A micro-nano composite substrate, its preparation method and application

Abstract

The present invention provides a micro-nano composite substrate and its preparation method and application. The micro-nano composite substrate is composed of a micro-grooved substrate and oriented nanofibers. The surface of the micro-grooved structure of the micro-grooved substrate is attached with oriented nanofibers, and the oriented nanofibers are maintained in a plane. The micro-nano composite substrate makes full use of the high porosity of the oriented nanofibers and the high support force of the micro-grooved substrate to achieve off-ground culture of cells. When it is used to culture hiPSC-CMs, it can show extremely high superiority in terms of cell morphology, cell structure, expression of genes related to cell maturation, and drug toxicity research, etc., providing a new way for constructing an in vitro myocardial model.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and particularly relates to a micro-nano composite substrate, a preparation method thereof, and an application thereof. Background Art

[0002] The oriented arrangement of cells has been widely observed, ranging from extracellular matrices (ECMs), concentric ECM waves in bone, aligned cells (vascular endothelial cells, skeletal muscle cells, and neuronal cells), to cytoskeletal fibers such as myofibrils in rod-shaped mature cardiomyocytes. The oriented arrangement of cells plays an important role in the maturation and regeneration of tissues and organs. The formation of cell orientation in vivo is usually accompanied by cell differentiation, proliferation, and changes in physical cues in the microenvironment around cells, further leading to the formation of various arrangements of subcellular structures, including the cytoskeleton, plasma membrane, and cell adhesion complexes. In addition, the oriented arrangement of cells combined with the proliferation, migration, and secretion of structural substances determines the hierarchy of cells and tissues, providing physical and mechanical properties and special biological functions at the tissue level. The aligned cell tissue also leads to the secretion and deposition of highly anisotropic ECM, which is related to tissue types and is crucial in determining tissue function. Therefore, mimicking the in vivo environment of these cells as much as possible during in vitro culture to enable them to maintain similar behaviors in vitro is beneficial for subsequent tissue engineering applications, in vitro model construction, and drug screening.

[0003] Among many oriented cells, spontaneously beating cardiomyocytes are the most special. Their dense, uniaxially arranged cell structure and the electrical and mechanical coupling between cells are the key factors for the synchronous contraction of the heart. Therefore, reproducing the anisotropic spatial arrangement of cardiomyocytes is a key design criterion for constructing functional myocardium. Currently, in the construction of in vitro myocardial models and the research and development of myocardial drugs, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a reliable source of cardiomyocytes. However, the initially differentiated hiPSC-CMs are in a fetal phenotype both structurally and functionally, that is, they are in an immature stage compared with adult cardiomyocytes, showing characteristics such as random growth directions, disordered sarcomeres, smaller contractile forces, and smaller action potentials, and cannot truly simulate the physiological structure and function of the human myocardium. Therefore, finding a method that can effectively promote the maturation of cardiomyocytes is quite necessary for constructing in vitro cell models.

[0004] Currently, methods for promoting the maturation of cardiomyocytes in vitro, such as surface patterning, fibrous scaffolds, etc., have been widely studied. Among them, the method of using surface patterning to promote cell maturation is the most extensive. The morphological functions of certain cells can be changed due to the topographical cues or chemical properties of the substrate, and both nano- and micro-topographical cues can endow the substrate with the characteristics of a high surface area and high volume ratio, which can simulate the fibrous structure of the ECM and thus regulate cell behavior. However, the existing surface patterning methods cannot effectively enhance the expression of genes related to cardiomyocyte maturation, nor can they effectively reduce the drug sensitivity of cardiomyocytes, resulting in limitations in the further development of myocardial models and myocardial drugs. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention aims to provide a micro-nano composite substrate, a preparation method and an application thereof. When used for cell culture, it can promote cell alignment, especially when culturing hiPSC-CMs, it can further promote the maturation of hiPSC-CMs, making their physiological structure and function closer to in vivo cardiomyocytes.

[0006] The primary object of the present invention is to provide a micro-nano composite substrate.

[0007] Another object of the present invention is to provide the application of the micro-nano composite substrate in cell culture.

[0008] Another object of the present invention is to provide a preparation method of the micro-nano composite substrate.

[0009] Another object of the present invention is to provide a method for cell culture.

[0010] The present invention realizes the above-mentioned invention objects through the following technical solutions:

[0011] The present invention provides a micro-nano composite substrate, which is composed of a microgrooved substrate and aligned nanofibers. The surface of the microgrooved structure of the microgrooved substrate is attached with aligned nanofibers, and the aligned nanofibers are maintained in a plane.

[0012] Preferably, the number of the microgrooved structures is several; the depth of each microgrooved structure is 10 - 100 μm, the width is 10 - 100 μm; the distance between two adjacent microgrooves is 10 - 100 μm.

[0013] Most preferably, the depth of each microgrooved structure is 40 μm, the width is 40 μm, and the distance between two adjacent microgrooves is 20 μm.

[0014] Preferably, the orientation of the aligned nanofibers is perpendicular to the axial direction of the microgrooved structure.

[0015] Preferably, the microgrooved substrate is made of PDMS.

[0016] Preferably, the oriented nanofibers are prepared from one or more of gelatin, lecithin, chitosan, collagen or sodium alginate.

[0017] The materials selected in the present invention all have good biocompatibility and low cost.

[0018] Preferably, the oriented nanofibers are monolayer.

[0019] The micro-nano composite substrate of the present invention provides a ground-free culture environment for cells. The multi-porous and micro-grooved substrate of the oriented nanofibers can significantly promote the exchange of cell nutrients. When using the micro-nano composite substrate of the present invention to culture cells, it can simulate the in-vivo microenvironment and regulate the behavior of cells, such as vascular endothelial cells and osteoblasts, etc., and further promote the oriented arrangement of cells; when used for culturing hiPSC-CMs in vitro, it can also make the structure and function of hiPSC-CMs closer to those of in-vivo cardiomyocytes. Specifically, it is manifested as: the sarcomere is extended, the expression of genes related to cardiomyocyte maturation is significantly increased, and the sensitivity to drugs is close to that of mature cardiomyocytes. Therefore, the application of the above micro-nano composite substrate in cell culture is also within the protection scope of the present invention.

[0020] Preferably, the cells are one or more of cardiomyocytes, fibroblasts, and endothelial cells.

[0021] More preferably, the cardiomyocytes are hiPSC-CMs.

[0022] More preferably, the endothelial cells are umbilical vein endothelial cells.

[0023] Preferably, the cell culture promotes cell maturation.

[0024] The present invention also provides a preparation method of the above micro-nano composite substrate. Oriented nanofibers are prepared on the surface of the micro-grooved structure of the micro-grooved substrate to obtain a micro-grooved substrate attached with oriented nanofibers, and the micro-nano composite substrate is obtained after cross-linking.

[0025] Preferably, the preparation process of the micro-grooved substrate is as follows: make a mask plate, use the mask plate to form micro-grooves on a silicon wafer through photolithography technology, and then pour a substrate material on the silicon wafer and cure it to obtain the micro-grooved substrate.

[0026] More preferably, the substrate material is a PDMS solution (the ratio of the PDMS main agent to the curing agent is 10-50:1). Most preferably, the ratio of the PDMS main agent to the curing agent is 10:1.

[0027] Among them, both the PDMS main agent and the curing agent are purchased from SYL GARD-184, DC184.

[0028] More preferably, the method for preparing the microgrooves is specifically as follows:

[0029] S1. Spin coat AZ40XT photoresist on the silicon wafer at 500 - 1000 rpm for 5 - 10 s, and then spin coat at 1500 - 2000 rpm for 10 - 15 s to form a photoresist layer with a thickness of 10 - 100 μm;

[0030] S2. Gradient pre-bake the photoresist layer at 55 - 65 °C for 30 - 40 s, 95 - 105 °C for 2 - 3 min, and 110 - 120 °C for 7 - 9 min. After cooling to 20 - 30 °C, perform back exposure on the photoresist layer with UV light through a mask, and then perform gradient post-bake on the photoresist layer (bake successively at 60 - 65 °C for 30 - 40 s, 85 - 95 °C for 1 - 2 min, and 100 - 105 °C for 2 - 3 min);

[0031] S3. Develop with AZ319MIF (AZ Electronic Materials) for 13 - 15 min to obtain the AZ40XT master mold;

[0032] S4. Treat the surface of the AZ40XT master mold with TMCS for anti-sticking for 1 - 10 min.

[0033] Most preferably, the conditions for the gradient pre-bake in S2 are 65 °C for 30 s, 95 °C for 2 min, and 120 °C for 8 min.

[0034] Most preferably, the conditions for the gradient post-bake in S2 are 65 °C for 30 s, 95 °C for 1 min, and 105 °C for 2 min.

[0035] More preferably, the curing is carried out at 60 - 80 °C for 1 - 4 h. Most preferably, it is cured at 60 °C for 2 h.

[0036] Preferably, the process for preparing the oriented nanofibers is as follows: By electrospinning, the spinning solution is prepared into oriented nanofibers.

[0037] More preferably, the solute of the spinning solution is one or more of gelatin, lecithin, chitosan, collagen, or sodium alginate.

[0038] More preferably, the solvent of the spinning solution is a mixed solution of acetic acid, ethyl acetate, and distilled water.

[0039] More preferably, the volume ratio of acetic acid, ethyl acetate, and distilled water is (18 - 25):(10 - 18):(8 - 15). Most preferably, the volume ratio of acetic acid, ethyl acetate, and distilled water is 21:14:10.

[0040] More preferably, the ratio of the solute to acetic acid is (0.2-1) g: (18-25) μL, and most preferably 0.2 g: 21 μL.

[0041] More preferably, during the preparation of the spinning solution, when the solute is added to the solvent, stirring is required at 22 to 28° C. for 6 to 12 hours, and most preferably, stirring is required at 25° C. for 9 hours.

[0042] More preferably, the method for preparing the oriented nanofibers is specifically as follows:

[0043] S1. Adjust the ambient temperature and humidity in the operating room to ensure that the spinning environment temperature is between 25 and 30°C and the humidity is between 40 and 75%;

[0044] S2. Take two iron sheets of the same length and size and place them against the drum collector at an inclined angle. The distance between the two iron sheets is 2 to 3 cm. Place the microgroove substrate between the two iron sheets and adjust the direction of the microgroove substrate to ensure that the orientation of the oriented nanofibers is perpendicular to the axial direction of the microgroove structure.

[0045] S3. Take a silicone tube with an inner diameter of 0.2 to 1 mm, both ends of which are covered with needles matching the inner diameter of the silicone tube, and remove the base of one of the needles;

[0046] S4. Use a 1-5 mL syringe to absorb the spinning solution, connect the syringe to the needle with a base, and slowly push the syringe until the spinning solution overflows from the needle without a base at the other end; then install the syringe containing the spinning solution on the syringe pump, making sure that the syringe pump supports the end of the syringe;

[0047] S5. Place the syringe between the syringe pump and the high-voltage DC power supply, with the needle without a base 15 to 20 cm away from the iron sheet, connect the positive electrode of the high-voltage DC power supply to the needle without a base, and connect the negative electrode to the drum collector;

[0048] S6. Adjust the parameters of the injection pump and adjust the spinning speed to 0.2-0.4 mL / h;

[0049] S7. Turn on the high-voltage DC power supply, rotate the voltage adjustment knob, and adjust the voltage to 13-15 kV;

[0050] S8. The spinning time is set to 3 to 10 minutes to obtain the oriented nanofibers;

[0051] Preferably, drying is also carried out before crosslinking.

[0052] More preferably, the drying is vacuum drying at 22-28°C for 6-12 hours. Most preferably, the drying is at 25°C for 12 hours.

[0053] Preferably, the crosslinking is carried out by crosslinking the microgrooved substrate with oriented nanofibers after drying in a crosslinking agent for 2 to 4 hours. More preferably, it is 4 hours.

[0054] Preferably, the crosslinking agent is an ethanol solution containing EDC and NHS.

[0055] More preferably, the concentrations of both EDC and NHS are 0.02 to 2 M. Most preferably, the concentrations of both EDC and NHS are 0.2 M.

[0056] Preferably, after crosslinking, it is rinsed with absolute ethanol 2 to 5 times and dried in vacuum for 6 to 12 hours to remove the remaining crosslinking agent on the surface. More preferably, it is rinsed with absolute ethanol 3 times and dried in vacuum for 12 hours.

[0057] The present invention also provides a method for cell culture, using the above-mentioned micro-nano composite substrate to culture cells.

[0058] Preferably, the cells are one or more of cardiomyocytes, endothelial cells, and fibroblasts.

[0059] More preferably, the cardiomyocytes are hiPSC-CMs.

[0060] More preferably, the endothelial cells are umbilical vein endothelial cells.

[0061] The technical solution of the present invention has the following beneficial effects:

[0062] The present invention provides a micro-nano composite substrate. The combination of oriented nanofibers and the microgrooved substrate provides a ground-free culture environment for cells. When it is used to culture hiPSC-CMs, the structure and function of hiPSC-CMs cultured in vitro are closer to those of cardiomyocytes in vivo. Specifically, it is manifested as: the sarcomere is elongated, the expression of genes related to cardiomyocyte maturation is significantly increased, and the sensitivity of cardiomyocytes to drug application is close to that of mature cardiomyocytes, indicating that the micro-nano composite substrate provided by the present invention can effectively promote the maturation of cardiomyocytes and has good biocompatibility. Description of the Drawings

[0063] Figure 1 It is a schematic structural diagram of the micro-nano composite substrate, wherein 1 - microgrooved substrate; 2 - oriented nanofibers.

[0064] Figure 2 It is a preparation flow chart of the micro-nano composite substrate.

[0065] Figure 3 It is an SEM image of the micro-nano composite substrate.

[0066] Figure 4 It is a fiber orientation distribution diagram of the micro-nano composite substrate.

[0067] Figure 5 It is a time roadmap for hiPSC-U1 culture and hiPSC-CMs reseeding.

[0068] Figure 6 A is the live / dead cell staining map of hiPSC-CMs, Figure 6 B is the statistical result of cell survival rate.

[0069] Figure 7 A is the Phalloidin fluorescence staining map of hiPSC-CMs; Figure 7 B is the CTnT immunofluorescence map of hiPSC-CMs; Figure 7 C is the α-actinin immunofluorescence map of hiPSC-CMs; Figure 7 D is the statistical graph of sarcomere length; 7E is the statistical graph of cell area; 7F is the statistical graph of cell roundness index.

[0070] Figure 8 A shows the expression of genes related to cardiomyocyte maturation, Figure 8 B is the ratio of the expression level of MYH6 gene to that of MYH7 gene, Figure 8 C is the ratio of the expression level of MLC2v gene to that of MLC2a gene.

[0071] Figure 9 A is the beating frequency graph of hiPSC-CMs after adding E-4031; Figure 9 B is the graph of the change in beating rate of hiPSC-CMs after adding E-4031; Figure 9 C is the beating frequency graph of hiPSC-CMs after adding ISP; Figure 9 D is the graph of the change in beating frequency of hiPSC-CMs after adding ISP; In the figure, control represents the blank control group, E-4031-50 represents the experimental group adding 50 nM E-4031, E-4031-100 represents the experimental group adding 100 nM E-4031, ISP-0.2 represents the experimental group adding 0.2 μM isoproterenol, and ISP-0.5 represents the experimental group adding 0.5 μM isoproterenol. Detailed implementation mode

[0072] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0073] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0074] Example 1 Preparation of micro-nano composite substrate

[0075] (1) Use Auto CAD 2015 software to draw a schematic diagram of the microgroove structure. The parameters of the microgroove structure are as follows: the depth is 40 μm, the width is 20 μm; the spacing between two adjacent microgrooves is 40 μm.

[0076] (2) Refer to soft lithography technology to form the shape of the microgroove on the silicon wafer:

[0077] S1. Use AZ40XT photoresist to spin-coat on the silicon wafer for 10 s at 500 rpm, and then spin-coat for 15 s at 2000 rpm to form a 40-μm photoresist layer.

[0078] S2. Gradient pre-bake the photoresist layer at 65 °C for 30 s, 95 °C for 2 min, and 120 °C for 8 min. After cooling to 25 °C, back-expose the photoresist layer with UV light through the mask, and then perform gradient post-baking on the photoresist layer (bake at 65 °C for 30 s, 95 °C for 1 min, and 105 °C for 2 min in sequence).

[0079] S3. Develop with AZ319MIF (AZ Electronic Materials) for 1 min to obtain the AZ40XT master mold.

[0080] S4. Treat the surface of the AZ40XT master mold with TMCS to prevent sticking for 1 min.

[0081] (3) Pour the PDMS solution (SYL GARD-184, DC184, the ratio of the PDMS main agent to the curing agent is 10:1) onto the AZ40XT master mold, cure it at 60 °C for 24 h, and then peel off the PDMS part from the mold to obtain the PDMS microgroove substrate.

[0082] (4) Prepare the spinning solution: At 25 °C, dissolve 0.2 g of gelatin in a mixed solution of acetic acid, ethyl acetate, and distilled water with a volume ratio of 21:14:10, add a magnetic stirrer, and stir on a magnetic stirrer at 25 °C for 8 h.

[0083] (5) Use the spinning solution prepared in (4) to prepare oriented nanofibers through an electrospinning machine. The specific steps are as follows:

[0084] S1. Adjust the environmental temperature and humidity in the operating room to ensure that the spinning environment temperature is 25-30 °C and the humidity is 65%.

[0085] S2. Take two pieces of iron sheets of the same length and size and place them against the drum collector at an inclined angle. The distance between the two iron sheets is 2 cm. Place the microgroove substrate between the two iron sheets and adjust the direction of the microgroove substrate to ensure that the orientation of the oriented nanofibers is perpendicular to the axial direction of the microgroove structure.

[0086] S3. Take a silicone tube with an inner diameter of 1 mm, both ends of which are covered with needles matching the inner diameter of the silicone tube, and remove the base of one of the needles;

[0087] S4. Use a 1mL syringe to absorb the spinning solution, connect the syringe to the needle with a base, and slowly push the syringe until the spinning solution overflows from the needle without a base at the other end; install the syringe containing the spinning solution on the syringe pump, making sure that the syringe pump supports the end of the syringe;

[0088] S5. The syringe is placed between the syringe pump and the high-voltage DC power supply, the needle without a base is 20 cm away from the iron sheet, the positive electrode of the high-voltage DC power supply is connected to the needle without a base, and the negative electrode is connected to the drum collector;

[0089] S6. Adjust the parameters of the injection pump and adjust the spinning speed to 0.3 mL / h;

[0090] S7. Turn on the high voltage DC power supply, rotate the voltage adjustment knob, and adjust the voltage to 13kV;

[0091] S8. The spinning time is set to 5 min;

[0092] S9. After spinning, adjust the voltage of the high-voltage DC power supply to zero, then turn off the high-voltage DC power supply, then turn off the injection pump, unplug both power plugs, use tweezers to remove the PDMS microgroove substrate with oriented nanofibers, place it in a vacuum dryer, and dry it at 25°C for 12 hours.

[0093] (6) The dried substrate was taken out and the oriented nanofibers on the surface were chemically cross-linked. The substrate was placed in an ethanol solution containing EDC and NHS (where anhydrous ethanol was used as the solvent and the concentrations of EDC and NHS were both 0.02 M) for 4 h. After the cross-linking was completed, the sample was rinsed three times with anhydrous ethanol and dried in a vacuum for 12 h to remove the chemical reagents remaining on the surface, and the following was obtained: Figure 1 The micro-nano composite substrate shown, wherein: 1- microgroove substrate; 2- oriented nanofibers.

[0094] The process flow diagram of Example 1 is as follows Figure 2 shown.

[0095] Example 2 Preparation of micro-nano composite substrate

[0096] The preparation method is the same as that of Example 1, except that the depth of the microgroove structure is 100 μm, the width is 100 μm, and the distance between adjacent two microgrooves is 10 μm.

[0097] Preparation of the micro-nano composite substrate in Example 3

[0098] The preparation method is the same as that of Example 1, except that the depth of the microgroove structure is 10 μm, the width is 10 μm, and the distance between adjacent two microgrooves is 100 μm.

[0099] Characterization of the surface morphology and microstructure of the micro-nano composite substrate in Example 4

[0100] I. Experimental method

[0101] The surface morphology and microstructure of the micro-nano composite substrate (AN-MG) prepared in Example 1 were characterized by a scanning electron microscope. The micro-nano composite substrate before and after crosslinking was fixed on the operating table of the microscope with double-sided tape, and the acceleration voltage was set to 15 kV, and the surface structure of the micro-nano composite substrate was observed under vacuum.

[0102] The fiber orientation of the micro-nano composite substrate before and after crosslinking was quantitatively statistically analyzed using Image J software to obtain the orientation distribution map of the oriented nanofibers.

[0103] II. Experimental results

[0104] The SEM image of the micro-nano composite substrate is as Figure 3 shown. It can be seen that the oriented nanofibers are arranged in an oriented manner on the surface of the groove, and the orientation of the oriented nanofibers is perpendicular to the axial direction of the microgroove structure, confirming the successful preparation of the micro-nano composite substrate.

[0105] The orientation distribution map of the oriented nanofibers is as Figure 4 shown. The results in the figure show that the orientation of the oriented nanofibers is consistent.

[0106] hiPSCs cell culture and reseeding of hiPSC-CMs in Example 5

[0107] I. hiPSCs cell culture:

[0108] (1) Passage of hiPSCs:

[0109] In the present invention, hiPSC-U1 (Gibco, USA) with a cell density of more than 80% was used for passage culture. The specific steps are as follows:

[0110] S1. Add 2 mL of Matrigel coating solution to a 60 mm A culture dish, shake it evenly back and forth, and place it in an incubator at 37 °C containing 5% CO 2 for 12 h;

[0111] S2. Equilibrate Dulbecco's Phosphate Buffered Saline (D-PBS, from Gibco, USA), PSC Digestive Solution (from Beijing Saibei Co., Ltd.), and PSC Complete Medium (from Beijing Saibei Co., Ltd.) to 25 °C.

[0112] S4. Wash hiPSC-U1 twice with 1 mL of D-PBS buffer and place it in Petri dish B.

[0113] S4. Add 2 mL of PSC Digestive Solution to Petri dish B and incubate it in an incubator at 37 °C with 5% CO 2 After 270 s of incubation, when it is observed under a microscope that the edges of most colonies of hiPSC-U1 begin to dissociate and gaps appear inside, terminate the digestion.

[0114] S5. Discard the PSC Digestive Solution, take 1 mL of PSC Complete Medium and pipette it in a fan-shaped swirling motion at the bottom of Petri dish B, repeat twice to suspend hiPSC-U1, and transfer the cell suspension into a 15 mL centrifuge tube.

[0115] S6. Gently pipette the 15 mL centrifuge tube 2 - 3 times, then take 350 μL of the cell suspension and inoculate it into a Matrigel-coated Petri dish A; gently shake it in an inverted "8" shape on the flat surface, let it stand in the laminar flow hood for 5 min and then transfer it to the incubator.

[0116] (2) Differentiation of hiPSCs:

[0117] When the confluence of hiPSC-U1 reaches 75% during subculture, induce the differentiation of hiPSCs with small molecule compounds. The specific steps are as follows:

[0118] Day 0: Discard the original medium in the well of the hiPSC-U1 subculture dish, and replace the medium in each well of the 12-well plate with 2 mL of RPMI / B-27 without insulin medium (hereinafter referred to as Medium A) containing 8 μM CHIR 99021 (from Gibco, USA), and place it in an incubator at 37 °C for 4 h.

[0119] Day 1: Discard Medium A in the well, and replace the medium in each well with 2 mL of RPMI / B-27 without insulin medium (hereinafter referred to as Medium B) and then culture for 48 h.

[0120] Day 3: Place the 12-well plate containing Medium B on the laminar flow bench and let it stand for 30 s. Use a pipette to collect 1 mL of the supernatant successively, and then mix it with 1 mL of RPMI / B-27 without insulin medium containing 5 μM IWP2 in an EP tube to obtain a combined medium. Then, gently shake the 12-well plate to suspend the cell debris, discard the waste liquid in the wells with a pipette, add 2 mL of the combined medium to each well, and incubate for 48 h.

[0121] Day 5: Discard the combined medium in the wells, and replace it with 2 mL of RPMI / B-27 without insulin medium (hereinafter referred to as Medium C) in each well, and incubate in the incubator for 48 h.

[0122] Day 7: Discard the Medium C in the wells, and replace it with 2 mL of RPMI / B-27 with insulin medium (Gibco, USA) in each well, and place it in a 5% CO 2 incubator and incubate for 48 h. Then, change the medium (RPMI / B-27 with insulin medium, hereinafter referred to as Medium D) every two days.

[0123] Day 10: Spontaneously beating hiPSC-CMs are observed. At the same time, discard the Medium D in the wells, and replace it with 2 mL of low-glucose RPM / B-27 medium (Gibco, USA) in each well, and place it in a 37 °C incubator containing 5% CO 2 and incubate for 72 h for the first purification.

[0124] Day 14: Discard the medium for the first purification, and replace it with 2 mL of low-glucose RPM / B-27 medium (Gibco, USA) in each well, and place it in a 37 °C incubator containing 5% CO 2 and incubate for 72 h for the second purification.

[0125] II. Re-seeding of hiPSC-CMs:

[0126] On Day 18, re-seed the cardiomyocytes, and the specific steps are as follows:

[0127] S1. After irradiating the coverslips (Glass), coverslips with oriented nanofibers (AN), and the micro-nano composite substrate obtained in Example 1 (AN-MG) with ultraviolet light for 6 h, soak them in D-PBS buffer for 2.5 h. Add 1 mL of Matrigel coating solution to each of the three substrates (Glass, AN, AN-MG) and place them in the incubator for 2 h.

[0128] S2. Wash the spontaneously beating hiPSC-CMs cells with 1 mL of D-PBS buffer; add 1 mL of trypsin, and place them in a 37 °C, 5% CO 2After incubating in the incubator for 330 s, it can be seen under a microscope that the edges of most hiPSCs-CMs colonies begin to dissociate and gaps appear inside.

[0129] S3. Add 1 mL of DMEM medium (Gibco, USA) to terminate digestion. Pipette the bottom of the culture dish in a fan-shaped swirling manner, repeat twice to suspend the hiPSCs-CMs cells, and transfer the cell suspension into a 15 mL centrifuge tube.

[0130] S4. Centrifuge the centrifuge tube containing the cell suspension at 1200 r / min for 5 min.

[0131] S5. After discarding the supernatant, add 1 mL of DMEM medium to the centrifuge tube containing the cell pellet, gently shake the centrifuge tube to evenly distribute the cells.

[0132] S6. Take out the three pre-treated substrates (Glass, AN, AN-MG), discard the Matrigel coating solution respectively, and then add 100 μL of cell suspension respectively, and try to re-seed all the cells on the substrates.

[0133] S7. Observe the cell morphology under a microscope to judge whether all the cells are re-seeded on the substrates; then place them in an incubator at 37 °C and 5% CO 2 for culture.

[0134] On the 25th day, mature hiPSC-CMs were observed.

[0135] The time roadmap of hiPSC-U1 culture and hiPSC-CMs re-seeding is as Figure 5 shown.

[0136] Example 6 Toxicity study of micro-nano composite substrates on hiPSC-CMs

[0137] S1. Dilute 100 μL of 10× assay buffer in the Live / dead kit (Abbkine) to 1 mL of 1× buffer with 900 μL of sterile water, and place it at 37 °C.

[0138] S2. Under light-proof conditions, take 2 μL of LiveDye and 1.5 μL of NucleDye from the Live / dead kit (Abbkine) and add them to 1 mL of 1× buffer respectively. After pipetting evenly, prepare the live / dead cell staining solution.

[0139] S3. Wash the hiPSC-CMs cell samples cultured on the three substrates with 1 mL of PBS, repeat the operation twice, then add the live / dead cell staining solution respectively, and place them in an incubator at 37 °C under light-proof conditions for 30 min for staining.

[0140] After staining, the cells were washed with 1 mL of PBS, and the cell viability was observed using an inverted fluorescence microscope and statistically analyzed using Image J.

[0141] The experimental results are as Figure 6 shown, where Figure 6 A is the live / dead cell staining image of hiPSC-CMs, Figure 6 B is the statistical result of cell survival rate. By statistically analyzing the green fluorescence and red fluorescence in the immunofluorescence images, it was found that the green fluorescence on the three substrates was significantly more, the cells all had good viability, and there was no significant difference among the three substrates, indicating that the micro-nano composite substrate of the present invention has good biocompatibility and no obvious cytotoxicity.

[0142] Example 7 Influence of the micro-nano composite substrate on the structure of hiPSC-CMs

[0143] In this example, immunofluorescence staining was used to evaluate the influence of the micro-nano composite substrate on the structure of hiPSC-CMs.

[0144] (1) The intracellular microfilament protein F-actin of hiPSC-CMs was stained with Phalloidin-TRITC staining solution (Abcam, ab235137). The specific steps are as follows:

[0145] S1. After the hiPSC-CMs replanted in Example 5 were continuously incubated on the three substrates for 24 h, they were washed 3 times with D-PBS for 5 min each time, and then 2 mL of 4% paraformaldehyde solution was added and fixed for 15 min;

[0146] S2. The paraformaldehyde solution was discarded, and the cells were washed repeatedly 3 times with D-PBS buffer for 5 min each time; then they were permeabilized with 0.2% TritonX-100 permeabilization solution (Solarbio, USA) and left standing at 25 °C for 10 min;

[0147] S3. After permeabilization, the cells were washed repeatedly 3 times with D-PBS buffer for 5 min each time; then PBS buffer containing 1% (v / v) Phalloidin-TRITC staining solution was added to the sample and incubated in the dark for 2 h;

[0148] S4. The cells were washed repeatedly 3 times with D-PBS buffer for 5 min each time; then 1 μg / mL DAPI dilution solution was added to the sample and incubated in the dark at room temperature for 20 min: finally, the cells were washed repeatedly three times with D-PBS buffer for 5 min each time;

[0149] S5. The samples were stored in D-PBS buffer to prevent them from drying out and deforming. The cell morphology was observed and imaged under an inverted fluorescence microscope at a magnification of 20 times to obtain the Phalloidin fluorescence staining map of hiPSC-CMs.

[0150] (2) The intracellular specific proteins of hiPSC-CMs were stained by the method of cellular immunofluorescence staining. The specific steps were as follows:

[0151] S1. Prepare the antibody dilution solution:

[0152] When staining hiPSC-CMs, a primary antibody dilution solution was prepared by mixing rabbit anti-α-actinin, mouse anti-CTnT with 1% blocking solution (MCE Rapid Blocking Buffer (TBS-T) Powder (100 mL of 1×)) at a volume ratio of 2:1:200. A secondary antibody dilution solution was prepared by mixing donkey anti-rabbit H&L IgG (Alexa Fluor488), goat anti-mouse H&L IgG (Alexa Fluor 594) with 1% blocking solution in the dark at a volume ratio of 2:1:500 and stored at -20 °C for later use. When staining the cell nucleus, 1 μL of DAPI stock solution was added to 1 mL of 1% blocking solution and mixed evenly in the dark, then stored at -20 °C for later use.

[0153] S2. After the re-seeded hiPSC-CMs in Example 5 were incubated on the three substrates for 24 h, they were washed 3 times with D-PBS buffer for 5 min each time, and then 2 mL of 4% paraformaldehyde solution was added to fix for 15 min.

[0154] S3. Discard the fixing solution, and repeat the washing 3 times with D-PBS buffer for 5 min each time; then permeabilize with 0.2% Triton X-100 permeabilizing solution (Solarbio Co., Ltd., USA) and let stand at 25 °C for 20 min.

[0155] S4. Discard the Triton X-100 permeabilizing solution, and repeat the washing 3 times with D-PBS buffer for 5 min each time; then block with 1% blocking solution and let stand at 25 °C for 1 h.

[0156] S5. Discard the blocking solution, and repeat the washing 3 times with D-PBS buffer for 5 min each time; add the primary antibody dilution solution and incubate at 4 °C for 12 h.

[0157] After the incubation with the primary antibody, wash the cells 3 times with D-PBS buffer for 5 minutes each time; add the secondary antibody dilution solution and incubate at 4°C in the dark for 12 hours; wash the cells 3 times with D-PBS buffer for 5 minutes each time; finally, add the DAPI dilution solution and incubate at 25°C for 20 minutes;

[0158] S7. Collect the DAPI dilution solution, wash the cells 3 times with D-PBS buffer for 5 minutes each time; record the staining results under an inverted fluorescence microscope under light-proof conditions to obtain the CTnT immunofluorescence images of hiPSC-CMs; and the α-actinin immunofluorescence images of hiPSC-CMs. Use Image J to statistically analyze the Phalloidin fluorescence staining images of hiPSC-CMs to obtain the sarcomere length, cell area, and cell roundness index of the cells.

[0159] The Phalloidin fluorescence staining images of hiPSC-CMs are as Figure 7 shown in A; the CTnT immunofluorescence images of hiPSC-CMs are as Figure 7 shown in B; the α-actinin immunofluorescence images of hiPSC-CMs are as Figure 7 shown in C; statistically analyze the Phalloidin fluorescence staining images of hiPSC-CMs using Image J to obtain the sarcomere length statistical chart as shown in Figure 7 D, Figure 7 the cell area statistical chart as shown in Figure 7 E, and

[0160] It can be seen that high expression was achieved in cardiomyocytes on all three substrates, and cardiomyocytes grew along the orientation of the oriented nanofibers on the AN and AN-MG substrates; from Figure 7 E, Figure 7 F, it can be seen that culturing cardiomyocytes on the AN and AN-MG substrates caused the cardiomyocytes to significantly elongate. However, from Figure 7 D (sarcomere length statistical chart), it can be seen that the sarcomeres of cardiomyocytes cultured on AN-MG are significantly longer than those of cardiomyocytes cultured on AN, indicating that this micro-nano composite substrate promotes the maturation of cardiomyocytes.

[0161] Example 8 Effect of the Micro-Nano Composite Substrate on the Expression of Genes Related to Cell Maturation in hiPSC-CMs

[0162] In this example, RT-PCR was used to evaluate the effect of the micro-nano composite substrate on the expression of genes related to cell maturation in hiPSC-CMs. The specific steps are as follows:

[0163] S1. After the hiPSC-CMs from Example 5 were replanted and cultured on the three substrates for 5 days, they were washed with D-PBS buffer, and then 500 μl of β-mercaptoethanol was added and incubated for 1 min to obtain cell lysates.

[0164] S2. The total RNA of hiPSC-CMs in the cell lysates was isolated using the FastPure@ Cell / Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., USA).

[0165] S3. Complementary DNA (cDNA) of the total RNA of hiPSC-CMs was synthesized using the SweScript RTⅠ First Strand cDNA Synthesis Kit (Servicebio Technology Co., Ltd., China).

[0166] S4. The gene expressions of MYH6, MYH7, MLC2v, MLC2a, and cTnI were quantified from 1 μL of the above cDNA using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., USA) and specific primers (Integrated DNA Technologies), and detected by the LightCycler@ 96 Real-Time PCR System (Roche).

[0167] The detection results of the gene expressions related to cell maturation in hiPSC-CMs are as Figure 8 shown, where Figure 8 A shows the expression of genes related to cardiomyocyte maturation (MYH6, MYH7, MLC2v, MLC2a, and cTnI), Figure 8 B shows the ratio of the expression level of MYH6 gene to that of MYH7 gene, Figure 8 C shows the ratio of the expression level of MLC2v gene to that of MLC2a gene. It can be seen that the expression levels of MYH7, MLC2v, and cTnI genes in cardiomyocytes cultured on AN-MG are significantly increased compared with those in cardiomyocytes cultured on AN, while the expression levels of MYH6 and MLC2a genes are significantly decreased. After calculation, it is found that the MYH6 / MYH6 value and the MLC2v / MLC2a value are significantly increased on AN-MG compared with the other two groups, indicating that the micro-nano composite substrate of the present invention can effectively enhance the expression of genes related to cardiomyocyte maturation and promote the maturation of cardiomyocytes.

[0168] Effect of the micro-nano composite substrate in Example 9 on the drug sensitivity of hiPSC-CMs

[0169] S1. Add 0.2 μM isoproterenol (ISP), 0.5 μM isoproterenol, 50 nM E-4031, and 100 nM E-4031 into 1 mL of RPMI / B-27 with insulin (Gibco) medium respectively, shake well to mix, and then culture hiPSC-CMs on three kinds of substrates for 30 min respectively, with a total of 12 experimental groups; at the same time, set up a blank control group without adding drugs, and culture hiPSC-CMs on three kinds of substrates with 1 mL of RPMI / B-27 with insulin (Gibco) medium for 30 min, with a total of 6 blank control groups.

[0170] S2. Assemble an imaging analysis system with an optical microscope, an image controller (charge coupled device, CCD), a laptop computer, and video analysis software MATLAB, monitor and record the beating conditions of hiPSC-CMs in real time, and save the generated videos in the hard disk for subsequent playback and analysis. The magnification of the CCD microscope is 10 times, and the resolution is 1024×968.

[0171] S3. Randomly select a local position in the video as the analysis object, extract 25 s as the time unit, and use the MATLAB program to analyze the changes in the heart rate per minute (beat per minute, BPM) and the beat interval, observe the changes in cardiac contraction and relaxation, and conduct statistics.

[0172] The experimental results are as Figure 9 shown, where Figure 9 A is the beating frequency diagram of hiPSC-CMs after adding E-4031; Figure 9 B is the diagram of the change in the beating frequency of hiPSC-CMs after adding E-4031; Figure 9 C is the beating frequency diagram of hiPSC-CMs after adding ISP; Figure 9 D is the diagram of the change in the beating frequency of hiPSC-CMs after adding ISP. In the figure, control represents the blank control group, E-4031-50 represents the experimental group added with 50 nM E-4031, E-4031-100 represents the experimental group added with 100 nM E-4031, ISP-0.2 represents the experimental group added with 0.2 μM isoproterenol, and ISP-0.5 represents the experimental group added with 0.5 μM isoproterenol.

[0173] It can be seen from Figure 9 A that as the concentration of E-4031 increases, the beating of cardiomyocytes on the three substrates gradually slows down; however, it can be seen from Figure 9B that among the two concentrations of E-4031, the difference in the decrease in the beating frequency of hiPSC-CMs cultured on AN-MG is the smallest; fromFigure 9 It can be seen that as the concentration of isoproterenol increases, the beating of cardiomyocytes on the three substrates gradually accelerates; however, as can be seen from Figure D, among the two concentrations of IPS, the difference in the increase in the beating frequency of hiPSC-CMs cultured on AN-MG is the smallest. This indicates that the cardiomyocytes cultured on the micro-nano composite substrate of the present invention have the lowest sensitivity to drugs, and the micro-nano composite substrate of the present invention can effectively reduce the sensitivity of cardiomyocytes to drugs and promote the maturation of cardiomyocytes.

[0174] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of a micro-nano composite substrate in promoting the maturation of cardiomyocytes hiPSC-CMs, It is characterized in that First, referring to the soft lithography technology, the shape of micro grooves is formed on the silicon wafer. The parameters of the micro groove structure are as follows: the depth is 40 μm, the width is 20 μm; the spacing between two adjacent micro grooves is 40 μm; The PDMS solution was poured onto the AZ40XT master mold and cured at 60°C for 24 hours, and then the PDMS part was peeled off from the mold to obtain a PDMS microgroove substrate; wherein, in the PDMS solution, the ratio of the PDMS main agent to the curing agent was 10:1; At 25°C, 0.2 g of gelatin was dissolved in a mixed solution of acetic acid, ethyl acetate and distilled water in a volume ratio of 21:14:10, and stirred at 25°C for 8 hours to prepare a spinning solution; The prepared spinning solution is then used to prepare oriented nanofibers through an electrospinning machine: S1. Adjust the ambient temperature and humidity in the operating room to ensure that the spinning ambient temperature is between 25 and 30°C and the humidity is 65%; S2. Take two pieces of iron sheets of the same length and size and place them against the drum collector at an inclined angle. The distance between the two iron sheets is 2 cm. Place the microgroove substrate between the two iron sheets and adjust the direction of the microgroove substrate to ensure that the orientation of the oriented nanofibers is perpendicular to the axial direction of the microgroove structure. S3. Take a silicone tube with an inner diameter of 1 mm, both ends of which are covered with needles matching the inner diameter of the silicone tube, and remove the base of one of the needles; S4. Use a 1mL syringe to absorb the spinning solution, connect the syringe to the needle with a base, and push the syringe until the spinning solution overflows from the needle without a base at the other end; install the syringe containing the spinning solution on the syringe pump, making sure that the syringe pump supports the end of the syringe; S5. The syringe is placed between the syringe pump and the high-voltage DC power supply, the needle without a base is 20 cm away from the iron sheet, the positive electrode of the high-voltage DC power supply is connected to the needle without a base, and the negative electrode is connected to the drum collector; S6. Adjust the parameters of the injection pump and adjust the spinning speed to 0.3 mL / h; S7. Turn on the high voltage DC power supply, rotate the voltage adjustment knob, and adjust the voltage to 13kV; S8. The spinning time is set to 5 min; S9. After the spinning is completed, the voltage of the high-voltage DC power supply is adjusted to zero, and then the high-voltage DC power supply is turned off, followed by the syringe pump, and the power plugs of both are unplugged. The PDMS micro-groove substrate with oriented nanofibers spun is removed with tweezers, and placed in a vacuum dryer and dried at 25°C for 12 hours; the dried substrate is taken out, and the oriented nanofibers on the surface are chemically cross-linked, and the substrate is placed in an ethanol solution containing EDC and NHS for cross-linking for 4 hours. After the cross-linking is completed, the sample is rinsed three times with anhydrous ethanol and dried in a vacuum for 12 hours to remove the chemical reagents remaining on the surface, thereby obtaining the micro-nano composite substrate; wherein, in the ethanol solution, anhydrous ethanol is used as the solvent, and the concentrations of EDC and NHS are both 0.02M.

2. The use according to claim 1, It is characterized in that There are several micro groove structures.

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