A stromal-enabled mesenchymal stem cell aggregate and uses thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a matrix-enabled mesenchymal stem cell aggregate and application. 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 subculturing mesenchymal stem cells in a cell culture matrix. The cell culture matrix is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The cell aggregate cells highly express endogenous epithelial cadherin and neural cadherin to promote transcription factor nuclear entry, up-regulate the expression level of anti-inflammatory immune regulation related factors and matrix degradation and cell migration related factors, and promote the polarization of activated macrophages to M2 type macrophages. The matrix-enabled mesenchymal stem cell aggregate has significantly improved effects of anti-inflammation, immune regulation and tissue damage repair in the treatment of liver fibrosis, diabetes and nerve injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a matrix- enabled mesenchymal stem cell aggregate and application thereof. BACKGROUND

[0002] Stem cell therapy is the most potential disease treatment method in the 21st century. It utilizes the unique characteristics of stem cells, including self-renewal, differentiation and paracrine properties of stem cells, to regenerate damaged cells and tissues in the human body by delivering exogenous stem cells to patients. With the increasing expectations of stem cell therapy for regenerative treatment, the number of applications of stem cells from various sources is increasing, and stem cell therapy is developing from autologous to allogeneic and induced pluripotent stem cells. Induced pluripotent stem cells are artificially prepared stem cells through cell gene reprogramming, which have wide application, but have genome instability, carcinogenicity and immune rejection. In 2006, the International Society for Cellular Therapy defined mesenchymal stem cells (MSC) as adherent cells that express CD73, CD105, CD90 (≥90%) and do not express hematopoietic markers CD34, CD45, CD14, CD19 and HLA-DR (≤2%), which can adhere to the surface of plastic. Such cells must have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. MSCs can be isolated from almost all tissues in the human body, but the origin of adult tissues may limit the proliferation and differentiation capacity of MSCs, and the shortage of quantity still limits its clinical application. The existing expansion and induction differentiation method of MSCs is mainly two-dimensional plate culture, which has the problems of reduced cell activity after long-term culture, low induction differentiation efficiency, unstable induction effect, and uncertain in vivo induction effect. Therefore, on the basis of ensuring the proliferation and differentiation of MSCs, optimizing the culture scheme is the top priority.

[0003] To obtain more performance and quantity of stem cells, those skilled in the art adjust the composition of the culture medium and the culture method. For example, the invention patent CN104830763B discloses the addition of Y-27632 in mesenchymal stem cell culture, after five generations of culture, compared with the control group without adding Y-27632, the number of umbilical cord mesenchymal stem cells in the experimental group with Y-27632 added increased by 31.87%; the number of amniotic membrane mesenchymal stem cells increased by 30%, and the number of bone marrow mesenchymal stem cells increased by 27.27%. The invention patent CN117904047A discloses a composition for hematopoietic stem cell culture, which is composed of components with 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 in the culture medium can effectively expand hematopoietic stem cells while better maintaining stem cell purity and cell state, and shorten the culture period. The invention patent CN114836372B discloses a culture method for the conversion of mouse epiblast stem cells to novel pluripotent stem cells, and the novel pluripotent stem cells obtained have high cell potential, fast proliferation rate, and can be stably passaged multiple times.

[0004] The research group of the inventors has been committed to the research of cadherin for a long time, and unexpectedly obtained a mesenchymal stem cell culture medium. The mesenchymal stem cells cultured by the cell culture medium have the ability to promote the nuclear transfer of β-catenin, enhance the in vitro proliferation activity, maintain good in vitro proliferation ability, maintain the ability of stemness, delay cell aging and apoptosis, induce the expression of multiple functions of hMSCs, and the hMSCs cultured by hENFc for the fifteenth generation do not have tumorigenicity and have no adverse effects on animals, which meets the standard of clinical application of hMSCs.

[0005] On the basis of the previous study, the inventors prepared the matrix-enabled mesenchymal stem cell into cell aggregates, and found that the matrix-enabled mesenchymal stem cell aggregates have smaller 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. The matrix-enabled mesenchymal stem cell aggregates have stronger anti-inflammatory gene expression, more CD206 positive cells and less CD86 positive cells of macrophages co-cultured with 3DENM, which shows that more macrophages are polarized from pro-inflammatory M1 type to M2 type; the matrix-enabled mesenchymal stem cell aggregates can promote cell migration. The implantation treatment of liver fibrosis shows that, compared with the single cell treatment group, the 3DENM has higher homing and planting efficiency to the fibrotic liver tissue, more non-fibrosis phenotype (LY6C-) macrophages in the fibrotic liver tissue, less collagen deposition, and significantly improved reversal of liver fibrosis treatment. The implantation treatment of diabetes shows that the matrix-enabled mesenchymal stem cell aggregates effectively improve the physiological and biochemical indicators of diabetes (including body weight, food intake, blood glucose, blood lipids, etc.) and the level 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 nephropathy (relieve glomerular fibrosis and kidney damage). SUMMARY

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

[0007] Preferably, the matrix-enabled mesenchymal stem cell aggregate is 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 the cell culture matrix hENFc matrixed cell culture plate, subculturing to obtain matrix-enabled mesenchymal stem cells;

[0009] (3) resuspending the matrix-enabled mesenchymal stem cells obtained in step (2), adding to a 3D cell culture system for culturing to obtain a mesenchymal stem cell aggregate.

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

[0011] The second object of the present application is to provide a preparation method of the stromal-primed mesenchymal stem cell aggregate, 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 substrate hENFc;

[0012] (2) inoculating mesenchymal stem cells into the cell culture substrate hENFc matrixed cell culture plate obtained in step (1), subculturing to obtain stromal-primed mesenchymal stem cells;

[0013] (3) resuspending the stromal-primed mesenchymal stem cells obtained in step (2) and adding to a 3D cell culture system for culture to obtain a mesenchymal stem cell aggregate.

[0014] The third object of the present application is to provide the use of the mesenchymal stem cell aggregate in the preparation of an anti-inflammatory drug.

[0015] The fourth object of the present application is to provide the use of the mesenchymal stem cell aggregate in the preparation of a drug for treating liver fibrosis.

[0016] The fifth object of the present application is to provide the use of the mesenchymal stem cell aggregate in the preparation of a drug for treating diabetes.

[0017] The beneficial effects of the present application are: (1) a stromal-primed mesenchymal stem cell is provided, which is obtained by subculturing mesenchymal stem cells on a cell culture substrate prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The stromal-primed mesenchymal stem cell obtained by culturing using the cell culture substrate of the present application can promote the nuclear translocation of β-catenin, maintain good in vitro proliferation ability and stemness, delay cell aging and apoptosis, promote the expression of various functions of hMSCs, and the hMSCs cultured to the fifteenth generation by hENFc priming do not have tumorigenicity and have no adverse effects on animals, meeting the clinical application standards of hMSCs. The stromal-primed mesenchymal stem cell aggregate is obtained by culturing the stromal-primed mesenchymal stem cell in a 3D cell culture system, which promotes cell aggregation and has 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 mode (2DM), mesenchymal stem cells cultured in two-dimensional mode on the hENFc matrix (2DENM) and aggregates of mesenchymal stem cells cultured in traditional two-dimensional mode (3DM), the matrix-enabled mesenchymal stem cell aggregates (3DENM) have stronger anti-inflammatory gene expression, and the macrophages co-cultured with 3DENM have more CD206 positive cells and fewer CD86 positive cells, which shows that more macrophages are transformed from pro-inflammatory M1 type to anti-inflammatory M2 type; and can promote cell migration homing.

[0019] (3) The matrix-enabled mesenchymal stem cell aggregates (3DENM) have better therapeutic effect on liver fibrosis, more cells migrate to the lesion liver tissue after 3DENM implantation, and more macrophages in the liver tissue are transformed into non-fibrosis phenotype (LY6C-), the expression of chemotactic factor receptor CX3CR1 is reduced, 3DENM has high expression of MMP2 / 9 and reduces collagen deposition in fibrotic liver, which shows that 3DENM has better therapeutic effect on 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 the glomerular fibrosis and kidney damage of diabetic rats.

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

[0022] Figure 1 Preparation of hENFc: (A) Fixed amount-protein concentration curve of hE-cad-Fc, hN-cad-Fc; (B) Fixed amount-time curve of hE-cad-Fc, hN-cad-Fc; (C) Interaction curve of hE-cad-Fc and hN-cad-Fc; (D) Fixed amount detection of hE-cad-Fc and hN-cad-Fc on different matrix surfaces Figure 2 Characterization of hENFc: (A) Element analysis of different matrix surfaces; (B) Stability detection of different matrices; (C) Distribution and structure detection of hE-cad-Fc and hN-cad-Fc on different matrix surfaces; (D) Hydrophilic and hydrophobic detection of different matrix surfaces

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

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

[0025] Figure 4 Statistical analysis of hMSCs volumes at different passages on different matrix surfaces

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

[0027] Note: (A) Real-time PCR detection; (B) Western blotting detection

[0028] Figure 6 OCT4 gene and protein expression in different generations of hMSCs on different matrix surfaces

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

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

[0031] Figure 8 Apoptosis detection of different generations of hMSCs on different matrix surfaces

[0032] Figure 9 Multiple differentiation capacity assays of different generations of hMSCs on different matrix surfaces. Note: (A) Oil Red O staining and adipogenic gene expression detection; (B) Alizarin Red staining and osteogenic gene expression detection; (C) Alcian Blue staining and chondrogenic gene expression detection.

[0033] Figure 10 MSC EN Oncogene and tumor suppressor gene expression point map

[0034] Figure 11 MSC EN karyotype analysis

[0035] Figure 12 MSC EN Tumorigenicity detection

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

[0037] Figure 13 Light microscopic observation of cell aggregates

[0038] Note: Optical mirror images before and after the formation of 3DM and 3DENM.

[0039] Figure 14 Light microscopic observation and particle size statistics of cell aggregates of different sizes

[0040] Figure 15Cell aggregate E-cadherin and N-cadherin protein expression and statistics in 3DM and 3DENM groups

[0041] Figure 16 Immune regulation function of cell aggregate was detected by co-culturing cell aggregate with activated PBMC. Cell aggregate inhibited PBMC proliferation (A), CD4+ PBMC proliferation (B), CD+8 PBMC proliferation (C), promoted activated PBMC apoptosis (D), and inhibited pro-inflammatory T lymphocytes, including TNF-α+CD4+ (E), Th1 (F) and Th17 (G).

[0042] Figure 17 Anti-inflammatory ability of cell aggregate was evaluated. (A) THP-1 induction schematic diagram; (B) light microscope images of M0 and M1 type THP-1 derived macrophages; (C) MSC co-culturing with THP-1 schematic diagram; (D) MSC anti-inflammatory gene qPCR results; (E) THP-1 macrophage WB results

[0043] Figure 18 Migration and homing ability of cell aggregate was evaluated. (A) qPCR detection of MSC migration and homing related genes; (B) MMP2 and MMP9 protein immunofluorescence staining; (C) CXCR4 protein immunofluorescence staining; (D) WB detection of MSC migration and homing related proteins; (E) crystal violet staining of MSC migration

[0044] Figure 19 Treatment of liver fibrosis mice and small animal imaging

[0045] Note: (A) MSC injection mouse model diagram; (B) small animal imaging results; (C) small animal imaging results statistics

[0046] Figure 20 Mouse liver immune cell flow cytometry

[0047] Figure 21 Sirius red staining and statistics of liver fibrosis mouse liver

[0048] Figure 22 Detection of biochemical and physiological indicators of diabetic rats. (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] Figure 23 Analysis of inflammatory response of diabetic rats

[0050] Figure 24Diabetic rats blood glucose and serum lipid profile detection Note: (A) blood glucose; (B) total cholesterol; (C) total triglyceride; (D) high density lipoprotein; (E) low density lipoprotein

[0051] Figure 25 Diabetic rat histopathology staining

[0052] Figure 26 Diabetic rat kidney function detection

[0053] Figure 27 BBB score of spinal cord injury treatment (A, B)

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

[0055] Figure 29 Rat grid crawling experiment analysis (A) and hot plate experiment analysis (B) after spinal cord injury treatment

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

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

[0058] In the following examples, the reagents used are commercially available unless otherwise specified.

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

[0060] In the following examples, the hE-cad-Fc modified matrix is referred to as hEFc, and the hN-cad-Fc modified matrix is referred to as hNFc. The hE-cad-Fc and hN-cad-Fc have been disclosed in the inventor's previous application for invention patent CN109337857B "Use of fusion protein E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc" and CN115040695B "Application of activity interface based on VE-cad-Fc / N-cad-Fc fusion protein", and the corresponding sequences are disclosed in detail, which will not be listed in this patent.

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

[0062] All data are presented as mean ± standard deviation; statistical differences between groups were assessed by one-way ANOVA. p < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ns means not significant. All in vitro experimental results are from 3 independent parallel experiments, and all animal experimental results are from 5 independent parallel experiments, unless otherwise stated. All results were statistically analyzed using GraphPad Prism 8.0 software.

[0063] Example One, Preparation of hENFc matrix and matrix-enabled mesenchymal stem cells

[0064] 1. Preparation of hENFc matrix

[0065] Purified hE-cad-Fc or / and hN-cad-Fc was diluted in PBS to reach a total protein concentration range of 0-30 pg / mL, and added to tissue culture treated cell culture plates (TCPS) at 150 pL protein solution per square centimeter; after 2 hrs incubation at 37 °C, further washing with PBS was performed 5 times.

[0066] 2. Adsorption amount-concentration and adsorption amount-time detection 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, 30 pg / mL, added to 96-well plates at 100 pL per well, and incubated at 37 °C for 2 hrs before further washing the plates 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 pg / mL, added to 96-well plates at 100 pL per well, and incubated at 37 °C for 0, 30, 60, 90, 120, 150, 180 min before further washing the plates 5 times with PBS. Finally, the modified surfaces were 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 non-specific interactions, then with mouse anti-E-cadherin antibody (extracellular domain, 1 :2000 dilution) or rabbit anti-N-cadherin antibody (extracellular domain, 1 :2000 dilution) for 2 h at 37 °C; after washing 3 times with PBS, the modified surfaces were incubated with HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1 :5000 dilution) for 2 h, 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 with a microplate reader.

[0068] 3. Hydrophilic / hydrophobic detection, elemental analysis and topography detection of hENFc substrates

[0069] The prepared TCPS, hEFc, hNFc and hENFc substrates were washed with ultrapure water for 5 times and air-dried, and the surface hydrophilicity of different surfaces was evaluated using a water contact angle (WCA) measuring instrument. The elemental changes before and after the modification of the substrate surface were evaluated using an X-ray photoelectron spectroscopy (XPS) instrument with a monochromatic Mg K a radiation source (1253.6 eV). C1s (binding energy 284.8 eV) 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 detection of hENFc substrates

[0071] Purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 5 μg / mL, added to 96-well plates at 100 μL per well, and incubated at 37°C for 2 hrs, followed by further washing of the plates 5 times with PBS; finally, the modified surfaces were determined by Elisa, briefly, each substrate was incubated with 1.0% (w / v) BSA for 1 hr to block non-specific interactions, then incubated with goat anti-human IgG (H+L) antibody (1:5000 dilution) for 2 hrs, followed by the addition of TMB solution and incubation at 37°C in the dark; after 15 min, a stop buffer was added, and the absorbance was measured at 450 nm with a microplate reader.

[0072] 5. Results

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

[0074] As shown in Figure 1 A, the surface immobilization of hE-cad-Fc and hN-cad-Fc on TCPS was related to the original solution concentration, and the amount of surface protein immobilization was proportional to the concentration when the Cad-Fc concentration was lower than 5 μg / mL, and reached saturation when the Cad-Fc concentration was 10 μg / mL. Figure 2 B demonstrated that the amount of surface protein immobilization was also affected by the immobilization time, and the maximum amount of protein immobilization was reached when incubated for 2 hrs. Figure 1 C showed that the fusion protein substrates with different ratios of hE-cad-Fc and hN-cad-Fc could be prepared by controlling the Cad-Fc concentration and immobilization time. To avoid the interaction of the two Cad-Fc in solution, I first observed the minimum concentration of the two Cad-Fc interaction by microthermal migration experiment, which was 400 μg / mL, much higher than the protein solution concentration of the saturated immobilization amount (10 μg / mL). To construct cadherin fusion protein substrates with different subtypes and ratios, we kept the total concentration of hE-cad-Fc and hN-cad-Fc constant at 5 μg / mL, while changing their ratios (hE-cad-Fc:hN-cad-Fc = 2:1, 1:1 and 1:2 to prepare hEFc, hNFc and hENFc substrates with different Cad-Fc ratios Figure 1 D).

[0075] 5.2 Physicochemical characterization of hENFc substrates

[0076] XPS analysis showed that the content of N1s element increased significantly after the surface modification of Cad-Fc, indicating that Cad-Fc was successfully immobilized on the TCPS surface Figure 2 A). Figure 2B shows that these Cad-Fc substrates can be stably maintained in the culture medium for 5 days. The topography analysis of these substrates by AFM shows that the nanorod-like structures of about 5-7 nm can be clearly observed on the surface of TCPS plate Figure 2 C). As shown in Figure 2 D, the hydrophilicity of these Cad-Fc substrates is significantly higher than that of TCPS. Cad-Fc is effectively immobilized on the TCPS plate, and the hydrophilicity of the culture plate surface is enhanced.

[0077] The inventors made the influence of hENFc substrates on the cell adhesion and proliferation of hMSCs. With the increase of the proportion of hE-cad-Fc in the hE-cad-Fc substrate, the expression of OCT4 of hMSCs increased. When the proportion of hE-cad-Fc was greater than or equal to hN-cad-Fc, the expression level of OCT4 gene of hMSCs was significantly higher than that of hMSCs cultured on TCPS surface. The subsequent experiment selected hENFc complex substrate (hE-cad-Fc: hN-cad-Fc = 1:1) as the preferred substrate for subsequent research.

[0078] Example Two, Preparation and Function of hENFc Substrate Enabled hMSCs

[0079] 1. Preparation of hENFc Substrate Enabled hMSCs

[0080] First, hMSCs were inoculated into hEFc, hNFc or hENFc substrateized T-75 cell culture bottles at 1x10 6 cells per bottle, and continuously cultured under conventional expansion conditions to the 15th generation to obtain hMSCs of different generations.

[0081] The following experiments used unmodified TCPS to prepare hMSCs of each generation as a control. hMSCs enabled on hEFc substrate were referred to as MSC E , hMSCs enabled on hNFc substrate were referred to as MSC N , hMSCs enabled on hENFc substrate were referred to as MSC EN , and hMSCs normally cultured on TCPS substrate were referred to as MSC T .

[0082] 2. Detection of the Proliferation Ability of hENFc Substrate Enabled hMSCs

[0083] All cells on different substrates were collected at the end of each passage and counted after excluding dead cells using trypan blue. Alternatively, MSCs were seeded onto TCPS, hEFc, hNFc or hENFc substrates and incubated for 24, 48 h before determining cell viability using a CCK-8 assay. That is, the cell culture supernatant was removed and the cells were washed three times with PBS; 100 μΐ^of CCK-8 (1 : 10 dilution) reagent was added to each well and incubated at 37 °C for 4 h; the absorbance was detected at 450 nm using a microplate reader and a calibration curve was established.

[0084] To compare the cell proliferation rate 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 cell number at passage, N(to) is the number of cells seeded 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.

[0088] 3. Detection of hENFc substrate-enabled hMSCs' stemness

[0089] Gene expression detection

[0090] ① RNA extraction: All culture medium was discarded for cultured MSCs, and the cells were washed three times with PBS. The cells were lysed by adding 1 mL of Trizol solution to each well and then transferred to a 1.5 mL centrifuge tube. Then, 200 μΐ^of chloroform was added and the mixture was shaken vigorously for 15 s. After incubation at room temperature for 5 min, the mixture was centrifuged at 4 °C and 12,000 rcf for 15 min. The supernatant was transferred to a new centrifuge tube and 500 μΐ^of isopropanol was added. After incubation at room temperature for 10 min, the mixture was centrifuged at 4 °C and 12,000 g for 10 min. After discarding the supernatant, 1 mL of 75% ethanol was added to each tube, mixed, and centrifuged at 4 °C and 7,500 g for 5 min. After discarding the supernatant, the tube was left to dry at room temperature. Finally, 20 μΐ^of sterile and enzyme-free ddH2O was added to dissolve the RNA, and the mixture was incubated at 55 °C for 10 min. The RNA was aliquoted and stored at -80 °C in an ultra-low temperature freezer.

[0091] ② Preparation of cDNA: Take RNase-free PCR tube, according to Vazyme instructions, add 4 μL RNase-free water, 4 μL gDNA wiper Mix, 8 μL template RNA to each tube, after sample addition, centrifuge for 5 sec, place in PCR instrument and follow the procedure of 42℃ for 2 min, 4℃∞ to remove genomic DNA; then add 4 μL 5×HiScriptⅡqRTSuperMixⅡ to each tube, follow the procedure of 50℃ for 15 min, 85℃ for 5 sec, 4℃∞ for reverse transcription; after reverse transcription, normalize the obtained cDNA and store at -20℃ for short-term preservation.

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

[0093]

[0094]

[0095] After sample addition, centrifuge for 5 sec, place in fluorescent quantitative PCR instrument and follow the procedure of 95℃ for 30 sec, 95℃ for 3 sec, 60℃ for 10 sec for 40 times, 95℃ for 15 sec, 60℃ for 60 sec, 95℃ for 15 sec for amplification; the obtained data takes the expression of β-actin of each group as internal reference, set three groups in parallel for each sample, and use 2 -ΔΔCt method for statistical calculation.

[0096] Protein expression detection

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

[0098] According to BCA kit instructions, determine the protein concentration in each sample; finally, add sample buffer (1:5 dilution) to each supernatant and boil for 10 min, then store in -80℃ ultra-low temperature refrigerator after aliquoting.

[0099] ② Western Blotting

[0100] The sample was added to a 10% SDS-polyacrylamide gel for electrophoretic analysis, and the electrophoresis was ended when the sample front migrated to a distance from the bottom of the glass plate; the water-absorbing sponge, PAGE gel, PVDF membrane, and water-absorbing sponge were clamped in order using a membrane transfer clamp, placed in an electroblotting instrument, and 100 V of electroblotting buffer was added for 90 min; after the membrane transfer was completed, the PVDF membrane was taken out and sealed with 5% BSA at room temperature for 2 h; then incubated with mouse anti-E-cadherin antibody (intracellular domain, 1:2000 dilution), rabbit anti-N-cadherin antibody (intracellular domain, 1:2000 dilution), mouse anti-a-catenin protein antibody (1:2000 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) at 37°C for 2 h or at 4°C overnight, then washed with TBST solution at room temperature for 5 min, repeated three times; HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1:2000 dilution) was added and incubated at room temperature for 2 h; then washed with TBST solution at room temperature for 5 min, repeated three times; finally, a suitable amount of color developing solution was added to the PVDF membrane, and a Western Blotting exposure instrument was used for observation and photography, and the graph was statistically analyzed using Image J.

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

[0102]

[0103] 4. Periodic detection of hMSCs enabled by hENFc matrix

[0104] 1x10 6 MSCs T and MSCs EN were harvested at the 6th, 11th, and 15th passages, respectively, and fixed in 70% cold ethanol for 2 h to overnight; after washing with PBS for 3 times, the cell precipitate was obtained by centrifugation at 1000 rpm for 10 min at room temperature; 100 μL of RNase A solution was added to each sample, and the cells were resuspended and incubated at 37°C for 30 min; 400 μL of PI staining solution was added and mixed, and incubated at 4°C for 30 min in the dark; the red fluorescence at 488 nm excitation wavelength was recorded by flow cytometry.

[0105] 5. Senescence detection of hMSCs by hENFc matrix

[0106] For senescence-associated β-galactosidase assay, MSCs were detected by SA-β-gal staining kit at passage 6, 11 and 15. T and MSCs EN . Briefly, cells were cultured on modified or non-modified 6-well plates to 80% confluence, fixed with 4% formaldehyde for 15 min after PBS washing; then, cells were incubated with freshly prepared β-galactosidase staining solution at 37°C overnight in a CO2-free oven; after PBS washing for 3 times, photographs were taken using an optical microscope, and the number of β-galactosidase positive staining cells was counted per 200 cells in randomly selected fields.

[0107] 6. Apoptosis detection of hMSCs by hENFc matrix

[0108] At passage 6, 11 and 15, 1 x 105 6 MSCs T and MSCs EN were harvested by trypsin without EDTA, respectively; cells were resuspended with Binding Buffer diluted with 1 ml deionized water (1:9 dilution); 5 μL Annexin V / FITC was added to each 100 μL cell suspension, and incubated at room temperature for 5 min in the dark; after adding 5 μL propidium iodide solution and 400 μL PBS, the cells were placed in a 5 ml flow tube, and immediately detected by flow cytometry. In addition, unstained cells were used as blank tubes; only Annexin V / FITC was added as single staining tubes.

[0109] 7. Differentiation potential detection of hMSCs by hENFc matrix

[0110] Passage 6 MSCs T , passage 11 MSCs T and passage 11 MSCs 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 DF12 medium 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 DF12 medium 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 nm Dex, and 10 ng / mL... Cells were cultured in DF12 containing TGF-β1, 1% human insulin transferrin, and sodium selenite to induce chondrogenesis. Mature cells were detected using toluidine blue chondrogenic solution. Simultaneously, PCR was performed using the following primers to detect the expression of triphase differentiation-related genes:

[0111]

[0112] 8. Tumorigenicity detection of hENFc matrix-empowered hMSCs

[0113] To examine the tumorigenicity of hENFc-enabled cultured 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 using the following formula. Mice were sacrificed after seven weeks of continuous feeding. The cells were injected with 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.

[0114]

[0115] 9. Results

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

[0117] like Figure 16 As shown, although the proliferative capacity of all cells gradually weakens with each passaging, the number of cells harvested from each passaging also gradually decreases. Figure 3 However, compared with hMSCs (MSCs) cultured on the surface of TCPS... TCompared 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, 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 remains at 2000μm 3 Within (approximately 16μm in diameter) Figure 4 ).

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

[0119] 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 )

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

[0121] 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.

[0122] 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 -P11), in the eleventh generation MSC EN (MSC EN -P11) and the sixth generation MSC EN (MSC T More significant Oil Red O-positive lipid droplets and expression of PPAR-γ, CEBPA, and LPL genes were found in P6. 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.

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

[0124] 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 T The 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 MSCEN with the sixth passage of normal cultured MSCs T There were no statistical differences between the cells in the expression of proto-oncogenes and tumor suppressor genes, and the expression levels of c-Myc, P21, P53 and P16 genes did not change significantly. Karyotype analysis did not find abnormal clones, and no chromosomal translocation, deletion or change in the number of chromosomes was observed. hENFc-enabled cultured hMSCs maintained chromosomal stability Figure 11

[0125] Tumorigenicity is another dangerous factor that needs to be considered for the clinical application of MSCs. Although MSCs themselves are not tumorigenic, genomic instability and the possibility of forming teratomas are low compared with iPSCs and ESCs, in order to ensure the safety of long-term clinical application of hENFc-enabled hMSCs, we injected MSCs T , MSCs EN enabled by hENFc to the fifteenth passage, and human brain astrocytoma cells (U87) into the subcutaneous tissue of nude mice, respectively. After seven weeks, we studied the possibility of these cells forming tumors. As shown in Figure 12 A, the mice inoculated with U87 developed yellow soybean-sized lumps at the injection site, while no lumps formed in the mice inoculated with MSCs T and MSCs EN . In order to monitor the overall health of the mice, we measured the body weight of the mice, and the results showed that there was no statistical difference in the body weight of the mice inoculated with PBS, MSCs T , MSCs EN and U87 Figure 12 B). No lumps formed at the injection site of the mice after inoculation with MSCs T and MSCs EN ( Figure 12 C), and these results showed that hMSCs enabled by hENFc to the fifteenth passage were not tumorigenic and had no adverse effects on animals, meeting the standard for the clinical application of hMSCs.

[0126] Example Three, Preparation of Matrix-enabled Mesenchymal Stem Cell Aggregates

[0127] 1. Experimental Methods

[0128] 1.13 Preparation and characterization of DENMs

[0129] (1) Preparation of 2DENMs

[0130] First, hMSCs were prepared according to 1x10 6 ​Cells were seeded into hENFc matrixed T-75 cell culture flasks and cultured continuously to passage 6 under regular expansion conditions to obtain the competent hMSCs (2DENM in the following examples). The above experiment was used unmodified TCPS matrix prepared hMSCs (2DM in the following examples) as a control.

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

[0132] 2DM, 2DENM were resuspended with 1 mL DMEM / F12 medium at 100, 400 and 800 cells per aggregate, respectively, and then uniformly dropped into AggreWell cell culture plates and centrifuged at 1000 rpm for 5 min at room temperature; placed in a cell culture incubator (5% CO2, saturated humidity, 37°C) for 12 h; observed and photographed under an inverted phase contrast microscope, and then gently blown up with a blunt gun head and transferred to a 15 mL centrifuge tube to obtain the corresponding cell aggregates 3DM and 3DENM. The cell aggregate diameters were outlined using ImageJ, the image was spatially calibrated (μm / pixel), and the aggregate size was calculated. For each independent experiment, 100 cell aggregates were measured under each condition.

[0133] 1.2 Western blotting

[0134] Proteins were extracted from cells using a cell scraper or ultrasonic disrupter under two-dimensional and three-dimensional culture conditions. The proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with non-specific binding sites using skim milk, followed by incubation of the primary antibody with the target protein, and then washed to remove unbound antibodies. Then, the secondary antibody was incubated to bind to the antibody immunoreacted with the primary antibody, and again washed to remove unbound secondary antibodies. Fluorescent substrate was added for chemiluminescence reaction to observe the signal of the target protein, and a Western blot imaging system was used to capture the protein signal, and image analysis software was used to quantitatively analyze the signal to compare the protein expression levels under different conditions.

[0135] 1.3 Fluorescent quantitative PCR

[0136] According to the method of Example 2, RNA was extracted and reverse transcribed into cDNA, and the following primers were used for PCR to detect the expression of related genes:

[0137]

[0138] 1.4 Immunofluorescence staining

[0139] The samples were fixed with 4% paraformaldehyde, followed by punching with PBS containing 3% BSA and 0.1% Triton X-100 for 10 min. Then, blocking was performed with PBS containing 3% BSA for 1 h, and the primary antibody solution was prepared with the 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 mounted with mounting 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 the buffy coat was obtained by Histopaque density gradient centrifugation (p = 1.077 g / cm3); TC-3D and EN-3D aggregates were then inoculated into 96-well plates treated for low adhesion; the DF12 culture medium was replaced with RPMI-1640 culture 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 phytohemagglutinin (PHA) was added to activate the PBMCs; after 3 days of co-culture, the entire culture medium was aspirated and centrifuged at 2000 rpm for 10 min, and the cell precipitate was collected for further testing; PBMCs cultured alone were used as a negative control, and each group of experiments was repeated 6 times.

[0142] For the PBMC proliferation test, the isolated PBMC suspension was centrifuged at 300 x g for 10 min, and all the supernatant was discarded; 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, 2 μM CFSE was added, and the mixture was thoroughly mixed and incubated at 37°C, 5% CO2 for 10 min; 3 times the volume of pre-cooled RPMI1640 medium was added, and the reaction was terminated at 4°C for 5 min; the mixture was centrifuged at 300 x g for 10 min, the supernatant was discarded, 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, and the mixture was centrifuged at 300 x g for 10 min, the supernatant was discarded, and the process was repeated once; then, the obtained cells were used for co-culture experiments; after the completion of co-culture, the cells in the culture medium were collected and analyzed using a flow cytometer.

[0143] For the PBMC apoptosis test, the cell precipitate obtained after co-culture was analyzed according to the apoptosis test.

[0144] For T lymphocyte and T lymphocyte subsets determination in PBMC cells, 1ml PBS was added to resuspend the cell pellet obtained after co-culture above, centrifuged at 300xg for 10min, the supernatant was discarded, and the PBMC subsets were characterized with the following antibodies: CD4+T cells (FITC-labeled anti-human CD4 antibody, 1:500 dilution), CD8+T cells (FITC-labeled anti-human CD8 antibody, 1:500 dilution), pro-inflammatory T lymphocytes (FITC-labeled anti-human CD4, APC-labeled anti-human TNF-a antibody, 1:500 dilution), Th1 (FITC-labeled anti-human CD4, APC-labeled anti-human IFN-γ antibody, 1:500 dilution), Th17 (FITC-labeled anti-human CD4, APC-labeled anti-human IL17A antibody, 1:500 dilution), and incubated at 4°C in the dark for 30min after adding the antibodies; 1ml PBS was added to wash the cells once, centrifuged at 300xg for 10min, the supernatant was discarded, 500μl PBS was added to resuspend the cells, and the cells were analyzed by flow cytometry after filtration.

[0145] 1.6 Culture of THP-1 cells, differentiation and polarization of macrophages

[0146] Culture was performed using RPMI1640 medium containing 10% fetal bovine serum, and the cells were passaged when the cell density reached 10 6 cells / mL. Subsequently, differentiation induction of THP1 macrophages was performed by placing the THP1 cells in a medium containing 100ng / mL 12-Phorbol-13-myristate acetate (PMA) for 24 to 48 hours. After induction, the THP1 cells were differentiated into macrophages, which could be observed by morphological changes under a microscope, and the differentiation effect was confirmed by detecting the expression of macrophage markers such as CD11b or CD14 by flow cytometry. Subsequently, the medium was replaced with a medium containing 20ng / mL IFN-γ and 100ng / mL lipopolysaccharide for 24h to form M1 type macrophages.

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

[0148] THP-1 cells were cultured in the well plate to differentiate into macrophages and polarize into M1 type. MSCs cell suspension was added to the upper chamber of the Transwell, and after the cells adhered, the Transwell was transferred to the THP-1 well. The assembled Transwell culture system was placed in the incubator for 48h, 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 expression of cadherins

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

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

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

[0154] TC-3D cell aggregates and EN-3D cell aggregates were co-cultured with PBMCs for 3 days, and the flow cytometry results (as shown in Figure 16 ) showed that compared with TC-3D, EN-3D cell aggregates significantly inhibited the proliferation of activated PBMCs, with an inhibition rate of about 17.5% Figure 16 (A), and among them, the inhibition rate of CD4+ PBMCs was about 19.9% Figure 16 (B), and the inhibition rate of CD8+ PBMCs was about 11.5% Figure 16 (C), EN-3D cell aggregates effectively promoted the apoptosis of activated PBMCs, with an apoptosis rate up to 7.7% Figure 16 (D), and at the same time, EN-3D cell aggregates significantly inhibited pro-inflammatory T lymphocytes, including TNF-α+ CD4+ Figure 16 (E), Th1 Figure 16 (F) and Th17 Figure 17 (G) cells.

[0155] Human mononuclear cells THP-1 were polarized into M1 pro-inflammatory macrophages Figure 17 (A, B), and then co-cultured with MSCs Figure 17 (C). The results showed that compared with 2DM and 2D cultured 2DENM cultured in the traditional way, 3DENM had stronger anti-inflammatory gene expression, such as COX2, TSG6, IL10 and IDO Figure 17D). Macrophages co-cultured with 3DENM have more CD206 positive cells and less CD86 positive cells Figure 18 E), i.e. more macrophages are converted from pro-inflammatory M1 type to M2 type.

[0156] 2.33DENM homing effect evaluation

[0157] CCR2 and CXCR4 are important molecules for MSCs to sense chemotactic factors in lesion site and cross endothelial layer, and the results of fluorescent quantitative PCR show that CCR2 and CXCR4 are significantly highly expressed in 3DENM, and 3DENM group more strongly expresses MMP2 and MMP9 genes that degrade extracellular matrix Figure 18 A). WB also shows the same results Figure 18 B), while FAK related to cell migration in 3DENM group is significantly activated. Immunofluorescence results show that 3DENM secretes more MMP2 Figure 18 C) ; cell staining results show that the number of cells successfully migrated in 3DENM group is more, which has significant difference compared with 3DM group Figure 19 D).

[0158] Example Four, Application of Matrix-enabled Mesenchymal Stem Cell Aggregates in Treating Liver Fibrosis

[0159] 1. Experimental method

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

[0161] A mouse liver fibrosis model was manufactured by intraperitoneal injection of 10% CCl4 solution, twice a week, 200 μL each time, and the control group was injected with the same volume of olive oil. After 8 weeks of injection, the stem cell aggregates prepared in Example Three were implanted through the tail vein (the experimental group was 3DENM, and the control group was 2DM and 2DENM), 10 6 MSCs, and the same volume of PBS was implanted in the sham operation group.

[0162] 1.2 Live animal imaging of liver fibrosis mice

[0163] Before small animal live imaging, MSCs were stained with DiR dye according to the steps in the instructions. Live imaging was performed on mice 24 h and 48 h after MSC injection. Before imaging, the mice were anesthetized with ether and fixed on the imaging table, the appropriate imaging parameters were set and the imaging process was started, and the fluorescence signals obtained in the imaging results were observed and counted.

[0164] 1.3 Flow cytometry of liver fibrosis mice

[0165] Mouse liver was collected for grinding, filtration and density gradient centrifugation to obtain immune cells. The cells were resuspended in PBS, incubated with primary antibody, and then washed to remove unbound antibodies. Fluorescently labeled secondary antibodies were added for incubation, and unbound antibodies were washed away. Injected into a flow cytometer, 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 liver fibrosis mouse liver

[0167] The mouse liver was fixed with 4% paraformaldehyde, embedded with paraffin, and then sectioned. After deparaffinization of the section, an appropriate amount of Sirius red staining reagent was prepared according to the proportion in the kit instructions. Stain for 30 minutes to 1 hour, adjust the staining time as needed. After washing off the excess staining solution with PBS, perform alcohol stepwise dehydration, transparentize in organic solvents, and mount with neutral resin. Photographed using a slide scanner and observed.

[0168] 2. Results

[0169] As shown in Figure 19 A, the constructed cell aggregates were implanted into liver fibrosis mice. Small animal imaging and statistical results showed that the 3DENM group had less lung retention and more homing to the liver Figure 20 B&C).

[0170] Flow cytometry identification results showed that 3DENM implantation caused macrophages to be more converted into non-promoting fibrosis phenotype (LY6C-), and macrophages expressed less chemokine receptor CX3CR1 Figure 21 ). Sirius red staining results showed that the 3DENM treatment group had less collagen deposition and had better therapeutic effect Figure 22 ).

[0171] Example Five, Application of Matrix-Enabled Mesenchymal Stem Cell Aggregates in the Preparation of Diabetic Drugs

[0172] 1. Experimental method

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

[0174] All 30 SD rats were adaptively fed for 1 week, and then were given high-fat diet (n=20) and normal diet (n=10) for 8 weeks, to ensure normal blood glucose (blood glucose <250 mg / dL); after 8 weeks, all rats were fasted for 12 h, and the high-fat diet-fed rats were injected intraperitoneally with STZ dissolved in sodium citrate solution at 35 mg / kg, while the normal diet-fed rats were injected intraperitoneally with the same amount of sodium citrate solution, and blood glucose was continuously detected for 3 d; when the blood glucose concentration of the high-fat diet-fed rats with STZ injection was ≥250 mg / dL, it was considered that the type 2 diabetes model was successfully constructed.

[0175] After the type 2 diabetes model was successfully constructed, the rats were gradually changed to normal diet, and the state of the rats was observed and the blood and urine indicators of the rats were detected; after 2 weeks of continuous culture, the 24 h urine of the rats was collected, and when the urine protein was >20 mg / 24 h and significantly higher than that of the normal control group, it was considered that kidney damage occurred at this time, and the diabetic kidney disease (DKD) rat model was successfully constructed. All animal feeding and experiments were strictly in accordance with the animal care and use guidelines approved by Nanjing University Drum Tower Hospital, and appropriate measures were taken to ensure the minimum pain and discomfort of the animals.

[0176] After the diabetes model was successfully prepared, the rats were randomly divided into disease group injected with normal saline (referred to as DKD), treatment group injected with MSC (referred to as KDK+2DM), treatment group injected with EN-MSC (referred to as KDK+2DENM), and treatment group injected with 3DENM (referred to as KDK+3DENM), and the normal rats without modeling were used as the control group (Health), with at least 5 rats in each group. The treatment groups were injected with 2×10 6 μL of 2DM, 2DENM or 3DENM of MSCs in the tail vein at the 11th week and the 12th week, and the modeling group was injected with 500 μL of normal saline.

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

[0178] The rats were weighed every week. The blood glucose was measured by taking blood from the end of the tail vein. One day before the end of the experiment, the urine and feces of the rats were collected in metabolic cages and stored in a refrigerator at -80°C. After two weeks of continuous treatment, the body weight of the rats was first recorded, and then the rats were anesthetized, the chest cavity was opened, and blood was taken from the heart at the left auricle with a 5 mL syringe and placed in an EP tube, centrifuged at 300 x g for 10 min, and the upper liquid was aspirated. After standing for 30 min, the supernatant was aspirated as serum and stored in a refrigerator at -80°C for later use. Then the abdominal cavity of the rat was opened, and the kidneys and spleen were removed for weighing. Part of the kidney was immediately placed in liquid nitrogen for storage; part of the kidney was placed in formaldehyde overnight, and the specimen was treated with 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol in low to high concentrations for 2 h, dehydrated with anhydrous ethanol twice for 1 h each time, and then the tissue block was placed in xylene for transparency. The transparent tissue was placed in melted paraffin, and after the paraffin completely immersed the tissue block, embedding was performed, and the cooled fixed block was fixed on a microtome, sliced into thin sections, placed in heated water to flatten, attached to a glass slide and dried in a constant temperature oven at 45°C, and stored at room temperature; part of the kidney was placed in formaldehyde overnight, and the kidney was dehydrated in 30% sucrose solution until it sank to the bottom. The tissue was removed, washed twice with pre-cooled PBS, and then immersed in a suitable amount of OCT embedding agent. The pre-cooled OCT was fixed on a microtome, sliced into thin sections, attached to a glass slide, and stored in a refrigerator at -80°C for later use.

[0179] 1.3 Detection of biochemical indicators of diabetic rats

[0180] ①Kidney index was calculated according to the following formula:

[0181]

[0182] ②Urine indicators:

[0183] After the rat urine was balanced to room temperature, the supernatant was centrifuged at 1000 x g for 10 min; 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 well, and stood for 2 min. The absorbance at 595 nm was measured, and the urine protein concentration was calculated.

[0184] ③Blood indicators:

[0185] The content of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) of rats was detected by using Nanjing Gulou Hospital automatic chemical analyzer; the content of TNF-α, IL-1β and TGF-β in rat serum was detected, first centrifuging all supernatants at 300xg for 10 min to remove precipitates; adding 300 μL of diluted washing solution (1:20 dilution) to soak the well plate for 30 sec; discarding all washing solution and patting dry on a blotting paper, adding 100 μL of medium or cell culture supernatant sample or diluted standard to each well; adding 50 μL of detection antibody (1:100 dilution) to each well; incubating at room temperature for 2 h; discarding the liquid, washing the plate with 300 μL of washing solution for 6 times, and discarding the washing solution after each washing and patting the well plate dry; adding 100 μL of horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well, and incubating at room temperature for 45 min; discarding the liquid, washing the plate with 300 μL of washing solution for 6 times, and discarding the washing solution after each washing and patting the well plate dry; adding 100 μL of color developing substrate TMB to each well, and incubating at room temperature for 30 min in the dark; adding 100 μL of stop solution to each well, at which time the color changed from blue to yellow; within 30 min, using an enzyme-labeled instrument to detect the OD values at 450 nm maximum absorption wavelength and 570 nm reference wavelength, and calculating the concentration.

[0186] 1.4 Kidney pathological staining of diabetic rats

[0187] The prepared frozen section was sequentially 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 for paraffin section dewaxing and hydration treatment.

[0188] ①For H&E staining: the treated section was placed into a hematoxylin staining box for dyeing for 5 min; the floating color was washed off in water, and gently washed with running water for 10 min; hydrochloric acid alcohol differentiation treatment was performed for 1 sec; the floating color was washed off in water, and washed with running water for 8 min; eosin was dyed for 90 sec; the floating color was washed off in water, and gently washed with running water for 10 min.

[0189] ②For PAS staining: the treated section was placed into a periodic acid solution, and oxidized at room temperature for 25 min; washed with running water for 2 times, each for 2 min, and gently washed with distilled water for 2 times, each for 2 min; Schiff Reagent was dyed at 37°C in the dark for 10 min; sodium sulfite solution was washed for 2 times, each for 2 min; washed with running water for 2 times, each for 2 min, and gently washed with distilled water for 2 times, each for 2 min; placed into a hematoxylin staining box for dyeing for 5 min; the floating color was washed off in water, and gently washed with running water for 10 min.

[0190] ③For Masson staining: put the prepared slice into Weigert iron hematoxylin staining solution and dye for 10 min; rinse with running water for 10 min; return to blue in Masson blue solution for 5 min; rinse with running water for 10 min, rinse with distilled water twice, each for 2 min; dye in ponceau red staining solution for 10 min; wash with weak acid working solution (distilled water: weak acid solution = 2:1) for 1 min; wash with 1% phosphomolybdate solution for 2 min; wash with weak acid working solution for 1 min; directly put into aniline blue staining solution and dye for 2 min; wash with weak acid working solution for 1 min.

[0191] After all the samples are dyed, dehydrate according to 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; dry in a ventilated place, soak in xylene for 5 min; soak in fresh xylene for 5 min; dry in a ventilated kitchen, then mount with neutral resin, and store at room temperature.

[0192] 1.5 Immunofluorescence staining of kidney of diabetic rats

[0193] The prepared frozen section is rinsed with running water for 5 min to wash away OCT, fixed with 4% (v / v) paraformaldehyde for 10 min, incubated with 0.1% Triton X-100 for 10 min, and blocked with 1% (w / v) BSA at 37°C for 30 min; 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) are added to each well, incubated at 4°C overnight, and then 200 μL of Alexa Fluor 488-conjugated goat anti-mouse antibody (1:250 dilution) and Alexa Fluor 594-conjugated goat anti-rabbit antibody (1:250 dilution) are added to each well, incubated at room temperature for 2 h; then 200 μL of mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) is added to each well, and observed under a laser confocal scanning microscope within a week. The expression and distribution of synaptopodin in different kidney tissues are detected.

[0194] 1.6 Western blotting of rat kidney protein

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

[0196] 2Experimental results

[0197] As Figure 23 shown, during the process of DKD model rats and their hMSCs intervention, we observed that compared with the healthy group, DKD rats significantly increased the amount of feed and water. Compared with DKD rats, the rats in the hMSCs treatment group reduced the amount of water and feed. Two weeks after hMSCs transplantation, the body weight of DKD rats decreased significantly. More importantly, the treatment effect of 3DENM was significantly improved compared with the treatment of 2DM and 2DENM.

[0198] Cell damage and inflammatory factor release caused by inflammatory response can induce the activation of fibroblasts, leading to fibrosis of renal interstitium. As Figure 24 shown, compared with the DKD group, the mRNA levels of IL-1β and TNF-α in the kidneys of the hMSCs treatment group rats 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 also further reduced; indicating that 3DENM can effectively alleviate the inflammatory response of DKD rats, thereby delaying the progression of diabetic nephropathy.

[0199] As Figure 25 shown, two weeks after MSCs transplantation, the blood glucose concentration of DKD rats improved slightly, in addition, the common complications of DKD also involve impaired lipid profile, manifested as elevated TC, TG, LDL-C and reduced 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 potentiation of hENFc matrix effectively improves the potential of hMSCs to slow down the kidney damage of diabetic rats, and improves the kidney function of DKD rats.

[0200] As Figure 26 shown, H&E and PAS staining showed that compared with the Health group, the glomeruli of rats in the DKD group were hypertrophic, and the mesangial area matrix was significantly proliferated; compared with the 2DM treatment group, the glomerular volume of 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 mesangial area of rats in the DKD group increased, while the transplantation of 3DENM effectively reduced the glomerular fibrosis; these results indicate that 3DENM effectively improves the kidney damage of DKD rats, and has a stronger protective effect on the kidneys of DKD rats.

[0201] Synaptopodin is involved in the regulation of podocyte morphology and dynamics and is essential for the maintenance of glomerular filtration function. Nephrin is closely related to the selective permeability of the glomerular filtration membrane. It interacts with other proteins to construct the interstitial membrane of the podocyte, maintain the structural integrity of the filtration membrane, and participate in the regulation of glomerular blood filtration and urine formation. In the glomerulus, Wilms tumor gene 1 (WT1) is related to the formation of glomerular mother cells and the differentiation of podocytes, while in the adult kidney, WT1 expression is related to the maintenance of podocyte function and the regulation of renal tubular and interstitial cell function. Through immunofluorescence staining and Western Blotting detection, we found that compared with 2DM treatment, 3DENM treatment increased the expression of Nephrin and WT1 proteins, restored the expression and distribution of Synaptopodin in the rat kidney (e.g. Figure 27 ). It is proved that 3DENM transplantation plays an important role in the repair of glomerular filtration function, the maintenance of filtration membrane integrity, and the regulation of glomerular structure and function in DKD rats.

[0202] Example Six: Application of Matrix-Enabled 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] The rats were anesthetized by inhaling isoflurane. A 1 cm longitudinal incision was made on the skin along the midline of the rat, with the T10 center point as the center. The muscles were bluntly separated to fully expose the T9-T11 spinous processes, and then a dorsal laminectomy was performed on the T10 vertebral body until the spinal cord was fully exposed. A 10g metal rod was dropped from a height of 25mm to simulate moderate spinal cord injury by using the NYU Impactor-III spinal cord injury impactor. The rat immediately showed involuntary convulsions and tail twisting, which met the model criteria, indicating successful modeling. Then the muscles, fascia and skin were sutured in turn, and the rats were placed on a constant temperature electric heating blanket for recovery. The sham operation group only underwent laminectomy without SCI. The modeled rats were randomly divided into three groups: SCI group, 2DM group and 3DENM group. Cefuroxime sodium was injected intramuscularly 3 days after the operation to prevent wound infection, and artificial urination was performed twice a day for 14 consecutive days. Two weeks later, 1*10 6 cells / rat of 2DM and 3DENM were given to the 2DM and 3DENM groups, respectively. The specific transplantation scheme was as follows: using a stereotactic injector, the cells were injected in situ at the injury center and 0.5 cm above and below it, with a cell concentration of 1*10 5 / μL.

[0206] 1.2 BBB locomotor rating

[0207] The hindlimb motor function was assessed by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. The BBB locomotor rating scale was initiated at day 1 after modeling and performed once a week until 8 weeks to evaluate the long-term recovery of hindlimb motor function in rats. The BBB locomotor rating scale ranges from 0 to 21, with 0 representing complete SCI, no spontaneous hindlimb movement in rats, less than 8 indicating only joint movement, 8-13 indicating coordinated movement, 14-20 indicating accurate and stable movement, and 21 representing normal movement of rats. Before each assessment, rats were allowed to freely move in an open area for 5 minutes, and then three researchers assessed and scored the movement of rats within 3 minutes according to the BBB locomotor rating scale.

[0208] 1.3 CatWalk gait analysis

[0209] At 8 weeks after SCI, the footprint, motor behavior, and body coordination of rats in different groups were objectively evaluated using the CatWalk XT system. The CatWalk XT system includes a platform made of a transparent glass plate, and a high-speed camera at the bottom of the platform can record the footprint through the green fluorescence intensity on the glass platform, and a red light-emitting diode at the top of the platform can measure the body profile of the rat. Each rat was trained for at least three days before the test to adapt to walking on the path, and a quiet environment should be ensured during the experiment to avoid disturbing the animals. The gait of each rat was recorded by the CatWalk XT system, and the related analysis was performed using the accompanying software. The analysis parameters include paw print shape, step sequence, etc.

[0210] 1.4 Grid test

[0211] Grid walking is a test to evaluate the ability to accurately control the placement of the hindpaws after spinal cord injury. It is used to assess the fine motor function and limb coordination of mice. Eight weeks after spinal cord injury modeling, rats were placed on a horizontal grid device composed of a 50 cm high, 45 x 45 cm grid with 1.5 x 1.5 cm openings. This test does not require pre-training, and the rat is placed in the center of the grid by lifting its tail. When all four paws are grasping the grid, the tail is released. One observer records the total number of steps of the bilateral hindlimbs for 120-150 steps, and two other observers record the total number of times the hindlimbs fall off the grid during this period. Finally, the average number is calculated to calculate the error rate of the hindlimbs.

[0212] Hindlimb error rate = total number of hindlimb falls / total number of hindlimb steps x 100%

[0213] 1.5 Hot plate test

[0214] The temperature of the hot plate was kept at 52°C, and a transparent acrylic cylinder was placed on it. After 6 weeks of injecting stem cells, the rats in each group were placed in the cylinder in turn. When the rat's hind limbs fully contacted the hot plate, the timing started immediately. When the mouse was observed to jump from the hot plate or start to lick the hind foot, it was considered that the mouse had pain, and the timing stopped. The time required for each mouse to produce a response was recorded. After spinal cord injury in rats, the pain threshold is reduced, and the perception of pain is more obvious. Stem cell treatment can improve the pain threshold of rats and prolong the reaction time.

[0215] 1.6 Louisville swim scale score

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

[0217] 1.7 Electrophysiological test of 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. After 8 weeks of spinal cord injury, the rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Motor evoked potentials (MEP) were measured by electrophysiological equipment. To detect MEP, a recording electrode was placed subcutaneously in the gastrocnemius muscle of the lower limb to record peak amplitude and latency, a grounding electrode was placed subcutaneously in the back, and a stimulating electrode and a reference electrode were placed subcutaneously between the two ears. A current of 5 mA was used to stimulate the motor cortex to obtain motor evoked potentials (MEP). MEP is used to represent the transmission of hind limb motor signals, representing descending conduction. The greater the potential difference between the peak and trough of the wave, the stronger the descending motor nerve conduction.

[0219] 2. Experimental results

[0220] As shown in Figure 28 The BBB scores of all rats decreased to 0 one day after successful SCI modeling, and recovered within 14 days but were all less than 8. Only joint movement was observed, and no foot weight-bearing was observed. After stem cell treatment, the scores of the treatment group improved significantly, and the 3DENM group recovered faster than the 2DM group. The 3DENM group reached 14 points first 3 weeks after cell transplantation, indicating that the rats in the 3DENM group could perform relatively stable and accurate movements. After 5 weeks of transplantation, they averaged 17-18 points, far exceeding the 12-13 points of the 2DM group, indicating that the 3DENM group had a more optimal treatment effect. Figure 29As shown in A, the footprints of gait analysis show that the footprints of the SCI group cannot be detected due to the inability of the hind limbs to bear weight, while the 3DENM group is observed to have more coordinated movement of the fore and hind limbs compared to the 2DM group, and is closer to the performance of the sham group, indicating that the 3DENM group has rescued more of the lost motor function after spinal cord injury; as Figure 29 As shown in A, the footprints of gait analysis show that the footprints of the SCI group cannot be detected due to the inability of the hind limbs to bear weight, while the 3DENM group is observed to have more coordinated movement of the fore and hind limbs compared to the 2DM group, and is closer to the performance of the sham group, indicating that the 3DENM group has rescued more of the lost motor function after spinal cord injury; as Figure 30 As shown in B, the results of the hot plate experiment show that after 6 weeks of cell transplantation treatment, the pain threshold of the stem cell treatment group is higher than that of the SCI group, the tolerance to pain is stronger, and the response time to heat stimulation is longer, indicating that the degree of thermal hyperalgesia in the stem cell treatment group is lower than that in the SCI group, which is due to the neuropathic pain of hyperalgesia caused by spinal cord injury; as Figure 30 As shown in A, in the swimming test, the 3DENM group has a swimming posture closer to the sham group than the 2DM group, Figure 30 B shows that the LSS score results show that the 3DENM group has better fore and hind limb coordination and trunk stability, ​ C uses motor evoked potentials (MEP) to evaluate the function of descending motor nerve conduction, and the results show that the motor potential difference of the 3DENM group is higher than that of the 2DM group, improving the nerve conduction function of the SCI rats.

[0221] In summary, the present application provides a stromal-competent mesenchymal stem cell, which is obtained by subculturing mesenchymal stem cells in a cell culture substrate prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The stromal-competent mesenchymal stem cell obtained by culturing in the cell culture substrate of the present application can promote the nuclear transfer of β-catenin, enhance the in vitro proliferation activity, maintain good in vitro proliferation ability, maintain stemness, delay cell aging and apoptosis, induce the expression of multiple functions of hMSCs, and the hMSCs cultured to the fifteenth generation by hENFc have no tumorigenicity and no adverse effects on animals, meeting the clinical application standards of hMSCs. The stromal-competent mesenchymal stem cell is subcultured in a 3D cell culture system to obtain stromal-competent mesenchymal stem cell aggregates, which have a smaller 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, 2D cadherin matrix-competent cultured 2DENM, and 3DM cultured by traditional methods, the stromal-competent mesenchymal stem cell aggregates have stronger anti-inflammatory gene expression, more CD206 and less CD86 expression of macrophages co-cultured with 3DENM, which shows that more macrophages are converted from pro-inflammatory M1 type to M2 type; and can promote cell migration. The stromal-competent mesenchymal stem cell aggregates have better effect on treating liver fibrosis, more macrophages are converted to non-fibrophenotype (LY6C-) after implantation, macrophages express less chemotactic factor receptor CX3CR1, and less collagen deposition. This shows that 3DENM has excellent therapeutic effect. The stromal-competent mesenchymal stem cell aggregates can better treat diabetes, effectively reduce the inflammatory response of DKD rats, thereby delaying the progression of diabetes and its nephropathy, reducing kidney damage in diabetic rats, and reducing glomerular fibrosis. The stromal-competent mesenchymal stem cell aggregates can also effectively improve the motor function, gait of spinal cord injury rats, and relieve neuropathic pain hypersensitivity, and can restore nerve conduction function.

Claims

1. A matrix-enabled mesenchymal stem cell aggregate, characterized in that, The mesenchymal stem cell aggregate is obtained by culturing mesenchymal stem cells in a 3D cell culture system after subculturing the mesenchymal stem cells in a cell culture substrate prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc, wherein the cell culture substrate is prepared by: (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 the solution to a tissue culture treated plate, incubating, and washing to obtain the cell culture substrate hENFc; (2) inoculating mesenchymal stem cells into the cell culture substrate hENFc obtained in step (1) to obtain the matrix-enabled mesenchymal stem cells by subculturing the cells; (3) resuspending the matrix-enabled mesenchymal stem cells obtained in step (2) and culturing the cells in a 3D cell culture system to obtain the mesenchymal stem cell aggregate.

2. The mesenchymal stem cell aggregate of claim 1, wherein, In step (1), the incubation time is 0.5-24 h and the incubation temperature is 4-37 °C.

3. The method for preparing matrix-empowered mesenchymal stem cell aggregates as described in claim 1, characterized in that, The method comprises 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 the solution to a tissue culture treated plate, incubating, and washing to obtain the cell culture substrate hENFc; (2) inoculating mesenchymal stem cells into the cell culture substrate hENFc obtained in step (1) to obtain the matrix-enabled mesenchymal stem cells by subculturing the cells; (3) resuspending the matrix-enabled mesenchymal stem cells obtained in step (2) and culturing the cells in a 3D cell culture system to obtain the mesenchymal stem cell aggregate.

4. Use of the mesenchymal stem cell aggregate according to any one of claims 1-2 in the preparation of a medicament for treating liver fibrosis.

5. Use of the mesenchymal stem cell aggregate according to any one of claims 1-2 in the preparation of a medicament for treating diabetes.

6. Use of the mesenchymal stem cell aggregate according to any one of claims 1-2 in the preparation of a medicament for repairing spinal cord injury.

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