Preparation method of cell sphere modularized bio-ink and application of cell sphere modularized bio-ink in myocardial repair material

Abstract

The invention belongs to the technical field of biomedical engineering, and provides a preparation method of cell sphere modularized bio-ink and application of the cell sphere modularized bio-ink in the field of myocardial repair treatment. Firstly, stem cell spheres are prepared on the basis of a microarray, a protein / polyphenol composite coating is introduced to the stem cell spheres, the stem cell spheres serve as a multifunctional armor, immune protection and immune regulation treatment aiming at a host immune system are provided, the hostile microenvironment is reversed, and the healing process of infarction myocardial tissue is promoted. Secondly, a biological material with good biocompatibility is selected to prepare gel microspheres carrying bioactive substances by adopting a micro-flow liquid drop control technology, the gel microspheres and the coated cell spheres are further closely packed to form cell sphere particle gel, disease treatment is performed in a direct injection or biological 3D printing mode, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to a method for preparing a cell sphere modular biological ink and its application in the field of myocardial infarction treatment. Background Art

[0002] Stem cell transplantation has been shown to be a promising approach for the treatment of damaged organs / tissues, such as myocardial tissue infarction, or complex diseases, including diabetes and osteoarthritis. Myocardial infarction (MI) caused by irreversible death of cardiomyocytes can impair myocardial function and lead to heart failure. Stem cell transplantation has been shown to be a promising treatment for reducing infarct size and restoring cardiac function after MI, due to its self-regenerative, immunomodulatory, and trophic effects, such as angiogenesis, anti-fibrosis, and anti-apoptosis. However, after MI, the initial stage of the pathological microenvironment is characterized by high levels of proteases and reactive oxygen species (ROS) induced by hypoxia-ischemia, which not only leads to a significant decrease in the cell viability of local cardiac cells and transplanted stem cells, but also reduces cell engraftment, thus affecting the therapeutic effect. In addition, trauma to cardiomyocytes triggers a severe inflammatory response, leading to the recruitment of monocytes, which can differentiate into macrophages at the infarct site. Since the initial macrophages (M0 type) can polarize toward pro-inflammatory M1 or anti-inflammatory M2 macrophages under environmental cues, the dangerous MI microenvironment usually leads to a series of severe inflammatory responses, ultimately leading to heart failure. This can also have harmful toxic effects on surrounding healthy cells or transplanted therapeutic cells. Therefore, correcting this severe inflammatory microenvironment at the site of myocardial infarction and triggering macrophage polarization toward the healing-favoring M2 phenotype may promote the healing of damaged cardiac tissue. Based on this, immunomodulatory therapy based on stem cell transplantation can correct macrophage polarization, thereby initiating a cascade reaction to reverse the pro-inflammatory microenvironment to a pro-healing microenvironment, which is considered a promising strategy for the treatment of myocardial infarction. Specifically, in order for stem cell therapy to reshape the pro-inflammatory microenvironment of myocardial infarction (such as hypoxia, high levels of ROS, excessive production of proteases and inflammatory factors, etc.), various biological events need to be precisely coordinated and managed during the treatment process, however, this remains a challenge in the field of myocardial repair.

[0003] Since direct transplantation of (allogeneic) stem cells for myocardial infarction faces low healing effects due to host immune clearance and adverse pathological microenvironment, researchers have worked hard to overcome these obstacles to improve the efficacy of stem cell therapy for myocardial repair. Considering that the early stage of myocardial infarction is characterized by hypoxia-ischemia, oxidative stress, and inflammation, researchers have proposed strategies to precondition stem cells with hypoxic / chemical conditions or genetic reprogramming to improve cell survival and / or function. In addition, studies have shown that three-dimensional (3D) structured stem cells or cell spheroid transplantation are more effective in treating myocardial infarction than traditional two-dimensional (2D) structured cell transplantation. This is because cell viability is retained under hypoxic conditions and the role of nutrient secretion for tissue repair is enhanced. Despite these advances, challenges remain in improving the duration of preconditioning on cells, providing protection from external stress, and shielding allogeneic cells from immune clearance. Therefore, efforts have been made to encapsulate cells in biomaterials, such as polysaccharides or proteins, to produce cell-loaded gels or patches that serve as a protective layer against external stress and the host immune system. Unfortunately, most encapsulation strategies can only provide a single function in mediating the donor-host response, but fail to recognize the importance of manipulating the surrounding environmental niche, and thus show inadequacy in terms of therapeutic efficacy. Therefore, innovative approaches that simultaneously improve donor cell function and reverse the adverse pathological microenvironment are considered to be more promising strategies for further application in the treatment of myocardial infarction.

[0004] The present invention proposes a method for preparing a cell spheroid modular bio-ink and its application strategy in the field of myocardial repair therapy. A protein / polyphenol composite coating is introduced on the cell spheroid and combined with a particle gel to form a printable bio-ink. The coating acts as a multifunctional armor to provide immune protection and immunomodulatory therapy for the host immune system to reverse the hostile microenvironment and promote the healing process of infarcted myocardial tissue. Summary of the invention

[0005] The present invention aims to provide a method for preparing a cell spheroid modular bio-ink and its application in the field of myocardial repair therapy.

[0006] The present invention generates a protein / polyphenol protective layer around the stem cell sphere by layer-by-layer self-assembly, wherein polyphenols are combined with proteins as cross-linking agents, and phenols have the ability to remove ROS. In addition, the coating composed of proteins and polyphenols can change the paracrine factors secreted by stem cells, promote angiogenesis and immunoregulation by activating glycolysis. The coated stem cell spheres are further combined with particle gels to form shear-thinning, injectable and printable bio-inks. When it was transplanted into rats with myocardial infarction, it was found that compared with the treatment with naked cell spheres, by mediating the polarization of macrophages to the pro-healing M2 phenotype, local inflammation and subsequent fibrosis were significantly reduced, and the cardiac function of the infarcted myocardial tissue was improved.

[0007] The present invention is implemented by adopting the following technical solutions.

[0008] The present invention provides a cell sphere modular bio-ink with tissue repair function, which consists of a cell sphere and a particle microgel; the dry weight mass fraction of the particle gel is 0.1-50%, and the volume fraction is greater than 0.5; the cell sphere introduces a layer of self-histone and polyphenol coating on the surface of the sphere; the particle gel provides injectable and printable properties; the diameter of the cell-carrying sphere is 40-1000um.

[0009] In the technical solution described above, further, the number of cells in the modular bio-ink is 2 to 10,000, preferably 100 to 3,000.

[0010] The present invention provides a method for preparing stem cell spheres with a coating, comprising the following steps:

[0011] (1) Fabrication of PDMS microporous device for culturing cell spheroids

[0012] There is a square container in the middle of the microporous PDMS device. There are 1024 holes at the bottom of the square container. Each trapezoidal well is 0.6mm thick, the square length of the upper side is 0.46mm, and the square length of the lower side is 0.25mm. The master mold is made by projection micro-stereolithography. Then, the main mold is embedded in the metal aluminum component. The cover plate is placed on the main mold and the four corners are fixed with screws to obtain the total assembly mold. Pour the degassed PDMS prepolymer on the mold and cure it at 85℃. Remove the cured microporous PDMS from the assembly mold, sterilize and hydrophilize it. After rinsing with deionized water and serum-free cell culture medium, it is used for cell sphere culture.

[0013] (2) The PDMS multi-microporous device described in step (1) is used for cell spheroid culture. The cell suspension will use 4 different cell densities to obtain spheroids of different sizes, and the diameter of each spheroid is measured.

[0014] (3) Collect the cell spheres described in step (2) and wash them. Incubate the collected spheres with natural polymers and then remove the supernatant by centrifugation. Add polyphenols to the coated spheres and gently mix them before separating them from the supernatant. The natural polymer layer and the polyphenol layer are alternately deposited on the spheres.

[0015] (4) The coated cell spheroids and microfluidic prepared particle gel described in step (3) are used as injectable or printable materials, and after dense stacking, the precise arrangement of the microgel carrying the bioactive substance is achieved by bioprinting.

[0016] In the technical solution described above, the other polymer material is one or a combination of two or more natural polymers or synthetic polymers, and the natural polymers include extracellular matrix, alginate, alginate derivatives, hyaluronic acid, chitosan, agarose, dextran; proteins include collagen, gelatin, gelatin derivatives, fibrin, agar, matrix glue, proteoglycan, glycoprotein, and laminin.

[0017] In the technical solution described above, specifically, the embedded living cells in step (3) can be selected by those skilled in the art according to different implementation purposes;

[0018] The living cells are generally one or a combination of two or more of primary culture cells, subculture cells, cell line culture cells and hybrids;

[0019] In the technical solution described above, the process of preparing microgel using an integrated microfluidic chip mainly includes the following steps:

[0020] ① The polymer is dissolved in water or PBS or culture medium to prepare a solution, and then a cross-linking reaction initiator is added to obtain a hydrogel prepolymer solution, which is used as the first phase aqueous solution for preparing an oil-in-water emulsion system. In another phase, cells are added to the above solution to obtain a hydrogel prepolymer solution, which is used as the second phase aqueous solution for preparing an oil-in-water emulsion system;

[0021] ② Use mineral oil (paraffin / dimethyl silicone oil) containing non-ionic surfactant as the oil phase of the oil-in-water emulsion system; or use fluorinated oil containing fluorinated surfactant as the oil phase of the oil-in-water emulsion system.

[0022] In the technical solution described above, the multi-channel microfluidic chip: the preferred flow rate and more preferably the flow rate of each channel is a multiple of that of a single channel. For example, the flow rate of a two-channel microfluidic chip is twice that of a single channel, the flow rate of a three-channel microfluidic chip is three times that of a single channel, and so on.

[0023] In the technical solution described above, specifically, the ratio of the sum of the flow rates of the first phase and the second phase water phase to the flow rate of the first heavy oil phase is between 1:0.01 and 1, preferably 1:0.05 and 0.5; the ratio of the flow rate of the first heavy oil phase to the flow rate of the second heavy oil phase is 1:0.1 to 50, preferably 1:0.5 to 10.

[0024] In the technical solution described above, the cross-linking reaction initiator includes ① a coordination reaction initiator (selected from a calcium-ethylenediaminetetraacetic acid chelate aqueous solution, a calcium-nitrilotriacetic acid chelate aqueous solution, calcium carbonate nanoparticles, calcium sulfate nanoparticles, calcium phosphate nanoparticles, or a combination thereof), and the final concentration of the initiator is 10-1000 mM in terms of calcium content; ② a photochemical reaction initiator with good biocompatibility (selected from 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, I2959), lithium phenyl-2,4,6-trimethylbenzoylphosphite (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP), azo initiator VA086 (2,2'-(Diazene-1,2-diyl)bis(N-(2-hydroxyethyl)-2-methylpropanamide), benzoin methyl ether (2-methoxy-2-phenyl-acetophenon), eosin Y (EosinY) or a combination of several thereof), the illumination wavelength is 365nm-780nm, and the illumination time is 0.00001s-600s; ③ a temperature-mediated cross-linking agent, and the cross-linking temperature is 0-40°C; ④ an enzyme used in an enzyme-catalyzed reaction (selected from one of dismutase, thrombin, and glutamine transaminase).

[0025] In the technical solution described above, the diameter of the particle gel product prepared by the microfluidic technology is 20 to 500 μm; the dispersion coefficient of the particle size distribution is 0.01 to 20%.

[0026] In the technical solution described above, step (4) is to use the coated cell spheroids and microfluidic prepared particle gel prepared in step (3) as printable bio-ink, and with the help of a three-dimensional (3D) mobile platform, achieve the purpose of accurately constructing a three-dimensional complex structure by point-by-point printing and layer-by-layer stacking on demand.

[0027] In the technical solution described above, the particle gel for injection or bioprinting is densely packed with microgel prepared by microfluidics, placed in a printing container, and bioprinted. The method of densely packing the cell spheroids with coating and the particle gel prepared by microfluidics can be a chemical operation unit such as centrifugation, filtration, and suction filtration.

[0028] In the technical solution described above, further, the material of the particle gel includes extracellular matrix, hyaluronic acid, chitosan, agarose, dextran or protein collagen, gelatin, gelatin derivatives, fibrin, agar, matrix glue, proteoglycan, glycoprotein, and laminin; the mixing method of the particle gel and the cell sphere includes convection mixing or shear mixing; the convection mixing uses mechanical stirring or flipping equipment to move or flip the particles as a whole, thereby achieving mixing; the shear mixing applies shear force to make the particles rub and squeeze each other during the mixing process, thereby breaking the agglomeration phenomenon between the particles and achieving a uniform mixing effect.

[0029] The beneficial effects of the present invention compared with the prior art are as follows:

[0030] (1) The present invention designs a new method for rapidly preparing cell spheroid modular bio-ink based on microchips, which realizes the mass production of cell spheroids and ensures high cell activity.

[0031] (2) The present invention proposes a myocardial infarction treatment strategy based on a biomaterial coating design combined with mesenchymal stem cell spheroids. The biomaterial coating can regulate the pathological myocardial infarction microenvironment and has multiple functions, including protecting donor cells from host immune clearance; by clearing ROS and stimulating MSC to promote healing paracrine remodeling of the pro-inflammatory microenvironment, the stem cell treatment effect is significantly improved. The potential therapeutic mechanism of the coating's effect on stem cells is further clarified.

[0032] (3) The present invention can precisely arrange the coated stem cell sphere particle gel by bioprinting technology. Among them, the coated stem cell sphere particle gel can be used as a tissue building block to create three-dimensional (3D) tissues and organs, forming single-cell precision and high-cell density biomimetic tissue microunits.

[0033] (4) The technology used in the present invention can effectively ensure cell activity; and the use of injectable direct in situ delivery of cells for disease treatment or bioprinting can achieve the construction of three-dimensional complex structures, which has good application prospects in the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0035] Figure 1 Fabrication of PDMS microporous device for culturing cell spheroids.

[0036] Figure 2 Schematic diagram of the process of polyphenol and protein encapsulation of cell spheroids.

[0037] Figure 3 Microfabrication of polyphenol- and protein-coated cell spheroids.

[0038] Figure 4 The coating protects stem cell spheroids.

[0039] Figure 5 To investigate the protective effect of coated stem cell spheroids on co-cultured cardiomyocytes in a ROS-rich microenvironment simulating inflammation.

[0040] Figure 6 Immunomodulatory effect of coated stem cell spheroids on macrophage polarization.

[0041] Figure 7 The coated stem cell spheroids have a more obvious healing ability in the early stage after myocardial infarction.

[0042] Figure 8 Injectability and printability of gels for coating stem cell spheroids and particles. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0044] The present invention first makes a PDMS multi-microporous device for culturing cell balls. There is a square container in the middle of the microporous PDMS device. There are 1024 holes at the bottom of the square container. Each trapezoidal well is 0.6 mm thick, the square length of the upper side is 0.46 mm, and the square length of the lower side is 0.25 mm. The main mold is made by projection micro-stereolithography technology. Then, the main mold is embedded in a metal aluminum component. The cover plate is placed on the main mold, and the four corners are fixed with screws to obtain a general assembly mold. The degassed PDMS prepolymer is poured on the mold and cured at 85°C. The cured microporous PDMS device is removed from the assembly mold, sterilized, and hydrophilized, such as Figure 1 . After rinsing with deionized water and serum-free cell culture medium, it was used for cell spheroid culture. The cell suspension was prepared at 4 different cell densities to obtain spheroids of different sizes, and the diameter of each spheroid was measured. The collected spheroids were incubated with natural polymers and then centrifuged to remove the supernatant. Polyphenols were added to the coated spheroids and gently mixed before separation from the supernatant. The natural polymer layers were alternately deposited on the spheroids with the polyphenol layers as shown in Figure 2. Figure 2 and Figure 3 .

[0045] The integrated microfluidic chip used is the integrated microfluidic chip disclosed in the invention patent (CN112275336A), which has at least two droplet production units on its substrate, and also includes multiple liquid phase input modules and a cleaning output module. The liquid phase input module can be divided into a dispersed phase distribution unit and a continuous phase distribution unit according to the type of liquid phase transported inside it, and its classification is only related to the type of liquid phase inside it, and has nothing to do with the relative position in the chip. Therefore, in actual production, the liquid phase input position in the channel can be arbitrarily replaced, so as to achieve the purpose of changing the droplet production mode according to actual needs and improve the flexibility of chip use. The present invention prepares microgels with an integrated microfluidic chip with a multi-layer structure that integrates 32 production units (the specific design method of the microfluidic chip is shown in CN112275336A), which can continuously and stably prepare cell-carrying microgel particles of various hydrogel materials, and can directly obtain active substance-carrying microgels through a continuous processing process. The coated cell spheroids and microfluidically prepared particle gels are used as injectable or printable materials. After dense stacking, the precise arrangement of microgels carrying bioactive substances is achieved by bioprinting.

[0046] The present invention is a myocardial infarction treatment strategy based on stem cell spheroids, which introduces a protein / polyphenol composite coating on the cell spheroids as a multifunctional armor to provide immune protection and immunomodulatory therapy against the host immune system to reverse the hostile microenvironment and promote the healing process of infarcted myocardial tissue. Specifically, a protein / polyphenol protective layer is generated around the stem cell spheroids through layer-by-layer self-assembly, in which polyphenols bind to proteins as cross-linkers and also provide the ability to scavenge ROS. In addition, the coating reconfigures the paracrine factors secreted by the coated stem cells to promote angiogenesis and immunomodulation by activating glycolysis. The coated stem cell spheroids were further transplanted into rats with myocardial infarction, and it was found that compared with the treatment with bare cell spheroids, the local inflammation and subsequent fibrosis were significantly reduced, and the cardiac function of the infarcted myocardial tissue was improved by mediating the polarization of macrophages to the pro-healing M2 phenotype.

[0047] Example 1

[0048] 1. Cell culture:

[0049] Taking the culture of umbilical cord mesenchymal stem cells (hUC-MSCs) as an example, the proliferation medium consists of α-MEM (α-minimum Eagle's medium), 10% fetal bovine serum (FBS, Gibco), and the culture conditions are 37°C, 95% relative humidity and 5% CO2. The cell culture medium is replaced every three days. Before use, the cells are washed with phosphate buffered saline (PBS), placed in trypsin / EDTA solution for 5 minutes, and suspended in the culture medium for use.

[0050] 2. Preparation of cell spheres:

[0051] hUC-MSCs cells isolated and extracted from the umbilical cord Wharton's jelly were cultured in a growth medium containing α-MEM, 10% fetal bovine serum and 1% penicillin / streptomycin at 5% CO2 and 37°C, and the medium was changed every 3 days. In a T75 cell culture flask, cells were passaged when they spread 70-80%, and all cells were used at the 5th generation. 0.8 mL of cell suspension was added to the PDMS microwell culture device for culture. The cell suspension will use 4 different cell densities (5.0×105, 1×106, 1.5×106, 2×106 cells / mL). After 12 hours, the hU-MSCs spheres were observed under an inverted fluorescence microscope (OLYMPUS IX73, Japan), and the diameter of each sphere was measured.

[0052] To characterize the cell morphology of hU-MSCs spheroids, f-actin and cell nuclei were stained using Alexa Fluor 594phalloidin and 4',6-diamidino-2-phenylindole (DAPI), respectively. Spheroid samples of different sizes were collected on days 1 and 7 and fixed overnight with 4% paraformaldehyde (PFA) solution. Then, the samples were permeabilized with 0.1% Triton X-100 for 20 minutes, incubated with 1% BSA for 45 minutes, and finally washed three times with PBS solution. These fixed samples were stained with Alexa Fluor594phalloidin for 45 minutes, then counterstained with DAPI for 10 minutes and washed three times with PBS. Fluorescence images were observed and acquired using an inverted confocal laser scanning microscope, as shown in Figure 5. Figure 3 .

[0053] Example 2

[0054] The hUC-MSCs cell spheres prepared in Example 1 were collected in a 15 mL centrifuge tube and washed twice with PBS. The collected spheres were incubated with 2 mg / mL fibrinogen (Fg) solution in α-MEM for 30 min, then centrifuged at 150 g for 1 min, and the supernatant was removed by gentle pipetting. 0.2 mg / mL EGCG was added to the Fg-coated spheres, gently mixed for 1 min, and centrifuged at 150 g for 1 min. The Fg / EGCG-coated spheres were incubated with 2 mg / mL Fg solution for 5 min and separated from the supernatant. The Fg layer was deposited alternately with the EGCG layer on the spheres ( Figure 2 ). Live and dead cells in Fg / EGCG-coated and uncoated constructs were labeled using the LIVE / DEAD assay kit according to the manufacturer’s instructions.

[0055] Example 3

[0056] After obtaining the coated stem cell spheres in Example 2, the DPPH method was used to detect the ability of the coated spheres to scavenge free radicals. 500 μL DPPH / methanol solution (100 μM) was added to a test tube of 1000 spheres. After incubation at 37°C for 1h and 4h, the supernatant was collected and measured at 570nm with a spectrophotometer. The viability of coated and uncoated cell spheres in the ROS microenvironment was further evaluated. 200 μM H2O2 was added to the culture medium to induce oxidative stress to simulate the ROS microenvironment. On days 1, 3, and 5, live and dead cells in Fg / EGCG-coated and uncoated spheres were labeled using the LIVE / DEAD viability assay kit. The proliferation of spheres was evaluated using Cell Counting Kit-8. 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining was used to detect intracellular ROS levels. The samples were incubated with 10 μM DCFH-DA (Beyotime, China) in the dark for 15-25 min, washed with PBS, and stained with DAPI. The samples were observed with a confocal microscope and the fluorescence intensity of DCFH-DA was quantified. Figure 4 .

[0057] Example 4

[0058] After obtaining cardiomyocytes, 200 μM H2O2 was added to the culture medium to induce oxidative stress for 3 days. Then, the coated and uncoated cell spheroids were incubated with neonatal rat cardiomyocytes in the ROS microenvironment for 3 consecutive days. The CMs culture medium was removed and the Cell Counting Kit-8 solution was added. After another 4 h of culture, the absorbance of the supernatant at 450 nm was measured by a microplate reader. DCFH-DA and dihydroethidium (DHE) were used to evaluate the levels of ROS and superoxide anions in cardiomyocytes, respectively. Briefly, CMs were cultured in a normal or ROS microenvironment induced by 200 μM H2O2, and their coated and uncoated spheroids were cultured. On the third day, 5 mM DHE or 10 μM DCFH-DA was added to the culture medium, respectively, and the culture medium was left in the dark for 15-25 min. The fluorescence intensity of DHE and DCFH-DA was quantified by a microplate reader. Real-time fluorescence quantitative PCR was used to detect the gene expression levels of CMs apoptosis markers caspase-3, α-actin 2 (ACTN2), cardiac troponin C type 1 (TNNC1), and connexin 43 (Cx43). Immunofluorescence staining was used to detect the expression levels of ACTN2 and Cx43 proteins, and an inverted confocal laser scanning microscope was used to observe and acquire fluorescence images. Total RNA was extracted using a microelution total RNA kit. Then, the RNA was reverse transcribed into cDNA using a PrimeScriptTM RT kit. Quantitative detection was performed using a fast SYBR Green Master Mix kit and a 7500Fast real-time fluorescence quantitative PCR instrument. The primer sequences used are shown in Table 2.3. Gene expression was further normalized (housekeeping gene GAPDH). The 2-ΔΔCt method was used to calculate the relative expression of genes, as shown in Figure 5 .

[0059] Example 5

[0060] Macrophage polarization was induced using the human monocytic leukemia cell line THP-1. THP-1 culture medium consisted of RPMI-1640 basal medium, 10% fetal bovine serum, and 1% penicillin / streptomycin. To polarize THP-1, 0.15 μM phorbol 12-myristate 13-acetate (PMA) was added to THP-1 culture medium for cell culture to obtain M0 macrophages. M0 macrophages were polarized to M1 phenotype in THP-1 culture medium with or without 0.01 ng / mL lipopolysaccharide (LPS) in a ROS microenvironment (200 μM H2O2) for 24 h (5×104 cells / cm2). Coated and uncoated spheres (2×105) were then added to the upper chamber of the transwell and cultured for 3 days. The relative expression of M1 maker (CD14, CD163, CCL-18 and CD206) and M2 maker (CD40, CCL-2, IL-1β, IL-6) genes was detected by qRT-PCR. The primer sequences used are shown in Table 2.4. The relative expression of CCR-7 and CD36 proteins was detected by immunofluorescence staining, and the fluorescence images were observed and acquired using an inverted confocal laser scanning microscope. Figure 6 .

[0061] Example 6

[0062] Heart samples were collected on the 4th day to evaluate the anti-inflammatory effect after treatment. The collected samples were fixed in 4wt% paraformaldehyde solution for 24 hours. Then, paraffin-embedded tissues were obtained according to standard procedures. The samples were cut into 5μm slices. For immunofluorescence staining, the samples were washed with PBS after fixation, treated with 0.3% Triton x-100 for 30 minutes, and then blocked with 5% bovine serum for 30 minutes. The macrophage surface markers CD68, Arg-1 and iNOS, the neutrophil surface marker MPO, the vascular marker α-smooth muscle actin (α-SMA), the cardiomyocyte markers, cardiac contractile proteins and gap junction proteins were incubated with primary antibodies. Subsequently, the sections were washed with PBS and then incubated with the corresponding secondary antibodies, and the cell nuclei were stained with DAPI, such as Figure 7 .

[0063] Example 7

[0064] 1. Preparation of microgel particles:

[0065] HAMA, GelMA and LAP were dissolved in distilled water to prepare an aqueous phase containing 2% (w / v) HAMA, 2.5% (w / v) GelMA and 0.1% (w / v) LAP. 0.5 w / v% triblock Krytox-PEG-Krytox surfactant was dissolved in electronic fluoride liquid 7100 to prepare an oil phase. The oil phase was stirred at 8000 rpm using a high-speed stirrer (stirring rod model S18N-19G), and the aqueous phase was added to the oil phase with a syringe at a ratio of 10:1 (v / v) of oil phase to aqueous phase. After the addition, stirring was continued for 1 min, and then ultraviolet light (power: 100%, wavelength 355 nm) was irradiated for 2 min to crosslink into spheres. The oil phase was removed by centrifugation, and the surfactant on the surface of the gel microspheres was removed by washing with 1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO), and PFO and the remaining oil phase were removed by centrifugation. The gel microspheres were then resuspended in distilled water.

[0066] 2. Dense packing of microgel particles:

[0067] Use a suction pump to filter, and drop the gel microspheres resuspended in water onto the filter membrane drop by drop through a dropper. After the filtration is completed, collect the gel microspheres in a syringe to obtain densely packed gel microspheres, and perform an injectability test on the filtered gel microspheres. The results are as follows: Figure 8 As shown, it can be seen that the printing obtains a relatively stable structure.

[0068] 3. Microgels mixed with coated cell spheroids as ink for printing biological structures and biological evaluation:

[0069] The prepared densely packed gel microspheres were soaked in DMEM culture medium containing 10% (v / v) dual antibody for 24 hours. After that, the gel microspheres were filtered in a sterile operating table to make them densely packed again and loaded into a screw-capped syringe. The 3T3 cells in the culture flask were digested with trypsin, and after digestion, an appropriate amount of culture medium was added, sucked into a 15mL centrifuge tube, and the supernatant was removed by centrifugation. Cell counting was performed using a hemocytometer. After counting, the required cells were resuspended in 100μL culture medium. The densely packed gel microspheres were loaded into a screw-capped syringe, and the resuspended cells were loaded into a screw-capped syringe. The two syringes were connected with an adapter, and the cells were pushed and pulled against each other to mix the cells and the densely packed gel microspheres. 1mL of densely packed gel microspheres were inoculated with 5×10 6 The densely packed gel microspheres seeded with cells were printed for testing. Then the culture medium was added and cultured in a cell culture incubator containing 5% CO2.

[0070] The biocompatibility of the printed microgels was investigated by using a live / dead assay. 2 mM calcein (green fluorescent dye to mark live cells) and 4 mM propidium iodide (red fluorescent dye to mark dead cells) were added to the microgel suspension and observed using a laser confocal scanning microscope after incubation for 20 minutes. The results are shown in Figure 2. Figure 8 As shown, the cell viability was only 85.3%, indicating that the cell microgels prepared by this method maintained cells at a high activity.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell spheroid modular bio-ink with tissue repair function, characterized in that: The modular bio-ink consists of cell spheres and particle microgels; the dry weight mass fraction of the particle gel is 0.1-50%, and the volume fraction is greater than 0.5; the cell sphere introduces a layer of self-organizing protein and polyphenol coating on the surface of the sphere; the particle gel provides injectable and printable properties; the diameter of the cell-carrying sphere is 40-1000um.

2. The cell spheroid modular bio-ink according to claim 1, characterized in that: The number of cells in the modular bio-ink is 2 to 10,000, preferably 100 to 3,000.

3. The method for preparing the cell spheroid modular biological ink according to claim 1, characterized in that: The preparation of the cell sphere comprises the following steps: (1) Cultivating cell spheroids in a PDMS microporous device: The cell suspension was prepared at four different cell densities to obtain spheroids of different sizes, and the diameter of each spheroid was measured; (2) collecting the cell spheres described in step (1), washing them, incubating the collected spheres with natural polymers, and removing the supernatant by centrifugation; adding polyphenols to the coated spheres, gently mixing them, and then separating them from the supernatant; and alternately depositing natural polymer layers and polyphenol layers on the spheres.

4. The cell spheroid modular bio-ink according to claim 3, characterized in that: The preparation method of the PDMS multi-microporous device includes: providing a square container with 1024 holes at the bottom of the square container, each of which is a trapezoidal well with a thickness of 0.6 mm, a square length of 0.46 mm on the upper side, and a square length of 0.25 mm on the lower side; using projection micro-stereolithography technology to make a main mold; embedding the main mold into a metal aluminum component; placing a cover plate on the main mold to fix the overall assembly mold; pouring degassed PDMS prepolymer liquid on the mold and curing it at 85°C; removing the cured microporous PDMS device from the assembly mold, sterilizing and hydrophilizing it; and using it for cell sphere culture after rinsing it with deionized water and serum-free cell culture medium.

5. The cell spheroid modular bio-ink according to claim 2, characterized in that: The coating provides a customized microenvironment for cells using natural polymers and / or small molecule polyphenols.

6. The cell spheroid modular bio-ink according to claim 5, characterized in that: Natural polymers include extracellular matrix, hyaluronic acid, chitosan, agarose, dextran; proteins include collagen, gelatin, gelatin derivatives, fibrin, agar, matrigel, proteoglycan, glycoprotein or laminin; small molecule polyphenols include anthocyanidins, catechins, quercetin, gallic acid, ellagic acid or arbutin.

7. The preparation method according to claim 2, characterized in that: The materials of the particle gel include extracellular matrix, hyaluronic acid, chitosan, agarose, dextran or protein collagen, gelatin, gelatin derivatives, fibrin, agar, matrix glue, proteoglycan, glycoprotein, and laminin; the mixing method of the particle gel and the cell ball includes convection mixing or shear mixing; the convection mixing uses mechanical stirring or flipping equipment to make the particles move or flip as a whole, thereby achieving mixing; the shear mixing applies shear force to make the particles rub and squeeze each other during the mixing process, thereby breaking the agglomeration phenomenon between the particles and achieving a uniform mixing effect.

8. Application of the cell spheroid modular bio-ink described in claim 1 in the field of preparing tissue repair materials.

9. The use according to claim 8, characterized in that The tissue repair is the repair of myocardial damage caused by acute myocardial infarction.

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