A chronic intermittent hypoxia muscle injury model and preparation and application thereof

Abstract

The present application relates to the technical field of muscle chip, in particular to a chronic intermittent hypoxia muscle injury model and its preparation and application. The present application first sets a channel layer on the surface of a polytetrafluoroethylene plate to obtain a male mold; then polydimethylsiloxane is poured on the male mold, and a female mold is obtained after post-processing; a silica gel frame matched with the female mold is prepared by using silica gel material; then the female mold and the silica gel frame are superimposed, and a muscle chip is obtained after post-processing; finally, myoblasts, cell culture solution and a substitute for extracellular matrix are mixed to obtain a suspension, and then the suspension is placed in the pretreated muscle chip to induce differentiation, and a chronic intermittent hypoxia muscle injury model is obtained through a precise oxygen control system. The present application provides a new research platform by using microfabrication technology, simulates the core tissue structure of muscle, realizes the key physiological functions of muscle, and can be more effectively applied in the research of obstructive sleep apnea hypopnea syndrome.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of muscle chip, in particular to a chronic intermittent hypoxia muscle injury model and preparation and application thereof. BACKGROUND

[0002] The genioglossus muscle is the most important dilator muscle of the upper airway, also known as the "upper airway safety muscle", and its dysfunction plays an important role in the pathogenesis of obstructive sleep apnea hypopnea syndrome (OSAHS). OSAHS leads to a chronic intermittent hypoxia (CIH) state of the body, which further aggravates the injury of the genioglossus muscle, forming a vicious cycle. If the injury of the genioglossus muscle can be completely or partially repaired, and its functional state is restored, it has important clinical significance for maintaining airway patency and improving the efficacy of OSAHS. However, due to the difficulty in obtaining clinical tissue samples and other problems, how to evaluate the influence of different degrees of CIH on the function and morphology of the human genioglossus muscle has become a key problem that needs to be solved in the field of basic research on OSAHS.

[0003] At present, the research models for muscle injury of OSAHS mainly include traditional cell 2D culture models, which have the disadvantages that cells grow adherently, cell polarity disappears, the 3D structure characteristics of in-vivo tissues are lost, the interaction between cells and the interaction between cells and matrix in a complex microenvironment cannot be reflected, and the biological characteristics reflected have certain differences from the real physiological and pathological conditions in vivo, and more importantly, the contraction function of muscle cannot be detected; the CIH mouse model is the most commonly used animal model in the field of basic research on OSAHS, but it has the disadvantages of high cost, long time consumption, and great species difference, which are irresistible. Therefore, constructing a muscle model that can effectively reflect the structure and function characteristics of in-vivo tissues can provide an effective technical platform for the research of muscle injury of OSAHS, thereby accelerating the rapid development of this field.

[0004] Organ-on-a-chip is one of the most advanced technologies for simulating human organs at present, and it is an important integration of four high-tech fields of medicine, microfluidics, tissue engineering and cell reprogramming / transdifferentiation. Its core is to construct the core tissue structure of the organ in vitro, and realize the key physiological functions of the organ. It has epoch-making significance in promoting the development of in-vitro disease models, drug screening and evaluation fields. Organ-on-a-chip is also increasingly recognized by the academic community, industry and national management levels. However, the construction of the muscle chip platform is relatively complex, and it mainly depends on special instruments and equipment, which is difficult to apply and popularize in general experimental platforms. SUMMARY

[0005] In order to solve the above problems, the application provides a chronic intermittent hypoxia muscle injury model and a preparation and application thereof. First, a channel layer is arranged on the surface of a polytetrafluoroethylene plate to obtain a male mold; then, polydimethylsiloxane is injected into the male mold, and post-processing is performed to obtain a female mold; a silica gel frame matched with the female mold is prepared by using silica gel material; then, the female mold and the silica gel frame are superimposed, and post-processing is performed to obtain a muscle chip; finally, myoblasts, cell culture solution and a substitute of extracellular matrix are uniformly mixed to obtain a suspension, and then the suspension is placed in the pretreated muscle chip to induce differentiation, and the chronic intermittent hypoxia muscle injury model is obtained through precise oxygen control system culture. The application provides a new research platform by using microfabrication technology, simulates the core tissue structure of muscle, realizes the key physiological function of muscle, and can be more effectively applied in the study of obstructive sleep apnea hypopnea syndrome.

[0006] The application obtains a male mold by preparing two parallel dumbbell-shaped channels on a polytetrafluoroethylene material through microfabrication technology; after the male mold is injected with polydimethylsiloxane (PDMS) and the film is removed, a patterned substrate to which cells can be addressed can be formed on the PDMS, so that the added myoblasts, cell culture solution and substitute of extracellular matrix (ECM) can form a shape similar to muscle after the suspension obtained by uniform mixing is solidified; then, silica gel frames are placed at both ends of the 3D cells as attachment points at both ends of the cells, and a specific directional uniaxial mechanical tension is provided for cell growth; finally, the myoblasts are induced to differentiate into muscle bundles by adding a culture medium containing 2% horse serum.

[0007] The muscle chip in the application is directly placed at the bottom of a small dish without sealing. After the muscle chip in the application is disinfected by ultraviolet irradiation, 0.2%-2% F-127 is first used to pretreat the bottom surface of the PDMS for 1 hour to prevent cell adhesion. After that, the myoblasts, cell culture solution and substitute of ECM are mixed at a certain ratio and inoculated in the groove; and the silica gel frames are connected outside the groove as attachment points at both ends of the cells to provide a specific directional uniaxial mechanical tension for cell growth. After the 3D cells are conventionally cultured for 3 days, the culture medium containing 2% horse serum is used to induce the myoblasts to differentiate into muscle bundles.

[0008] The purpose of the application can be achieved by the following technical solutions:

[0009] The first purpose of the application is to provide a preparation method of a chronic intermittent hypoxia muscle injury model, which comprises the following steps:

[0010] (1) Preparation of a male mold: a channel layer is arranged on the surface of a polytetrafluoroethylene plate to obtain a male mold;

[0011] (2) Preparation of a female mold: polydimethylsiloxane is injected into the male mold prepared in step (1), and post-processing is performed to obtain a female mold;

[0012] (3) Preparation of the frame: a silica gel frame matching the negative mold obtained in step (2) is prepared by using a silica gel material;

[0013] (4) Preparation of the muscle chip: the negative mold prepared in step (2) is superimposed with the silica gel frame prepared in step (3), and a muscle chip is obtained after post-processing;

[0014] (5) Preparation of the chronic intermittent hypoxia muscle injury model: the myoblasts, cell culture solution and substitute of extracellular matrix are mixed to obtain a suspension, and then the suspension is placed in the pretreated muscle chip to induce differentiation, and the chronic intermittent hypoxia muscle injury model is obtained by precise oxygen control system culture.

[0015] In an embodiment of the present application, in step (1), the channel layer is two parallel dumbbell-shaped channels;

[0016] The dumbbell-shaped channel comprises a connecting channel and end channels on both sides of the connecting channel;

[0017] The height of the channel is 1-2 mm.

[0018] In an embodiment of the present application, the specific preparation process of the positive mold is as follows: two parallel dumbbell-shaped structures are designed by 3D CAD software Solidworks; according to the parameters designed by the software, two parallel dumbbell-shaped protruding channels are prepared on the surface of a polytetrafluoroethylene plate by microfabrication technology, forming a positive mold.

[0019] In an embodiment of the present application, in step (2), the post-processing is to dry after standing overnight, and then cool, peel off the film to obtain a negative mold;

[0020] The negative mold is a polydimethylsiloxane plate with two parallel dumbbell-shaped grooves;

[0021] The dumbbell-shaped groove comprises a connecting groove and end grooves on both sides of the connecting groove; the dumbbell-shaped groove matches the dumbbell-shaped channel;

[0022] The length and width of the polydimethylsiloxane plate are the same as the length and width of the polytetrafluoroethylene plate, respectively;

[0023] The height of the two parallel dumbbell-shaped grooves is 1-2 mm.

[0024] In an embodiment of the present application, the specific preparation process of the negative mold is as follows: polydimethylsiloxane (PDMS) is used as the negative mold material, the PDMS is poured into the positive mold, and after standing overnight at low temperature, it is taken out and placed in an oven for baking, and finally placed at room temperature for cooling and then peeled off. At this time, a negative mold with two parallel dumbbell-shaped groove structures is formed on the PDMS plate.

[0025] In one embodiment of the present application, in step (3), the silica gel frame is a "hui" type frame provided with square holes;

[0026] The length of the square hole is the same as the length of the connecting groove;

[0027] The distance between the square hole and the length direction of the outer side of the silica gel frame is the same as the width of the end channel;

[0028] The width of the square hole is 0.5-1.5mm larger than the farthest distance of the two dumbbell-shaped grooves in the width direction;

[0029] The height of the silica gel frame is 0.4-0.5mm.

[0030] In one embodiment of the present application, the specific preparation process of the silica gel frame is as follows: the square appearance is designed by 3D CAD software Solidworks; according to the parameters designed by the software, the silica gel frame is prepared by micro-machining technology with silica gel material.

[0031] In one embodiment of the present application, in step (4), the superposition mode is specifically that the silica gel frame is placed on one side of the female mold with dumbbell-shaped grooves, and fixed by a sterile needle;

[0032] The post-treatment is disinfection treatment.

[0033] In one embodiment of the present application, the specific preparation process of the muscle chip is as follows: the female mold and the frame are well docked, and are connected by a sterile fine needle and placed in a cell culture dish or a 6-well plate, and after sterilization under ultraviolet lamp, it is ready for use.

[0034] In one embodiment of the present application, in step (5), the pretreatment is to immerse the muscle chip in 0.2%-2% F-127 to prevent cell adhesion, and after standing, it is dried;

[0035] The cell culture solution is a culture medium containing 10% FBS;

[0036] The extracellular matrix substitute includes Matrigel glue, fibrinogen, thrombin and aprotinin;

[0037] The dosage ratio of myoblasts, cell culture solution, Matrigel glue, fibrinogen, thrombin and aprotinin is 7.5x10 5 6-18μL:10μL:10μg:0.05U:0.08μg;

[0038] During the induction and differentiation process, the induction and differentiation time is 7-14 days.

[0039] In one embodiment of the present application, the pretreatment process is as follows: to form 3D muscle bundles, the muscle chip is immersed in 0.2%-2% F-127 to prevent cell adhesion, and left at room temperature for 1 hour, after which the F-127 in the muscle chip is completely absorbed without washing. F-127 is completely absorbed without washing.

[0040] In one embodiment of the present application, in step (5), the low-oxygen culture and normal-oxygen culture in the precise oxygen control system are a cycle, and then the cycle culture is performed.

[0041] In the low-oxygen culture process, the O2 content in the culture environment is maintained at 1-10%, and the CO2 content is maintained at 5%.

[0042] In the normal-oxygen culture process, the O2 content in the culture environment is maintained at 21%, and the CO2 content is maintained at 5%.

[0043] In one embodiment of the present application, in each cycle, the low-oxygen culture time is maintained at 30-40 minutes, and the normal-oxygen culture time is maintained at 20-30 minutes.

[0044] The entire cycle process lasts for 3-96 hours.

[0045] The second object of the present application is to provide a chronic intermittent hypoxia muscle injury model prepared by the above method.

[0046] The third object of the present application is to provide an application of the above chronic intermittent muscle injury model in the study of obstructive sleep apnea hypopnea syndrome.

[0047] In one embodiment of the present application, first, when the myoblast cell density reaches more than 80%, 0.25% trypsin is used for digestion, counting is performed, and finally the number of cells inoculated in each groove is ensured to be 7.5×10 5 Preparation of ECM substitutes: Matrigel, fibrinogen, thrombin and aprotinin. The suspension is prepared according to the following ratio: 17.2 μL of cell suspension (the number of cells is 7.5×10 5 ​The 3D cell culture suspension is prepared as follows: 50 μL of the cell suspension is mixed with 17.2 μL of cell culture solution, 10 μL of Matrigel glue, 10 μL of fibrinogen (20 mg / mL), 1 μL of thrombin (50 U / mL), and 1 μL of aprotinin (80 μg / mL). After the preparation of the suspension, the suspension is inoculated on the bottom surface of the pre-processed PDMS and placed in a cell culture box at 37°C and 5% CO2 for 1 hour. After the Matrigel solidifies, DMEM cell culture solution is added for 3D cell culture. The DMEM cell culture solution contains 10% FBS and 1% double-antibiotic agents.

[0048] In one embodiment of the present application, the differentiation of myoblasts to form muscle bundles is induced as follows: after the 3D cell culture is stable for 3 days, the culture medium is replaced with a culture medium containing 2% horse serum for induction of differentiation. The culture medium for induction of differentiation is replaced every other day. The culture medium containing 2% horse serum contains 97% DMEM and 1% double-antibiotic agents.

[0049] In one embodiment of the present application, the morphology of muscle bundles is detected as follows: in order to prove the differentiation and maturation of myoblasts, the 3D cells are subjected to immunofluorescence staining of sarcomeric α-actinin (SAA) and myosin heavy chain (MHC) on day 0, 3, 5, 7, 10, and 14 of induction. The morphology of muscle bundles and the expression of maturation markers are observed, and the maturation of muscle bundles is evaluated by quantitative analysis.

[0050] In one embodiment of the present application, the contraction function of muscle bundles is detected as follows: the two ends of the 3D muscle bundle induced and matured as described above are placed in a platinum electrode device. The 3D muscle bundle is stimulated to contract by an electrical stimulation device. The contraction function of muscle bundles is evaluated by quantifying the displacement of the 3D muscle bundle.

[0051] The CIH muscle injury pathological model based on the physiological model of the muscle chip of the present application can be used to evaluate the damage to muscle caused by different degrees of CIH conditions by placing the muscle chip obtained as described above in a CIH environment.

[0052] Compared with the prior art, the present application has the following beneficial effects:

[0053] (1) The application provides a chronic intermittent hypoxia muscle injury model preparation method which is simple to operate and good in repeatability, and the innovation is embodied in that: from cells, ECM to mechanical force, the muscle microenvironment is more bionic; the muscle bundle morphology is more similar to the muscle tissue in vivo; the contraction function of the muscle bundle can be detected. Compared with the traditional CIH mouse model, the chronic intermittent hypoxia muscle injury model provided by the application can simulate human muscle, and has the advantages of low price and short experimental period.

[0054] (2) The application applies the chronic intermittent hypoxia muscle injury model to the research of OSAHS, can accurately evaluate the damage of muscle in morphology and function under different degrees of CIH, and further reveals the correlation between the severity of OSAHS and muscle damage. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 It is a physical map of the male die in the muscle chip of the application;

[0056] Figure 2 It is a physical map of the silica gel frame in the muscle chip of the application;

[0057] Figure 3 It is a flow chart of the preparation of the chronic intermittent hypoxia muscle injury model of the application;

[0058] Figure 4 It is a physical map of the formation of 3D muscle bundle in the muscle chip of the application;

[0059] Figure 5 It is the situation of the formation of muscle bundle after the myoblasts are inoculated in the muscle chip of the application and induced to differentiate for different days (the immunofluorescence staining maps of SAA of the muscle after the induction and differentiation for 0, 1, 3, 5, 7, 10 and 14 days are sequentially shown in the figure);

[0060] Figure 6 It is a quantitative analysis map of the muscle fiber diameter and differentiation index of the 3D muscle bundle after the induction and differentiation of the 3D muscle bundle in the muscle chip for different times in the application; wherein *P<0.05; **P<0.01; ***P<0.001; ns: no significance;

[0061] Figure 7 It is a contraction displacement map of the 3D muscle bundle in the muscle chip in the application;

[0062] Figure 8 It is a pathological model of CIH injury muscle constructed by placing the 3D muscle bundle formed in the muscle chip in the CIH environment in the application;

[0063] Figure 9 It is a quantitative analysis statistical map of the muscle fiber diameter and length in the muscle chip under the CIH condition; wherein *P<0.05; **P<0.01; ***P<0.001;

[0064] Reference numerals in the figure: 1, male mold; 11, end channel; 12, connecting channel; 13, polytetrafluoroethylene plate; 2, female mold; 21, end groove; 22, connecting groove; 23, polydimethylsiloxane plate; 3, silica gel frame; 31, square hole; 4, suspension; 5, muscle bundle. DETAILED DESCRIPTION

[0065] The application provides a preparation method of a chronic intermittent hypoxia muscle injury model, comprising the following steps:

[0066] (1) Preparation of a male mold: a channel layer is arranged on a polytetrafluoroethylene plate to obtain the male mold;

[0067] (2) Preparation of a female mold: polydimethylsiloxane is injected into the male mold prepared in step (1), and post-processing is performed to obtain the female mold;

[0068] (3) Preparation of a frame: a silica gel frame matched with the female mold obtained in step (2) is prepared from silica gel material;

[0069] (4) Preparation of a muscle chip: the female mold prepared in step (2) and the silica gel frame prepared in step (3) are superimposed, and post-processing is performed to obtain the muscle chip;

[0070] (5) Preparation of a chronic intermittent hypoxia muscle injury model: myoblasts, cell culture solution and a substitute for extracellular matrix are uniformly mixed to obtain a suspension, and then the suspension is placed in the pretreated muscle chip to induce differentiation, and the chronic intermittent hypoxia muscle injury model is obtained through a precision oxygen control system.

[0071] In an embodiment of the application, in step (1), the channel layer is two parallel dumbbell-shaped channels.

[0072] The dumbbell-shaped channel comprises a connecting channel and end channels on both sides of the connecting channel.

[0073] The height of the channel is 1-2 mm.

[0074] In an embodiment of the application, the specific preparation process of the male mold is as follows: two parallel dumbbell-shaped structures are designed through 3D CAD software Solidworks; according to the parameters designed by the software, two parallel dumbbell-shaped protruding channels are prepared on the surface of the polytetrafluoroethylene plate through microprocessing technology to form the male mold.

[0075] In an embodiment of the application, in step (2), the post-processing is drying after standing overnight, and then cooling and removing the film to obtain the female mold.

[0076] The female mold is a polydimethylsiloxane plate with two parallel dumbbell-shaped grooves.

[0077] The dumbbell-shaped groove comprises a connecting groove and end grooves on both sides of the connecting groove; the dumbbell-shaped groove matches with a dumbbell-shaped channel;

[0078] The length and width of the polydimethylsiloxane plate are the same as the length and width of the polytetrafluoroethylene plate.

[0079] The height of the two parallel dumbbell-shaped grooves is 1-2 mm.

[0080] In an embodiment of the present application, the specific preparation process of the negative mold is as follows: using polydimethylsiloxane (PDMS) as the negative mold material, pouring the PDMS on the positive mold, placing it in a low temperature environment overnight, taking it out and placing it in an oven for baking, and finally placing it in a room temperature environment for cooling and then removing the film, at this time, the negative mold with the groove structure of two parallel dumbbell-shaped structures is formed on the PDMS plate.

[0081] In an embodiment of the present application, in step (3), the silica gel frame is a "h" type frame provided with a square hole.

[0082] The length of the square hole is the same as the length of the connecting groove.

[0083] The distance between the square hole and the outside of the silica gel frame in the length direction is the same as the width of the end channel.

[0084] The width of the square hole is 0.5-1.5 mm larger than the farthest distance of the two dumbbell-shaped grooves in the width direction.

[0085] The height of the silica gel frame is 0.4-0.5 mm.

[0086] In an embodiment of the present application, the specific preparation process of the silica gel frame is as follows: designing a square appearance through 3D CAD software Solidworks; and preparing the silica gel frame through micro-machining technology according to the parameters designed by the software.

[0087] In an embodiment of the present application, in step (4), the superposition mode is specifically that the silica gel frame is placed on one side of the negative mold with the dumbbell-shaped groove, and fixed through a sterile needle.

[0088] The post-processing is disinfection treatment.

[0089] In an embodiment of the present application, the specific preparation process of the muscle chip is as follows: the negative mold and the frame are well docked, and the two are connected through a sterile fine needle and placed in a cell culture dish or a 6-well plate, and then sterilized under a UV lamp.

[0090] In one embodiment of the present application, in step (5), the pretreatment is to immerse the muscle chip into 0.2%-2% Pluronic F-127 to prevent cell adhesion, and then to absorb the Pluronic F-127 in the muscle chip after standing for 1 hour at room temperature. F-127 to prevent cell adhesion, and then to absorb the Pluronic F-127 in the muscle chip after standing for 1 hour at room temperature.

[0091] The cell culture solution is a culture medium containing 10% FBS.

[0092] The extracellular matrix substitute includes Matrigel, fibrinogen, thrombin and aprotinin.

[0093] The use amount ratio of myoblasts, cell culture solution, Matrigel, fibrinogen, thrombin and aprotinin is 7.5×10 5 : 16-18 μL: 10 μL: 10 μg: 0.05 U: 0.08 μg.

[0094] During the induction differentiation process, the induction differentiation time is 7-14 days.

[0095] In one embodiment of the present application, the pretreatment process is as follows: in order to form a 3D muscle bundle, the muscle chip is immersed into 0.2%-2% Pluronic F-127 to prevent cell adhesion, and then to absorb the Pluronic F-127 in the muscle chip after standing for 1 hour at room temperature. F-127 to prevent cell adhesion, and then to absorb the Pluronic F-127 in the muscle chip after standing for 1 hour at room temperature. F-127 to prevent cell adhesion, and then to absorb the Pluronic F-127 in the muscle chip after standing for 1 hour at room temperature.

[0096] In one embodiment of the present application, in step (5), in the precise oxygen control system, the low-oxygen culture and the normal-oxygen culture are a cycle, and then the cycle culture is performed.

[0097] During the low-oxygen culture, the O2 content in the culture environment is maintained at 1-10%, and the CO2 content is maintained at 5%.

[0098] During the normal-oxygen culture, the O2 content in the culture environment is maintained at 21%, and the CO2 content is maintained at 5%.

[0099] In one embodiment of the present application, in each cycle, the low-oxygen culture time is maintained at 30-40 minutes, and the normal-oxygen culture time is maintained at 20-30 minutes.

[0100] The whole cycle process lasts for 3-96 h.

[0101] The present application provides a chronic intermittent hypoxia muscle injury model prepared by the above method.

[0102] The present application provides an application of the above chronic intermittent muscle injury model in the study of obstructive sleep apnea hypopnea syndrome.

[0103] In one embodiment of the present application, when the myoblasts reach a density of more than 80%, they are digested with 0.25% trypsin, counted, and finally, the number of cells inoculated in each groove is ensured to be 7.5 x 10 5 The second step is to prepare the ECM substitutes: Matrigel, fibrinogen, thrombin and aprotinin. The suspension is prepared according to the following proportions: 17.2 μL of cell suspension (7.5 x 10 5 The suspension is prepared immediately before being inoculated on the pre-treated PDMS and placed in a cell culture incubator at 37°C and 5% CO2 for 1 hour. After the matrix is solidified, DMEM cell culture medium is added for 3D cell culture. The DMEM cell culture medium contains 10% FBS and 1% double-antibiotic by volume. The next day, the 3D cells are observed to be detached from the surrounding and adhered to the frame at both ends.

[0104] In one embodiment of the present application, the differentiation of myoblasts to form muscle bundles is induced as follows: after 3 days of stable 3D cell culture, the medium is replaced with a medium containing 2% horse serum for induction of differentiation. The medium for induction of differentiation is replaced every other day. The medium containing 2% horse serum contains 97% DMEM and 1% double-antibiotic by volume.

[0105] In one embodiment of the present application, the morphology of muscle bundles is detected as follows: to prove the differentiation and maturation of myoblasts, the 3D cells are subjected to immunofluorescence staining of sarcomeric α-actinin (SAA) and myosin heavy chain (MHC) at 0, 3, 5, 7, 10 and 14 days of induction. The morphology of muscle bundles and the expression of maturation markers are observed, and the maturation of muscle bundles is evaluated by quantitative analysis.

[0106] In one embodiment of the present application, the contraction function of muscle bundles is detected as follows: the two ends of the 3D muscle bundles induced and matured as described above are placed in a platinum electrode device, and the 3D muscle bundles are stimulated to contract by an electrical stimulation device. The contraction displacement of 3D muscle bundles is quantified to evaluate the contraction function of muscle.

[0107] In one embodiment of the present application, a CIH muscle injury pathological model is constructed based on a muscle chip physiological model. The muscle chip obtained as described above is placed in a CIH environment to evaluate the damage to muscle under different degrees of CIH conditions.

[0108] The application will be described in detail below with reference to the drawings and specific embodiments.

[0109] In the following examples, if no special instructions, the reagents used are commercially available reagents; the detection means and methods used are conventional detection means and methods in the art.

[0110] Example 1: Preparation of muscle chip model.

[0111] As shown in Figures 1-3 , first, two parallel dumbbell-shaped structures were designed by 3D CAD software Solidworks; according to the parameters designed by the software, the dumbbell-shaped channels (including the connecting channels 12 and the end channels 11 on both sides of the connecting channels 12) were prepared on the surface of the polytetrafluoroethylene plate 13 by microfabrication technology to form the male mold 1; the length, width and depth of the two dumbbell-shaped channels are the same. Among them, the length of the connecting channel 12 is 7 mm, the width is 2 mm, and the depth is 1.5 mm; the length of the end channel 11 perpendicular to the connecting channel 12 is 4 mm, the length parallel to the connecting channel 12 is 2 mm, and the depth is 1.5 mm. The distance between adjacent dumbbell-shaped channels is 4 mm.

[0112] PDMS was used as the material of the female mold 2, and the PDMS was poured into the chip male mold 1 and placed in a 4℃ refrigerator overnight to exclude air bubbles in the PDMS. The next day, it was taken out and placed in an oven at 80℃ for 1 hour, and the PDMS was solidified. After cooling at room temperature, the film was removed, and at this time a polydimethylsiloxane plate 23 (PDMS plate) with two parallel dumbbell-shaped groove structures (including connecting grooves 22 and end grooves 21 on both sides of the connecting grooves 22) was formed, i.e. the female mold 2.

[0113] A square appearance was designed by 3D CAD software Solidworks; according to the parameters designed by the software, a silica gel frame 3 (a "hui" type frame with a square hole 31) was prepared by microfabrication technology using silica gel material. The silica gel frame parameters are: the outer edge width is 12 mm, the length is 11 mm; the inner edge 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 groove.

[0114] The silica gel frame 3 was placed on one side of the female mold 2 with a dumbbell-shaped groove, and connected by a sterile No. 3 needle, placed in a cell culture dish or 6-well plate without sealing, and sterilized under ultraviolet light for at least 1 hour, then sterilized, to obtain a muscle chip.

[0115] Example 2: Inducing C2C12 myoblast cells to differentiate into muscle fibers based on the muscle chip platform.

[0116] The specific operation flow chart of the muscle chip is as shown in Figure 3As shown. After the complete muscle chip is prepared, in order to form 3D muscle bundles within the muscle chip, the muscle chip needs to be immersed in 0.2% water before seeding C2C12 cells onto the chip. F-127, to prevent cell adhesion. 0.2% The F-127 was left to stand at room temperature for 1 hour to completely dry out; no washing was required.

[0117] Preparation of suspension 4: First, when the myoblast density reaches over 80%, trypsin digestion is performed, and the cells are counted. Finally, ensure that the number of cells seeded in each groove is 7.5 × 10⁻⁶. 5 Next, alternatives to ECM were prepared: Matrigel, fibrinogen, thrombin, and aprotinin. Finally, each groove was seeded with 4 units of suspension, with the following concentrations: 17.2 μL cell suspension (cell count 7.5 × 10⁻⁶). 5 The mixture consisted of 17.2 μL of cell culture medium, 10 μL of Matrigel gel, 10 μL of fibrinogen (20 mg / mL), 1 μL of thrombin (50 U / mL), and 1 μL of aprotinin (80 μg / mL). After preparing the suspension of cells and ECM substitute, it was immediately seeded onto pre-treated PDMS substrates and placed in a 37°C, 5% CO2 cell culture incubator for 1 hour. After the substrate gel solidified, DMEM cell culture medium containing 10% FBS and 1% penicillin antibodies was added for 3D cell culture. The next day, 3D cells were observed to detach from their surroundings and adhere to the frame at both ends.

[0118] Inducing myoblast differentiation into muscle bundles 5: After 3 days of stable culture, the culture medium was replaced with one containing 2% horse serum for further differentiation induction. The induction medium was changed every other day. The 2% horse serum culture medium contained 97% DMEM and 1% penicillin-dextrose antibody. Figure 4 This is an image of the muscle bundles induced to form over 14 days in a muscle chip.

[0119] Example 3: Detection of the morphology of 3D muscle bundles formed in a muscle chip.

[0120] To demonstrate the induction of myoblast differentiation and maturation, C2C12 cells formed in the muscle chip can be subjected to immunofluorescence staining of SAA on muscle bundles at 0, 3, 5, 7, 10, and 14 days of induction. First, place the muscle bundles induced to differentiate at different time points in 2% PFA at 4°C overnight for fixation; the next day, discard the PFA and rinse with PBS for 3 times, 10 minutes each time; then add goat serum blocking solution containing 0.2% Triton-100 X and block at room temperature for 1 hour; recover the blocking solution without washing, add SAA primary antibody diluent (1:100, Sigma), and incubate at 4°C overnight. The next day, place the sample at room temperature for 1 hour, recover the primary antibody diluent, and rinse with PBS for 3 times, 10 minutes each time. Add goat anti-mouse secondary antibody (1:1000, Abeam) and incubate at 4°C overnight. The next day, recover the secondary antibody, rinse with PBS for 3 times, 10 minutes each time, add DAPI (1:10000, Biyun) and incubate at room temperature for 15 minutes, and rinse with PBS for 3 times, 10 minutes each time. After staining, place under a confocal microscope to take pictures and record the morphology of the muscle bundles, and evaluate the maturation of the muscle bundles by quantitative analysis. From Figures 5-6 It can be seen that compared with 0, 3, 5, 7, and 10 days, the diameter of the muscle fibers is the largest and the cell differentiation index is the highest at 14 days of induction differentiation of C2C12 cells.

[0121] Example 4: Detection of contraction of 3D muscle bundles formed in the muscle chip.

[0122] After C2C12 cells are induced to differentiate in the muscle chip for 14 days, they are placed in an electrical stimulation device with platinum electrodes at both ends, and a certain electrical stimulation is applied. When the field strength is 20V / cm and the pulse width is 10ms, the muscle contraction can be effectively stimulated. From Figure 7 It can be seen that 3D muscle bundles can undergo single contraction when subjected to electrical stimulation at 0.5Hz, and can undergo tetanic contraction when subjected to electrical stimulation at 10Hz.

[0123] Example 5: Construction of CIH injury muscle chip pathological model based on muscle chip.

[0124] After C2C12 cells are induced to differentiate in the muscle chip for 14 days, they are placed in a Biospherix (Oxycycler, C42, USA) precision oxygen control system for pathological modeling. The CIH condition is: 1% O2+5% CO2(40min) and 21% O2+5% CO2(20min).

[0125] To detect the damage of 3D muscle bundle in CIH environment, the 3D muscle bundle placed in CIH environment for 1st day, 2nd day, 3rd day and 4th day were stained by immunofluorescence for MHC and SAA. The method of immunofluorescence staining was as described above, and the concentration of MHC used was 1:10 (DSHB). After staining, the 3D muscle bundle was placed under confocal microscope to take pictures and record the morphology of muscle bundle, and the damage of muscle bundle was quantitatively analyzed.

[0126] By Figure 8 The morphological changes of 3D muscle bundle exposed to CIH environment for different days were shown. As in the 1st day of CIH environment, the muscle fibers began to appear obvious edema and rupture; with the passage of time, the muscle fibers gradually became thin and short, and finally the morphology of muscle fibers almost completely disappeared; Figure 9 Quantitative analysis of the diameter and length of muscle fibers showed that the longer the 3D muscle bundle was exposed to CIH environment, the greater the damage was.

[0127] The above description of the embodiments is to facilitate the ordinary skilled in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a chronic intermittent hypoxia muscle injury model, characterized by, It includes the following steps: (1) Preparation of the male mold: A channel layer is set on the surface of a polytetrafluoroethylene plate to obtain the male mold; The channel layer is two parallel dumbbell-shaped channels; The dumbbell-shaped channels include connecting channels and end channels on both sides of the connecting channels; The height of the channels is 1 - 2 mm; (2) Preparation of the female mold: Polydimethylsiloxane is poured onto the male mold prepared in step (1), and post-treatment is carried out to obtain the female mold; The post-treatment is to let it stand overnight and then dry, and then cool and remove the film to obtain the female mold; The female mold is a polydimethylsiloxane plate with two parallel dumbbell-shaped grooves; The dumbbell-shaped grooves include connecting grooves and end grooves on both sides of the connecting grooves; the dumbbell-shaped grooves match the dumbbell-shaped channels; The length and width of the polydimethylsiloxane plate are the same as those of the polytetrafluoroethylene plate respectively; The height of the two parallel dumbbell-shaped grooves is 1 - 2 mm; (3) Preparation of the frame: A silicone frame matching the female mold obtained in step (2) is prepared using a silicone material; The silicone frame is a "return" - shaped frame with square holes; the silicone frame serves as the attachment points at both ends of the cells and provides uniaxial mechanical tension in a specific direction for cell growth; The length of the square holes is the same as the length of the connecting grooves; The distance between the square holes and the outer edge of the silicone frame along the length direction is the same as the width of the end channels; The width of the square holes is 0.5 - 1.5 mm larger than the farthest distance between the two dumbbell-shaped grooves along the width direction; The height of the silicone frame is 0.4 - 0.5 mm; (4) Preparation of the muscle chip: The female mold prepared in step (2) and the silicone frame prepared in step (3) are superimposed, and disinfection treatment is carried out to obtain the muscle chip; The specific superimposing method is to place the silicone frame on the side of the female mold with dumbbell-shaped grooves and fix it with a sterile needle; ( ) Preparation of the chronic intermittent hypoxia muscle injury model: Myoblasts, cell culture medium and a substitute for the extracellular matrix are mixed evenly to obtain a suspension, and then the suspension is placed on the pretreated muscle chip and placed in a cell incubator at 37 °C and 5% CO₂ for 1 hour. After the Matrigel gel coagulates, DMEM cell culture medium is added for 3D cell culture. After 3D cell culture is stable for 3 days, the medium containing 2% horse serum is replaced for induced differentiation, and a chronic intermittent hypoxia muscle injury model is obtained through a precision oxygen control system culture; In the precision oxygen control system, hypoxic culture and normoxic culture are taken as one cycle, and then cycle culture is carried out; During the hypoxic culture process, the O₂ content in the culture environment is maintained at 1 - 10%, and the CO₂ content is 5%; During the normoxic culture process, the O₂ content in the culture environment is maintained at 21%, and the CO₂ content is 5%; In each cycle, the hypoxic culture time is maintained at 30 - 40 minutes, and the normoxic culture time is maintained at 20 - 30 minutes; The entire cycle process lasts for 3 - 96 h; The pretreatment is to immerse the muscle chip in 0.2% - 2% Pluronic F - 127 to prevent cell adhesion, and after standing, it is blotted dry; The cell culture solution is a medium with 10% FBS by volume fraction; The extracellular matrix substitute comprises Matrigel glue, fibrinogen, thrombin and aprotinin; The amounts of myoblasts, cell culture fluid, 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 DMEM cell culture medium comprises 10% FBS and 1% double-antibody by volume concentration; the medium comprising 2% horse serum comprises 97% DMEM and 1% double-antibody by volume concentration; During the induction differentiation process, the induction differentiation time is 7-14 days.

2. A chronic intermittent hypoxia muscle injury model prepared by the method of claim 1.

3. Application of the chronic intermittent muscle injury model of claim 2 in the study of obstructive sleep apnea hypopnea syndrome.

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