Aggregate of matrix-enabled mesenchymal stem cells and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a matrix-enabled mesenchymal stem cell aggregate and application, and the matrix-enabled mesenchymal stem cell aggregate is obtained by subculturing matrix-enabled mesenchymal stem cells in a 3D cell culture system. The matrix-enabled mesenchymal stem cells are obtained by subculture of mesenchymal stem cells in a cell culture matrix, and the cell culture matrix is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. Endogenous epithelium cadherin and nerve cadherin are highly expressed in cell aggregation cells, so that transcription factors are promoted to enter nuclei, the expression levels of anti-inflammatory immune regulation related factors and matrix degradation and cell migration related factors are up-regulated, and activated macrophages are promoted to be polarized to M2 type macrophages; the effects of anti-inflammation, immune regulation, tissue damage repair and the like of intravenous implantation of the matrix-enabled mesenchymal stem cell aggregate for treating diseases such as hepatic fibrosis, diabetes mellitus, nerve injury and the like are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an aggregate of matrix-enabled mesenchymal stem cells and applications thereof. Background Art

[0002] Stem cell therapy is the most promising disease treatment method in the 21st century. It utilizes the unique characteristics of stem cells, including their self-renewal, differentiation and paracrine properties, and regenerates damaged cells and tissues in the human body by delivering exogenous stem cells to patients. As people's expectations for stem cell therapy for regenerative treatments are getting higher and higher, the number of applications of stem cells from various sources is increasing. Stem cell therapy has also shown a diversified development from autologous to allogeneic, and induced pluripotent stem cells to allogeneic. Induced pluripotent stem cells are artificially made by cell gene reprogramming. They have a wide range of applications, but they have genomic instability, carcinogenicity and immune rejection reactions. In 2006, the International Society for Cellular Therapy defined mesenchymal stem cells (MSCs) as adherent cells that express CD73, CD105, CD90 (≥90%) and do not express hematopoietic markers CD34, CD45, CD14, CD19 and HLA-DR (≤2%) and can adhere to plastic surfaces. Such cells must have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. MSCs can be isolated from almost all tissues of the human body, but their origin from adult tissues may limit their proliferation and differentiation capabilities. Their shortage in number still limits their clinical applications. Existing methods for the expansion and induced differentiation of MSCs are mainly two-dimensional plate culture, which has the disadvantages of reduced cell activity after long-term culture, low induced differentiation efficiency, unstable induction effect, and uncertain induction effect in vivo. Therefore, optimizing the culture program is of utmost importance while ensuring the efficacy of MSCs after proliferation and differentiation.

[0003] In order to obtain stem cells with better performance and larger numbers, technicians in this field adjust the composition of the culture medium and the culture method. For example, invention patent CN104830763B discloses that Y-27632 is added to the culture of mesenchymal stem cells. After five generations of culture, compared with the control group without Y-27632, the number of umbilical cord mesenchymal stem cells in the experimental group with Y-27632 increased by 31.87%; the number of amniotic mesenchymal stem cells increased by 30%, and the number of bone marrow mesenchymal stem cells increased by 27.27%. Invention patent CN117904047A discloses a composition for hematopoietic stem cell culture, which is composed of the following components at the following concentrations: SR1750-1000nM, TPO 40-160ng / mL, SCF 40-120ng / mL, Flt-3L 40-60ng / mL and IGFBP250-100ng / mL. Adding the above composition to the culture medium can effectively amplify hematopoietic stem cells while better maintaining the purity and cell state of stem cells and shortening the culture cycle. Invention patent CN114836372B discloses a culture method for converting mouse epiblast stem cells into new pluripotent stem cells. The obtained new pluripotent stem cells have high cell potential, fast proliferation rate, and can be stably passaged multiple times.

[0004] The research group to which the inventor belongs has long been committed to the research of cadherin, and unexpectedly obtained a mesenchymal stem cell culture matrix. The matrix-empowered mesenchymal stem cells obtained by culturing mesenchymal stem cells using the cell culture matrix can promote the nuclear transfer of β-catenin, enhance in vitro proliferation activity, maintain good in vitro proliferation ability, stemness maintenance ability, delay cell aging and apoptosis, and induce the expression of multiple functions of hMSCs. The hMSCs cultured to the fifteenth generation through hENFc empowerment are not tumorigenic and have no adverse effects on animals, which meets the clinical application standards of hMSCs.

[0005] On the basis of previous studies, the inventors prepared the cultured matrix-empowered mesenchymal stem cells into cell aggregates and found that the matrix-empowered mesenchymal stem cell aggregates have a small particle size and can be widely used in multiple scenarios including intravenous injection, local treatment, tissue engineering, in vitro organ model construction and systemic treatment. With stronger anti-inflammatory gene expression, macrophages co-cultured with 3DENM have more CD206-positive cells and fewer CD86-positive cells, which shows that more macrophages are polarized from the pro-inflammatory M1 type to the M2 type; it can promote cell migration. The study of implantation treatment of liver fibrosis showed that compared with the single cell treatment group, the homing and colonization efficiency of 3DENM to fibrotic liver tissue was improved, the number of non-fibrogenic phenotype (LY6C-) macrophages in fibrotic liver tissue increased, collagen deposition was reduced, and the reversal of liver fibrosis treatment was significantly improved. Studies on implantation therapy for diabetes have shown that it can effectively improve the physiological and biochemical indicators of diabetes (including body weight, food intake, blood sugar, blood lipids, etc.) and the levels of blood inflammatory factors (IL-1β, TNF-α and TGF-β), reduce the inflammatory response of DKD rats, and delay the progression of diabetes and its kidney disease (alleviate glomerular fibrosis and kidney damage). Summary of the invention

[0006] The primary purpose of the present invention is to provide a matrix-enabled mesenchymal stem cell aggregate, which is obtained by subculturing mesenchymal stem cells in a cell culture matrix and then culturing them in a 3D cell culture system. The cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc.

[0007] Preferably, the mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated cell culture plate, incubating, and washing to obtain a cell culture matrix hENFc;

[0008] (2) inoculating mesenchymal stem cells into a cell culture plate matrixed with hENFc, and subculturing to obtain matrix-empowered mesenchymal stem cells;

[0009] (3) resuspending the matrix-empowered mesenchymal stem cells obtained in step (2), adding them to a 3D cell culture system for culture, and obtaining mesenchymal stem cell aggregates.

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

[0011] The second object of the present invention is to provide a method for preparing the matrix-enabled mesenchymal stem cell aggregates, comprising the following steps: (1) diluting 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated cell culture plate, incubating, and washing to obtain a cell culture matrix hENFc;

[0012] (2) inoculating the mesenchymal stem cells into the cell culture plate matrixed with hENFc obtained in step (1), and subculturing to obtain matrix-empowered mesenchymal stem cells;

[0013] (3) resuspending the matrix-empowered mesenchymal stem cells obtained in step (2), adding them to a 3D cell culture system for culture, and obtaining mesenchymal stem cell aggregates.

[0014] The third object of the present invention is to provide the use of the mesenchymal stem cell aggregates in the preparation of anti-inflammatory drugs.

[0015] The fourth object of the present invention is to provide the use of the mesenchymal stem cell aggregates in the preparation of drugs for treating liver fibrosis.

[0016] The fifth object of the present invention is to provide the use of the mesenchymal stem cell aggregates in the preparation of drugs for treating diabetes.

[0017] The beneficial effects of the present invention are as follows: (1) providing a matrix-enabled mesenchymal stem cell, wherein the matrix-enabled mesenchymal stem cell is obtained by subculturing mesenchymal stem cells on a cell culture matrix, wherein the cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The matrix-enabled mesenchymal stem cells obtained by culturing the cell culture matrix of the present invention can promote the nuclear transfer of β-catenin, maintain good in vitro proliferation ability and stemness, delay cell aging and apoptosis, and promote the expression of multiple functions of hMSCs. The hMSCs cultured to the fifteenth generation by hENFc empowerment are not tumorigenic and have no adverse effects on animals, and meet the clinical application standards of hMSCs. The matrix-enabled mesenchymal stem cells are cultured in a 3D cell culture system to obtain matrix-enabled mesenchymal stem cell aggregates, which promote cell aggregation and have a small particle size, and can be widely used in multiple scenarios including intravenous injection, local treatment, tissue engineering, in vitro organ model construction, and systemic treatment.

[0018] (2) Compared with mesenchymal stem cells cultured in traditional two-dimensional method (2DM), mesenchymal stem cells cultured in two dimensions on hENFc matrix (2DENM) and mesenchymal stem cell aggregates cultured in traditional two-dimensional method (3DM), matrix-enabled mesenchymal stem cell aggregates (3DENM) have stronger anti-inflammatory gene expression. Macrophages co-cultured with 3DENM have more CD206-positive cells and fewer CD86-positive cells, which shows that more macrophages have transformed from pro-inflammatory M1 type to anti-inflammatory M2 type; it can promote cell migration and homing.

[0019] (3) The matrix-enabled mesenchymal stem cell aggregates (3DENM) described above have better therapeutic effects in treating liver fibrosis. After 3DENM implantation, more cells migrate to the diseased liver tissue, and the macrophages in the liver tissue are more likely to transform into a non-fibrogenic phenotype (LY6C-), reducing the expression of the chemokine receptor CX3CR1. 3DENM highly expresses MMP2 / 9 and reduces collagen deposition in fibrotic livers, indicating that 3DENM has better therapeutic effects in treating liver fibrosis.

[0020] (4) The matrix-enabled mesenchymal stem cell aggregates (3DENM) can better treat diabetes, effectively reduce the inflammatory response of DKD rats, delay the pathological indicators and progression of diabetes, and slow down glomerular fibrosis and kidney damage in diabetic rats.

[0021] (5) The matrix-enhanced mesenchymal stem cell aggregates (3DENM) can better treat spinal cord injury, effectively improve various pathological indicators related to lower limb movement in rats with spinal cord injury, and quickly and efficiently repair the lower limb movement function of rats with spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Preparation of hENFc Note: (A) The fixed amount-protein concentration curve of hE-cad-Fc and hN-cad-Fc; (B) The fixed amount-time curve of hE-cad-Fc and hN-cad-Fc; (C) The interaction curve between hE-cad-Fc and hN-cad-Fc; (D) The fixed amount detection of hE-cad-Fc and hN-cad-Fc on different matrix surfaces Figure 2 Characterization of hENFc Note: (A) Element analysis on different substrate surfaces; (B) Stability test on different substrates; (C) Distribution and structure test of hE-cad-Fc and hN-cad-Fc on different substrate surfaces; (D) Hydrophilicity test on different substrate surfaces

[0023] Figure 3 Detection of the proliferation ability of hMSCs at different passages on different matrix surfaces

[0024] Note: (A) cell doubling time; (B) cell number

[0025] Figure 4 Volume statistics of hMSCs at different generations on different matrix surfaces

[0026] Figure 5 Detection of endogenous cadherin expression in hMSCs of different passages on different matrix surfaces

[0027] Note: (A) Fluorescence quantitative PCR detection; (B) Western Blotting detection

[0028] Figure 6 Expression of OCT4 gene and protein in hMSCs of different passages on different matrices

[0029] Figure 7 Senescence detection of hMSCs at different passages on different matrix surfaces

[0030] Note: (A) Cell cycle detection; (B) β-galactosidase staining

[0031] Figure 8 Apoptosis detection of hMSCs at different passages on different matrices

[0032] Fig. 9 Multiple differentiation ability tests of hMSCs of different generations on different matrix surfaces Note: (A) Oil red O staining and adipogenic gene expression test; (B) Alizarin red staining and osteogenic gene expression test; (C) Alcian blue staining and chondrogenic gene expression test

[0033] Fig.10 MSC EN Oncogene and tumor suppressor gene expression dot plots

[0034] Fig.11 MSC EN Karyotype analysis

[0035] Fig.12 MSC EN Tumorigenicity assay

[0036] Note: (A) Light microscopic image of mouse tumor size; (B) Mouse weight; (C) Tumor volume

[0037] Fig.13 Light microscopic observation of cell aggregates

[0038] Note: 3DM and 3DENM before and after light microscopy

[0039] Fig.14 Preparation of cell aggregates of different sizes, light microscopic observation and particle size statistics

[0040] Fig.15Expression of E-cadherin in cell aggregates Note: (A) Protein expression and statistics of endogenous E-cadherin and N-cadherin in 3DM and 3DENM groups; (B) Immunofluorescence staining of E-cadherin and N-cadherin in 3DM and 3DENM groups

[0041] Fig.16 The cell aggregates were co-cultured with activated PBMCs to detect the immunoregulatory function of the cell aggregates. The aggregates inhibited the proliferation of PBMCs (A), inhibited the proliferation of CD4+PBMCs (B), inhibited the proliferation of CD+8PBMCs (C), promoted the apoptosis of activated PBMCs (D), and inhibited proinflammatory T lymphocytes, including TNF-α+CD4+ (E), Th1 (F), and Th17 (G).

[0042] Fig.17 Evaluation of the anti-inflammatory ability of cell aggregates Note: (A) Schematic diagram of THP-1 induction; (B) Light microscopy of M0 and M1 THP-1 derived macrophages; (C) Schematic diagram of MSC and THP-1 co-culture; (D) qPCR results of MSC anti-inflammatory genes; (E) WB results of THP-1 macrophages

[0043] Fig.18 Evaluation of cell aggregate migration and homing ability Note: (A) qPCR detection of genes related to MSC migration and homing; (B) immunofluorescence staining of MMP2 and MMP9 proteins; (C) immunofluorescence staining of CXCR4 protein; (D) WB detection of proteins related to MSC migration and homing; (E) crystal violet staining of MSC migration

[0044] Fig.19 Treatment of mice with liver fibrosis and small animal imaging

[0045] Note: (A) Schematic diagram of MSC-injected mice; (B) Small animal imaging results; (C) Statistics of small animal imaging results

[0046] Fig. 20 Mouse liver immune cell flow cytometry

[0047] Fig.21 Sirius red staining and statistics of liver in mice with hepatic fibrosis

[0048] Fig. 22 Biochemical and physiological indexes of diabetic rats Note: (A) 24-hour food intake; (B) 24-hour water intake; (C) 24-hour urine protein; (D) rat body weight; (E) kidney weight; (F) kidney index

[0049] Fig.23 Analysis of inflammatory response in diabetic rats

[0050] Fig.24Blood glucose and serum lipid profile detection in diabetic rats Note: (A) blood glucose; (B) total cholesterol; (C) total triglycerides; (D) high-density lipoprotein; (E) low-density lipoprotein

[0051] Fig.25 Pathological staining of diabetic rats

[0052] Fig.26 Renal function test in diabetic rats

[0053] Fig. 27 BBB score for spinal cord injury treatment (A, B)

[0054] Fig.28 Gait analysis of rats after spinal cord injury treatment

[0055] Fig.29 Analysis of the grid crawling test (A) and hot plate test (B) in rats after spinal cord injury treatment

[0056] Fig.30 Swimming posture (A), LSS score (B) and motor evoked potential analysis (C) of rats after spinal cord injury treatment DETAILED DESCRIPTION

[0057] Non-limiting embodiments of the present invention will be further described in greater detail by reference to specific examples, which should not be construed as limiting the scope of the present invention in any way.

[0058] In the following examples, unless otherwise specified, all reagents used can be purchased from the market.

[0059] In the following examples, unless otherwise specified, the methods used are all conventional methods.

[0060] 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, wherein hE-cad-Fc and hN-cad-Fc have been disclosed in the invention patents CN109337857B "Use of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc" and CN115040695B "Application of a fusion protein active interface based on VE-cad-Fc / N-cad-Fc" previously applied by the inventor, respectively, and the corresponding sequences are disclosed in detail, which will not be listed in this patent.

[0061] The matrix jointly modified by hE-cad-Fc and hN-cad-Fc is referred to as hENFc.

[0062] All data are expressed as mean ± standard deviation; statistical differences between multiple groups were evaluated by one-way analysis of variance (ANOVA). p<0.05 was considered statistically significant, *p<0.05, **p<0.01, ***p<0.001, ns indicated no significant difference. Unless otherwise specified, all in vitro experimental results were from 3 independent parallel experiments, and all animal experimental results were from 5 independent parallel experiments. All results were statistically analyzed using GraphPad Prism version 8.0 software.

[0063] Example 1. Preparation of hENFc matrix and matrix-empowered mesenchymal stem cells

[0064] 1. Preparation of hENFc Matrix

[0065] The purified hE-cad-Fc and / or hN-cad-Fc were diluted in PBS to a total protein concentration range of 0-30 μg / mL, and 150 μL of protein solution per square centimeter was added to tissue culture treated cell culture plates (TCPS); after incubation at 37°C for 2 hrs, the plates were further washed 5 times with PBS.

[0066] 2. Detection of adsorption amount-concentration and adsorption amount-time of hE-cad-Fc and hN-cad-Fc

[0067] To evaluate the amount-concentration relationship of hE / N-cad-Fc immobilized on TCPS, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to reach a concentration range of 0, 1, 3, 5, 10, 20, and 30 μg / mL, and 100 μL was added to each well of a 96-well plate. After incubation at 37°C for 2 hrs, the plate was further washed 5 times with PBS. To evaluate the amount-time relationship of hE / N-cad-Fc immobilized on TCPS, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to 5 μg / mL, and 100 μL was added to each well of a 96-well plate. After incubation at 37°C for 0, 30, 60, 90, 120, 150, and 180 min, the plate was further washed 5 times with PBS. Finally, the modified surface was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, each substrate 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 h; after washing three times with PBS, the modified surface was incubated with HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1:5000 dilution) for 2 h, and then TMB solution was added and incubated at 37°C in the dark; after 15 min, stop buffer was added, and the absorbance was measured at 450 nm using a microplate reader.

[0068] 3. Hydrophilicity detection, elemental analysis and morphology detection of hENFc matrix

[0069] The prepared TCPS, hEFc, hNFc and hENFc substrate surfaces were washed with ultrapure water for 5 times and then dried. The surface hydrophilicity of different surfaces was evaluated using a water contact angle (WCA) meter. The elemental changes before and after substrate surface modification were evaluated using an X-ray photoelectron spectroscopy (XPS) device using a monochromatic MgKα radiation source (1253.6eV). C1s (binding energy 284.8eV) was used as a reference for charge correction, and the high-resolution spectra of C1s, N1s, O1s and S2p were analyzed using Avantage software. The morphology and roughness of the modified surface were characterized by atomic force microscopy (AFM).

[0070] 4. Stability test of hENFc matrix

[0071] Purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 5 μg / mL, and 100 μL per well was added to a 96-well plate. After incubation at 37°C for 2 hrs, the plate was further washed 5 times with PBS. Finally, the modified surface was determined by Elisa. Briefly, each matrix was incubated with 1.0% (w / v) BSA for 1 hr to block nonspecific interactions, and then incubated with goat anti-human IgG (H+L) antibody (1:5000 dilution) for 2 hrs, and then TMB solution was added and incubated at 37°C in the dark. After 15 minutes, stop buffer was added and the absorbance was measured at 450 nm using an ELISA reader.

[0072] 5. Results

[0073] 5.1 Identification of hN-cad-Fc, hE-cad--Fc and preparation of hENFc

[0074] like Figure 1 As shown in 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 lower than 5 μg / mL, the surface protein immobilization amount is proportional to the concentration and reaches saturation when the Cad-Fc concentration is 10 μg / mL. Figure 2 B proves that the amount of surface protein immobilization is also affected by the fixation time, and the maximum amount of protein immobilization is reached when incubated for 2 hrs. Figure 1 C shows that fusion protein matrices with different ratios of hE-cad-Fc and hN-cad-Fc can be prepared by controlling the Cad-Fc concentration and fixation time. In order to avoid the interaction between the two Cad-Fc in solution, I first observed through micro-thermophoresis experiments that the minimum concentration of the two Cad-Fc interactions was 400μg / mL, which was much greater than the saturated fixed amount of protein solution concentration (10μg / mL). In order to construct cadherin fusion protein matrices with different subtypes and ratios, we kept the total concentration of hE-cad-Fc and hN-cad-Fc constant at 5μg / 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).

[0075] 5.2 Physicochemical Characterization of hENFc Matrix

[0076] 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 2B shows that these Cad-Fc matrices can be stably maintained in the culture medium for 5 days. The morphological analysis of these matrices by AFM showed that nanorod-like structures of about 5-7 nm can be clearly observed on the surface of the TCPS plate ( Figure 2 C) Figure 2 As shown in Figure D, the hydrophilicity of these Cad-Fc matrices is significantly higher than that of TCPS. Cad-Fc was effectively immobilized on the TCPS plate and enhanced the hydrophilicity of the culture plate surface.

[0077] The inventors studied the effect of hENFc matrix on hMSCs cell adhesion and proliferation. As the proportion of hE-cad-Fc in the cadherin fusion protein matrix increased, the expression of OCT4 in hMSCs increased. Among them, when the proportion of hE-cad-Fc was greater than or equal to hN-cad-Fc, the level of OCT4 gene expression in hMSCs was significantly higher than that of hMSCs cultured on the TCPS surface. In subsequent experiments, the hENFc composite matrix (hE-cad-Fc: hN-cad-Fc = 1:1) was selected as the preferred matrix representative for subsequent research.

[0078] Example 2: Preparation and function of hENFc matrix-enabled hMSCs

[0079] 1. Preparation of hENFc matrix-enhanced hMSCs

[0080] First, hMSCs were cultured at a rate of 1 × 10 per vial. 6 The cells were seeded into T-75 cell culture flasks with hEFc, hNFc or hENFc matrix and cultured continuously to the 15th generation under conventional expansion conditions to obtain empowered hMSCs of different generations.

[0081] In the following experiments, hMSCs of various generations were prepared using unmodified TCPS as a control. E hNFc-enhanced hMSCs are referred to as MSCs N hENFc matrix-enabled hMSCs are referred to as MSCs. EN hMSCs cultured normally on TCPS matrix are referred to as MSCs. T .

[0082] 2. Detection of the proliferation ability of hENFc matrix-enabled hMSCs

[0083] At the end of each passage, all cells on the surface of different matrices were collected and counted after excluding dead cells using trypan blue. Another way is to inoculate MSCs into TCPS, hEFc, hNFc or hENFc matrices, and use CCK-8 to determine cell viability after incubation for 24 and 48 hours. That is, remove the supernatant of cell culture, wash the cells three times with PBS; add 100 μL of CCK-8 (1:10 dilution) reagent to each well, incubate at 37°C for 4 hours, and use an enzyme reader to detect the absorbance at 450nm to establish a calibration curve.

[0084] In order to compare the cell proliferation rates at different passages, the doubling time (DT) and population doubling level (PDL) were calculated according to the following formula:

[0085]

[0086]

[0087] Where N(t) is the number of cells at the time of subculture, N(to) is the number of cells seeded at the previous subculture, A(48) is the cell absorbance value at 48 h, and A(24) is the cell absorbance value at 24 h.

[0088] 3. Detection of stemness of hMSCs empowered by hENFc matrix

[0089] Gene expression detection

[0090] ① RNA extraction: For cultured MSCs, discard all culture medium, wash cells with PBS for 3 times, add 1mL Trizol solution to each well to lyse cells, transfer to a 1.5mL centrifuge tube, add 200μL chloroform and shake vigorously for 15sec, incubate at room temperature for 5min, and centrifuge at 4℃, 12000rcf for 15min; aspirate the supernatant to a new centrifuge tube, add 500mL isopropanol, incubate at room temperature for 10min, and centrifuge at 4℃, 12000g for 10min; discard the supernatant, add 1mL 75% ethanol to each tube, mix well, and centrifuge at 4℃, 7500g for 5min, discard the supernatant and dry at room temperature; finally, add 20μL sterile and enzyme-free ddH2O to each tube to dissolve RNA, bathe at 55℃ for 10min, and store in a -80℃ ultra-low temperature refrigerator after aliquoting.

[0091] ② Preparation of cDNA: Take an RNase-free PCR tube and add 4μL RNase-free water, 4μL gDNA wiper Mix and 8μL template RNA to each tube according to the Vazyme instructions. After the addition is completed, centrifuge it instantly for 5 seconds in a handheld centrifuge, place it in a PCR instrument and remove genomic DNA according to the program of 42℃2min, 4℃∞; then add 4μL 5×HiScriptⅡqRTSuperMixⅡ to each tube and reverse transcribe it according to the program of 50℃15min, 85℃5sec, 4℃∞; after the reverse transcription is completed, the obtained cDNA is normalized and stored at -20℃ for short-term storage.

[0092] ③ Fluorescence quantitative PCR: After reverse transcription, add 2 μL of cDNA of each group as template to each tube according to the Vazyme instructions, add 1 μL of primers of E-cadherin, N-cadheirn, α-catenin, β-catenin, p120-catenin and β-actin respectively, and each group is supplemented with 10 μL 5×ChamQ Universal SYBR qPCR Master Mix and 7 μL RNase-free water;

[0093]

[0094]

[0095] After the sample was added, it was centrifuged for 5 seconds using a handheld centrifuge, and then placed in a fluorescent quantitative PCR instrument and amplified according to the program of 95℃30sec, 95℃3sec, 60℃10sec repeated 40 times, 95℃15sec, 60℃60sec, 95℃15sec; the data obtained used the expression of β-actin in each group as the internal reference, and three groups were set up for each sample in parallel. -ΔΔCt Method for statistical calculation.

[0096] Protein expression detection

[0097] ① Extraction of total cell protein: For cell culture samples, discard all culture medium, then wash the cells with PBS, add 100 μL of strong lysis buffer to each well and incubate on ice for 10 minutes; use cell scraper to extract all liquid and transfer to a centrifuge tube, centrifuge at 4°C, 13000 rpm for 15 minutes, and take the supernatant;

[0098] The protein concentration in each sample was determined according to the instructions of the BCA kit; finally, the loading buffer (1:5 dilution) was added to the supernatant of each tube and boiled for 10 minutes, and the aliquots were stored in a -80°C ultra-low temperature refrigerator.

[0099] ②Western Blotting

[0100] The samples were added to 10% SDS-polyacrylamide gel for electrophoresis analysis. When the sample front migrated to the distance from the bottom of the glass plate, the electrophoresis was terminated. The absorbent sponge, PAGE gel, PVDF membrane, and absorbent sponge were clamped in order using a transfer clip, placed in an electrotransfer instrument, and electrotransfer buffer was added for 90 min at 100 V. After the transfer was completed, the PVDF membrane was taken out and blocked with 5% BSA at room temperature for 2 h. Then, mouse anti-E-cadherin antibody (intracellular domain, 1:2000 dilution), rabbit anti-N-cadherin antibody (intracellular domain, 1:2000 dilution), mouse anti-α-catenin protein antibody (1:20 00 dilution), mouse anti-β-catenin protein antibody (1:2000 dilution), mouse anti-p120-catenin protein antibody (1:2000 dilution) and mouse anti-human β-actin (1:5000 dilution) were incubated at 37°C for 2h or 4°C overnight, and then washed with TBST solution at room temperature for 5min, repeated three times; HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1:2000 dilution) was added and incubated at room temperature for 2h; then washed with TBST solution at room temperature for 5min, repeated 3 times; finally, an appropriate amount of color developing solution was added to the PVDF membrane, and the membrane was observed and photographed using a Western Blotting exposure instrument, and the graphics were statistically analyzed using Image J.

[0101] MSCs were harvested at passages 6, 11, and 15. T and MSC EN Total RNA and total protein were extracted using the above method; the expression of E-cadherin, N-cadherin and OCT4 genes and proteins were detected by fluorescent quantitative PCR and Western Blotting.

[0102]

[0103] 4. Cycle detection of hENFc matrix-enhanced hMSCs

[0104] At the 6th, 11th and 15th passages, 1×10 6 MSC T and MSC EN The cells were fixed in 70% cold ethanol for 2 h to overnight; after washing with PBS three times, the cells were centrifuged at 1000 rpm for 10 min at room temperature to obtain a cell pellet; 100 μL RNase A solution was added to each sample, the cells were resuspended and then placed in a 37°C water bath for 30 min; 400 μL PI staining solution was added and mixed, and incubated at 4°C in the dark for 30 min; the red fluorescence at an excitation wavelength of 488 nm was recorded by flow cytometry.

[0105] 5. Senescence detection of hENFc matrix-enhanced hMSCs

[0106] For senescence-associated β-galactosidase assay, MSCs were detected using the SA-β-gal staining kit at passages 6, 11, and 15. T and MSC EN Briefly, cells were cultured on modified or non-modified 6-well plates until 80% confluence, washed with PBS and fixed with 4% formaldehyde for 15 min; then, cells were incubated with freshly prepared β-galactosidase staining solution in a CO2-free oven overnight at 37°C; after washing with PBS three times, photos were taken using an optical microscope, and the number of cells positively stained for β-galactosidase was counted for every 200 cells randomly selected in the field of view.

[0107] 6. Apoptosis detection of hENFc matrix-enhanced hMSCs

[0108] At passages 6, 11, and 15, 1 × 10 6 MSC T and MSC EN Cells; resuspend the cells with 1ml of Binding Buffer diluted with deionized water (diluted 1:9); add 5μL of AnnexinV / FITC to every 100μL of cell suspension and mix well, incubate at room temperature in the dark for 5min; add 5μL of propidium iodide solution and 400μL of PBS, place in a 5ml flow tube, and immediately perform flow cytometry detection. In addition, the unstained cells are used as blank tubes; the tubes without propidium iodide and only AnnexinV / FITC are used as single staining tubes.

[0109] 7. Detection of differentiation potential of hMSCs empowered by hENFc matrix

[0110] The sixth generation MSC T 11th generation MSC T and 11th generation MSC ENThe cells were cultured in differentiation medium for 14 days, and the tri-lineage differentiation ability of hMSCs was detected. For osteogenic differentiation: osteogenesis was induced by culturing cells in DF12 supplemented with 50 μg / mL ascorbic acid 2-phosphate, 10 nM dexamethasone (Dex), 10 mM β-glycerophosphate and 10% FBS, 1% penicillin-streptomycin, and mature cells after differentiation were stained with Alizarin Red S solution; for adipogenic differentiation: adipogenesis was induced by culturing cells in DF12 supplemented with 10% FBS, 50 μg / mL ascorbic acid 2-phosphate, 1 μM Dex, 0.1 mM indomethacin and 4 U / L insulin, and mature cells were detected using Oil Red O staining kit; for chondrogenic differentiation: chondrogenic differentiation was induced by culturing cells in DF12 supplemented with 2% FBS, 50 μg / mL ascorbic acid 2-phosphate, 50 μg / mL L-proline, 100 nm Dex, 10 ng / mL TGF-β1, 1% human insulin, transferrin and sodium selenite were added to DF12 cells to induce chondrogenesis, and toluidine blue chondrogenic solution was used to detect mature cells. At the same time, PCR was used to detect the expression of genes related to the three-phase differentiation using the following primers:

[0111]

[0112] 8. Detection of tumorigenicity of hENFc matrix-enhanced hMSCs

[0113] To examine the tumorigenicity of hENFc-enabled hMSCs, 1×10 7 6th generation MSC T (n=3) and 15th generation MSC EN (n=6) The cells were subcutaneously transplanted into the abdomen of 4-week-old NOD-SCID mice; the weight of the mice was measured every 3 days and the volume of the tumor was measured according to the following formula. The mice were killed after seven weeks of continuous feeding. 7 Mice bearing human glioblastoma (U87) (n=3) served as positive controls, and mice injected with an equal volume of PBS (n=3) served as negative controls.

[0114]

[0115] 9. Results

[0116] 9.1 Effect of hENFc matrix on the proliferation of hMSCs expanded in vitro

[0117] like Fig.16 As shown in Figure 2, although the proliferation capacity of all cells gradually weakened with the passage of cells, the number of cells harvested in each passage also gradually decreased ( Figure 3 However, hMSCs (MSC T), the proliferation ability of hMSCs cultured in the cadherin fusion protein matrix was well maintained, especially hMSCs cultured in the hENFc matrix (MSC EN ), the cell doubling time remained within 25 hrs until the eleventh generation, and the proliferation capacity did not change significantly. This indicates that the hENFc matrix effectively maintains the in vitro proliferation activity of hMSCs through the cell-cell interaction mediated by biomimetic cell E-cadherin and N-cadherin. In addition, the size of hMSCs cultured in the hENFc matrix did not change with the passage expansion. EN The volume is always 2000μm 3 (diameter is about 16μm) Figure 4 ).

[0118] 9.2 Effect of hENFc matrix on the maintenance of stemness of in vitro expanded hMSCs

[0119] like Figure 5 As shown in the figure, hMSCs cultured in vitro lost the expression of E-cadherin. Although the expression of N-cadherin was retained, it was gradually lost as the culture progressed. The use of biomimetic hENFc matrix culture restored the expression of endogenous E-cadherin in hMSCs and maintained the presence of N-cadherin. E-cadherin plays a key role in maintaining the stemness of stem cells. We continued to investigate the expression of hMSCs stemness indicator OCT4 in long-term passage. Gene and protein detection results showed that, compared with MSC T In comparison, MSC EN The stemness of MSCs was maintained in serial passages, and the sixth, eleventh and fifteenth passages EN The stemness is higher than that of MSC T . More importantly, the eleventh generation MSC EN The stemness is significantly better than that of the sixth generation MSCs T ( Figure 6 )

[0120] 9.3 Effects of hENFc matrix on senescence and apoptosis of hMSCs expanded in vitro

[0121] Replicative senescence is the biggest obstacle to hMSCs culture in vitro, which leads to the loss of cell proliferation ability and changes in secretion profile, limiting the therapeutic efficacy of cells. Through cell cycle detection, we found that each generation of MSCs EN The number of cells in S phase was significantly higher than that of MSCs T β-galactosidase staining further confirmed that MSC EN The percentage of senescent cells in the MSCs was always below 10% and significantly less than that in the T ( Figure 7 ). The result is Figure 8 As shown, after long-term in vitro culture, MSC T A large number of apoptosis occurred, and MSC EN The proportion of apoptotic cells did not change significantly with the increase in passage number, which made the eleventh generation MSCs EN The proportion of senescent and apoptotic cells in the sixth generation MSCs T Basically the same.

[0122] We further investigated the differentiation capacity of hMSCs at each generation and compared it with the eleventh generation MSCs. T (MSC T -P11) compared to the eleventh generation MSC EN (MSC EN -P11) and the sixth generation of MSC EN (MSC T -P6) found more significant Oil Red O-positive lipid droplets and PPAR-γ, CEBPA, and LPL gene expressions ( Fig. 9 A), MSC EN -P11 and MSC T There was no statistical difference between P6 and P7. This proves that hENFc matrix can enhance the ability of cultured MSCs. EN -P11 and conventionally cultured MSCs T -P6 has the same adipogenic differentiation properties. EN -P11 cells were also detected in the cells after directed differentiation by alizarin red S staining and toluidine blue staining. Fluorescence quantitative PCR detection of the expression of mature bone and cartilage markers also confirmed that MSC EN -P11 and MSC T -P6 has the same osteogenic ( Fig. 9 B) and cartilage ( Fig. 9 C) differentiation potential.

[0123] 9.3 Safety Analysis of hENFc Matrix-Enabled hMSCs

[0124] The in vitro expansion and culture of MSCs inevitably leads to cell replicative senescence, which in turn leads to cell genomic instability, a serious defect that hinders the development of MSC-based cell therapy industry. Therefore, we examined the use of hENFc matrix to empower MSCs cultured to the eleventh generation. EN Compared with conventional culture to the sixth generation of MSC T The results of the analysis of the expression levels of genes related to genome stability transformation in transcriptome sequencing data showed that ( Fig.10 ), 11th generation MSCEN MSCs cultured normally at the sixth generation T There was no statistical difference in the expression of oncogenes and tumor suppressor genes between cells, and there was no significant change in the expression levels of c-Myc, P21, P53 and P16 genes. Karyotype analysis did not find clones with chromosomal abnormalities, and no chromosomal translocation, deletion or change in chromosome number was observed. hENFc-enabled hMSCs maintained chromosomal stability ( Fig.11 ).

[0125] Tumorigenicity is another risk factor that needs to be considered in the clinical application of MSCs. Compared with iPSCs and ESCs, although MSCs themselves do not have tumorigenicity, genomic instability and the possibility of forming teratomas are very low, in order to ensure the safety of hENFc matrix for long-term empowerment of hMSCs in clinical applications, we will use MSCs that meet clinical standards T hENFc empowers MSC cultured to the fifteenth generation EN and human astrocytic glioblastoma cells (U87) were injected subcutaneously into nude mice. Seven weeks later, the potential for these cells to form tumors was studied. Fig.12 As shown in A, mice inoculated with U87 developed soybean-sized lumps at the injection site, while mice inoculated with MSC T and MSC EN In order to monitor the overall health of the mice, we measured the body weight of the mice. The results showed that the nude mice inoculated with PBS, MSC T MSC EN There was no statistical difference in body weight between the mice of the U87 group and the mice of the Fig.12 B) Seeding MSCs T and MSC EN No bulge formed at the inoculation site of the mice. Fig.12 C) These results indicate that hMSCs cultured to the fifteenth generation after hENFc empowerment are not tumorigenic and have no adverse effects on animals, which meets the clinical application standards of hMSCs.

[0126] Example 3: Preparation of matrix-empowered mesenchymal stem cell aggregates

[0127] 1. Experimental Methods

[0128] 1.13 Preparation and characterization of DENM

[0129] (1) Preparation of 2DENM

[0130] First, hMSCs were cultured at a rate of 1 × 10 per vial. 6The cells were inoculated into a hENFc matrix T-75 cell culture flask and cultured continuously to the 6th generation under conventional expansion conditions to obtain empowered hMSCs (hereinafter referred to as 2DENM). The above experiment used hMSCs prepared with unmodified TCPS matrix (hereinafter referred to as 2DM) as a control.

[0131] (2) Preparation and characterization of 3DENM

[0132] According to 100, 400 and 800 cells per aggregate, 2DM and 2DENM were resuspended in 1mL DMEM / F12 medium, and then evenly added to the AggreWell cell culture plate, centrifuged at room temperature for 5 minutes at 1000rpm; placed in a cell culture incubator (5% CO2, saturated humidity, 37°C) and cultured for 12 hours; observed and photographed under an inverted phase contrast microscope, then gently blown up with a blunt gun tip and transferred to a 15mL centrifuge tube to obtain the corresponding cell aggregates 3DM and 3DENM. ImageJ was used to outline the diameter of the cell aggregates, spatially calibrate the images (μm / pixel), and count the size of the aggregates. For each independent experiment, 100 cell aggregates were measured under each condition.

[0133] 1.2 Western blotting

[0134] Proteins were extracted from cells, and cells in two-dimensional culture and three-dimensional culture were extracted using a cell scraper or ultrasonic disruptor. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. Nonspecific binding sites of the PVDF membrane were blocked with skim milk, followed by incubation of the primary antibody with the target protein, and then washed to remove unbound antibodies. Secondary antibodies were then incubated to bind to the antibodies immunoreactive with the primary antibody, and washed again to remove unbound secondary antibodies. Fluorescent substrates were added for chemiluminescent reaction to observe the signal of the target protein, and the protein signal was captured using a Western blot imaging system, and the signal was quantitatively analyzed using image analysis software to compare the protein expression levels under different conditions.

[0135] 1.3 Fluorescence quantitative PCR

[0136] Referring to the method of Example 2, RNA was extracted and converted into cDNA, and PCR was performed using the following primers to detect the expression of related genes:

[0137]

[0138] 1.4 Immunofluorescence staining

[0139] The samples were fixed with 4% paraformaldehyde, and then perforated with PBS containing 3% BSA and 0.1% TritonX-100 for 10 minutes. Then, the samples were blocked with PBS containing 3% BSA for one hour, and the primary antibody solution was prepared with blocking solution and incubated overnight. After washing away the unbound primary antibody, the fluorescent secondary antibody was incubated overnight. Finally, after washing away the unbound secondary antibody, the samples were sealed with a sealing agent containing DAPI and observed under a fluorescence microscope.

[0140] 1.5 Co-culture of aggregates with activated PBMCs from peripheral blood

[0141] PBMCs were isolated from whole blood donated by healthy donors, and these lipid layers were obtained by Histopaque density gradient centrifugation (ρ=1.077 g / cm3); TC-3D and EN-3D aggregates were then inoculated into low-adhesion treated 96-well plates; DF12 medium was replaced with RPMI-1640 medium supplemented with 10% FBS, and PBMCs were added to the culture medium of TC-3D and EN-3D aggregates at a ratio of 10:1, respectively; 50 μg / mL of plant lectin (PHA) was added to activate PBMCs; after 3 days of co-culture, all the culture medium was aspirated and centrifuged at 2000 rpm for 10 min, and the cell pellet was collected for further detection; PBMCs cultured alone were used as negative controls, and each group of experiments was repeated 6 times.

[0142] For the PBMC proliferation test, the separated PBMC suspension was centrifuged at 300×g for 10 min, and all the supernatant was discarded; 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, and 2 μM CFSE was added. After thorough mixing, the cells were cultured at 37°C, 5% CO2 for 10 min; 3 volumes of pre-cooled RPMI1640 culture medium were added and the culture was terminated at 4°C for 5 min; centrifuged at 300×g for 10 min, the supernatant was discarded, 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, and the cells were centrifuged at 300×g for 10 min, the supernatant was discarded, and the co-culture experiment was then carried out with the obtained cells; after the co-culture was completed, the cells in the culture medium were collected and analyzed by flow cytometer.

[0143] For PBMC apoptosis detection, the cell pellet obtained after the above co-culture was analyzed according to the cell apoptosis experiment.

[0144] For the determination of T lymphocytes and T lymphocyte subsets in PBMC cells, 1 ml of The cells were resuspended in PBS, centrifuged at 300×g for 10 min, and the supernatant was discarded. The PBMC subpopulations were characterized using the following antibodies: CD4+T cells (FITC-labeled anti-human CD4 antibody, diluted 1:500), CD8+T cells (FITC-labeled anti-human CD8 antibody, diluted 1:500), pro-inflammatory T lymphocytes (FITC-labeled anti-human CD4, APC-labeled anti-human TNF-a antibody, diluted 1:500), Th1 (FITC-labeled anti-human CD4, APC-labeled anti-human IFN-γ antibody, diluted 1:500), Th17 (FITC-labeled anti-human CD4, APC-labeled anti-human IL17A antibody, diluted 1:500). After adding antibodies, the cells were incubated at 4°C in the dark for 30 min; 1 ml PBS was added to wash the cells once, centrifuged at 300×g for 10 min, the supernatant was discarded, 500 μL PBS was added to resuspend, and the cells were filtered and analyzed by flow cytometry.

[0145] 1.6 THP-1 cell culture, macrophage differentiation and polarization

[0146] The cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. 6 cells / mL. Subsequently, THP1 macrophage differentiation induction was performed by placing THP1 cells in a culture medium containing 100ng / mL12-Phorbol-13-myristateacetate (PMA) for 24 to 48 hours. After induction, THP1 cells will differentiate into macrophages, and the morphological changes can be observed under a microscope, and the differentiation effect can be confirmed by detecting the expression of macrophage markers such as CD11b or CD14 by flow cytometry. The culture medium was then replaced with 20ng / mL IFN-γ and 100ng / mL lipopolysaccharide for polarization for 24h to form M1 macrophages.

[0147] 1.7 Co-culture of hMSCs and THP-1 differentiated M1 macrophages

[0148] THP-1 cells were cultured in a well plate to differentiate into macrophages and polarize into M1 type. MSCs cell suspension was added to the upper chamber of Transwell, and after it adhered to the wall, the Transwell was transferred to the THP-1 well. The assembled Transwell culture system was placed in an incubator and cultured for 48 hours, and samples were taken from the upper and lower chambers for subsequent analysis.

[0149] 2. Results

[0150] 2.13 Preparation of DENM cell aggregates and their cadherin expression

[0151] like Fig.13 As shown in the figure, light microscopy results showed that both groups were able to form smooth round cell aggregates after 12 hours. Fig.14 As shown in the figure, all cells aggregated to form cell aggregates with complete structures and clear boundaries after 12 hours of culture. The diameter of the aggregates (3DENM) formed by 800 2DENM cells is about 117μm, the diameter of the aggregates formed by 450 2DENM cells is about 101μm, and the diameter of the aggregates formed by 100 2DENM cells is only about 70μm. Aggregates of different sizes can be suitable for different application scenarios, such as intravenous injection, local treatment, tissue engineering, in vitro organ model construction and systemic treatment.

[0152] WB results showed that the expression of endogenous E-cadherin in cells was significantly increased in the 3DENM group ( Fig.15 A), immunofluorescence results showed that E-cadherin in the 3DENM group was distributed in clusters and N-cadherin between cells ( Fig.15 B).

[0153] 2.23 Evaluation of DENM immune regulation and anti-inflammatory function

[0154] TC-3D cell aggregates and EN-3D cell aggregates were co-cultured with PBMC for 3 days, and the flow cytometry results (such as Fig.16 As shown in Figure 2, EN-3D cell aggregates significantly inhibited the proliferation of activated PBMCs compared with TC-3D, with an inhibition rate of approximately 17.5% ( Fig.16 A), and the CD4+PBMC inhibition rate was about 19.9% ​​( Fig.16 B), CD8+PBMC inhibition rate was about 11.5% ( Fig.16 C), EN-3D cell aggregates effectively promoted the apoptosis of activated PBMCs, with the apoptosis rate increased by 7.7% ( Fig.16 D), and at the same time, EN-3D cell aggregates significantly inhibited pro-inflammatory T lymphocytes, including TNF-α+CD4+( Fig.16 E), Th1( Fig.16 F) and Th17( Fig.16 G) Cells.

[0155] Polarization of human THP-1 monocytes into M1 pro-inflammatory macrophages ( Fig.17 A, B), and then co-cultured with MSCs ( Fig.17 C). The results showed that compared with 2DM cultured in the traditional way and 2DENM cultured in 2D, 3DENM had stronger expression of anti-inflammatory genes, such as COX2, TSG6, IL10 and IDO ( Fig.17D). Macrophages co-cultured with 3DENM had more CD206-positive cells and fewer CD86-positive cells ( Fig.17 E), that is, more macrophages transform from the pro-inflammatory M1 type to the M2 type.

[0156] 2.33DENM homing effect evaluation

[0157] CCR2 and CXCR4 are important molecules for MSC to sense chemokines in the lesion site and cross the endothelium. Fluorescence quantitative PCR results showed that CCR2 and CXCR4 were significantly overexpressed in 3DENM. The 3DENM group more strongly expressed the MMP2 and MMP9 genes that degrade the extracellular matrix ( Fig.18 A). WB also showed the same results ( Fig.18 B), while FAK related to cell migration was significantly activated in the 3DENM group. Immunofluorescence results showed that 3DENM secreted more MMP2 ( Fig.18 C); Cell staining results showed that the number of cells in the 3DENM group that successfully migrated was greater than that in the 3DM group, with significant differences ( Fig.18 D).

[0158] Example 4: Application of matrix-empowered mesenchymal stem cell aggregates in the treatment of liver fibrosis

[0159] 1. Experimental Methods

[0160] 1.1 Preparation of mouse fibrosis model and cell implantation

[0161] The mouse liver fibrosis model was established by intraperitoneal injection of 10% CCl4 solution twice a week, 200 μL each time, and the control group was injected with an equal volume of olive oil. Eight weeks after the injection, the stem cell aggregates prepared in Example 3 (3DENM in the experimental group and 2DM and 2DENM in the control group) were implanted through the tail vein. Each mouse was implanted with 10 6 MSCs and sham-operated group were implanted with an equal volume of PBS.

[0162] 1.2 In vivo imaging of mice with liver fibrosis

[0163] Before in vivo imaging of small animals, MSCs were stained with DiR dye according to the instructions. In vivo imaging of mice was performed 24h and 48h after MSCs injection. Before imaging, mice were anesthetized with ether and fixed on the imaging table, and appropriate imaging parameters were set and the imaging process was started. The fluorescence signals of the imaging results were observed and counted.

[0164] 1.3 Flow cytometry of livers from mice with hepatic fibrosis

[0165] Mouse livers were collected for grinding, filtration, and density gradient centrifugation to obtain immune cells. The cells were resuspended in PBS, incubated with primary antibodies, and unbound antibodies were washed away. Fluorescently labeled secondary antibodies were added for incubation, and unbound antibodies were washed away. Injected into a flow cytometer and flow cytometry analysis was performed according to the instrument settings. After obtaining the data, FlowJo software was used for statistical analysis and chart drawing.

[0166] 1.4 Sirius red staining of livers of mice with hepatic fibrosis

[0167] Mouse liver was fixed with 4% paraformaldehyde, embedded in paraffin and sliced. After dewaxing, an appropriate amount of Sirius red staining reagent was taken and the staining working solution was prepared according to the ratio in the kit instructions. Staining was performed for 30 minutes to 1 hour, and the staining time was adjusted as needed. After washing away the excess staining solution with PBS, alcohol was used for step-by-step dehydration, transparentized in an organic solvent, sealed with a neutral resin, and observed after taking pictures with a film scanner.

[0168] 2. Results

[0169] like Fig.19 As shown in A, the constructed cell aggregates were implanted into mice with liver fibrosis. Small animal imaging and statistical results showed that the 3DENM group had less lung retention and more homing to the liver ( Fig.19 B&C).

[0170] Flow cytometry results showed that 3DENM implantation caused more macrophages to transform into a non-fibrogenic phenotype (LY6C-), and macrophages expressed less chemokine receptor CX3CR1 ( Fig. 20 ). Sirius red staining results showed that the 3DENM treatment group had less collagen deposition and better therapeutic effect ( Fig.21 ).

[0171] Example 5: Application of matrix-empowered mesenchymal stem cell aggregates in the preparation of drugs for treating diabetes

[0172] 1. Experimental Methods

[0173] 1.1 Preparation of rat diabetic model and cell implantation

[0174] All 30 SD rats were adaptively fed for 1 week, and then fed with a high-fat diet (n=20) and a normal diet (n=10) for 8 consecutive weeks to ensure that the rats had normal blood sugar (blood sugar <250 mg / dL); after eight weeks, all rats were fasted for 12 hours, and rats fed with a high-fat diet were intraperitoneally injected with STZ dissolved in sodium citrate solution at 35 mg / kg, while rats fed with a normal diet were intraperitoneally injected with an equal amount of sodium citrate solution. Blood sugar was continuously tested for 3 days. When the blood sugar concentration of rats in the high-fat diet-fed and STZ-injected group was ≥250 mg / dL, it indicated that the type 2 diabetes model was successfully established.

[0175] After the type 2 diabetes model was successfully established, the rats were gradually fed a normal diet, the rats were observed, and their blood and urine indicators were tested; after 2 weeks of continuous culture, the rats' 24-hour urine was collected. When the urine protein was >20 mg / 24 hours and significantly higher than that of the normal control group, it was considered that renal damage occurred at this time, and the diabetic nephropathy (DKD) rat model was successfully established. All animal husbandry and experiments strictly followed the animal care and use guidelines approved by Nanjing University Drum Tower Hospital, and appropriate measures were taken to ensure that the animals had minimal pain and discomfort.

[0176] After the successful establishment of the diabetes model, the rats were randomly divided into a disease group injected with normal saline (DKD), a treatment group injected with MSC (KDK+2DM), a treatment group injected with EN-MSC (KDK+2DENM), and a treatment group injected with 3DENM (KDK+3DENM). Normal rats without modeling were used as the control group (Health). There were at least 5 rats in each group. The treatment groups were injected with 2×10 6 The modeling group was injected with 500 μL of normal saline.

[0177] 1.2 Collection of blood, urine and tissue specimens from diabetic rats

[0178] Rats were weighed weekly. Blood was collected from the end of the tail vein to measure blood glucose. One day before the end of the experiment, urine and feces of rats were collected in a metabolic cage and frozen in a -80°C refrigerator. After two weeks of continuous treatment, the weight of the rats was first recorded, and then the rats were anesthetized, the chest cavity of the rats was opened, and the heart was punctured at the left atrial appendage with a 5mL syringe to collect blood and placed in an EP tube. Centrifuged at 300×g for 10 minutes, the upper liquid was aspirated, and the supernatant was aspirated as serum after standing for 30 minutes, and stored in a -80°C refrigerator for later use. After that, the abdominal cavity of the rats was opened, and the kidneys and spleen were taken out for weighing. Some kidneys were immediately placed in liquid nitrogen for storage; some kidneys were placed in formaldehyde for fixation overnight, and the specimens were treated with 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol in order from low concentration to high concentration for 2 hours, dehydrated with anhydrous ethanol twice, each time for 1 hour, and then the tissue block was placed in xylene for transparency, and the transparent tissue was placed in molten paraffin. After the paraffin was completely immersed in the tissue block, it was embedded, cooled and fixed into blocks, and the pre-cooled wax blocks were fixed on the microtome, cut into thin slices, put in heated water to flatten, attached to the slide, dried in a 45℃ constant temperature box, and stored at room temperature; some kidneys were placed in formaldehyde for fixation overnight, dehydrated with 30% sucrose solution until the kidney sank to the bottom, the tissue was removed, washed twice with pre-cooled PBS, and an appropriate amount of OCT embedding agent was added to immerse the tissue, the pre-cooled OCT was fixed on the microtome, cut into thin slices, attached to the slide, and stored in a -80℃ refrigerator for later use.

[0179] 1.3 Detection of biochemical indicators in diabetic rats

[0180] ① The renal index is calculated as follows:

[0181]

[0182] ②Urine indicators:

[0183] After the rat urine was equilibrated to room temperature, it was centrifuged at 1000×g for 10 min to obtain the supernatant; the rat urine was collected using a metabolic cage, and the mouse urine to be tested was diluted to an appropriate concentration, so that the total volume of the sample was diluted to 5 μL, 195 μL of Coomassie Brilliant Blue G250 solution was added, mixed thoroughly, and allowed to stand for 2 min; the absorbance at 595 nm was measured, and the urine protein concentration was calculated.

[0184] ③ Blood indicators:

[0185] The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) contents of rats were detected by using the fully automatic chemical analyzer of Nanjing Drum Tower Hospital; the contents of TNF-α, IL-1β and TGF-β in rat serum were detected. All supernatants were centrifuged at 300×g for 10 min to remove the precipitate; 300 μL of diluted washing solution (1:20 dilution) was added to soak the plate for 30 seconds; all washing solution was discarded and patted dry on absorbent paper, and 100 μL of culture medium or cell culture supernatant sample or diluted standard was added to each well; 50 μL of detection antibody (1:100 dilution) was added to each well; Incubate at room temperature for 2 hours; discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times, discard the washing solution after each wash and pat the plate dry; add 100 μL of horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well and incubate at room temperature for 45 minutes; discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times, discard the washing solution after each wash and pat the plate dry; add 100 μL of chromogenic substrate TMB to each well and incubate at room temperature in the dark for 30 minutes; add 100 μL of stop solution to each well, at which time the color changes from blue to yellow; within 30 minutes, use an enzyme reader for dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm, and calculate the concentration.

[0186] 1.4 Pathological staining of diabetic rat kidneys

[0187] The prepared frozen sections were treated with xylene for 20 min, anhydrous ethanol for 3 min, 95% ethanol for 3 min, 95% ethanol for 3 min, 90% ethanol for 3 min, 80% ethanol for 3 min, and 70% ethanol for 3 min to dewax and hydrate the paraffin sections.

[0188] ① For H&E staining: Place the processed sections in a hematoxylin staining box and stain for 5 minutes; wash off the floating color in water and rinse gently with running water for 10 minutes; treat with hydrochloric acid and alcohol for 1 second; wash off the floating color in water and rinse with running water for 8 minutes; stain with eosin for 90 seconds; wash off the floating color in water and rinse gently with running water for 10 minutes.

[0189] ② For PAS staining: Place the processed sections in periodic acid solution and oxidize at room temperature for 25 minutes; rinse twice with running water for 2 minutes each time, and gently rinse twice with distilled water for 2 minutes each time; stain with Schiff Reagent at 37℃ in the dark for 10 minutes; wash twice with sodium sulfite solution for 2 minutes each time; rinse twice with running water for 2 minutes each time, and gently rinse twice with distilled water for 2 minutes each time; stain in a hematoxylin staining box for 5 minutes; wash off floating color in water, and gently rinse with running water for 10 minutes.

[0190] ③ For Masson staining: Place the processed sections in Weigert iron hematoxylin staining solution for 10 minutes; rinse with running water for 10 minutes; return to blue with Masson bluing solution for 5 minutes; rinse with running water for 10 minutes, rinse twice with distilled water, 2 minutes each time; stain with Ponceau fuchsin staining solution for 10 minutes; wash with weak acid working solution (distilled water: weak acid solution = 2:1) for 1 minute; wash with 1% phosphomolybdic acid solution for 2 minutes; wash with weak acid working solution for 1 minute; directly place in aniline blue staining solution for 2 minutes; wash with weak acid working solution for 1 minute.

[0191] After all samples were stained, they were dehydrated by treating with 70% alcohol for 1 sec, 80% alcohol for 1 sec, 85% alcohol for 1 sec, 90% alcohol for 1 sec, 95% alcohol for 30 sec, and anhydrous ethanol for 30 sec; dried in a ventilated place, immersed in xylene for 5 min; immersed in fresh xylene for 5 min; air-dried in a fume hood, sealed with neutral resin, and stored at room temperature.

[0192] 1.5 Immunofluorescence staining of diabetic rat kidneys

[0193] The prepared frozen sections were rinsed with running water for 5 minutes to wash off the OCT, fixed with 4% (v / v) paraformaldehyde for 10 minutes, incubated with 0.1% TritonX-100 for 10 minutes, and blocked with 1% (w / v) BSA for 30 minutes at 37°C; then 200 μL of mouse anti-human-E-cadherin (intracellular domain, 1:250 dilution) and rabbit anti-human-N-cadherin (intracellular domain, 1:250 dilution) were added to each well and incubated overnight at 4°C, and then 200 μL of AlexaFluor488-conjugated goat anti-mouse antibody (1:250 dilution) and AlexaFluor594-conjugated goat anti-rabbit antibody (1:250 dilution) were added to each well and incubated at room temperature for 2 hours; then 200 μL of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was added to each well to mount the sections and observed under a laser confocal scanning microscope within one week. The expression and distribution of synaptopodin in different renal tissues were detected.

[0194] 1.6 Western blotting of rat kidney

[0195] First, weigh 50 mg of kidney tissue and place it in a 1.5 mL centrifuge tube, add 0.5 mL of protein lysis buffer; homogenize with a tissue homogenizer in an ice bath; then centrifuge at 4°C and 12000 rpm for 15 min, take the supernatant and measure the protein concentration and Western Blotting experiment according to the method in 2.3.7.2, and detect the protein level of Nephrin, WT1 and Podocalyxin expression in the kidneys of different treatments.

[0196] 2 Experimental results

[0197] like Fig. 22 As shown in the results, during the diabetic rat model (DKD) and its hMSCs intervention, we observed that DKD rats significantly increased their feed and water intake compared with the healthy group. Rats in the hMSCs-treated group reduced their water and feed intake compared with DKD rats. Two weeks after hMSCs transplantation, the body weight of DKD rats was significantly reduced. More importantly, the therapeutic effect of 3DENM was significantly improved compared with the treatment of 2DM and 2DENM.

[0198] Cell damage and release of inflammatory factors caused by inflammatory response induce fibroblast activation, leading to renal interstitial fibrosis. Fig.23 As shown in the figure, compared with the DKD group, the mRNA levels of IL-1β and TNF-α in the kidneys of rats in the hMSCs treatment group were reduced. Compared with the treatment of 2DM and 2DENM, the expression of IL-1β and TNF-α in the kidneys of rats in the 3DENM treatment group was reduced, and the expression of TGF-β was further reduced, indicating that 3DENM can effectively reduce the inflammatory response of DKD rats, thereby delaying the progression of diabetic nephropathy.

[0199] The results are as follows Fig.24 As shown, two weeks after MSCs transplantation, blood glucose concentration in DKD rats was slightly improved. In addition, common complications of DKD also involve impaired lipid profiles, specifically increased TC, TG, LDL-C and decreased HDL-C. Compared with rats in the untreated group and the 2DM treatment group, the blood lipid levels of rats in the 3DENM treatment group tended to be normal. These results indicate that the empowered culture of hENFc matrix effectively enhances the potential of hMSCs to slow down kidney damage in diabetic rats and improves renal function in DKD rats.

[0200] like Fig.25 As shown in the figure, H&E and PAS staining showed that compared with the Health group, the rats in the DKD group had glomerular hypertrophy and obvious mesangial matrix proliferation; compared with the 2DM treatment group, the glomerular volume of the rats in the 3DENM treatment group was basically normal, and the mesangial matrix was significantly reduced. Masson staining results showed that the collagen deposition in the glomerular mesangial area of ​​the rats in the DKD group increased, and the transplantation of 3DENM effectively reduced glomerular fibrosis; these results indicate that 3DENM effectively improved the renal injury of DKD rats and had a stronger protective effect on the kidneys of DKD rats.

[0201] Synaptopodin is related to maintaining the structure and function of the glomerular filtration membrane. It is involved in regulating the morphology and dynamic structure of podocytes and is essential for maintaining the filtration function of the glomerulus. Uremia-related protein (Nephrin) is closely related to the selective permeability of the glomerular filtration membrane. It constructs the interstitial membrane of podocytes by interacting with other proteins, maintains the structural integrity of the filtration membrane, and participates in regulating glomerular blood filtration and urine formation. In the glomerulus, the expression of Wilms tumor gene 1 (WT1) is related to the formation of glomerular blasts and the differentiation of podocytes, while in the adult kidney, the expression of WT1 is related to maintaining the function of podocytes and regulating the function of renal tubules and interstitial cells. Through immunofluorescence staining and Western Blotting detection results, we found that compared with the treatment of 2DM, the treatment of 3DENM increased the expression of Nephrin and WT1 proteins and restored the expression and distribution of Synaptopodin in the kidneys of rats (such as Fig.26 ). It was demonstrated that 3DENM transplantation plays an important role in repairing the glomerular filtration function, maintaining the integrity of the filtration membrane, and regulating the glomerular structure and function of DKD rats.

[0202] Example 6: Application of matrix-empowered mesenchymal stem cell aggregates in spinal cord injury repair

[0203] 1. Experimental Methods

[0204] 1.1 Preparation of spinal cord injury model and cell implantation

[0205] Rats were anesthetized by inhalation of isoflurane. The hair on the back was shaved off, and a 1-cm longitudinal incision was made on the skin along the midline of the rat, with the center point of T10 as the center. The muscles were bluntly separated to fully expose the spinous processes of T9-T11, and then the dorsal laminectomy of the T10 vertebra was performed until the spinal cord was completely exposed. A 10-g metal rod was allowed to fall freely from a height of 25 mm to hit T10 to simulate moderate spinal cord injury using the NYU Impactor-III spinal cord injury impactor. The rats then developed involuntary spasms and tail torsion, and the physical signs met the model criteria, indicating that the model was successful. Then the muscles, fascia, and skin were sutured in turn, and the rats were placed on a constant temperature electric blanket for resuscitation. For the sham operation group, only laminectomy was performed without SCI. The model rats were randomly divided into three groups: SCI group, 2DM group, and 3DENM group. Cefuroxime sodium was injected intramuscularly 3 days after surgery to prevent wound infection. Artificial urination was performed twice a day for 14 consecutive days. After two weeks, the 2DM group and the 3DENM group were given 1*10 6 The specific transplantation plan was as follows: the 2DM and 3DENM were injected in situ at the injury center and 0.5 cm above and below the injury center using a stereotaxic injection device, with a cell concentration of 1*10 5 / μL.

[0206] 1.2 BBB motor function score

[0207] Hindlimb motor function was evaluated by Basso, Beattie and Bresnahan (BBB) ​​motor score, which started on the first day after modeling and was performed once a week until 8 weeks to evaluate the long-term recovery of rat hindlimb motor function. The BBB motor function score ranged from 0 to 21 points, with 0 representing complete SCI and no spontaneous hindlimb movement in the rat, less than 8 points indicating only joint movement, 8-13 points indicating coordinated movement, 14-20 points indicating more accurate and stable movement, and 21 points representing normal movement in the rat. Before each evaluation, the rats were allowed to move freely in an open area for 5 minutes, and then three researchers jointly evaluated and scored the rats' movement status within 3 minutes according to the BBB motor scoring scale.

[0208] 1.3CatWalk gait analysis

[0209] Eight weeks after SCI, the CatWalk XT system was used to objectively evaluate the footprints, motor behavior, and body coordination of rats in different groups. The CatWalk XT system includes a platform made of a transparent glass plate. The high-speed camera at the bottom of the platform can record footprints through the green fluorescence intensity on the glass platform. The red light-emitting diode on the top of the platform can measure the body contour of the rat. Each rat was trained for at least three days before the test to adapt to walking on the path. During the experiment, a quiet environment should be maintained to avoid disturbing the animals. The gait of each rat was recorded with the CatWalk XT system and related analysis was performed using the supporting software. The analysis parameters included paw print morphology, step sequence, etc.

[0210] 1.4 Grid Experiment

[0211] Grid crawling is a test to assess the ability to accurately control the placement of the hind paws after spinal cord injury. It examines the fine motor function and limb coordination ability of mice. Eight weeks after spinal cord injury modeling, the rats were placed in sequence on a horizontal grid device with a height of 50 cm, a grid of 45×45 cm, and a grid opening of 1.5×1.5 cm. This test does not require pre-training. Just lift the mouse's tail and place it in the center of the grid. When all four paws grab the grid, release the tail. One observer records the total number of bilateral hind limb steps from 120 to 150 steps, and the other two observers record the total number of times the hind limbs on either side slip and fall off the grid during this period. Finally, the average is taken to calculate the hind limb error rate.

[0212] Hind limb error rate = total number of hind limb falls / total number of hind limb steps × 100%

[0213] 1.5 Hot plate experiment

[0214] The temperature of the hot plate was maintained at 52°C, and a transparent acrylic cylinder was placed on it. Six weeks after the injection of stem cells, rats from each group were placed in the cylinder in turn. The timing was started immediately when the rats' hind limbs were completely in contact with the hot plate. When the rats were observed to have reaction movements such as jumping off the hot plate or licking their hind feet, it was considered that the rats felt pain, and the timing was stopped. The time required for each rat to react was recorded. The pain threshold of rats is lowered after spinal cord injury, and the pain perception will be more obvious. Stem cell treatment can increase the pain threshold of rats and prolong the reaction time.

[0215] 1.6 Louisville Swim Scale Score

[0216] The swimming test used a 0-15 LSS scoring system to assess forelimb dependence, hindlimb coordination, body angels, and trunk stability. Rats were acclimated to the water environment before testing and trained to swim from one end to the other in a glass tank filled with water. Each rat received two double-blind tests before surgery and once a week for up to one month after injury.

[0217] 1.7 Electrophysiological test: Motor evoked potential (MEP)

[0218] Electrophysiological testing can monitor the functional recovery of the descending motor nerve conduction system in rats after spinal cord injury. Eight weeks after spinal cord injury, rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Motor evoked potentials (MEPs) were measured by electrophysiological equipment. In order to detect MEPs, recording electrodes were placed subcutaneously in the gastrocnemius muscle of the lower limb to record the peak amplitude and latency, a ground electrode was placed subcutaneously in the midline of the back, and a stimulating electrode and a reference electrode were placed subcutaneously between the two ears. A current of 5 mA / time was used to stimulate the motor area of ​​the cerebral cortex to obtain motor evoked potentials (MEPs). MEPs are used to indicate the transmission of hindlimb motor signals and represent descending conduction. The greater the potential difference between the peaks and troughs, the stronger the descending motor nerve conduction.

[0219] 2. Experimental results

[0220] like Fig. 27 As shown in A and 27B, the BBB scores dropped to 0 points one day after the successful SCI modeling, and recovered within 14 days but were all less than 8 points. Only joint movement was seen, and there was no plantar weight bearing. After stem cell treatment, the scores of the treatment group were significantly improved, and the 3DENM group recovered faster than the 2DM group. Three weeks after cell transplantation, the 3DENM group was the first to reach 14 points, indicating that the rats in the 3DENM group were able to perform relatively stable and accurate movements. Five weeks after transplantation, the average score reached 17-18 points, far exceeding the 12-13 points of the 2DM group, indicating that the 3DENM group had a better therapeutic effect; Fig.28As shown in the figure, the footprints of the gait analysis showed that the footprints of the SCI group could not be detected because the hindlimbs could not bear weight, while the 3DENM group observed more coordinated movements of the front and back limbs compared with the 2DM group, which was closer to the performance of the sham group, indicating that the 3DENM group had more recovered the motor function lost after spinal cord injury; Fig.29 As shown in the results of A, in the grid crawling experiment, the 3DENM group had a lower walking error rate than the 2DM group, indicating that they had a stronger ability to control the placement of their hind paws and exhibited good fine motor function and limb coordination ability; Fig.29 As shown in B, the results of the hot plate experiment showed that 6 weeks after cell transplantation, the stem cell treatment group had a higher pain threshold, a stronger tolerance to pain, and a longer reaction time to thermal stimulation than the SCI group, indicating that the degree of thermal hyperalgesia in the stem cell treatment group was lower than that in the SCI group. This is because spinal cord injury can cause hyperalgesic neuropathic pain; Fig.30 As shown in the results of A, in the swimming test, the 3DENM group had a swimming posture closer to that of the sham group than the 2DM group. Fig.30 The LSS score results of B showed that the 3DENM group had better coordination of forelimbs and hindlimbs and trunk stability. Fig.30 Motor evoked potential (MEP) was used to evaluate the descending motor nerve conduction function in C, and the results showed that the motor potential difference in the 3DENM group was higher than that in the 2DM group, which improved the nerve conduction function of SCI rats.

[0221] In summary, the present invention provides a matrix-enabled mesenchymal stem cell, wherein the matrix-enabled mesenchymal stem cell is obtained by subculture of mesenchymal stem cells in a cell culture matrix, and the cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The matrix-enabled mesenchymal stem cells obtained by culture using the cell culture matrix of the present invention can promote the nuclear transfer of β-catenin, enhance in vitro proliferation activity, maintain good in vitro proliferation ability, stemness maintenance ability, delay cell aging and apoptosis, induce the expression of multiple functions of hMSCs, and the hMSCs cultured to the fifteenth generation by hENFc empowerment are not tumorigenic and have no adverse effects on animals, meeting the clinical application standards of hMSCs. The matrix-enabled mesenchymal stem cells are subcultured in a 3D cell culture system to obtain matrix-enabled mesenchymal stem cell aggregates, which have a small particle size and can be widely used in multiple scenarios including intravenous injection, local treatment, tissue engineering, in vitro organ model construction and systemic treatment. Compared with 2DM cultured in the traditional way, 2DENM cultured in the 2D-cadherin matrix-enabled culture, and 3DM cultured in the traditional way, matrix-enabled mesenchymal stem cell aggregates have stronger anti-inflammatory gene expression, and macrophages co-cultured with 3DENM have more CD206 and less CD86 expression, which shows that more macrophages have changed from pro-inflammatory M1 type to M2 type; it can promote cell migration. The matrix-enabled mesenchymal stem cell aggregates have better effects on the treatment of liver fibrosis. After implantation, macrophages are more transformed into non-fibrogenic phenotypes (LY6C-), macrophages express less chemokine receptor CX3CR1, and less collagen deposition. This shows the excellent therapeutic effect of 3DENM. The matrix-enabled mesenchymal stem cell aggregates can better treat diabetes, can effectively reduce the inflammatory response of DKD rats, thereby delaying the progression of diabetes and its nephropathy, slowing down kidney damage in diabetic rats, and reducing glomerular fibrosis. The matrix-empowered mesenchymal stem cell aggregates can also effectively improve the motor function and gait of rats with spinal cord injury, relieve neuropathic pain hypersensitivity, and restore nerve conduction function.

Claims

1. A matrix-enabled mesenchymal stem cell aggregate, characterized in that: The matrix-enabled mesenchymal stem cell aggregates are obtained by subculturing mesenchymal stem cells in a cell culture matrix and then culturing them in a 3D cell culture system. The cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc.

2. The mesenchymal stem cell aggregate according to claim 1, characterized in that The mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated plate, incubating, and washing to obtain a cell culture matrix hENFc; (2) inoculating the mesenchymal stem cells into the cell culture flask containing the cell culture matrix hENFc obtained in step (1), and subculturing to obtain matrix-empowered mesenchymal stem cells; (3) resuspending the matrix-empowered mesenchymal stem cells obtained in step (2), adding them to a 3D cell culture system for culture, and obtaining mesenchymal stem cell aggregates.

3. The mesenchymal stem cell aggregate according to claim 2, characterized in that The incubation time in step (1) is 0.5-24 hours, and the incubation temperature is 4-37°C.

4. The method for preparing matrix-empowered mesenchymal stem cell aggregates according to claim 1, characterized in that: The steps include: (1) diluting 1-3 portions of hE-cad-Fc and 1-3 portions of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated plate, incubating, and washing to obtain a cell culture matrix hENFc; (2) inoculating the mesenchymal stem cells into the cell culture flask containing the cell culture matrix hENFc obtained in step (1), and culturing them in a conventional manner to obtain matrix-empowered mesenchymal stem cells; (3) resuspending the matrix-empowered mesenchymal stem cells obtained in step (2), adding them to a 3D cell culture system for culture, and obtaining mesenchymal stem cell aggregates.

5. Use of the mesenchymal stem cell aggregates according to any one of claims 1 to 3 in the preparation of anti-inflammatory, immunomodulatory and tissue damage repair drugs.

6. Use of the mesenchymal stem cell aggregates according to any one of claims 1 to 3 in the preparation of anti-inflammatory, immunomodulatory and liver fibrosis reversal drugs for treating liver fibrosis.

7. Use of the mesenchymal stem cell aggregates according to any one of claims 1 to 3 in the preparation of metabolic / immune regulation drugs for treating diabetes and tissue repair drugs for related syndromes.

8. Use of the mesenchymal stem cell aggregates according to any one of claims 1 to 3 in the preparation of drugs for anti-inflammatory, immunomodulatory and tissue repair of damaged nerves / blood vessels.

Images