Cell microsphere for maintaining cell activity by resisting shearing force and application of cell microsphere in disease treatment

Abstract

The invention relates to the technical field of cell biology, in particular to cell activity maintenance of cell microspheres by resisting shear force and application of the cell microspheres in disease treatment. The invention provides a novel strategy, the cells, especially mesenchymal stem cells (MSCs), are wrapped by the sodium alginate hydrogel microspheres, so that the cells can be effectively protected from being influenced by external mechanical stimulation, and the activity and functions of the cells are maintained. The cell microspheres prepared by the preparation method provided by the invention not only can protect cells from mechanical stimulation and maintain the activity and function of the cells, but also simplify the operation process, avoid the use of tedious chemical cross-linking agents, and ensure the stability and biocompatibility of the cells. The strategy solves the problems of survival and function maintenance of cells in a complex mechanical environment in a breakthrough manner, has wide application potential, and particularly has important application value in cell therapy, regenerative medicine, tissue repair and treatment of diseases (such as arthritis and cartilage injury) related to mechanical stimulation.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to cell microspheres that maintain cell activity by resisting shear force and their application in disease treatment. Background Art

[0002] In the fields of regenerative medicine and cell therapy, stem cells (particularly mesenchymal stem cells (MSCs)) are widely used in tissue repair, disease treatment, and regenerative medicine. Stem cells possess robust self-renewal capacity and multidirectional differentiation potential, making them a research hotspot in many clinical applications. However, during in vitro expansion and transport, stem cells are often exposed to a variety of complex physical and mechanical stimuli, such as FSS. These mechanical stimuli can impair stem cell function, thereby affecting their therapeutic efficacy and even causing cell apoptosis, limiting their potential for clinical application.

[0003] FSS, as a common mechanical stimulus, usually occurs in blood circulation, body fluid flow, or in vitro culture environments. Studies have shown that FSS has a significant impact on cell survival and function, especially in stem cell therapy, where FSS may lead to cell morphological changes, abnormal gene expression, dysfunction, and even cell apoptosis. Many studies have attempted to improve cell survival in mechanical environments by changing culture conditions, using chemical additives, or other external stimuli, but these methods are often unable to effectively avoid the negative effects of mechanical stimulation and may have adverse effects on the biological properties of cells.

[0004] To overcome this problem, scientists have begun to explore the use of different cell carriers and protection strategies. Among them, microsphere carriers have become a promising technology because they can provide structural protection and cell function support. Microsphere carriers can effectively isolate external adverse factors and provide cells with a stable microenvironment by encapsulating cells in polymer materials. However, most existing cell carrier technologies focus on nutritional support and anti-apoptosis effects, and the protective effect on cells against mechanical stimulation (especially FSS) is still limited.

[0005] Sodium alginate hydrogel, a natural polysaccharide material, has been widely used in the biomedical field due to its good biocompatibility, strong degradability, and tunability. However, how to effectively integrate this material with cells and enhance the cells' ability to resist external mechanical stimuli (such as FSS) remains a major challenge in current technology.

[0006] Therefore, a new technology is urgently needed that can provide more effective protection against the complex mechanical stimuli to which cells are subjected during in vitro and in vivo treatments through the rational design of cell carriers, particularly microspheres encapsulating stem cells. This technology should be able to maintain cell viability, minimize the negative effects of mechanical damage on cells, and potentially play a greater clinical role in regenerative medicine and cell therapy. Summary of the Invention

[0007] The present invention aims to at least partially address one of the technical problems in the related art. To this end, one object of the present invention is to provide cell microspheres and a method for preparing the same, which not only protect cells from physical damage from the external environment but also effectively maintain cell activity and prevent cell apoptosis under the action of FSS.

[0008] To this end, the first aspect of the present invention provides a method for preparing cell microspheres. In some embodiments of the present invention, the preparation method comprises:

[0009] S1: Mixing sodium alginate solution, mannitol, and cells to obtain a mixed solution;

[0010] S2: injecting the mixed solution into an electrospray device to obtain microsphere precursors with uniform cell distribution;

[0011] S3: mixing the microsphere precursor with a calcium chloride solution and performing a gelation treatment to obtain cell microspheres.

[0012] The present invention proposes a novel strategy to encapsulate cells, especially mesenchymal stem cells (MSCs), by encapsulating them with sodium alginate hydrogel microspheres, thereby effectively protecting the cells from external mechanical stimulation (such as fluid shear force FSS), thereby maintaining the activity and function of the cells. The cell microspheres prepared by the preparation method of the present invention can not only protect the cells from mechanical stimulation and maintain their activity and function, but also simplify the operation process, avoid the use of cumbersome chemical cross-linking agents, and ensure the stability and biocompatibility of the cells. This strategy has a breakthrough solution to the problem of cell survival and maintenance of function in complex mechanical environments, and has wide application potential, especially in cell therapy, regenerative medicine, tissue repair and the treatment of diseases related to mechanical stimulation (such as arthritis, cartilage damage, etc.). It has important application value.

[0013] In some embodiments of the present invention, the working concentration of sodium alginate in the sodium alginate solution is 2 to 5 wt %.

[0014] In some embodiments of the present invention, the working concentration of mannitol is 3-6 wt %.

[0015] In some embodiments of the present invention, the concentration of the cells in the mixed solution is 1×10^1 to 1×10^8 cells / mL.

[0016] In some embodiments of the present invention, the working concentration of the calcium chloride solution is 0.05-0.5M.

[0017] In some embodiments of the present invention, in step S2, when using an electrospray device, the voltage and liquid flow rate are controlled so that the mixed liquid forms a Taylor cone at the nozzle of the electrospray device, thereby obtaining a microsphere precursor with uniform cell distribution.

[0018] By controlling the electrospray process, a uniform and controllable microsphere structure is obtained, which significantly improves the cell survival rate and therapeutic effect.

[0019] The second aspect of the present invention provides a cell microsphere. In some embodiments of the present invention, the cell microsphere is obtained by the preparation method described in the first aspect.

[0020] The cell microspheres of the present invention can effectively resist the effect of fluid shear force, reduce the negative impact of FSS on cells, and thus significantly improve the survival rate and activity of cells in a dynamic fluid environment.

[0021] In some embodiments of the present invention, the diameter of the cell microspheres is 0.03-2 mm.

[0022] In some embodiments of the present invention, the density of cells in the cell microspheres is 10 to 100 cells per microsphere.

[0023] The third aspect of the present invention provides a composition. In some embodiments of the present invention, the composition comprises the cell microspheres described in the second aspect.

[0024] The fourth aspect of the present invention provides use of the cell microspheres described in the second aspect and the composition described in the third aspect in preparing a drug. In some embodiments of the present invention, the drug is used to treat diseases requiring cell regeneration.

[0025] In some embodiments of the present invention, the disease includes burns, scalds, osteoarthritis, joint injuries, fractures, chronic pain, osteoporosis, autoimmune diseases, metabolic diseases, neurodegenerative diseases and cardiovascular diseases.

[0026] A fifth aspect of the present invention provides a method for alleviating mechanical stimulation of cells. In some embodiments of the present invention, the method comprises:

[0027] The cells are prepared into the cell microspheres described in the second aspect.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] The present invention effectively solves the problem in the prior art that stem cells are easily damaged in complex mechanical environments (especially FSS) by using sodium alginate hydrogel microspheres to encapsulate cells (such as MSCs). Compared with traditional encapsulation technology, the microspheres of the present invention can not only protect cells from mechanical stimulation and maintain their activity and function, but also simplify the operation process, avoid the use of cumbersome chemical cross-linking agents, and ensure the stability and biocompatibility of cells. In addition, this technology obtains a uniform and controllable microsphere structure by precisely controlling the electrospray process, significantly improving the survival rate and therapeutic effect of cells, especially showing an efficacy superior to traditional MSCs injection in a rat knee osteoarthritis model. Therefore, the present invention has a high application potential, especially in cell therapy, regenerative medicine and the treatment of diseases related to mechanical stimulation, showing stronger advantages and broad application prospects than the existing technology.

[0030] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0032] Figure 1 The optical fiber structure of the cell microsphere is shown;

[0033] Figure 2 shows the microscopic images of cell microspheres obtained under a scanning electron microscope;

[0034] Figure 3 Shows the live and dead cell staining of cell microspheres in static culture state;

[0035] Figure 4 Shows the live and dead cell staining of cell microspheres under FSS stimulation;

[0036] Figure 5 The flow cytometry results of cell mitochondria staining under FSS stimulation are shown;

[0037] Figure 6 The flow cytometry results of cell apoptosis under FSS stimulation are shown;

[0038] Figure 7 The figure shows the EDU staining results of rat BMSCs treated with the secretion supernatant of cell microspheres under FSS stimulation;

[0039] Figure 8The graph shows the CCK-8 results of rat BMSCs proliferation treated with the supernatant of cell microsphere secretion under FSS stimulation;

[0040] Figure 9 The figure shows the β-gal staining results of rat BMSCs treated with the secretion supernatant of cell microspheres under FSS stimulation;

[0041] Figure 10 The figure shows the results of Transwell staining of rat BMSCs treated with the secretion supernatant of cell microspheres under FSS stimulation;

[0042] Figure 11 Shows the schematic diagram of the construction of the SD rat osteoarthritis model and the grouping of in vivo animal experiments;

[0043] Figure 12 Shown are the micro-CT results of SD rat knee OA after treatment;

[0044] Figure 13 The figure shows the HE staining results of SD rat knee joint OA after treatment;

[0045] Figure 14 The figure shows the immunohistochemical staining results of type II collagen (COL2) after treatment of OA in the knee joint of SD rats;

[0046] Figure 15 The figure shows the results of aggrecan staining after treatment of OA in the knee joint of SD rats;

[0047] Figure 16 The figure shows the results of MMP13 staining after treatment of OA in SD rat knee joints;

[0048] Figure 17 The figure shows the results of ADAMTS5 staining after treatment of SD rat knee OA. DETAILED DESCRIPTION

[0049] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0050] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0051] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0052] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0053] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0054] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0055] According to a specific embodiment of the present invention, the present invention provides a method for preparing cell microspheres, comprising:

[0056] S1: Mixing sodium alginate solution, mannitol, and cells to obtain a mixed solution;

[0057] S2: injecting the mixed solution into an electrospray device to obtain microsphere precursors with uniform cell distribution;

[0058] S3: mixing the microsphere precursor with a calcium chloride solution and performing a gelation treatment to obtain cell microspheres.

[0059] It should be noted that there are no particular limitations on the structure of the electrospray device. By controlling the conditions, any device capable of spraying the mixed solution to obtain microsphere precursors with uniform cell distribution is encompassed within the scope of protection of the present invention. For example, the electrospray device described in the present invention may also be the electrospray device described in patent CN115582079A.

[0060] According to a specific embodiment of the present invention, the working concentration of sodium alginate in the sodium alginate solution is 2-5 wt %. For example, the working concentration of sodium alginate in the sodium alginate solution can be 2 wt %, 3 wt %, 4 wt %, 5 wt %, etc.

[0061] According to a specific embodiment of the present invention, the working concentration of the mannitol is 3 to 6 wt%. For example, the working concentration of the mannitol can be 3 wt%, 4 wt%, 5 wt%, 6 wt%, etc. According to a specific embodiment of the present invention, the concentration of the cells in the mixed solution is 1×10^1 to 1×10^8 cells / mL. For example, the concentration of the cells in the mixed solution can be 1×10^1 cells / mL, 1×10^2 cells / mL, 1×10^3 cells / mL, 1×10^4 cells / mL, 1×10^5 cells / mL, 1×10^6 cells / mL, 1×10^7 cells / mL, 1×10^8 cells / mL, etc.

[0062] According to a specific embodiment of the present invention, the working concentration of the calcium chloride solution is 0.05 to 0.5 M. For example, the working concentration of the calcium chloride solution can be 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, etc.

[0063] According to a specific embodiment of the present invention, the working concentration of sodium alginate in the sodium alginate solution is 2-5wt%; the working concentration of mannitol is 3-6wt%; the concentration of the cells in the mixed solution is 1×10^1-1×10^8 cells / mL; and the working concentration of the calcium chloride solution is 0.05-0.5M.

[0064] According to a specific embodiment of the present invention, in step S2, when using an electrospray device, the voltage and liquid flow rate are controlled so that the mixed liquid forms a Taylor cone at the nozzle of the electrospray device, thereby obtaining a microsphere precursor with uniform cell distribution. Thus, by controlling the electrospray process, a uniform and controllable microsphere structure is obtained, significantly improving cell survival rate and therapeutic efficacy.

[0065] According to a specific embodiment of the present invention, the present invention provides a cell microsphere obtained by the above-mentioned preparation method.

[0066] The cell microspheres of the present invention can effectively resist the effect of fluid shear force, reduce the negative impact of FSS on cells, and thus significantly improve the survival rate and activity of cells in a dynamic fluid environment.

[0067] According to a specific embodiment of the present invention, the diameter of the cell microsphere is 0.03-2 mm. For example, the diameter of the cell microsphere is 0.03 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, etc.

[0068] According to a specific embodiment of the present invention, the cell density in the cell microspheres is 10 to 100 cells per microsphere. Within this cell density range, cell survival and function can be further maintained, and the survival rate and activity of cells in a dynamic fluid environment can be further improved.

[0069] According to a specific embodiment of the present invention, the present invention provides a composition comprising the cell microspheres described above.

[0070] According to a specific embodiment of the present invention, the cell microspheres and compositions provided by the present invention can be used to prepare drugs for treating diseases requiring cell regeneration.

[0071] According to a specific embodiment of the present invention, the diseases requiring cell regeneration include burns, scalds, osteoarthritis, joint injuries, fractures, chronic pain (including neuralgia and joint pain), osteoporosis, autoimmune diseases (such as rheumatoid arthritis), metabolic diseases (such as diabetes, obesity), neurodegenerative diseases (such as Alzheimer's disease) and cardiovascular diseases (such as atherosclerosis).

[0072] According to a specific embodiment of the present invention, the present invention also provides a method for alleviating mechanical stimulation of cells, the method comprising:

[0073] The cells were prepared into cell microspheres as described above.

[0074] The present invention aims to solve the problem that the functional activity of stem cells is impaired under complex mechanical stimulation environments, especially under the action of fluid shear stress (FSS). This technology uses sodium alginate hydrogel microspheres constructed based on an electrospray device, and uses electrospray technology to evenly wrap mesenchymal stem cells (MSCs) inside the microspheres. This type of microsphere not only protects the cells from physical damage in the external environment through solid wrapping, but also effectively maintains the activity of the cells and avoids cell apoptosis under the action of FSS. Specifically, the design of the cell microspheres can effectively resist the action of fluid shear force, reduce the negative impact of FSS on cells, and thus significantly improve the survival rate and activity of cells in a dynamic fluid environment. The cell microsphere culture supernatant after FSS stimulation can significantly promote the proliferation, migration and anti-apoptosis of rat bone marrow mesenchymal stem cells (BMSCs), demonstrating its potential in tissue repair. In addition, experimental studies using a rat knee osteoarthritis (OA) model have shown that the therapeutic effect of using the cell microspheres is significantly better than that of simply injecting MSCs. The cell microspheres not only increased the survival rate of MSCs but also enhanced their repair function in pathological conditions, effectively alleviating cartilage damage and inflammatory responses caused by arthritis. This technology provides a new solution for stem cell therapy, especially in large FSS application environments, and has broad application prospects.

[0075] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0076] Example 1: Preparation of cell microspheres

[0077] 1. Prepare cell suspension:

[0078] 1) Prepare a 2 wt% sodium alginate solution (Aladdin, #S100127) by dissolving it in an appropriate amount of deionized water, ensuring that the sodium alginate is completely dissolved.

[0079] 2) Add 4.5 wt% mannitol (Aladdin, M108829-100g) to improve solution stability and reduce cell damage. Mix thoroughly until the solution becomes transparent.

[0080] 3) Adjust the cell concentration in the suspension to 2 × 10 cells / mL. The cells used are MSCs, ensuring that the cells are evenly dispersed.

[0081] 4) Using a syringe pump, the cell-containing sodium alginate suspension is transported through a polytetrafluoroethylene (Teflon) tube to the nozzle of an electrospray device (the electrospray device in patent CN115582079A).

[0082] 2. Electrospray process:

[0083] 1) Connect the high voltage module of the electrospray device to the syringe pump and set the voltage to gradually increase from 0 kV to 11.5 kV;

[0084] 2) During the application of voltage, the sodium alginate solution forms a Taylor cone at the nozzle, which gradually extends into a jet. At this point, droplets are ejected from the nozzle, forming an aerosol containing MSCs.

[0085] 3) The spray module of the electrospray device can ensure the stable formation of microsphere precursors with uniform cell distribution.

[0086] 3. Gelation and collection of microspheres:

[0087] 1) After spraying, the microparticles enter a metal collection tray filled with a 0.1 M calcium chloride (CaCl) solution. The calcium chloride solution promotes the gelation process of sodium alginate, thereby forming a strong microsphere structure.

[0088] 2) During the electrospray process, 4 mL of calcium chloride solution was periodically added to the collection plate to ensure uniform gelation.

[0089] 3) This process needs to be carried out in a sterile environment to prevent cell contamination.

[0090] 4. Microsphere collection, cleaning and culture:

[0091] 1) After about 10 minutes of spraying and gelation, the formed cell-encapsulated microspheres are gently removed from the collection plate;

[0092] 2) The microspheres were transferred to sterile culture medium and washed twice with culture medium to remove residual calcium chloride and avoid adverse effects of calcium chloride on the cells;

[0093] 3) After washing, the encapsulated cell microspheres were transferred to culture medium (human mesenchymal stem cell expansion medium, Wuhan Vinocell Biotechnology Co., Ltd.) for culture. The culture medium was changed every two days.

[0094] 4) When changing the culture medium, slightly tilt the culture plate to allow the microspheres to settle to the bottom, which helps to efficiently remove the old culture medium without disturbing the microsphere structure.

[0095] Note: The above human mesenchymal stem cell expansion medium was supplemented with 5% FBS and 1% double antibody (FBS and double antibody were purchased from Wuhan Vinocell Biotechnology Co., Ltd.).

[0096] Example 2: Cell microsphere light microscopy and scanning electron microscopy (SEM) imaging

[0097] The cell microspheres were placed in fresh culture medium (human mesenchymal stem cell expansion medium) and their morphology and structure were observed under an optical microscope. Meanwhile, a scanning electron microscope (SEM) (Inspect S50) was used to observe the microscopic morphology and internal structure of the cell microspheres.

[0098] Optical microscopy results ( Figure 1 ) shows the spherical structure of the cell microspheres, with a diameter of approximately 250 μm. The microspheres are composed of a solid spherical matrix formed by sodium alginate, which encapsulates MSCs. MSCs are evenly or locally distributed within the sodium alginate matrix, forming granular or uneven optical reflections. SEM image ( Figure 2 ) shows the complete appearance and internal structure of sodium alginate microspheres. The left image shows the intact microspheres, which exhibit a regular spherical morphology and a textured surface, likely formed during the gelation process. The right image shows the internal structure of the microspheres, revealing their porous three-dimensional network matrix. This pore structure facilitates cell encapsulation and nutrient exchange. These features demonstrate the good structural integrity and internal porosity of the sodium alginate microspheres.

[0099] Example 3: Live or dead staining of cell microspheres in a resting state and after FSS stimulation

[0100] After one day of static culture and one day of FSS stimulation, the cell microspheres were stained for viability. The specific method was as follows: 1 μL of calcein-AM and 4 μL of ethidium dimer-1 were added to 2 mL of sterile PBS solution. The cell microspheres were soaked in the solution at room temperature for 30 minutes. The microspheres were then gently washed three times with sterile PBS, the PBS solution was aspirated, and the cells were photographed under a laser confocal microscope.

[0101] Dead / live staining results ( Figure 3 ) shows that after the cell microspheres were cultured in a static state for 1 day, the living cells were labeled with green fluorescence and showed a relatively concentrated distribution inside the microspheres; the dead cells were labeled with red fluorescence and were fewer in number and scattered. The merged image shows the overall distribution of living and dead cells, further verifying the good biocompatibility of the microspheres. After 1 day of FSS stimulation ( Figure 4), live cells, labeled with green fluorescence, are evenly distributed and concentrated, indicating a high survival rate; the number of dead cells labeled with red fluorescence is extremely small, indicating a very low cell mortality rate. The combined image further confirms the protective effect of the microspheres on cells. Under FSS (Fluid Shear Stress) stimulation, MSCs still maintain good activity and survival. This shows that sodium alginate encapsulation can provide a stable microenvironment for cells and effectively resist the influence of external mechanical forces.

[0102] Example 4: Detection of mitochondrial membrane potential of cells under FSS stimulation (TMRM staining flow cytometry analysis)

[0103] Collect the cells or cell microspheres and wash them with an appropriate amount of PBS to remove residual culture medium; then add a staining solution containing TMRM (tetramethylrhodamine methyl ester) (concentration of 100-200nM); incubate the cells and staining solution at 37°C in the dark for 20-30 minutes to allow TMRM to fully enter the mitochondria and bind to the negatively charged membrane; after staining, wash the cells with PBS to remove unbound dye; finally, detect the TMRM fluorescence signal (red) by flow cytometry or fluorescence microscopy to evaluate changes in mitochondrial membrane potential.

[0104] Flow cytometry results of TMRM staining of MSCs and their encapsulated cell microspheres under resting state and FSS stimulation conditions ( Figure 5 ) showed that the proportion of cells with TMRM staining was higher in MSCs and cell microspheres that were not stimulated by shear force (93.5% and 90.2%), indicating that the mitochondrial membrane potential was well maintained under quiescent conditions, indicating high cell activity. Under FSS stimulation, the TMRM-positive proportion of isolated MSCs dropped significantly to 60.6%, indicating that FSS caused significant damage to bare MSCs. However, under the same stimulation conditions, the TMRM-positive proportion of MSCs encapsulated in cell microspheres remained at 90.8%, close to the quiescent state, indicating that the microspheres played an effective protective role for the cells.

[0105] Example 5: Flow cytometric staining detection of cell apoptosis under FSS stimulation

[0106] First, naked MSCs and MSCs encapsulated in sodium alginate microspheres were collected and washed separately after quiescence and FSS stimulation. Subsequently, the cells were stained using the Annexin V-FITC / PI double staining kit: 205 μL of working solution (190 μL of buffer, 5 μL of Annexin V-FITC, and 10 μL of PI) was added and incubated at room temperature for 10-15 minutes in the dark according to the reagent instructions. After staining, the sample was diluted and immediately detected by flow cytometry, and the fluorescence signals of the cells in the FITC and PI channels were recorded to distinguish the distribution ratios of early apoptotic, late apoptotic, and dead cells. Finally, the data were processed using analysis software to generate scatter plots and perform quantitative statistical analysis.

[0107] The flow cytometry results of apoptosis and death of MSCs and their encapsulated cell microspheres in resting state and FSS stimulation conditions are as follows Figure 6 As shown, in MSCs and cell microspheres not stimulated by shear stress, the majority of cells were located in the Q3 quadrant (early apoptotic and viable cells), with low percentages of apoptotic and dead cells, approximately 7.97% (MSCs) and 7.45% (cell microspheres), respectively, indicating good cell survival in a resting state. Under FSS stimulation, exposed MSCs underwent significant apoptosis and death (in the Q2 quadrant, the percentage of late apoptotic and dead cells increased significantly to 30.1%), indicating significant damage to MSCs by FSS. However, after FSS stimulation, the percentage of apoptotic and dead cells in MSCs encapsulated in cell microspheres only increased to 11.3%, far lower than that in bare MSCs, demonstrating the protective effect of cell microspheres against FSS. The percentage of non-viable cells in bare MSCs was significantly higher than that in encapsulated cell microspheres under FSS stimulation (P < 0.0004), further confirming the effectiveness of cell microspheres in protecting MSCs from mechanical damage.

[0108] Example 6: EdU staining of rat BMSCs treated with different cell culture supernatants

[0109] The experiment first collected the culture supernatant of bare MSCs, FSS-stimulated MSCs (FSS+MSCs), cell microspheres, and FSS-stimulated cell microspheres (FSS+cell microspheres), and centrifuged to remove residual cells and particles. Rat BMSCs were then inoculated into culture plates and treated with culture supernatants of different states for 48 hours. Afterwards, 10 μM EdU working solution was added and incubated for 2 hours to mark proliferating cells. After the culture was completed, the cells were washed and fixed with PBS, and then treated with permeabilization solution to increase permeability. Finally, fluorescent labeling was performed with EdU color development reaction solution, and the cell nuclei were stained with DAPI. After color development, the proportion of EdU-positive cells was observed and counted by fluorescence microscopy to evaluate cell proliferation.

[0110] EdU staining was used to detect the effects of different culture supernatant treatments on the proliferation capacity of rat BMSCs. The results are as follows ( Figure 7 ): The proportion of EdU-positive cells in the control group was low, indicating that the proliferation level of BMSCs under basic conditions (DMEM low glucose medium (Vicente) + 10% FBS (GIBCO) + 1% double antibody (GIBCO)) was limited. The group treated with the culture supernatant of naked MSCs (MSCs group) significantly increased the EdU-positive proportion of BMSCs, indicating that factors secreted by MSCs can promote the proliferation of BMSCs. However, treatment with the culture supernatant of MSCs after FSS stimulation (FSS+MSCs group) significantly reduced the proliferation ability of BMSCs, and the proportion of EdU-positive cells was significantly decreased compared with that of MSCs in the resting state. In contrast, the group treated with the culture supernatant of cell microspheres (cell microsphere group) and the group treated with the culture supernatant of cell microspheres stimulated by FSS (FSS+cell microsphere group) both showed a higher proliferation ability of BMSCs, and there was no significant difference between the two. The results show that cell microspheres can protect the function of MSCs under FSS stimulation conditions, so that the factors they secrete can still significantly promote the proliferation of BMSCs.

[0111] Example 7: CCK8 detection of rat BMSCs treated with different cell culture supernatants

[0112] The experiment first collected the culture supernatants of naked MSCs, FSS-stimulated MSCs (FSS+MSCs), cell microspheres, and FSS-stimulated cell microspheres (FSS+cell microspheres). Rat BMSCs were seeded in 96-well plates, and an appropriate amount of cell suspension (5000 cells / well) was added to each well. The culture supernatants of different states were used instead of conventional culture medium for culture. At specific time points on days 1, 3, and 5, 10 μL of CCK-8 reagent (according to the reagent instructions) was added to each well. After incubation at 37°C for 2 hours in the dark, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The proliferation capacity of BMSCs under different conditions was evaluated based on the changes in OD values, and the results were statistically analyzed to compare the differences between the groups.

[0113] CCK-8 test results ( Figure 8) showed that culture supernatants in different states had a significant effect on the proliferation ability of rat BMSCs. On the first day of the experiment, there was no statistically significant difference in the OD values ​​of the groups, indicating that the starting conditions were the same. Starting from the 5th day, the culture supernatant of the naked MSCs group significantly increased the proliferation ability of BMSCs (P<0.0001), while the culture supernatant of the MSCs group stimulated by FSS (FSS+MSCs) significantly reduced the proliferation effect, and there was no significant difference compared with the control group (NS). The cell microsphere group and the cell microsphere group stimulated by FSS (FSS+cell microsphere) both showed higher OD values ​​on the 5th day, which were comparable to the MSCs group (P>0.05) and significantly higher than the control group (P<0.0001). The results show that FSS weakens the promoting effect of factors secreted by naked MSCs on the proliferation of BMSCs, while cell microsphere encapsulation can effectively protect the function of MSCs, so that they can still secrete factors to promote the proliferation of BMSCs under FSS stimulation.

[0114] Example 8: β-gal staining of rat BMSCs after treatment with different cell culture supernatants

[0115] The experiment first collected the culture supernatants of naked MSCs, FSS-stimulated MSCs (FSS+MSCs), cell microspheres, and FSS-stimulated cell microspheres (FSS+cell microspheres). Rat BMSCs were seeded in 6-well plates, and an appropriate amount of cell suspension was added to each well and cultured to approximately 70% confluence. Subsequently, the culture medium was replaced with the culture supernatants of different states and cultured for another 48 hours. After treatment, the cells were washed twice with PBS and freshly prepared β-gal staining working solution (including X-gal, iron ion and magnesium ion buffer) was added and incubated at 37°C in the dark for 12 to 16 hours. After incubation, the cells were observed under a microscope and β-gal-positive cells (appearing as blue-stained areas) were photographed. The ratio of β-gal-positive area to total cell area was calculated using image analysis software, the senescence level of different groups was statistically analyzed, and the effects of culture supernatants on BMSCs were compared.

[0116] β-gal staining results ( Figure 9) showed that culture supernatants in different states had a significant effect on the cell senescence level of rat BMSCs. In the control group, the area ratio of β-gal-positive cells was low, indicating that the senescence level of BMSCs under basal conditions was low. In the BMSCs treated with the MSCs group, the number of β-gal-positive cells was significantly reduced, indicating that the culture supernatant of bare MSCs can inhibit the senescence of BMSCs. In the FSS+MSCs group, the number of β-gal-positive cells further increased significantly, indicating that the culture supernatant of bare MSCs stimulated by FSS played a significant role in promoting the senescence of BMSCs. In contrast, in the BMSCs treated with the culture supernatant of the cell microsphere group, the area ratio of β-gal-positive cells was close to that of the control group, indicating that the culture supernatant of the cell microsphere did not significantly induce the senescence of BMSCs. More importantly, in the FSS+cell microsphere group, the area ratio of β-gal-positive cells was not significantly different from that of the cell microsphere group, but was significantly lower than that of the FSS+MSCs group, indicating that the cell microspheres can effectively protect the function of MSCs under FSS stimulation conditions and prevent it from inducing the senescence of BMSCs.

[0117] Example 9: Transwell experiment of rat BMSCs treated with different cell culture supernatants

[0118] The experiment first examined the migration ability of rat BMSCs using Transwell chambers. Culture supernatants from bare MSCs, FSS-stimulated MSCs (FSS+MSCs), cell microspheres, and FSS-stimulated cell microspheres (FSS+cell microspheres) were collected and centrifuged to remove residual particles before use. The culture supernatants from different conditions were added to the lower chamber of the Transwell as a chemoattractant. An appropriate amount of rat BMSC suspension (e.g., 2×10 cells / well) was seeded into the upper chamber, and serum-free culture medium was used to maintain the upper chamber environment. Subsequently, the Transwells were incubated in a 37°C, 5% CO incubator for 24 hours to allow BMSCs to migrate to the underside of the membrane. After incubation, the Transwell chambers were washed with PBS to remove non-migrated cells, and the upper surface was gently wiped with a cotton swab. After fixation with 4% paraformaldehyde for 10 minutes, cells that had migrated to the underside of the membrane were stained with crystal violet. After staining, migrating cells were observed and photographed using a microscope. Image analysis was used to calculate the area ratio of migrating cells to assess the effect of culture supernatants from different groups on the migration ability of BMSCs.

[0119] Transwell assay results ( Figure 10) showed that culture supernatants in different states had a significant effect on the migration ability of rat BMSCs. In the control group, the number of BMSCs that migrated to the bottom of the Transwell membrane was small, and the migration area accounted for the lowest proportion, indicating that the migration ability of BMSCs under basal conditions was low. The culture supernatant of the MSCs group significantly promoted the migration of BMSCs, and the migration area ratio increased significantly, indicating that the factors secreted by naked MSCs had a strong pro-migratory effect. However, in the FSS+MSCs group, the migration area ratio decreased significantly, close to that of the control group, indicating that FSS weakened the pro-migratory effect of naked MSCs on BMSCs. In contrast, the migration area ratios of the cell microsphere group and the FSS+cell microsphere group were significantly higher than that of the control group, and there was no significant difference with the MSCs group, indicating that the cell microsphere culture supernatant has a stable promoting effect on the migration of BMSCs, and can protect the function of MSCs under FSS stimulation conditions, so that the factors secreted by them can still effectively promote the migration of BMSCs. The overall results verified the effectiveness of cell microspheres in protecting the function of MSCs and maintaining their pro-migratory ability.

[0120] Example 10: Experimental grouping of SD rat osteoarthritis model

[0121] In order to explore whether cell microspheres can protect MSCs from complex mechanical stimulation in vivo and achieve better therapeutic effects, an animal experiment based on a rat knee OA model was designed. Figure 11 As shown in the results, an OA model was established by anterior cruciate ligament transection (ACLT), and a sham operation group was set up as a control. The experiment was divided into four groups: normal saline group, blank microsphere group, naked MSCs group, and cell microsphere group. The first intra-articular injection was performed 2 weeks after ACLT surgery, and normal saline, blank microspheres, naked MSCs, or MSCs encapsulated in cell microspheres were injected respectively; the second injection was performed 2 weeks later. The experimental endpoint was set at 6 weeks after surgery, and knee joint samples were collected for analysis, including Micro-CT detection of bone structure changes, H&E and specific staining (such as COL2, Aggrecan, MMP13 and ADAMTS5) to evaluate cartilage damage repair and matrix degradation. By comparing the cartilage repair and protection effects among the groups, it was verified whether the cell microspheres can effectively protect the function of MSCs in the complex environment in vivo and exert a therapeutic effect superior to naked MSCs.

[0122] Example 11: Micro-CT scan results of SD rat knee joints after treatment

[0123] After fixation, the specimens were scanned using a high-resolution Micro-CT device, with appropriate parameters (including resolution, scanning voltage, and current) set to obtain three-dimensional images of the joint. After scanning, the overall morphology of the knee joint bone structure was observed using three-dimensional reconstruction technology. Cross-sectional, sagittal, and coronal images were obtained to analyze subchondral bone structure, osteophyte formation, and trabecular integrity to assess the effectiveness of different treatments on knee bone protection and repair.

[0124] Micro-CT results of the knee joint after treatment ( Figure 12 ) showed that there were significant differences in the protection and repair of bone and joint structures among different treatment groups. In the normal saline group and the blank microsphere group, obvious subchondral bone destruction, trabecular structure disorder and osteophyte formation were observed, indicating that these two groups failed to effectively prevent joint degenerative lesions. The naked MSCs group slowed down the destruction of bone and joints to a certain extent, but there was still obvious damage to the bone structure. In contrast, the cell microsphere group showed the best bone structure protection effect, with the subchondral bone basically intact, the trabeculae had good continuity, and the formation of joint osteophytes was significantly reduced, which was close to that of the sham operation group. This shows that MSCs encapsulated in cell microspheres can better exert their therapeutic effects under complex mechanical stimulation environments, effectively protect articular cartilage and bone structure, and are superior to naked MSCs and other treatment groups.

[0125] Example 12: HE staining of SD rat knee joints after treatment

[0126] H&E staining was used to evaluate the protective effects of different treatment groups on knee cartilage. The specimens were fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and tissue sections (about 6 μm thick) were made. The sections were dewaxed with xylene, hydrated with gradient ethanol, and then stained with hematoxylin (H) for nuclei, and eosin (E) for cytoplasm and matrix. After staining, the sections were dehydrated with gradient ethanol and transparentized with xylene, and then observed under an optical microscope after sealing. Slice images were collected and analyzed for cartilage surface integrity, cartilage layer thickness, and subchondral bone structure to evaluate the protective and repair effects of different treatments on knee cartilage.

[0127] H&E staining results ( Figure 13) showed that there were significant differences in the protective effects of articular cartilage in different treatment groups. In the normal saline group and the blank microsphere group, severe wear of the cartilage surface was observed, the cartilage layer became thinner or even disappeared, accompanied by significant destruction of the subchondral bone, and obvious joint degeneration. The exposed MSCs group alleviated cartilage damage to a certain extent, but there were still surface irregularities and slight destruction of the subchondral bone. In contrast, the cell microsphere group showed a better cartilage protection effect, the cartilage surface was basically smooth, the thickness of the cartilage layer was close to normal, and the subchondral bone structure was relatively complete, similar to the sham operation group. The results show that MSCs encapsulated in cell microspheres can more effectively protect knee cartilage tissue under complex mechanical stimulation environments, which is better than exposed MSCs and other control groups.

[0128] Example 13: COL2 staining of SD rat knee joints after treatment

[0129] The effects of different treatments on type II collagen (COL2) expression in knee articular cartilage were assessed by immunohistochemical staining for COL2. After sections were dewaxed and hydrated, antigens were retrieved using a citrate buffer at high temperature. Endogenous peroxidase was subsequently blocked with 3% hydrogen peroxide, and nonspecific binding sites were blocked with normal serum. Sections were incubated with an anti-type II collagen primary antibody overnight at 4°C, followed by a secondary antibody incubation at room temperature for 30 minutes, and developed with DAB. After staining, cell nuclei were counterstained with hematoxylin, and sections were dehydrated, transparentized, and mounted. Microscopic observation and images were captured, and the proportion of COL2-positive cells was calculated using image analysis software to assess the protective effects of different treatments on type II collagen expression in the cartilage matrix.

[0130] Immunohistochemical staining results ( Figure 14 ) showed that there were significant differences in the effects of different treatment groups on the expression of cartilage type II collagen (COL2). The saline group and the blank microsphere group had the lowest proportion of COL2-positive cells, and type II collagen was almost absent in the cartilage matrix, indicating that the cartilage was severely degenerated. The naked MSCs group showed a certain degree of recovery of COL2 expression, but the proportion of positive cells was still significantly lower than that of the cell microsphere group. The proportion of COL2-positive cells in the cell microsphere group increased significantly, approaching that of the sham operation group, indicating that the cell microspheres significantly protected the expression of type II collagen in the cartilage matrix. The sham operation group had the highest proportion of COL2-positive cells, showing the characteristics of normal cartilage. Quantitative analysis showed that the cell microsphere group had a better protective effect on COL2 expression than the naked MSCs group, and significantly better than the saline group and the blank microsphere group. The results show that cell microspheres can more effectively protect chondrocyte function and maintain the expression of type II collagen under complex mechanical environments.

[0131] Example 14: Aggrecan staining of SD rat knee joints after treatment

[0132] Immunohistochemical staining was used to evaluate the effects of different treatments on the expression of knee cartilage matrix proteoglycan (Aggrecan). Sections were dewaxed in xylene and hydrated with graded ethanol. Antigens were then retrievaled using high-temperature citric acid buffer. Endogenous peroxidases were then blocked with 3% hydrogen peroxide, and nonspecific binding sites were blocked with normal serum. Primary antibody against Aggrecan was added, and the sections were incubated overnight at 4°C. Secondary antibody was then added and incubated for 30 minutes at room temperature. Color was developed using DAB. After color development, cell nuclei were counterstained with hematoxylin, and sections were dehydrated with graded ethanol, transparentized, and mounted. Finally, sections were observed and photographed under a microscope. Image analysis software was used to calculate the proportion of Aggrecan-positive cells to assess the protective effects of different treatments on cartilage matrix proteoglycan expression.

[0133] Immunohistochemical staining results ( Figure 15 ) showed that different treatment groups had a significant effect on the expression of Aggrecan in the cartilage matrix. The proportion of Aggrecan-positive cells in the normal saline group and the blank microsphere group was the lowest, and the expression of proteoglycans in the cartilage matrix was significantly reduced, indicating that the cartilage degradation was more serious. The naked MSCs group significantly increased the expression of Aggrecan, and the proportion of positive cells was higher, but still lower than that of the cell microsphere group. The proportion of Aggrecan-positive cells in the cell microsphere group further increased, close to that of the sham operation group, indicating that cell microsphere-encapsulated MSCs can more effectively protect and restore the expression of proteoglycans in the cartilage matrix. The sham operation group had the highest expression of Aggrecan, maintaining normal cartilage characteristics. These results show that cell microsphere encapsulation technology is superior to naked MSCs and other control groups in protecting the cartilage matrix and restoring cartilage function.

[0134] Example 15: MMP13 staining of SD rat knee joints after treatment

[0135] Immunohistochemical staining was used to evaluate the effects of different treatments on the expression of the knee cartilage-degrading enzyme MMP13. After sections were dewaxed and hydrated, antigens were retrieved using a citrate buffer at high temperature. Endogenous peroxidases were then blocked with 3% hydrogen peroxide, followed by blocking of nonspecific binding sites with normal serum. Primary antibodies against MMP13 were added, incubated overnight at 4°C, followed by treatment with a secondary antibody, and development with DAB. Following staining, nuclei were counterstained with hematoxylin, dehydrated, and mounted. Sections were observed under a microscope, and images were captured. Image analysis was used to calculate the proportion of MMP13-positive cells to assess the inhibitory effects of different treatments on cartilage-degrading enzyme expression.

[0136] Immunohistochemical staining results ( Figure 16) showed that different treatment groups had a significant effect on the expression of the cartilage-degrading enzyme MMP13. In the normal saline group and the blank microsphere group, the proportion of MMP13-positive cells was the highest, showing extensive cartilage degradation activity, indicating that the joint degeneration was the most serious. The naked MSCs group significantly reduced the expression of MMP13, but the proportion of positive cells was still higher than that of the cell microsphere group. The proportion of MMP13-positive cells in the cell microsphere group was further reduced, close to that of the sham operation group, indicating that the cell microsphere-encapsulated MSCs can significantly inhibit the expression of cartilage-degrading enzymes, thereby reducing the destruction of the cartilage matrix. The sham operation group had the lowest MMP13 expression, maintaining a normal cartilage state. The results show that cell microsphere encapsulation technology is superior to naked MSCs and other control groups in inhibiting MMP13 expression and protecting the cartilage matrix, and can more effectively prevent joint degeneration.

[0137] Example 16: ADAMTS5 staining of SD rat knee joints after treatment

[0138] ADAMTS5 immunohistochemical staining was used to evaluate the effects of different treatments on the expression of the cartilage-degrading enzyme ADAMTS5 in the knee joint. After sections were dewaxed and hydrated, antigens were retrieved using high-temperature citrate buffer. Endogenous peroxidases were blocked with 3% hydrogen peroxide, and nonspecific binding sites were blocked with normal serum. Subsequently, an ADAMTS5 primary antibody was applied, and the sections were incubated overnight at 4°C. Following this, the sections were incubated with a secondary antibody and developed with DAB. After development, cell nuclei were counterstained with hematoxylin, and the sections were dehydrated with graded ethanol, transparentized, and mounted. Sections were observed and photographed under a microscope, and the proportion of ADAMTS5-positive cells was calculated using image analysis software to assess the inhibitory effects of different treatments on the expression of the cartilage matrix-degrading enzyme ADAMTS5.

[0139] Immunohistochemical staining results ( Figure 17 ) showed that different treatment groups had a significant effect on the expression of ADAMTS5, an enzyme related to cartilage degradation. The proportion of ADAMTS5-positive cells was the highest in the normal saline group and the blank microsphere group, showing obvious cartilage matrix degradation activity, indicating that the cartilage degeneration was severe. The naked MSCs group significantly reduced the expression of ADAMTS5, but the proportion of positive cells was still higher than that in the cell microsphere group. The cell microsphere group showed a lower ADAMTS5-positive cell ratio, which was close to that of the sham operation group, indicating that MSCs encapsulated by cell microspheres can more effectively inhibit the expression of enzymes related to cartilage matrix degradation, thereby protecting cartilage tissue. The sham operation group had the lowest ADAMTS5 expression, showing the characteristics of normal joints. These results show that cell microspheres can effectively protect the function of MSCs under complex mechanical environments, significantly inhibit the expression of ADAMTS5, an enzyme related to cartilage degradation, and thus alleviate cartilage degeneration.

[0140] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0141] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing cell microspheres, characterized in that: include: S1: Mixing sodium alginate solution, mannitol, and cells to obtain a mixed solution; S2: injecting the mixed solution into an electrospray device to obtain microsphere precursors with uniform cell distribution; S3: mixing the microsphere precursor with a calcium chloride solution and performing a gelation treatment to obtain cell microspheres.

2. The preparation method according to claim 1, characterized in that The working concentration of sodium alginate in the sodium alginate solution is 2-5 wt %.

3. The preparation method according to claim 1, characterized in that The working concentration of the mannitol is 3-6 wt %.

4. The preparation method according to claim 1, characterized in that The concentration of the cells in the mixed solution is 1×10^1 to 1×10^8 cells / mL.

5. The preparation method according to claim 1, characterized in that The working concentration of the calcium chloride solution is 0.05-0.5M.

6. The preparation method according to claim 1, characterized in that In step S2, when using an electrospray device, the voltage and liquid flow rate are controlled so that the mixed liquid forms a Taylor cone at the nozzle of the electrospray device, thereby obtaining a microsphere precursor with uniform cell distribution.

7. A cell microsphere, characterized in that: The method according to any one of claims 1 to 6 is used to obtain the present invention.

8. The cell microsphere according to claim 7, characterized in that The diameter of the cell microspheres is 0.03-2 mm.

9. The cell microsphere according to claim 7, characterized in that In the cell microspheres, the density of cells is 10 to 100 per microsphere.

10. A composition, characterized in that The cell microsphere comprises the cell microsphere according to any one of claims 7 to 9.

11. Use of the cell microspheres according to any one of claims 7 to 9 or the composition according to claim 10 in preparing a drug, characterized in that: The medicine is used for treating diseases requiring cell regeneration.

12. The use according to claim 11, characterized in that The diseases include burns, scalds, osteoarthritis, joint injuries, fractures, chronic pain, osteoporosis, autoimmune diseases, metabolic diseases, neurodegenerative diseases and cardiovascular diseases.

13. A method for alleviating mechanical stimulation of cells, characterized in that: The method comprises: The cells are prepared into the cell microspheres according to any one of claims 7 to 9.

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