A vascularized muscle model and preparation and application thereof

Abstract

The present application relates to the technical field of muscle chip, in particular to a vascularized muscle model and its preparation and application. The present application first mixes myoblasts, endothelial cells, cell culture solution and extracellular matrix substitute to obtain a suspension; then the suspension is placed in a pretreated muscle chip to induce differentiation to obtain a vascularized muscle model. The present application realizes the co-culture of muscle and blood vessels in the chip, which is more bionic muscle microenvironment, so that the formed tissue is more similar to in vivo in terms of morphological structure and key physiological function, and can be more effectively applied to the basic research and clinical drug screening of muscle injury related diseases, so that the obtained research results are more accurate and reliable. The vascularized muscle model prepared by the present application can be widely applied to the basic research and drug evaluation system of muscle related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of muscle chip, in particular to a vascularized muscle model and preparation and application thereof. BACKGROUND

[0002] Skeletal muscle is the most abundant muscle type in the human body, accounting for about 40% of body weight, and its main function is to generate contractile force, allowing people to breathe freely and move, etc. Therefore, skeletal muscle is crucial to human health. Under pathological conditions, many diseases are accompanied by muscle dysfunction, such as Duchenne muscular dystrophy, juvenile dermatomyositis, myasthenia gravis, muscular dystrophy, and obstructive sleep apnea hypopnea syndrome, etc. Exploring the pathogenesis and evaluating drug efficacy are the key to treating muscle injury-related diseases. However, at present, the discovery, development and toxicity analysis of new drugs in traditional clinical practice are mostly based on 2D cell culture and small animal models for preclinical trials, but only 11.8% of the drugs entering clinical trials are approved, resulting in an average cost of $2.5 billion for newly approved drugs. One of the reasons for this low efficacy is that animal disease models cannot truly replicate human diseases, and there are differences in drug response and toxicity between animals and humans. Therefore, we need to develop more effective human bionic muscle models to accelerate the rapid development of this field.

[0003] Human organ chip is an important integration of four high-tech fields of medicine, microfluidics, tissue engineering and cell reprogramming / transdifferentiation, and has important application value in promoting the development of in vitro disease models and drug screening and evaluation. The core of organ chip is to construct the core tissue structure of the organ in vitro to realize the key physiological functions of the organ. The core component of skeletal muscle is muscle fiber (muscle cell), and there are multiple capillaries accompanying the muscle fiber, so muscle is a highly vascularized tissue. Physiologically, blood vessels not only provide oxygen for muscle tissue to support muscle contraction, but also interact with adjacent cells to regulate skeletal muscle microenvironment homeostasis. For example, endothelial cells (ECs) can interact with surrounding cells to promote myogenesis and angiogenesis by secreting a series of growth factors, such as insulin-like growth factor 1, hepatocyte growth factor, basic fibroblast growth factor and vascular endothelial growth factor, etc. However, in the process of building muscle chips, researchers have paid more attention to muscle fibers themselves, often ignoring the important role of blood vessels in maintaining muscle contraction and regulating muscle microenvironment homeostasis. SUMMARY

[0004] To solve the above problems, the purpose of the present application is to provide a vascularized muscle model and its preparation and application. The present application first mixes myoblasts, endothelial cells, cell culture solution and extracellular matrix substitute to obtain a suspension; then the suspension is placed in a pretreated muscle chip to induce differentiation to obtain a vascularized muscle model. The present application realizes the co-culture of muscle and blood vessels in the chip, more bionic muscle microenvironment, so that the formed tissue is more similar to in vivo in terms of morphological structure and key physiological functions, which can be more effectively applied to the basic research and clinical drug screening of muscle injury related diseases, and the obtained research results are more accurate and reliable. The vascularized muscle model prepared by the present application can be widely applied to the basic research and drug evaluation system of muscle related diseases.

[0005] The purpose of the present application can be achieved by the following technical solutions:

[0006] The first purpose of the present application is to provide a preparation method of a vascularized muscle model, comprising the following steps:

[0007] (1) mixing myoblasts, endothelial cells, cell culture solution and extracellular matrix substitute to obtain a suspension;

[0008] (2) placing the suspension prepared in step (1) in a pretreated muscle chip to induce differentiation to obtain a vascularized muscle model.

[0009] In an embodiment of the present application, in step (1), the cell culture solution is a culture medium with a volume fraction of 10% FBS and 1% ECGS.

[0010] In an embodiment of the present application, in step (1), the extracellular matrix substitute includes Matrigel glue, fibrinogen, thrombin and aprotinin.

[0011] In an embodiment of the present application, in step (1), the amount ratio of myoblasts, endothelial cells, cell culture solution, Matrigel glue, fibrinogen, thrombin and aprotinin is 7.5x10 5 6-18μL:10μL:10μg:0.05U:0.08μg;

[0012] In an embodiment of the present application, the number ratio of myoblasts to endothelial cells is 3-6:1.

[0013] In an embodiment of the present application, in step (2), the pretreatment is to immerse the muscle chip in 0.2%-2% F-127 to prevent cell adhesion, and then dry after standing.

[0014] In an embodiment of the present application, the The volume fraction of F-127 is 0.2%-2%.

[0015] In one embodiment of the present application, in step (2), the differentiation induction time is 7-14 days.

[0016] A second object of the present application is to provide a vascularized muscle model prepared by the above method.

[0017] A third object of the present application is to provide an application of the vascularized muscle model in the basic research and drug evaluation system of muscle-related diseases.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application provides a simple and repeatable method for preparing a human vascularized muscle chip, which has the advantages of simple chip manufacturing method, no need for complex instruments and equipment and technology integration, and convenience for popularization in general research platform; the chip can be generated by mold injection processing, and the processing cost is low; the chip can be disassembled, cleaned and sterilized for repeated use, which greatly increases the number of reusable organ chips. In addition, the present application realizes the co-culture of muscle and blood vessels in the chip, which is more bionic muscle microenvironment, so that the formed tissue is more similar to in vivo in terms of morphological structure and key physiological functions, and can be more effectively applied to the basic research and clinical drug screening of muscle injury related diseases, so that the obtained research results are more accurate and reliable. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a physical picture of a negative mold needed for preparing a vascularized muscle chip in the present application;

[0021] Figure 2 It is a physical picture of a silica gel frame needed for preparing a vascularized muscle chip in the present application;

[0022] Figure 3 It is a physical picture of placing the silica gel frame on one side of the negative mold with dumbbell-shaped grooves and fixing it with a sterile needle in the present application;

[0023] Figure 4 It is a physical picture of a vascularized 3D muscle bundle formed in the present application;

[0024] Figure 5 It is an immunofluorescence staining picture of muscle tissue formed by the vascularized muscle chip of the present application in cross section and longitudinal section; thick arrows represent muscle, and thin arrows represent blood vessels;

[0025] Figure 6 It is an MHC immunofluorescence staining picture of muscle formed by myoblasts and endothelial cells in the chip at the 7th day of differentiation induction in the present application;

[0026] Figure 7 Quantitative analysis chart of muscle fiber diameter and analysis index of the vascularized muscle tissue formed in the application after 7 days of induced differentiation, and comparison with simple muscle tissue; wherein **P<0.01; ***P<0.001;

[0027] Figure 8 SAA immunofluorescence staining of the vascularized muscle formed after 7 days of induced differentiation in the application, to observe the striation formed in the muscle;

[0028] Figure 9 Quantitative analysis chart of striation frequency in the vascularized muscle tissue formed in the application, and comparison with simple muscle chip; wherein **P<0.01;

[0029] Figure 10 Quantitative analysis chart of contraction displacement of the vascularized muscle formed after 7 days of induced differentiation; and comparison with simple muscle chip; wherein **P<0.01;

[0030] Figure 11 Detection of muscle injury in the vascularized muscle chip under chronic intermittent hypoxia condition;

[0031] Figure 12 Quantitative analysis chart of muscle fiber diameter and length in the vascularized muscle chip under chronic intermittent hypoxia condition, and comparison with simple muscle tissue; wherein *P<0.05; **P<0.01;

[0032] Figure 13 Construction of a strain model of the vascular muscle chip. DETAILED DESCRIPTION

[0033] The application provides a preparation method of a vascularized muscle model, comprising the following steps:

[0034] (1) mixing myoblast cells, endothelial cells, cell culture solution and extracellular matrix substitutes to obtain a suspension;

[0035] (2) placing the suspension prepared in step (1) in a pretreated muscle chip to induce differentiation to obtain a vascularized muscle model.

[0036] In an embodiment of the application, in step (1), the cell culture solution is a culture medium with a volume fraction of 10% FBS and 1% ECGS.

[0037] In an embodiment of the application, in step (1), the extracellular matrix substitutes comprise Matrigel glue, fibrinogen, thrombin and aprotinin.

[0038] In one embodiment of the present application, in step (1), the amount of myoblasts, endothelial cells, cell culture medium, Matrigel, fibrinogen, thrombin and aprotinin is 7.5 x 10 5 16-18 μL: 10 μL: 10 μg: 0.05 U: 0.08 μg;

[0039] In one embodiment of the present application, the ratio of the number of myoblasts to endothelial cells is 3-6: 1.

[0040] In one embodiment of the present application, in step (2), the pretreatment is to immerse the muscle chip into 0.2%-2% F-127 to prevent cell adhesion, and then dry after standing.

[0041] In one embodiment of the present application, the volume fraction of the F-127 is 0.2%-2%.

[0042] In one embodiment of the present application, in step (2), during the induction differentiation process, the induction differentiation time is 7-14 days.

[0043] The present application provides a vascularized muscle model prepared by the above method.

[0044] The present application provides an application of the vascularized muscle model in the basic research of muscle-related diseases and the drug evaluation system.

[0045] The present application will be described in detail below in conjunction with the drawings and specific examples.

[0046] In the following examples, if not specifically stated, the reagents used are commercially available reagents; the detection means and methods used are conventional detection means and methods in the art.

[0047] Example 1: Construction of a vascularized muscle chip model.

[0048] Fabrication of Chip Frame: First, design two parallel dumbbell-shaped structures using 3D CAD software Solidworks; according to the parameters designed by the software, fabricate dumbbell-shaped channels (including connecting channels and end channels on both sides of the connecting channel) on the surface of a polytetrafluoroethylene plate through microfabrication technology to form a male mold; the length, width, and depth of the two dumbbell-shaped channels are the same. Among them, the length of the connecting channel is 7 mm, the width is 2 mm, and the depth is 1.5 mm; the length of the end channel perpendicular to the connecting channel is 4 mm, the length parallel to the connecting channel is 2 mm, and the depth is 1.5 mm. The distance between adjacent dumbbell-shaped channels is 4 mm. Use PDMS as the negative mold material, pour PDMS onto the chip male mold, and place it in a 4°C refrigerator overnight to remove the bubbles in the PDMS. The next day, take it out and bake it in an oven at 80°C for 1 hour. After the PDMS solidifies and cools at room temperature, remove the film. At this time, two parallel dumbbell-shaped groove structures (including connecting grooves and end grooves on both sides of the connecting groove) are formed on the polydimethylsiloxane plate (PDMS plate), which is the negative mold (as Figure 1 shown). Design a square appearance using 3D CAD software Solidworks; according to the parameters designed by the software, fabricate a silica gel frame (a "hui"-shaped frame with square holes) using silica gel material through microfabrication technology, as Figure 2 shown. The parameters of the silica gel frame are: the outer width is 12 mm, the length is 11 mm; the inner width is 10 mm, the length is 7 mm; the above parameter settings can ensure that the silica gel frame can match the dumbbell-shaped grooves. Place the silica gel frame on the side of the negative mold with dumbbell-shaped grooves and connect them through a No. 3 sterile fine needle (as Figure 3 shown), place it in a cell culture dish or well plate, without sealing, and place it under an ultraviolet lamp for at least 1 hour for sterilization. Then immerse the chip in a solution containing 0.2% F-127 for 1 hour to prevent cell adhesion.

[0049] Preparation of Suspension: First, when the density of myoblasts and human umbilical vein endothelial cells (HUVEC) reaches more than 80%, digest them with trypsin and count them. The ratio of C2C12 to HUVEC is 6:1, and ensure that the total number of inoculated cells in each groove is 7.5×10 5 cells. Secondly, prepare an alternative to ECM: Matrigel, fibrinogen, thrombin, and aprotinin. Finally, inoculate the suspension into each groove, and the content of each substance is as follows: 17.2 μL of cell suspension (the number of cells is 7.5×10 5C2C12:HUVEC=6:1)+17.2 μL cell culture medium+10 μL Matrigel+10 μL fibrinogen (20 mg / mL)+1 μL thrombin (50 U / mL)+1 μL aprotinin (80 μg / mL). After the cell and ECM substitute suspension was prepared, it was immediately seeded on the bottom pretreated PDMS and placed in a cell culture incubator at 37 °C, 5% CO2for 1 hour. After the matrix was solidified, DMEM and ECM cell mixed culture medium (volume ratio of two culture media was 1:1) was added for 3D cell culture. The DMEM cell culture medium contained 10% FBS and 1% double-antibiotic by volume concentration, and the ECM culture medium contained 5% FBS, 1% ECGS and 1% double-antibiotic by volume concentration. The next day, 3D cells were seen to be detached from the surrounding and adhered to the frame at both ends.

[0050] Inducing myoblast differentiation to form muscle bundles: after 3D cell culture was stable for 3 days, the medium containing 2% horse serum was used to induce differentiation. The medium for inducing differentiation was changed every other day. The 2% horse serum medium contained 96% DMEM, 1% ECGS and 1% double-antibiotic by volume concentration. Among them Figure 4 The actual picture of the vascularized muscle bundle formed in the chip for 7 days of induction, and compared with simple muscle tissue.

[0051] Example 2: Detection of the morphology of 3D muscle bundles and blood vessels formed in the vascularized muscle chip.

[0052] To observe the maturation of muscle fibers, the morphology of blood vessels and the location relationship between muscle fibers and blood vessels, we performed immunofluorescence staining of MHC and CD31 on muscle fibers and vascular endothelial cells in the vascularized muscle chip. First, the vascularized muscle tissue induced to differentiate for 7 days was placed in 2% PFA at 4°C overnight for fixation. The next day, the PFA was discarded, and the PBS was washed for 3 times, 10 minutes each time. Then the tissue was embedded with OCT, and the vascularized muscle tissue was sliced from the transverse and longitudinal directions using a frozen tissue section machine and placed in a -80°C refrigerator. The next day, the sections were taken out and placed in a 37°C oven for 1 hour, washed in PBS for 3 times, 5 minutes each time, and then the tissue was added with 0.2% Triton-100 X containing goat serum blocking solution for 1 hour at room temperature. The blocking solution was recovered without washing, and MHC primary antibody diluent (1:10, DSHB) was added and incubated at 4°C overnight. The next day, the sample was placed at room temperature for 1 hour, the primary antibody diluent was recovered, and the PBS was washed for 3 times, 10 minutes each time. CD31 primary antibody diluent (1:100, Abeam) was added and incubated at 4°C overnight. Then the sample was placed at room temperature for 1 hour, the primary antibody diluent was recovered, and the PBS was washed for 3 times, 10 minutes each time. Goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody (Abeam) were added with a dilution ratio of 1:1000 and incubated at 4°C overnight. The next day, the secondary antibody was recovered, the PBS was washed for 3 times, 10 minutes each time, DAPI (1:10000, Biyun Tian) was added and incubated at room temperature for 15 minutes, and the PBS was washed for 3 times, 10 minutes each time. After staining, the sample was placed under a confocal microscope to take pictures and record the maturation of muscle fibers, the morphology of blood vessels and the location relationship between them. From the results, it can be seen that the myoblasts formed mature muscle fibers after 7 days of induction and differentiation, the vascular endothelial cells expressed CD31 positively, and were arranged in sequence and attached to the muscle fibers. Even the morphology of a blood vessel-like structure can be seen, where the thick arrow indicates the muscle fiber and the thin arrow indicates the blood vessel. Figure 5 As can be seen, the myoblasts formed mature muscle fibers after 7 days of induction and differentiation, the vascular endothelial cells expressed CD31 positively, and were arranged in sequence and attached to the muscle fibers. Even the morphology of a blood vessel-like structure can be seen, where the thick arrow indicates the muscle fiber and the thin arrow indicates the blood vessel.

[0053] Example 3: Detection of the maturation of muscle formed in the vascularized muscle chip.

[0054] To detect the effect of blood vessels in promoting myoblast differentiation and maturation, the tissues formed in the vascularized muscle chip were subjected to immunofluorescence staining of MHC after 7 days of induction differentiation, and compared with simple muscle chip. First, the muscle bundles at different time points of induction differentiation were placed in 2% PFA at 4°C overnight for fixation; the next day, the PFA was discarded, and the PBS was washed for 3 times, 10 minutes each time; then, goat serum containing 0.2% Triton-100 X blocking solution was added for blocking at room temperature for 1 hour; the blocking solution was recovered without washing, and MHC primary antibody diluent (1:10, DSHB) and SAA primary antibody diluent (1:100, Sigma) were added, and incubated at 4°C overnight. The next day, the sample was incubated at room temperature for 1 hour, the primary antibody diluent was recovered, and the PBS was washed for 3 times, 10 minutes each time. Goat anti-mouse secondary antibody (1:1000, Abeam) was added, and incubated at 4°C overnight. The next day, the secondary antibody was recovered, the PBS was washed for 3 times, 10 minutes each time, DAPI (1:10000, Biyun) was added, and incubated at room temperature for 15 minutes, and the PBS was washed for 3 times, 10 minutes each time. After the staining was completed, the sample was placed under a confocal microscope to take pictures to record the differentiation of myoblasts, and the maturation of muscle bundles was evaluated by quantitative analysis. From Figures 6-9 As can be seen, compared with the simple muscle chip, the vascularized muscle chip was well differentiated at 7 days of induction differentiation, mature muscle fibers were formed, the diameter of the muscle fibers was larger, the cell differentiation index was higher, and the frequency of cross striation was higher, which indicated that blood vessels played an important role in inducing muscle fiber maturation.

[0055] Example 4: Detection of the contraction function of the muscle formed in the vascularized muscle chip.

[0056] After the C2C12 cells and HUVEC cells were induced to differentiate in the chip for 7 days, they were placed in an electric stimulation device with platinum electrodes at both ends, and a certain electric stimulation was applied. When the field strength was 20V / cm and the pulse width was 10ms, the muscle contraction could be effectively stimulated. When the vascularized muscle chip was given an electric stimulation at 1Hz, the vascularized muscle bundle tissue could undergo single contraction, and the displacement of the muscle tissue movement was quantified by Image J software. From Figure 10 As can be seen, compared with the simple muscle chip, the muscle tissue formed in the vascularized muscle chip had a larger contraction displacement, which indicated that the vascularized muscle tissue had a larger contraction force, and the vascular endothelial cells played an important role in maintaining the muscle contraction function.

[0057] Example 5: Application of the vascularized muscle chip model in the study of obstructive sleep apnea hypopnea syndrome.

[0058] After 7 days of differentiation, C2C12 cells and HUVEC cells were placed in a Biospherix (Oxycycler, C42, USA) precision oxygen control system to construct a chronic intermittent hypoxia (CIH) model to model the pathophysiological conditions of obstructive sleep apnea hypopnea syndrome. The CIH conditions were 1% O2+5% CO2(40 min) and 21% O2+5% CO2(20 min).

[0059] To detect the damage of vascularized muscle bundles in the CIH environment, they were placed in the CIH environment for one day, and MHC immunofluorescence staining was performed on the vascularized 3D muscle bundles. The immunofluorescence staining method is as described above, and the concentration of MHC used is 1:10 (DSHB). After staining, the vascularized 3D muscle bundles were placed under a confocal microscope to take pictures and record the morphology of the muscle bundles, and the damage of the muscle bundles was quantitatively analyzed. Figure 11 The changes in the morphology of the vascularized 3D muscle bundles exposed to the CIH environment for one day are shown, and compared with simple 3D muscle bundles. As in the first day of the CIH environment, the muscle fibers of the 3D muscle bundles began to show obvious edema and rupture, while the muscle fibers of the vascularized 3D muscle bundles did not show changes in morphology. Figure 12 The diameter and length of the muscle fibers were quantitatively analyzed, and the results showed that compared with the vascularized 3D muscle bundles, the damage caused by CIH to the simple 3D muscle bundles was greater, which suggests that the application of vascularized muscle chips to obstructive sleep apnea hypopnea syndrome may result in more accurate results and be more similar to in vivo.

[0060] Example 6: Application of the vascularized muscle chip model to the study of muscle strain model.

[0061] The vascularized muscle chip was placed under an electrical stimulation device containing platinum electrodes, and 20Hz electrical stimulation was given to the vascularized muscle every 15 seconds to cause tetanic contraction of the vascularized muscle, thereby constructing a muscle strain model. After 1 hour, SAA immunofluorescence staining was performed. The steps are as described above. Figure 13 The results show that after continuous tetanic contraction, the muscle fibers of the vascularized muscle bundles have reduced cross striations, and the muscle fiber morphology has swelled, and some have even dissolved and disappeared. The left panel shows the SAA staining results of the normal vascularized muscle chip, and the right panel shows the damage of the muscle fibers in the muscle strain model.

[0062] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as a number of alternatives. It is not intended to limit the application to the precise form described.

Claims

1. A method for preparing a vascularized muscle model, characterized by, It comprises the following steps: (1) mixing myoblasts, endothelial cells, cell culture solution and extracellular matrix substitute to obtain a suspension; The cell culture solution is a culture medium containing 10% FBS and 1% ECGS by volume fraction; The extracellular matrix substitute comprises Matrigel, fibrinogen, thrombin and aprotinin; The amounts of myoblasts and endothelial cells, cell culture medium, Matrigel gel, fibrinogen, thrombin and aprotinin were 7.5 x 10 5 6-18 μL: 10 μL: 10 μg: 0.05 U: 0.08 μg; The number ratio of myoblasts to endothelial cells is 3-6:1; (2) placing the suspension prepared in step (1) in a pretreated muscle chip and placing it in a cell culture incubator at 37℃ and 5% CO2 for 1 hour, and after the Matrigel solidifies, adding DMEM cell culture medium and ECM culture medium in a volume ratio of 1:1 for 3D cell culture, and after 3 days of stable 3D cell culture, replacing the culture medium containing 2% horse serum for induction differentiation to obtain a vascularized muscle model; The pretreatment is to immerse the muscle chip in Pluronic F-127 with a volume fraction of 0.2%-2% to prevent cell adhesion, and then dry it after standing; the DMEM cell culture medium contains 10% FBS and 1% double-antibody by volume concentration, and the ECM culture medium contains 5% FBS, 1% ECGS and 1% double-antibody by volume concentration; the culture medium containing 2% horse serum contains 96% DMEM, 1% ECGS and 1% double-antibody by volume concentration; during the induction differentiation process, the induction differentiation time is 7-14 days; The muscle chip is prepared by the following method: Two parallel dumbbell-shaped channels are prepared on the surface of a polytetrafluoroethylene plate, the dumbbell-shaped channels comprise a connecting channel and end channels on both sides of the connecting channel, forming a positive mold; the length, width and depth of the two dumbbell-shaped channels are the same; the length of the connecting channel is 7 mm, the width is 2 mm, and the depth is 1.5 mm; the length of the end channel perpendicular to the connecting channel is 4 mm, the length parallel to the connecting channel is 2 mm, and the depth is 1.5 mm; the distance between adjacent dumbbell-shaped channels is 4 mm; PDMS is poured into the positive mold and placed in a 4℃ refrigerator overnight to exclude air bubbles in the PDMS; the next day, it is taken out and placed in an oven at 80℃ for 1 hour, and the PDMS solidifies; after cooling at room temperature, the film is removed to obtain a negative mold; A silica gel frame is prepared using silica gel material, and the outer edge width of the silica gel frame is 12 mm and the length is 11 mm; the inner edge width is 10 mm and the length is 7 mm; The silica gel frame is placed on one side of the negative mold with a dumbbell-shaped groove, connected by a sterile 3-gauge needle, placed in a cell culture dish or well plate without sealing, and sterilized under a UV lamp for more than 1 hour to obtain a muscle chip.

2. A vascularized muscle model prepared by the method of claim 1.

3. Use of the vascularized muscle model of claim 2 in the basic research and drug evaluation system of muscle-related diseases.

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