mixture was immediately transferred to a 5 ml flow cytometry tube for flow cytometry analysis. Unstained cells served as blank tubes; cells containing only Annexin V / FITC without propidium iodide were single-stained tubes.

[0117] 7. Detection of differentiation potential of hENFc matrix-empowered hMSCs

[0118] The 6th generation MSC T 11th generation MSC T and the 11th generation MSC ENCells were cultured in differentiation medium for 14 days, and the trilineage differentiation capacity of hMSCs was assessed. For osteogenic differentiation: osteogenicity was induced by culturing cells in DMEM / F12 supplemented with 50 μg / mL ascorbic acid 2-phosphate, 10 nM dexamethasone (Dex), 10 mM β-glycerophosphate, 10% FBS, and 1% penicillin-streptomycin. Mature cells after differentiation were stained with Alizarin Red S solution. For adipogenic differentiation: adipogenesis was induced by culturing cells in DMEM / F12 supplemented with 10% FBS, 50 μg / mL ascorbic acid 2-phosphate, 1 μM Dex, 0.1 mM indomethacin, and 4 U / L insulin. Mature cells were detected using an Oil Red O staining kit. For chondrogenic differentiation: chondrogenic differentiation was induced by supplementing cells with 2% FBS, 50 μg / mL ascorbic acid 2-phosphate, 50 μg / mL L-proline, 100 nmDex, and 10 ng / mL... Cells were cultured in DMEM / F12 containing TGF-β1, 1% human insulin transferrin, and sodium selenite to induce chondrogenesis. Mature cells were detected using toluidine blue chondrogenic solution. Simultaneously, following the method in Example 2, RNA was extracted and reverse-engineered into cDNA. PCR was performed using the following primers to detect the expression of genes related to triphase differentiation:

[0119]

[0120] 8. Detection of tumorigenicity of hENFc matrix-empowered hMSCs

[0121] To examine the tumorigenicity of hENFc-enabled hMSCs, 1 × 10⁻⁶ cells were used. 7 A 6th generation MSC T (n = 3) and the 15th generation MSC EN (n = 6) Cells were subcutaneously transplanted into the abdomen of 4-week-old NOD-SCID mice; mouse body weight and tumor volume were measured every 3 days according to the following formula. Mice were sacrificed after seven weeks of continuous feeding. Injection of 1 × 10⁻⁶ cells... 7 Mice with individual glioblastoma (U87) (n = 3) served as positive controls, while mice injected with an equal volume of PBS (n = 3) served as negative controls.

[0122]

[0123] 9. Results

[0124] 9.1 Effect of hENFc matrix on the proliferation ability of in vitro expanded hMSCs

[0125] Although the proliferative capacity of all cells gradually decreases with each passage, the number of cells harvested each time also gradually decreases. Figure 3However, compared with hMSCs (MSCs) cultured on the TCPS surface... T Compared to cadherin fusion protein matrix, hMSCs cultured in this matrix maintained their proliferation capacity well, especially hMSCs cultured in the hENFc matrix. EN The cell doubling time remained within 25 hours throughout the eleventh generation of passages, and the proliferative capacity showed no significant change. This indicates that the hENFc matrix effectively maintained the in vitro proliferative activity of hMSCs through cell-cell interactions mediated by E-cadherin and N-cadherin. Furthermore, the size of hMSCs cultured in the hENFc matrix did not change with passage expansion. EN The volume remained at 2000 μm 3 (within approximately 16 μm in diameter) Figure 4 ).

[0126] 9.2 Effect of hENFc matrix on the maintenance of stemness in in vitro expanded hMSCs

[0127] like Figure 5 As shown, in vitro cultured hMSCs lost E-cadherin expression, although N-cadherin expression was preserved, but it was gradually lost during culture. However, culture using a biomimetic hENFc matrix re-conjugated endogenous E-cadherin expression in hMSCs and maintained the presence of N-cadherin. E-cadherin plays a crucial role in maintaining stem cell stemness, and we further investigated the expression of OCT4, a stemness marker in hMSCs, during long-term passage. Gene and protein analysis results showed that, compared with MSCs… T In comparison, MSC EN The stemness was maintained in successive generations of MSCs, particularly in the sixth, eleventh, and fifteenth generations. EN The dryness of all of them was higher than that of MSC. T Most importantly, the eleventh generation MSC EN Its dryness is significantly better than that of the sixth generation MSC. T ( Figure 6 )

[0128] 9.3 Effects of hENFc matrix on senescence and apoptosis of in vitro expanded hMSCs

[0129] Replicative senescence is the biggest obstacle to the in vitro culture of hMSCs, leading to loss of cell proliferation capacity and alterations in secretory profiles, thus limiting the therapeutic efficacy of the cells. Cell cycle analysis revealed that MSCs at different passages... EN The number of cells in S phase was significantly higher than that in MSCs. T β-galactosidase staining further confirmed the presence of MSCs.EN The percentage of senescent cells was consistently below 10% and significantly lower than that of MSCs. T ( Figure 7 The result is as follows: Figure 8 As shown, after long-term in vitro culture, MSCs T A large number of apoptosis occurred, while MSCs EN The proportion of apoptotic cells did not change significantly with increasing passage number, making the eleventh generation MSCs... EN In terms of the ratio of senescent and apoptotic cells, compared with sixth-generation MSCs T Basically the same.

[0130] We further examined the differentiation capacity of each generation of hMSCs, comparing it with that of the eleventh generation MSCs. T (MSC) T Compared to the 11th generation MSC, -P11) EN (MSC) EN -P11) and the sixth generation MSC EN (MSC) T More significant Oil Red O-positive lipid droplets were found in -P6) and PPAR-γ, CEBPA, LPL Gene expression ( Figure 9 A), MSC EN -P11 and MSC T There was no statistically significant difference between -P6. This demonstrates that hENFc matrix-encapsulated MSCs... EN -P11 and conventionally cultured MSCs T -P6 exhibits the same adipogenic differentiation characteristics. Similarly, in MSCs EN Alizarin Red S and Toluidine Blue positive cells were also detected in cells after P11 directed differentiation. Quantitative real-time PCR detection of mature bone and cartilage markers also confirmed the presence of MSCs. EN -P11 and MSC T -P6 has the same osteogenic properties ( Figure 9 B) and cartilage ( Figure 9 C) The potential for differentiation.

[0131] 9.3 Safety Analysis of hENFc Matrix-Enabled hMSCs

[0132] In vitro expansion and culture of MSCs inevitably leads to replicative senescence, resulting in genomic instability, a serious drawback hindering the development of MSC-based cell therapy. Therefore, we examined MSCs cultured to the eleventh generation using hENFc matrix empowerment. EN Compared with conventional culture to the sixth generation of MSCs TThe expression of genes related to genome stability and chromosome karyotype were analyzed to investigate potential genetic instability. Analysis of gene expression levels related to genome stability transformation in transcriptome sequencing data showed that ( Figure 10 ), eleventh generation MSC EN MSCs cultured normally at the sixth generation T There were no statistically significant differences in the expression of proto-oncogenes and tumor suppressor genes among cells, and the expression levels of c-Myc, P21, P53, and P16 genes did not change significantly. Karyotype analysis did not reveal any clones with chromosomal abnormalities; no chromosomal translocations, deletions, or changes in chromosome number were observed. hENFc-enabled hMSCs maintained chromosomal stability. Figure 11 ).

[0133] Tumorigenicity is another risk factor to consider in the clinical application of MSCs. Compared to iPSCs and ESCs, although MSCs themselves do not possess tumorigenicity, have low genomic instability, and a low likelihood of forming teratomas, to ensure the safety of long-term hENFc matrix-encapsulated hMSCs in clinical applications, we will use MSCs that meet clinical criteria... T hENFc-enhanced MSCs cultured to the 15th generation EN Human astrocytoma cells and U87 cells were injected subcutaneously into nude mice. Seven weeks later, the potential for these cells to form tumors was investigated. Figure 12 As shown in Figure A, mice inoculated with U87 developed a pea-sized lump at the injection site, while those inoculated with MSCs... T and MSC EN No tumors formed in any of the nude mice. To monitor the overall health of the mice, we measured their weight, and the results showed that mice inoculated with PBS and MSCs showed no abnormalities. T MSC EN There was no statistically significant difference in body weight between the mice and the U87 mice. Figure 12 B). MSC vaccination T and MSC EN No bulge formed at the injection site in the mice afterward. Figure 12 These results indicate that hMSCs cultured to the 15th generation with hENFc empowerment do not exhibit tumorigenicity and have no adverse effects on animals, meeting the standards for clinical application of hMSCs.

[0134] Example 3: Preparation of cell aggregates pretreated with cadherin and IGF-1

[0135] 1. Method

[0136] 1.1 Preparation of cells pretreated with cadherin and IGF-1:

[0137] MSCs with cadherin matrix were cultured in complete medium. After 48-72 hours, when the cells reached 50-60% confluence, they were pretreated with medium containing 0-15 ng / mL human recombinant IGF-1 for 10-24 hours. When the cells reached 70-80% confluence, the pretreated cells were collected. The control group consisted of complete medium without human recombinant IGF-1, supplemented with IL-1β, IFN-γ, and TNF-α (at a concentration of 10 ng / mL, which is commonly used in the literature for MSC activation).

[0138] 1.2 Preparation of cell aggregates pretreated with cadherin and IGF-1:

[0139] MSCs with cadherin matrix were cultured in complete medium. After 48-72 hours, the cells reached 50-60% confluence. They were then pretreated with 0-15 ng / mL human recombinant IGF-1 medium for 10-24 hours until the cells reached 70-80% confluence. The cells were then digested and collected. E / N-cad+IGF-1-3D cell aggregates (E / N-IGF-3D aggregates) of 360,000 cells / well (equivalent to 300 cells / aggregate) were prepared in AggreWell cell culture plates.

[0140] 1.3 Western blotting

[0141] Proteins were extracted from cells in a three-dimensional culture state using an ultrasonic homogenizer. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The PVDF membrane was then subjected to non-specific binding site blocking with skim milk, followed by incubation with primary antibody to bind the target protein, and washing to remove unbound antibody. Next, secondary antibody incubation was performed to bind the antibody to the primary antibody-mediated immunoreaction, and washing again removed unbound secondary antibody. A fluorescent substrate was added to induce a chemiluminescent reaction to observe the target protein signal. The protein signal was captured using a Western blot imaging system, and quantitative analysis was performed using image analysis software to compare protein expression levels under different conditions.

[0142] 1.4 Quantitative Real-Time PCR

[0143] Following the method in Example 2, RNA was extracted and reversed into cDNA. PCR was then performed using the following primers to detect the expression of genes related to anti-inflammation, tissue regeneration, stemness, metabolism, and exosome secretion:

[0144]

[0145] 2. Results

[0146] 2.1 Optimization of IGF-1 Pretreatment Conditions

[0147] Pretreatment of MSCs with different factors in combination with E / N-cad showed that IGF-1 significantly improved MSC secretory function compared to other factors. Figure 13 .

[0148] When cells reached 50-60% confluence, the addition of different concentrations of recombinant human IGF-1 to the culture medium showed that as the IGF-1 concentration increased to 10 ng / mL, cell proliferation was significantly and stably enhanced. Figure 14 .

[0149] IGF-1 pretreatment can regulate the expression of anti-inflammatory and stem genes in cells. Further optimization of IGF-1 concentration in combination with E / N-cad showed that, compared to the system without IGF-1, an IGF-1 concentration of 10 ng / mL upregulated the Nanog gene level by approximately 2.5 times and the anti-inflammatory gene IDO by approximately 70%. Figure 15 Furthermore, compared to the TCPS and E / N-cad groups without IGF-1, the E / N-cad combined with 10 ng / ml IGF-1 group showed significantly upregulated expression of endogenous cadherin, IGF-1 receptor, anti-inflammatory, chemotactic, repair / regeneration, and homing-related mRNAs, such as... Figure 16 .

[0150] Cells pretreated with E / N-cad and 10 ng / mL IGF-1 were prepared into cell aggregates. The aggregates had intact structures and clear boundaries, such as... Figure 17 .

[0151] After E / N-cadherin combined with IGF-1 was used to prepare aggregates, compared with aggregates of 2D and TCPS cultured cells, E / N-IGF-3D cells showed higher expression of endogenous E-cadherin and N-cadherin, as well as IGFR, and significantly increased expression of the cell stem gene Nanog. Figure 18 .

[0152] Compared with TC-3D, TC-IGF-3D, and E / N-3D, the expression of IGF-1 receptor and its phosphorylated receptor in E / N-IGF-3D cell aggregates was significantly increased. In particular, compared with TC-IGF-3D, the formation of 3D cell aggregates after E / N-cad combined with IGF-1 pretreatment significantly upregulated the cellular response to IGF-1 stimulation, achieving a non-linear upregulation of IGF-1 stimulation responsiveness. Figure 19 .

[0153] Compared with the TC-3D group, the E / N-IGF-3D group showed that the expression of the anti-inflammatory gene TSG-6 was upregulated by approximately 50%, and the expression of IL-10 and HGF was significantly upregulated (both greater than 4-fold). The expression of glycolysis-related genes PFKP and PKM2 was upregulated by approximately 50%, and the expression of the mitochondrial functional gene MCU was upregulated by approximately 50%. Figure 20 Compared to TC-3D, E / N-IGF-3D cell aggregates show significantly upregulated anti-inflammatory activity, and their metabolic pathway is more biomimetic to the metabolic form of MSCs in vivo, which is beneficial for improving the therapeutic function expression after cell reinfusion.

[0154] Example 4: Exosome Extraction and Identification

[0155] 1. Method

[0156] 1.1 Collection of exosomes:

[0157] E / N-IGF-3D MSC aggregates were cultured continuously in basal medium for 48 hours, and the supernatant was collected. Exosomes were collected using a gradient centrifugation method: 300 g, centrifuged at 4℃ for 10 min to remove cells and collect the supernatant; 10,000 g, centrifuged at 4℃ for 30 min to remove cell debris and collect the supernatant; 100,000 g, centrifuged at 4℃ for 90 min, and the precipitate was collected as exosomes.

[0158] 1.2 Identification and detection of exosomes:

[0159] ① Dynamic light scattering (DLS) measurement of particle size: Take 20 μL of exosome solution resuspended in PBS, dilute it 100 times with pure water to 2 mL, place it in a test dish, and analyze it in a dynamic light scattering instrument. Plot the obtained data into a particle size distribution map.

[0160] ② Transmission electron microscopy (TEM) morphology determination of exosomes: 10 μL of exosome solution resuspended in PBS was added to a copper grid and allowed to stand at room temperature for 1-2 min. The sample liquid was then blotted dry along the outside of the copper grid with filter paper. 10 μL of 20 mg / mL phosphotungstic acid staining solution was added to the copper grid and allowed to stand for 1-2 min before being blotted dry. The sample was observed and photographed under a TEM microscope at 100,000 magnification.

[0161] ③ Protein biomarker assay: After BCA assay of protein concentration, 40 μg of total exosome protein WB was taken for the detection of characteristic proteins CD9, CD63, and CD81.

[0162] ④ Exosome function detection: Following the method in Example 2, miRNA was extracted and reversed into cDNA. PCR was then performed using the following primers to detect the expression of related genes:

[0163]

[0164] 2. Results

[0165] 2.1 Exosomes produced by E / N-IGF-3D MSC aggregates have uniform particle size, intact membrane structure, and a distribution range of 143-184 nm. Figure 21 .

[0166] 2.2 Compared with the TC-3D group, the exosomes produced by E / N-IGF-3D cell aggregates met industry standards for exosomes, exhibiting high expression of CD9 / CD63 / CD81, Calnexin negativity, and slightly upregulated cadherin expression in the exosomes. Figure 22 .

[0167] 2.3 Anti-inflammatory and anti-fibrotic miRNAs from exosomes derived from E / N-IGF-3D cell aggregates were significantly upregulated, including approximately 60-fold upregulation of mir-21, approximately 150-fold upregulation of mir-181, approximately 25-fold upregulation of mir-133b-5p, approximately 100-fold upregulation of mir-19b-3p, approximately 4-fold upregulation of mir-23b-5p, and approximately 40-fold upregulation of mir-223-5p. Figure 23 .

[0168] Example 5: Systemic sclerosis

[0169] 1. Experimental Methods

[0170] (1) Establishment and treatment of a mouse model of systemic sclerosis:

[0171] ① Six- to eight-week-old SFP-grade female C57BL mice were randomly divided into four groups: normal control group (Control group), disease control group (BLM group), TC-3D treatment group (TC-3D group), and E / N-IGF-3D treatment group (E / N-IGF-3D group) (a total of 24 mice, 6 mice in each group). Mice in the normal control group were injected with 100 μL of physiological saline in their backs daily, while mice in the disease control group and the cell aggregate treatment group were injected with 100 μL of bleomycin at a dose of 1 mg / mL daily for 28 consecutive days.

[0172] ② TC-3D and E / N-IGF-3D cell aggregates were administered to mice with systemic sclerosis via tail vein injection at a dose of 100,000 cells / mouse, once a week for two consecutive weeks. Samples were collected 28 days after the first injection, and the treatment effect was tested according to clinical specimens.

[0173] (2) Study on the therapeutic function of MSC aggregates by in vitro cell co-culture

[0174] ① Co-culture with mouse fibroblasts: Mouse fibroblasts were seeded in the lower chamber of a transwell. When the cells reached 70-80% confluence, a fibrotic cell model was constructed using 10 ng / mL TGF-β. Cell aggregates were then seeded in the upper chamber. PCR and Western blot were used to investigate its fibrosis reversal function. The PCR method followed the procedure in Example 2, using the following primers to detect the expression of relevant genes:

[0175]

[0176] ② Co-culture with human umbilical vein endothelial cells (HUVECs): HUVECs were seeded in the lower chamber of a transwell. When the cells reached 70-80% confluence, an endothelial cell injury model was constructed using 0.1 U BLM, and the cell aggregates were seeded in the upper chamber. PCR and Western blot methods were used to study its endothelial repair-promoting function. The PCR method followed the procedure in Example 2, using the following primers to detect the expression of relevant genes:

[0177]

[0178] ③ Co-culture with PBMCs: The bottom surface of the cell culture plate was treated with low adhesion, and the aggregates and PBMCs were seeded in 48-well plates at a ratio of 1:10. The inhibitory effect of cell aggregates on the proliferation of PBMCs was detected after 3 days.

[0179] The above implementation follows the method described in Example 2, where RNA is extracted, reversed to cDNA, and PCR is performed using the following primers to detect the expression of relevant genes:

[0180] 2. Results

[0181] Compared with the TC-3D, TC-IGF-3D, and E / N-3D groups, under inflammatory conditions (30 ng / mL IL-1β), staining results and CCK8 cell viability assays showed that the survival rate and cell activity of E / N-IGF-3D cell aggregates were significantly improved. Figure 24 Western blot results showed that the expression of the anti-apoptotic protein BCL-2 in E / N-IGF-3D cell aggregates was significantly upregulated, while the expression of the pro-apoptotic protein BAX was significantly downregulated, indicating that the anti-apoptotic ability of E / N-IGF-3D cell aggregates was significantly enhanced under inflammatory conditions. Figure 25 .

[0182] E / N-IGF-3D cell aggregates can effectively inhibit the proliferation of activated T cells, with an inhibition efficiency of up to approximately 83%. Figure 26In the co-culture experiment of hMSCs and damaged endothelial cells (pretreated with 0.1U BLM for 72 hours), flow cytometry, Western blotting, and PCR results showed that in the E / N-IGF-3D cell aggregate co-culture group, the apoptosis rate of damaged endothelial cells decreased by 23.1%, and the expression levels of endothelial barrier-related intercellular adhesion proteins VE-cadherin and ZO-1 were upregulated. Apoptosis-related BCL-2 (anti-regulation) was significantly upregulated, and BAX (pro-regulation) was significantly downregulated. Figure 27 E / N-IGF-3D cell aggregates have the effect of promoting the repair of damaged endothelial cells and improving the integrity of vascular intima. Compared with the TC-3D group, E / N-IGF-3D cell aggregates significantly inhibited the expression of fibrosis-related genes in skin fibroblasts, including a downregulation of COL1A1 of approximately 18.8% and a downregulation of α-SMA of approximately 50%, such as... Figure 28 The results showed that E / N-IGF-3D cell aggregates have the function of efficiently reversing the fibrotic phenotype of fibroblasts.

[0183] Animal model treatment experiments showed that, compared with the TC-3D group, the E / N-IGF-3D cell aggregate group effectively inhibited the immune infiltration of lymphocytes and monocytes at the lesion site of systemic sclerosis, reduced pro-inflammatory CD4+ lymphocytes by 19.8%, including a 0.72% reduction in Th1 lymphocytes, and an increase in anti-inflammatory regulatory T lymphocytes (Tregs) by 0.5%. Figure 29 This study revealed that E / N-IGF-3D cell aggregates possess highly effective immunomodulatory and anti-inflammatory therapeutic effects. Simultaneously, the E / N-IGF-3D cell aggregate group showed a reduction of approximately 16.7% in dermal layer thickness and an increase of approximately 100% in subcutaneous fat layer thickness, indicating that E / N-IGF-3D cell aggregates have the therapeutic effect of reversing dermal fibrosis. Figure 30 Gene analysis of lung fibrosis in E / N-IGF-3D cell aggregates showed that COL1A1, COL1A2, and α-SMA gene expression were downregulated by approximately 50%, and COL3A1 gene expression was downregulated by approximately 35%. Histological sections also showed reduced collagen deposition in the lung tissue. Figure 31 This study revealed that E / N-IGF-3D cell aggregates have a significant therapeutic effect in reversing pulmonary fibrosis. Furthermore, immunofluorescence assays of lung tissue vessels in the E / N-IGF-3D cell aggregate treatment group showed the vascular integrity of fibrotic lung tissue (e.g., ...). Figure 32 ) and permeability, (such as Figure 33 Significant improvement was observed. Overall, E / N-IGF-3D cell aggregates demonstrated the function and efficacy of reversing the fibrotic process of damaged tissues.

[0184] Example 6: Dryness of the Skin

[0185] 1. Experimental Methods

[0186] 1.1 Establishment and treatment of Sjögren's syndrome in mice:

[0187] ① Select SFP-grade female NOD mice, confirm saliva flow rate at 8 weeks of age, and establish a Sjögren's disease mouse model (a total of 24 mice, 6 mice in each group), and house them in an SFP-grade animal facility for future use.

[0188] ② TC-3D and E / N-IGF-3D cell aggregates were administered via tail vein injection at a dose of 100,000 cells / mouse. Samples were collected 21 days after the first injection and analyzed according to clinical specimen testing.

[0189] 2. Results

[0190] Compared with the TC-3D treatment group, the E / N-IGF-3D cell aggregate group significantly improved salivary gland function in patients with Sjögren's syndrome, including upregulating salivary gland flow rate by approximately 30%, increasing the salivary gland / body weight ratio, reducing inflammatory cell infiltration, decreasing acinar and ductal atrophy, and reducing collagen deposition and fibrosis. Simultaneously, it increased the expression levels of the aquaporin AQP5 and the ion transporter NKCC1 in damaged salivary glands, resulting in relief of salivary gland function. Figure 34 .

[0191] Example 7: Systemic lupus erythematosus

[0192] 1. Experimental Methods

[0193] 1.1 Treatment of systemic lupus erythematosus model mice (MRL / Lpr mice) with E / N-IGF-3D cell aggregates

[0194] Eighteen 8-week-old female MRL / lpr lupus rats were housed in an SPF environment for 2 weeks to acclimatize; the treatment group received a tail vein injection of 1 × 10⁻⁶ ozontally at 10 weeks of age. 6 One TC-3D and 1 × 10 6 E / N-IGF-3D cell aggregates were collected from the PBS group after injection of 200µL of PBS. Samples were collected two weeks after treatment and analyzed according to clinical sample testing.

[0195] 2. Results

[0196] 2.1 Morphological study of E / N-IGF-3D cell aggregates in mice with systemic lupus erythematosus

[0197] Two weeks after treatment, the fur of the mice in the treatment group gradually returned to normal. Analysis of images of the perirenal lymph nodes and spleen, as well as their weight-to-body weight ratio, showed that the organ size and organ index of the perirenal lymph nodes and spleen in the E / N-IGF-3D cell aggregate treatment group were significantly lower than those in the other two groups. Figure 35As shown in the figure. These results indicate that intravenous implantation of E / N-IGF-3D cell aggregates inhibits perirenal lymph node and splenomegaly, which is closely related to the inhibitory function of E / N-IGF-3D cell aggregates in inhibiting lymphocyte proliferation.

[0198] 2.2 Study on physiological and biochemical indicators of E / N-IGF-3D cell aggregates in mice with systemic lupus erythematosus

[0199] like Figure 36 As shown, the levels of protein, creatinine, and urea nitrogen in the urine of mice treated with E / N-IGF-3D cell aggregates, as well as the levels of anti-dsDNA antibodies, TNF-α, and IgG in the blood, were significantly lower than those in mice treated with PBS and TC-3D. This indicates that the E / N-IGF-3D group was more effective in improving the physiological and biochemical indicators of mice with systemic lupus erythematosus.

[0200] 2.3 Kidney tissue pathology and antibody deposition in mice with systemic lupus erythematosus treated with E / N-IGF-3D cell aggregates

[0201] like Figure 37 As shown in Figures A and B, the histopathological results of MRL / Lpr mouse kidney tissue observed and analyzed using H&E, PAS, and Masson staining showed that, compared with the PBS treatment group and the TC-3D treatment group, the E / N-IGF-3D cell aggregate treatment group showed significantly reduced glomerular, interstitial, and perivascular damage, effectively reversing glomerular damage. Notably, compared with the PBS treatment group and the TC-3D cell aggregate treatment group, the E / N-IGF-3D cell aggregate treatment group showed significantly reduced characteristics of lupus nephritis fibrosis, including thickening of the glomerular mesangial and basement membrane, inflammatory cell infiltration, interstitial fibrosis, and occasional crescent formation.

[0202] Antibody deposition plays a crucial role in the pathogenesis of systemic lupus erythematosus. Immunofluorescence results are as follows... Figure 37 As shown, the deposition of C3 and IgG antibodies in the kidneys of MRL / Lpr mice transplanted with E / N-IGF-3D cell aggregates was significantly lower than that in the PBS treatment group and the TC-3D cell aggregate treatment group. These results indicate that in vivo treatment with E / N-IGF-3D cell aggregates can more effectively improve the lupus-like phenotype in MRL / Lpr mice.

[0203] 2.4 Analysis of immune cell subsets in spleen and perirenal lymph nodes of mice treated with E / N-IGF-3D cell aggregates

[0204] Compared with the PBS and TC-3D aggregate treatment groups, the E / N-IGF-3D cell aggregate treatment group showed a significant increase in the ratio of Treg cells in the spleen and lymph nodes. In addition to Treg cells, CD4+ effector T cells secreting IFNγ and TNFα, as well as CD11b+ cells, were significantly reduced in the E / N-IGF-3D cell aggregate treatment group. Figure 38 As shown in the figure. These results indicate that the E / N-IGF-3D cell aggregate treatment group can modulate immune cells, more effectively suppress inflammation, and further improve MRL / Lpr kidney injury.

[0205] In summary, this invention provides a mesenchymal stem cell aggregate jointly empowered by matrix and cytokines. This aggregate is obtained by passage-culturing novel mesenchymal stem cells in a 3D cell culture system. Specifically, the novel mesenchymal stem cells are obtained by passage-culturing mesenchymal stem cells in cadherin-rich culture medium and IGF-1. The cadherin-rich culture medium is prepared from 1-3 parts hE-cad-Fc and 1-3 parts hN-cad-Fc, and pretreated with E / N-cad and 10 ng / mL IGF-1. The resulting E / N-IGF-3D aggregates have intact structures and clear boundaries. Compared with untreated TC-3D aggregates, E / N-IGF-3D aggregates showed significantly upregulated expression of endogenous cadherin, as well as IGF-1 receptor and its phosphorylated receptor. Anti-inflammatory gene expression was also upregulated, including approximately 50% upregulation of TSG-6 gene expression, and significant upregulation (greater than 4-fold) of IL-10 and HGF gene expression. Furthermore, the cell metabolic pathways more closely mimicked the in vivo MSC metabolic patterns, which is beneficial for improving the therapeutic effect after cell reinfusion. In vitro cell studies showed that E / N-IGF-3D aggregates can effectively inhibit the proliferation of activated T cells, efficiently reverse the fibrotic phenotype of skin fibroblasts, and promote the repair of damaged endothelial cells and improve the integrity of the vascular endothelium. The E / N-IGF-3D aggregates described in this invention can treat systemic sclerosis. Compared with the untreated TC-3D cell therapy group, E / N-IGF-3D cells show significantly improved survival and anti-apoptotic ability in inflammatory environments, effectively inhibiting the immune infiltration of lymphocytes and monocytes at the lesion site, reducing pro-inflammatory CD4+ lymphocytes, and increasing anti-inflammatory Treg cells. At the same time, the E / N-IGF-3D aggregates significantly improve the efficacy of reversing dermal and pulmonary fibrosis. Furthermore, the E / N-IGF-3D aggregates significantly improve the vascular integrity and permeability of fibrotic lung tissue. The E / N-IGF-3D aggregate described in this invention can treat Sjögren's syndrome. Compared with TC-3D, the E / N-IGF-3D aggregate treatment group significantly upregulated salivary gland flow rate, increased the salivary gland / body weight ratio, reduced inflammatory cell infiltration, reduced acinar and duct atrophy, reduced salivary gland collagen deposition and fibrosis, and increased the expression of aquaporins and ion transporters in damaged salivary glands, effectively improving salivary gland function in Sjögren's syndrome.The E / N-IGF-3D cell aggregate therapy for systemic lupus erythematosus (SLE) mice described in this invention significantly inhibited perirenal lymph node and spleen enlargement and suppressed lymphocyte proliferation compared to the TC-3D treatment group. It also significantly reduced physiological and biochemical indicators in SLE mice, including urinary protein, creatinine, blood urea nitrogen levels, and various pathological parameters such as anti-dsDNA antibody levels, TNF-α and IgG concentrations in the blood. Furthermore, the E / N-IGF-3D aggregate therapy group showed a marked reduction in glomerular mesangial and basement membrane thickening, inflammatory cell infiltration, interstitial fibrosis, and occasional crescent formation. Simultaneously, the E / N-IGF-3D aggregate therapy, by regulating immune cells, more effectively suppressed inflammation and further improved MRL / Lpr renal injury.

Claims

1. A mesenchymal stem cell aggregate jointly energized by matrix and cytokines, characterized in that, The aggregates are obtained by culturing pretreated mesenchymal stem cells in a culture medium supplemented with cell culture medium and IGF-1, and then passaged in a 3D cell culture system. The cell culture medium is prepared from 1-3 parts hE-cad-Fc and 1-3 parts hN-cad-Fc. The mesenchymal stem cell aggregates jointly empowered by the matrix and cytokines were prepared by the following method: (1) Dilute 1-3 hE-cad-Fc and 1-3 hN-cad-Fc to a total protein concentration of 1-30 μg / mL, add to tissue culture plates, incubate, and wash with PBS to obtain cell culture medium; (2) Seed mesenchymal stem cells into cell culture plates cultured in step (1) and passage them to obtain matrix-empowered mesenchymal stem cells. (3) The matrix-energized mesenchymal stem cells obtained in step (2) are cultured until the cells reach 50-60% confluence. Human recombinant IGF-1 is added to the culture medium and cultured until 70-80% confluence. The cells are then digested and collected to obtain mesenchymal stem cells pretreated with cadherin and IGF-1. (4) The cells obtained in step (3) are added to a 3D cell culture system for culture to obtain mesenchymal stem cell aggregates that are jointly empowered by matrix and cytokines.

2. The mesenchymal stem cell aggregates jointly energized by matrix and cytokines as described in claim 1, characterized in that, The incubation time in step (1) is 0.5-24hrs, and the incubation temperature is 4-37℃.

3. The mesenchymal stem cell aggregates jointly empowered by matrix and cytokines as described in claim 1, characterized in that, The concentration of IGF-1 added in step (3) is 5-15 ng / mL.

4. The mesenchymal stem cell aggregates jointly energized by matrix and cytokines as described in any one of claims 1-3, characterized in that, The cells and cell aggregates highly express endogenous cadherin and phosphorylated IGF-1 receptors.

5. The method for preparing mesenchymal stem cell aggregates pretreated with cadherin and IGF-1 as described in claim 1, characterized in that, The mesenchymal stem cell aggregates jointly empowered by the matrix and cytokines were prepared by the following method: (1) Dilute 1-3 hE-cad-Fc and 1-3 hN-cad-Fc to a total protein concentration of 1-30 μg / mL, add to tissue culture plates, incubate, and wash with PBS to obtain cell culture medium; (2) Seed mesenchymal stem cells into cell culture plates cultured in step (1) and passage them to obtain matrix-empowered mesenchymal stem cells. (3) The matrix-energized mesenchymal stem cells obtained in step (2) are cultured until the cells reach 50-60% confluence. Human recombinant IGF-1 is added to the culture medium and cultured until 70-80% confluence. The cells are then digested and collected to obtain mesenchymal stem cells pretreated with cadherin and IGF-1. (4) The cells obtained in step (3) are added to a 3D cell culture system for culture to obtain mesenchymal stem cell aggregates that are jointly empowered by matrix and cytokines.

6. The use of the mesenchymal stem cell aggregates jointly empowered by matrix and cytokines as described in any one of claims 1-3 in the preparation of drugs for treating immune diseases, wherein the immune diseases are autoimmune diseases and inflammatory immune diseases, wherein the autoimmune diseases are one or more of systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, hyperthyroidism, and diabetic nephropathy, and wherein the inflammatory immune diseases are one or more of lupus nephritis, rheumatoid arthritis, ulcerative colitis, and chronic hepatitis.

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