Preparation method of cell sphere module biological ink and application thereof in myocardial repair material

Abstract

This invention belongs to the field of biomedical engineering technology, providing a method for preparing modular bio-ink for cell spheres and its application in the field of myocardial repair therapy. First, a method for preparing stem cell spheres based on microarrays is described. By introducing a protein / polyphenol composite coating onto the cell spheres, a multifunctional armor is created, providing immune protection and immunomodulatory therapy against the host's immune system to reverse the hostile microenvironment and promote the healing process of infarcted myocardial tissue. Second, using microfluidic droplet control technology, gel microspheres loaded with bioactive substances are prepared from biocompatible biomaterials. These microspheres are then densely packed and coated to form cell sphere particle gels. These gels can be used for disease treatment via direct injection or bio-3D printing, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and specifically relates to a method for preparing modular bio-ink for cell spheres and its application in the treatment of myocardial infarction. Background Technology

[0002] Stem cell transplantation has proven to be a promising approach for treating damaged organs / tissues, such as myocardial infarction, or complex diseases including diabetes and osteoarthritis. Myocardial infarction (MI), caused by irreversible death of cardiomyocytes, can impair myocardial function, leading to heart failure. Stem cell transplantation, with its self-regenerative, immunomodulatory, and nutritional effects, such as angiogenesis, anti-fibrosis, and anti-apoptosis, has shown promise as a treatment for reducing infarct size and restoring cardiac function after myocardial infarction. However, the initial phase of the pathological microenvironment after myocardial infarction is characterized by high levels of proteases and reactive oxygen species (ROS) induced by hypoxia and ischemia. This not only leads to a significant decrease in the viability of local cardiac cells and transplanted stem cells but also reduces cell engraftment, thus affecting treatment efficacy. Furthermore, cardiomyocyte trauma triggers a severe inflammatory response, leading to the recruitment of monocytes, which can differentiate into macrophages at the infarct site. Since naïve macrophages (M0 type) can polarize into pro-inflammatory M1 or anti-inflammatory M2 macrophages upon environmental cues, the dangerous myocardial infarction microenvironment often results in 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 towards the healing-promoting 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 that reverses 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 remodel the pro-inflammatory microenvironment of myocardial infarction (such as hypoxia, high levels of ROS, excessive production of proteases and inflammatory factors), precise coordination and management of various biological events are required during treatment; however, this remains a challenge in the field of myocardial repair.

[0003] Because direct transplantation of (allogeneic) stem cells for myocardial infarction faces challenges such as low healing rates due to host immune clearance and adverse pathological microenvironment, researchers are striving to overcome these obstacles to improve the efficacy of stem cell therapy for myocardial repair. Considering that the early stages of myocardial infarction are characterized by hypoxia-ischemia, oxidative stress, and inflammation, researchers have proposed strategies to pretreat stem cells with hypoxic / chemical conditions or genetic reprogramming to improve cell viability and / or function. Furthermore, studies have shown that three-dimensional (3D) stem cell or cell spheroid transplantation is more effective in treating myocardial infarction than traditional two-dimensional (2D) structured cell transplantation because it preserves cell viability under hypoxic conditions and enhances the secretion of nutrients for tissue repair. Despite these advances, challenges remain in improving the duration of pretreatment on cells, providing protection against external stress, and shielding allogeneic cells from immune clearance. Therefore, efforts are being made to encapsulate cells in biomaterials, such as polysaccharides or proteins, to create cell-bearing gels or patches as a protective layer against external stress and the host immune system. Unfortunately, most encapsulation strategies only provide a single function in mediating the donor-host response, failing to recognize the importance of manipulating the surrounding ecological niche, and thus showing inadequacy in terms of therapeutic efficacy. Therefore, innovative approaches that simultaneously improve donor cell function and reverse the adverse pathological microenvironment are considered more promising strategies for further application in the treatment of myocardial infarction.

[0004] This invention proposes a method for preparing modular bio-ink for cell spheres and its application strategy in the field of myocardial repair therapy. By introducing a protein / polyphenol composite coating on cell spheres and combining it with particulate gel to form a printable bio-ink, the coating acts as a multifunctional armor, providing immune protection and immunomodulatory therapy against the host immune system to reverse the hostile microenvironment and promote the healing process of infarcted myocardial tissue. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing modular bio-ink for cell spheres and its application in the field of myocardial repair therapy.

[0006] This invention generates a protein / polyphenol protective layer around stem cell spheroids through layer-by-layer self-assembly. Polyphenols act as cross-linking agents, binding to proteins, and phenols possess the ability to scavenge reactive oxygen species (ROS). Furthermore, the protein and polyphenol coating alters paracrine factors secreted by stem cells, promoting angiogenesis and immune regulation by activating glycolysis. The coated stem cell spheroids are then combined with a particulate gel to form a shear-thinning, injectable, and printable bio-ink. Transplantation into rats with myocardial infarction revealed that, compared to treatment with naked cell spheroids, it significantly reduced local inflammation and subsequent fibrosis and improved cardiac function in infarcted myocardial tissue by mediating macrophage polarization towards the pro-healing M2 phenotype.

[0007] The present invention is implemented using the following technical solution.

[0008] This invention provides a modular bio-ink with tissue repair function, comprising cell spheres and particulate microgels; the dry weight mass fraction of the particulate gel is 0.1-50%, and the volume fraction is greater than 0.5%; the cell spheres have an autologous protein and polyphenol coating on their surface; the particulate gel provides injectable and printable properties; the diameter of the cell-carrying spheres is 40-1000 μm.

[0009] In the above-described technical solution, the modular bio-ink contains 2 to 10,000 cells, preferably 100 to 3,000 cells.

[0010] This invention provides a method for preparing coated stem cell spheres, comprising the following steps:

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

[0012] The microporous PDMS device has a square container in the center. The bottom of the square container has 1024 holes. Each hole is a trapezoidal well with a thickness of 0.6 mm, a square on the upper side with a length of 0.46 mm, and a square on the lower side with a length of 0.25 mm. A master mold was fabricated using projection micro-stereolithography. The master mold was then embedded into an aluminum component. A cover plate was placed on the master mold, and the four corners were secured with screws to obtain the final assembly mold. Degassed PDMS prepolymer solution was poured onto the mold and cured at 85°C. The cured microporous PDMS device was removed from the assembly mold, sterilized, and subjected to hydrophilic treatment. After rinsing with deionized water and serum-free cell culture medium, it was used for cell sphere culture.

[0013] (2) The PDMS multi-micropore device described in step (1) is used for cell sphere culture. The cell suspension will be prepared using four different cell densities to obtain spheres of different sizes, and the diameter of each sphere will be measured.

[0014] (3) Collect the cell spheres described in step (2) and wash them. Incubate the collected spheres with the natural polymer, and then centrifuge to remove the supernatant. Add the polyphenolic substance to the coated spheres, mix gently, and then separate from the supernatant. The natural polymer layer and the polyphenolic substance layer are deposited alternately on the spheres.

[0015] (4) The coated cell spheres and microfluidically prepared particulate gels described in step (3) are used as injectable or printable materials. After dense stacking, the precise arrangement of microgels carrying bioactive substances is achieved by bioprinting.

[0016] In the above-described technical solution, the other polymer material is one or more combinations of natural polymers or synthetic polymers. The natural polymers include extracellular matrix, alginate, alginate, alginate derivatives, hyaluronic acid, chitosan, agarose, dextran; protein-based collagen, gelatin, gelatin derivatives, fibrin, agar, matrix gum, proteoglycans, glycoproteins, and laminin.

[0017] In the above-described technical solution, specifically, the embedded living cells mentioned 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 the following: primary cultured cells, passaged cultured cells, cell line cultured cells, and hybrids.

[0019] The process of preparing microgels using an integrated microfluidic chip, as described above, mainly includes the following steps:

[0020] ① A solution is prepared by dissolving the polymer in water, PBS, or culture medium, and then a cross-linking initiator is added to obtain a hydrogel prepolymer solution, which serves as the first aqueous phase solution for preparing a water-in-oil emulsion system. Another phase is prepared by adding cells to the above solution to obtain a hydrogel prepolymer solution, which serves as the second aqueous phase solution for preparing a water-in-oil emulsion system.

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

[0022] In the technical solution described above, the multi-channel microfluidic chip has a preferred flow rate and a more preferred flow rate that 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 velocities of the first phase and the second phase water phase to the flow velocity of the first heavy oil phase is between 1:0.01 and 1, preferably between 1:0.05 and 0.5; the ratio of the flow velocity of the first heavy oil phase to the flow velocity of the second heavy oil phase is between 1:0.1 and 50, preferably between 1:0.5 and 10.

[0024] In the technical solution described above, the crosslinking reaction initiator includes ① a coordination reaction initiator (selected from one or more of the following: calcium-ethylenediaminetetraacetic acid chelate aqueous solution, calcium-aminotriacetic acid chelate aqueous solution, calcium carbonate nanoparticles, calcium sulfate nanoparticles, and calcium phosphate nanoparticles), wherein the final concentration of the initiator is 10-1000 mM based on the calcium content; ② a photochemical reaction initiator with good biocompatibility (selected from 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (lithium)). The following are the components of the cross-linking agent: phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), azo initiator VA086 (one or a combination of 2,2'-(Diazene-1,2-diyl)bis(N-(2-hydroxyethyl)-2-methylpropanamide), benzoin methyl ether (2-methoxy-2-phenyl-acetophenon), and eosin Y (Eosin Y), with a light wavelength of 365nm-780nm and a light exposure time of 0.00001s-600s; ③ a temperature-mediated cross-linking agent with a cross-linking temperature of 0-40℃; ④ an enzyme used in the enzyme-catalyzed reaction (selected from one of superoxide dismutase, thrombin, and transglutaminase).

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

[0026] In the above-described technical solution, step (4) uses the coated cell spheres prepared in step (3) and the microfluidically prepared particulate gel as printable bio-inks. With the help of a three-dimensional (3D) mobile platform, the three-dimensional complex structure is precisely constructed by printing point by point on demand and layer by layer.

[0027] In the above-described technical solution, the particulate gel for injection or bioprinting is formed by densely packing microgels prepared under microfluidic control and placing them in a printing container for bioprinting. The method for densely packing the coated cell spheres and the microfluidically prepared particulate gel can be a chemical processing unit such as centrifugation, filtration, or vacuum filtration.

[0028] In the above-described technical solution, the material of the particulate gel further includes extracellular matrix, hyaluronic acid, chitosan, agarose, dextran or protein-based collagen, gelatin, gelatin derivatives, fibrin, agar, matrix gel, proteoglycans, glycoproteins, and laminin; the mixing method of the particulate gel with the cell spheres includes convective mixing or shear mixing; the convective mixing is achieved by mechanical stirring or turning equipment to move or turn the particles as a whole, thereby achieving mixing; the shear mixing is achieved by applying shear force to make the particles rub and squeeze against each other during the mixing process, thereby breaking the aggregation phenomenon between particles and achieving a uniform mixing effect.

[0029] The advantages of this invention over the prior art are as follows:

[0030] (1) This invention designs a new method for rapid preparation of modular bio-ink for cell spheres based on microchips, which enables the mass production of cell spheres and ensures high cell activity.

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

[0032] (3) This invention can precisely arrange coated stem cell sphere gel particles using bioprinting technology. The coated stem cell sphere gel particles can be used as tissue building blocks to create three-dimensional (3D) tissues and organs, forming biomimetic tissue micro-units with single-cell precision and high cell density.

[0033] (4) The technology used in this invention can effectively ensure cell viability; and the use of injectable direct in situ delivery of cells for disease treatment or bioprinting can realize the construction of three-dimensional complex structures, which has good application prospects in the field of regenerative medicine. Attached Figure Description

[0034] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0035] Figure 1 Fabrication of a PDMS multi-microporous device for culturing cell spheres.

[0036] Figure 2 This is a schematic diagram illustrating the process by which polyphenols and proteins encapsulate cell spheres.

[0037] Figure 3 Microfabrication of cell spheres encapsulated with polyphenols and proteins.

[0038] Figure 4 This is to demonstrate the protective effect of the coating on stem cell spheres.

[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 This refers to the immunomodulatory effect of coated stem cell spheroids on macrophage polarization.

[0041] Figure 7 The coated stem cell spheres exhibit a more pronounced healing-promoting ability in the early stages following myocardial infarction.

[0042] Figure 8 To enhance the injectability and printability of coated stem cell spheres and particulate gels. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0044] This invention first fabricates a PDMS microporous device for culturing cell spheres. The microporous PDMS device has a square container in the center. The bottom of the square container has 1024 pores. Each pore is a trapezoidal well with a thickness of 0.6 mm, a square on the upper side with a length of 0.46 mm, and a square on the lower side with a length of 0.25 mm. A master mold is fabricated using projection micro-stereolithography. Then, the master mold is embedded into a metal aluminum component. A cover plate is placed on the master mold, and the four corners are fixed with screws to obtain the final assembly mold. Degassed PDMS prepolymer solution is poured onto the mold and cured at 85°C. The cured microporous PDMS device is removed from the assembly mold, sterilized, and subjected to hydrophilic treatment, such as... Figure 1 After rinsing with deionized water and serum-free cell culture medium, the cells were used for cell spheroid culture. Four different cell densities were used to obtain spheroids of different sizes, and the diameter of each spheroid was measured. The collected spheroids were incubated with a natural polymer, followed by centrifugation to remove the supernatant. Polyphenols were added to the coated spheroids, gently mixed, and then separated from the supernatant. The natural polymer layer was alternately deposited with the polyphenol layer on the spheroids as follows: Figure 2 and Figure 3 .

[0045] The integrated microfluidic chip used is the one disclosed in the invention patent (CN112275336A). Its substrate has at least two droplet production units and includes multiple liquid phase input modules and a cleaning output module. The liquid phase input modules can be divided into dispersed phase distribution units and continuous phase distribution units based on the type of liquid phase they transport. This classification is only related to the type of liquid phase within the chip and not to their relative position within the chip. Therefore, the liquid phase input position within the channel can be arbitrarily changed in actual production, achieving the goal of changing the droplet production method according to actual needs and improving the flexibility of chip use. This invention uses a multi-layered integrated microfluidic chip with 32 production units (the specific design method of the microfluidic chip is described in CN112275336A) to prepare microgels. It can continuously and stably prepare cell-loaded microgel particles of various hydrogel materials and can directly obtain microgels loaded with active substances through a continuous processing step. Coated cell spheres and microfluidically prepared particulate gels are used as injectable or printable materials. After dense stacking, the precise arrangement of microgels carrying bioactive substances is achieved by bioprinting.

[0046] This invention presents a stem cell spheroid-based therapeutic strategy for myocardial infarction. By introducing a protein / polyphenol composite coating onto the spheroids as a multifunctional armor, it provides immunoprotection and immunomodulatory therapy against the host immune system, reversing the hostile microenvironment and promoting 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. Polyphenols act as cross-linking agents, binding to proteins and providing the ability to scavenge reactive oxygen species (ROS). Furthermore, the coating reconfigures paracrine factors secreted by the coated stem cells, promoting angiogenesis and immunomodulation by activating glycolysis. Further transplantation of the coated stem cell spheroids into rats with myocardial infarction revealed that, compared to treatment with naked spheroids, it significantly reduced local inflammation and subsequent fibrosis and improved cardiac function in the infarcted myocardial tissue by mediating macrophage polarization towards the pro-healing M2 phenotype.

[0047] Example 1

[0048] 1. Cell culture:

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

[0050] 2. Preparation of cell spheroids:

[0051] hUC-MSCs isolated from Wharton's jelly of the umbilical cord were cultured in growth medium containing α-MEM, 10% fetal bovine serum, and 1% penicillin / streptomycin at 37°C with 5% CO2, and the medium was changed every 3 days. Cells were passaged in T75 cell culture flasks when they reached 70-80% confluence, and all cells were used at passage 5. 0.8 mL of cell suspension was added to PDMS microwell culture apparatus for culture. Four different cell densities were used (5.0 × 10⁵, 1 × 10⁶, 1.5 × 10⁶, and 2 × 10⁶ cells / mL). After 12 h, the hU-MSCs spheroids were observed under an inverted fluorescence microscope (OLYMPUS IX73, Japan), and the diameter of each spheroid was measured.

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

[0053] Example 2

[0054] The hUC-MSCs cell spheres prepared in Example 1 were collected in 15 mL centrifuge tubes and washed twice with PBS. The collected spheres were incubated with α-MEM in 2 mg / mL fibrinogen (Fg) solution for 30 min, then centrifuged at 150 g for 1 min, and the supernatant was gently removed. 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 and EGCG layer were deposited alternately on the spheres. Figure 2 Following the manufacturer's instructions, live and dead cells in Fg / EGCG-coated and uncoated constructs were labeled using the LIVE / DEAD assay kit.

[0055] Example 3

[0056] In Example 2, after obtaining coated stem cell spheres, the ability of the coated spheres to scavenge free radicals was assessed using the DPPH assay. 500 μL of DPPH / methanol solution (100 μM) was added to a test tube containing 1000 spheres. After incubation at 37°C for 1 h and 4 h, the supernatant was collected and measured at 570 nm using a spectrophotometer. The viability of coated and uncoated cell spheres in a ROS microenvironment was further evaluated. Oxidative stress was induced by adding 200 μM H2O2 to the culture medium to simulate a ROS microenvironment. On days 1, 3, and 5, live and dead cells within Fg / EGCG-coated and uncoated spheres were labeled using the LIVE / DEAD viability assay kit. Spheroid proliferation was assessed using the Cell Counting Kit-8. Intracellular ROS levels were detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. Incubate 10 μM DCFH-DA (Beyotime, China) in the dark for 15-25 minutes, wash with PBS, and stain with DAPI. Observe the sample using a confocal microscope and quantify the fluorescence intensity of DCFH-DA, such as... Figure 4 .

[0057] Example 4

[0058] After obtaining cardiomyocytes, oxidative stress was induced for 3 days by adding 200 μM H2O2 to the culture medium. Then, coated and uncoated cell spheroids were continuously incubated with neonatal rat cardiomyocytes in a ROS microenvironment for 3 days. The culture medium for the cardiomyocytes was removed, and Cell Counting Kit-8 solution was added. After culturing for another 4 hours, the absorbance of the supernatant at 450 nm was measured using a microplate reader. The levels of ROS and superoxide anion in the cardiomyocytes were evaluated using DCFH-DA and dihydroethidium (DHE), respectively. In short, cardiomyocytes were cultured in a normal or ROS microenvironment induced by 200 μM H2O2, and both coated and uncoated spheroids were cultured. On day 3, 5 mM DHE or 10 μM DCFH-DA was added to the culture medium, and the cells were incubated in the dark for 15-25 min. The fluorescence intensity of DHE and DCFH-DA was quantified using a microplate reader. Real-time quantitative PCR was used to detect the gene expression levels of apoptosis markers caspase-3, α-actin 2 (ACTN2), cardiac troponin C1 (TNNC1), and connexin 43 (Cx43) in cardiac cytokines (CMs). Immunofluorescence staining was used to detect the protein expression levels of ACTN2 and Cx43, and fluorescence images were obtained using an inverted confocal laser scanning microscope. Total RNA was extracted using a micro-elution total RNA kit. Then, RNA was reverse transcribed into cDNA using the PrimeScript™ RT kit. Quantitative detection was performed using the Rapid SYBR Green Master Mix kit and a 7500Fast real-time quantitative PCR instrument. The primer sequences used are shown in Table 2.3. Gene expression was further normalized (housekeeping gene GAPDH). The relative gene expression levels were calculated using the 2-ΔΔCt method, such as... 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 cells, M0 macrophages were obtained by adding 0.15 μM phorbol 12-myristate 13-acetate (PMA) to the THP-1 medium. M0 macrophages were polarized to the M1 phenotype in THP-1 medium with or without 0.01 ng / mL lipopolysaccharide (LPS) under a ROS microenvironment (200 μM H2O2) for 24 h (5 × 10⁴ cells / cm²). Then, coated and uncoated spheroids (2 × 10⁵ cells) were added to the upper cavity of a transwell and co-cultured for 3 days. The relative expression of M1 maker (CD14, CD163, CCL-18, and CD206) and M2 maker (CD40, CCL-2, IL-1β, and IL-6) genes was detected by qRT-PCR. Primer sequences used are shown in Table 2.4. The relative expression of CCR-7 and CD36 proteins was detected by immunofluorescence staining. Fluorescence images were observed and acquired using an inverted confocal laser scanning microscope. Figure 6 .

[0061] Example 6

[0062] Heart samples were collected on day 4 to assess the anti-inflammatory effect after treatment. Collected samples were fixed in 4 wt% paraformaldehyde solution for 24 hours. Paraffin-embedded tissue was then obtained according to standard procedures. Samples were cut into 5 μm sections. Immunofluorescence staining was performed, followed by washing with PBS, treatment with 0.3% Triton X-100 for 30 min, and then blocking with 5% bovine serum for 30 min. Primary antibody binding incubation was performed for macrophage surface markers CD68, Arg-1, and iNOS; neutrophil surface marker MPO; vascular marker α-smooth muscle actin (α-SMA); and cardiomyocyte markers, cardiac contractile proteins, and gap junction proteins. Subsequently, sections were washed with PBS and incubated with appropriate secondary antibodies. Cell nuclei were stained with DAPI. 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% of the triblock Krytox-PEG-Krytox surfactant was dissolved in electronic fluorination 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), while simultaneously adding the aqueous phase dropwise to the oil phase at a ratio of 10:1 (v / v) using a syringe. After the addition was complete, stirring was continued for 1 min, followed by UV irradiation (power: 100%, wavelength: 355 nm) for 2 min to crosslink the microspheres. The oil phase was removed by centrifugation, and the surface surfactant of the gel microspheres was removed by washing with 1H,1H,2H,2H-perfluoro-1-octanol (PFO). The PFO and remaining oil phase were then removed by centrifugation, and the gel microspheres were resuspended in distilled water.

[0066] 2. Dense packing of microgel particles:

[0067] Vacuum filtration was performed using a vacuum pump, and water-suspended gel microspheres were added dropwise onto the filter membrane using a dropper. After filtration, the gel microspheres were collected in a syringe to obtain densely packed gel microspheres. An injectability test was then performed on the filtered gel microspheres, and the results are as follows: Figure 8 As shown, the printing process yields a relatively stable structure.

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

[0069] The prepared close-packed gel microspheres were soaked in DMEM medium containing 10% (v / v) penicillin and antibiotics for 24 hours. Afterwards, the microspheres were filtered under sterile conditions to re-cluster and loaded into a screw-type syringe. 3T3 cells in the culture flask were digested with trypsin. After digestion, an appropriate amount of culture medium was added, and the mixture was aspirated into a 15 mL centrifuge tube. The supernatant was removed by centrifugation. Cell counting was performed using a hemocytometer. After counting, the required cells were resuspended in 100 μL of culture medium. The close-packed gel microspheres and resuspended cells were loaded into a screw-type syringe, connected by an adapter. The syringes were then pushed and pulled to mix the cells with the close-packed gel microspheres. 1 mL of close-packed gel microspheres was seeded at a rate of 5 × 10⁶ cells / mL. 6 Cells were seeded. Densely packed gel microspheres containing these cells were then printed for testing. Culture medium was added, and the microspheres were cultured in a cell culture incubator containing 5% CO2.

[0070] Biocompatibility of the printed cells was assessed using a live / dead fluorescence assay. 2 mM calcein (a green fluorescent dye for live cells) and 4 mM propidium iodide (a red fluorescent dye for dead cells) were added to the microgel suspension. After incubation for 20 minutes, the cells were observed using a laser confocal scanning microscope. The results are as follows: Figure 8 As shown, the cell survival rate was only 85.3%, indicating that the cell microgel prepared by this method maintains the cells in a high state of activity.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 modular bio-ink with tissue repair function, characterized in that, The modular bio-ink consists of cell spheres and particulate gels; the particulate gels have a dry weight mass fraction of 0.1-50% and a volume fraction greater than 0.5%; the cell spheres have an autologous protein and polyphenol coating on their surface; the particulate gels provide injectable and printable properties; the cell spheres have a diameter of 40-1000 μm. The preparation of the cell spheres includes the following steps: (1) Cell spheres were cultured in a PDMS multi-micropore device: cell suspensions were prepared with four different cell densities to obtain spheres of different sizes, and the diameter of each sphere was measured. (2) Collect the cell spheres described in step (1), wash them, incubate the collected spheres with the natural polymer, and centrifuge to remove the supernatant; add polyphenolic substances to the coated spheres, mix gently, and then separate the supernatant; the natural polymer layer and the polyphenolic substance layer are deposited alternately on the spheres in sequence; The method for preparing a PDMS microporous device includes: a square container with 1024 holes at the bottom, each hole being a trapezoidal well with a thickness of 0.6 mm, a square on the upper side with a length of 0.46 mm, and a square on the lower side with a length of 0.25 mm; fabricating a master mold using projection micro-stereolithography; embedding the master mold into an aluminum metal component; placing a cover plate on the master mold to fix it into a final assembly mold; pouring degassed PDMS prepolymer onto the mold and curing it at 85°C; removing the cured microporous PDMS device from the assembly mold, sterilizing and hydrophilically treating it; rinsing it with deionized water and serum-free cell culture medium, and then using it for cell sphere culture.

2. The modular bio-ink for cells according to claim 1, characterized in that, The modular bio-ink contains 2 to 10,000 cells.

3. The modular bio-ink for cells according to claim 1, characterized in that, The coating provides cells with materials that are natural polymers and / or small molecule polyphenols.

4. The modular bio-ink for cells according to claim 3, characterized in that, Natural polymers include extracellular matrix, hyaluronic acid, chitosan, agarose, dextran, protein collagen, gelatin, gelatin derivatives, fibrin, agar, matrix gum, proteoglycans, glycoproteins or laminin; small molecule polyphenols include anthocyanins, catechins, quercetin, gallic acid, ellagic acid or arbutin.

5. The modular bio-ink for cells according to claim 1, characterized in that, The materials of the particulate gel include extracellular matrix, hyaluronic acid, chitosan, agarose, dextran or protein-based collagen, gelatin, gelatin derivatives, fibrin, agar, matrix gel, proteoglycans, glycoproteins, and laminin; the mixing method of the particulate gel with cell spheres includes convective mixing or shear mixing; the convective mixing is achieved by mechanical stirring or tumbling equipment to move or tumble the particles as a whole, thereby achieving mixing; the shear mixing is achieved by applying shear force to make the particles rub and squeeze against each other during the mixing process, thereby breaking the aggregation between particles and achieving a uniform mixing effect.

6. The application of the modular bio-ink of cell spheres as described in claim 1 in the field of preparing tissue repair materials.

7. The application according to claim 6, characterized in that, The tissue repair mentioned refers to the repair of myocardial injury in acute myocardial infarction.

Images