Self-assembled ventricle-like structures, systems, and methods based on stress-induced coiled membranes

Abstract

The application discloses a self-assembled ventricle-like structure based on stress-induced curling film, a system and a method, and belongs to the technical field of heart-like researches.The ventricle-like structure disclosed by the application is composed of a life-like unit and a mechanical unit, wherein the life-like unit is composed of a stent layer, a fibronectin layer and a myocardial cell layer and is used for providing driving force of the overall structure; the mechanical unit is composed of a pipeline and an end face flat plug and is used for realizing efficient operation of one-way pumping; the structure can spontaneously generate movement and can exhibit different contraction capacities in different electric stimulation environments; and the pump-out flow rate control of the ventricle-like structure can be simply realized by changing the external electric stimulation environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of heart-like research, and particularly relates to a self-assembled heart-like ventricular structure based on stress-induced coiled membranes, a system and a method. BACKGROUND

[0002] Organoids are a new type of in vitro cell model, which have complex three-dimensional cell structures and can simulate various aspects of organ development, composition and function. Different cell types can be randomly or directionally differentiated and assembled into organoids in a 3D culture system from embryonic stem cells, human induced pluripotent stem cells, adult stem cells or even tumor cells. So far, healthy and cancer organoid models of various organs have been successfully established, such as lung, stomach, intestine, liver, pancreas, kidney, prostate and brain. Organoids show great potential in basic research and clinical applications. First, organoids can be used to simulate and study organ development and related diseases. Second, organoids have a wide application space in drug experiments and disease treatment. Although corresponding organoids have been developed in various organs, the progress in the field of heart-like organoids is obviously lagging behind, partly due to the complexity of early heart development. The heart is the first functional organ to form during embryonic development and is also the key point of in vitro organoid culture.

[0003] In the heart, the left ventricle is a major component of the heart, which is the most powerful ventricle responsible for pumping blood from the heart to the whole body. The formation of the ventricle is a key step in heart development. In current heart-like research, in vitro myocardial models with chambers have been generated through geometric constraints, scaffolds, molds and other tissue engineering techniques. Valves, diaphragms or asymmetric structures are designed to utilize muscle contraction to drive unidirectional fluid movement. However, current research lacks a simple assembly method for the multi-cellular heart model obtained by stem cell differentiation. In addition, there are few studies on the heart's blood circulation delivery capacity, and the performance of existing studies is far from the ability of the real natural heart. Therefore, it is of great significance to design and manufacture a heart-like organ that is closer to the delivery capacity of the real heart and to realize dynamic culture of tissues based on this, laying a foundation for the development of new organoids and culture. SUMMARY

[0004] The present application aims to provide a self-assembled heart-like ventricular structure based on stress-induced coiled membranes, a system and a method, which solves the technical problem that the existing heart-like organ is far from the ability of the real natural heart.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application discloses a self-assembled heart-like ventricular structure based on stress-induced coiled membranes, comprising:

[0007] The life-like unit and the mechanical unit are connected; the life-like unit comprises a scaffold layer, a fibronectin layer and a myocardial cell layer; the fibronectin layer is arranged on the surface of the scaffold layer, and the myocardial cell layer is directionally grown on the fibronectin layer; the life-like unit is curled to form a three-dimensional conical ventricular structure;

[0008] The mechanical unit comprises a pipe and an end face flat plug;

[0009] One end of the pipe is connected to the small end of the three-dimensional conical ventricular structure, and the end face flat plug is connected to the large end of the three-dimensional conical ventricular structure.

[0010] Further, the scaffold layer comprises a top layer, an intermediate layer and a bottom layer arranged in sequence;

[0011] The top layer is composed of a patterned area; the patterned area and the upper surface of the intermediate layer are provided with microgrooves.

[0012] Further, the fibronectin layer is in the form of a film covering the upper surface of the top layer;

[0013] The myocardial cell layer is obtained by directionally growing myocardial cells on the surface of the fibronectin layer along the direction of the microgrooves on the upper surface of the top layer;

[0014] The protrusion height of the microgrooves is 5-10 μm, the protrusion width is 4-6 μm, and the interval between the protrusions is 20-50 μm.

[0015] Further, the material of the bottom layer is polydimethylsiloxane, and the material of the fibronectin layer is fibronectin;

[0016] The myocardial cells are primary neonatal rat myocardial cells;

[0017] The materials of the scaffold layer and the end face flat plug are both PDMS materials; the diameter of the end face flat plug is 10-15 mm, the thickness is 1-1.5 mm, and the diameter of the central coaxial hole is 2 mm.

[0018] Further, the material of the pipe is PEEK material; the inner diameter of the pipe is 1-1.9 mm;

[0019] The diameter of the end face flat plug and the diameter of the central coaxial hole change isometrically with the change of the size of the scaffold layer.

[0020] Further, the elongation rates of the top layer, the intermediate layer and the bottom layer are 8%-10%, 0% and 12%-15%, respectively.

[0021] The application further discloses a self-assembled ventricle structure driving system based on stress-induced coiled membranes, which comprises a stimulator, an electrode and a solution environment.

[0022] The self-assembled ventricle structure based on stress-induced coiled membranes is arranged in the solution environment.

[0023] Further, the solution environment is a culture solution; the electrode is made of any one of graphite, gold, platinum and silver; the electric field driving voltage of the stimulator is 4-10 V, and the driving frequency is 0.5-2 Hz.

[0024] The application further discloses a preparation method of the self-assembled ventricle structure based on stress-induced coiled membranes.

[0025] The support layer is prepared by a spin coating and curing method, and then fibronectin is coated on the support layer to form a fibronectin layer.

[0026] Myocardial cells are planted on the fibronectin layer to obtain a myocardial cell layer, and then the self-assembled ventricle structure based on stress-induced coiled membranes is obtained by connecting the myocardial cell layer with a pipeline and a flat end plug respectively.

[0027] Further, the support layer comprises a top layer, a middle layer and a bottom layer, and the materials of the three layers are all PDMS materials; the top layer is composed of a patterned area.

[0028] The preparation method of the support layer is as follows:

[0029] PDMS is spin coated on a silicon mold to prepare the patterned area in the top layer, the spin-coated and cured PDMS is separated after the preparation of the patterned area is completed, and is placed on a stretching clamp and stretched to a certain proportion by a stretching mechanism; then PDMS is spin coated on the middle layer, the stretched patterned area in the top layer is adhered to one side of the middle layer, after solidification and adhesion, the middle layer is turned over, PDMS is spin coated on the other side of the middle layer, and the stretched non-patterned bottom layer is adhered to the other side.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] The application discloses a self-assembled ventricle structure based on stress-induced coiled membranes, which is composed of a life-like unit and a mechanical unit, wherein the life-like unit is composed of a support layer, a fibronectin layer and a myocardial cell layer, and is used to provide driving force of the overall structure; the mechanical unit is composed of a pipeline and a flat end plug, and is used to realize one-way pumping and efficient operation; the structure can spontaneously generate movement, and can exhibit different contraction capacities in different electric stimulation environments; the pumping flow rate control of the ventricle structure can be realized by changing the external electric stimulation environment.

[0032] Further, the support layer releases stress by setting pre-stretching and non-stretching PDMS film after bonding to realize the construction of three-dimensional shape, realizes self-assembly of the ventricular structure; micro-grooves are set on the surface as cell directional topographic clues, and the myocardial cells grow along the directional clues to generate anisotropic driving force, which also helps to improve the contraction performance of the ventricular structure, and the structure is composed of only one layer of myocardial cells, one layer of fibronectin and one layer of PDMS substrate, which is the life movement part of the ventricular structure, and the structure is simple and convenient to manufacture.

[0033] The application further discloses a driving system of the self-assembled ventricular structure based on the stress-induced curling film, which comprises a stimulator, electrodes and a solution environment, an electric stimulation signal is given through the output end of the stimulator, the electric stimulation signal forms an electric field in the solution environment through the paired electrodes, and the electric field provides an electric signal to the myocardial cell layer, so that the contraction behavior of the myocardial cells in the myocardial cell layer is controlled, and the overall contraction of the ventricular structure is further controlled; in the application, the radial contraction and the torsional contraction of the ventricular structure are combined, the contraction of the cell muscle tissue makes the overall volume of the cavity smaller, so that the liquid in the cavity is squeezed out from one port, when the cell relaxes, the liquid flows into the cavity from another port, and the net flow is generated in the next cycle, so that the pumping effect is presented. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a whole structure diagram of the self-assembled ventricular structure based on the stress-induced curling film;

[0035] Figure 2 It is a detail diagram of the self-assembled ventricular structure based on the stress-induced curling film;

[0036] Figure 3 It is a size schematic diagram of the self-assembled ventricular structure based on the stress-induced curling film;

[0037] Figure 4 It is a stretching mechanism schematic diagram of the stress-induced curling film;

[0038] Figure 5 It is a living body preparation flow chart of the self-assembled ventricular structure based on the stress-induced curling film;

[0039] Figure 6 It is a contraction mechanism diagram of the self-assembled ventricular structure based on the stress-induced curling film;

[0040] Figure 7 It is a driving schematic diagram of the self-assembled ventricular structure based on the stress-induced curling film;

[0041] Figure 8Schematic diagram of dynamic culture of self-assembly ventricular structure based on stress-induced crimped membrane and other tissues connected together;

[0042] Wherein: 1-myocardial cell layer; 2-fibronectin layer; 3-scaffold layer; 4-top layer; 5-middle layer; 6-bottom layer; 7-pipe; 8-end face flat plug; 9-tissue to be cultured; 10-stretching mechanism; 11-stimulator; 12-electrode; 13-solution environment. DETAILED DESCRIPTION

[0043] In order to make the personnel in the technical field better understand the present application scheme, the technical scheme in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the scope of protection of the present application.

[0044] It should be noted that the terms "first", "second" and the like in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily describe a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily limit to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0045] The present application discloses a self-assembly ventricular structure based on stress-induced crimped membrane, comprising a life-like unit and a mechanical body unit, wherein the life-like unit comprises a scaffold layer 3, a fibronectin layer 2 and a myocardial cell layer 1; the scaffold layer 3 is composed of three layers, namely a top layer 4, a middle layer 5 and a bottom layer 6, the top layer 4 is composed of a patterned area, the patterned area and the middle layer 5 are provided with microgrooves, the fibronectin layer 2 is in the form of a film covering the upper surface of the scaffold layer 3 containing microgrooves, the myocardial cells in the myocardial cell layer 1 grow directionally above the fibronectin layer 2 along the microgrooves, and the myocardial tissue in the myocardial cell layer contains myofibrils; the mechanical body unit is composed of a pipe 7 and an end face flat plug 8; one end of the pipe 7 is connected with the small end of a three-dimensional conical ventricular structure; the end face flat plug 8 is connected with the large end of the three-dimensional conical ventricular structure, and surgical sutures are used to tighten the connection position to make the connection tight.

[0046] Preferably, the top layer 4, the middle layer 5 and the bottom layer 6 have different stretch rates, the top layer 4, the middle layer 5 and the bottom layer 6 are 10%, 0% and 15% respectively, so as to form different degrees of bending, the top layer 4 and the middle layer 5 are bonded to form a smaller spiral structure of the inner layer, and the bottom layer 6 and the middle layer 5 are bonded to form a larger spiral structure of the outer layer.

[0047] The application further discloses a driving system of the self-assembled ventricle-like structure based on the stress-induced coiled film, which comprises a stimulator 11, electrodes 12 and a solution environment 13; an electric stimulation signal is given through an output end of the stimulator 11, the electric stimulation signal forms an electric field in the solution environment 13 through the paired electrodes 12, and the electric field provides an electric signal for the myocardial cell layer 1, so as to control the contraction behavior of the myocardial cells of the myocardial cell layer 1 and further control the overall contraction of the self-assembled ventricle-like structure based on the stress-induced coiled film.

[0048] Preferably, the solution environment 13 is a culture solution, and the material of the electrodes 12 is any one of graphite, gold, platinum and silver.

[0049] Preferably, the electric field driving voltage of the ventricle-like structure is 4-10V, and the driving frequency is 0.5-2Hz.

[0050] Preferably, the material of the bottom layer 6 is polydimethylsiloxane, and the material of the fibronectin layer 2 is fibronectin.

[0051] Preferably, the material of the myocardial cell layer 1 is primary neonatal rat myocardial cells.

[0052] Preferably, the protrusion height of the micro-groove is 5-10μm, the protrusion width is 4-6μm, and the interval between the protrusions is 20-50μm.

[0053] Preferably, in the 2D combined PDMS support layer 3, the micro-groove direction forms an angle of 45 degrees with the symmetry axis; and the 3D myocardial spiral fiber after release forms an angle of 45 degrees.

[0054] Preferably, the end face flat plug 8 is made of PDMS material, has a diameter of 11mm and a thickness of 1mm, and a coaxial hole in the center has a diameter of 2mm.

[0055] Preferably, the pipeline 7 is made of PEEK material, and the inner diameter of the pipeline is 1.9mm.

[0056] The self-assembled ventricle structure based on stress-induced coiled membrane disclosed in the application is driven by a driving system, and the contraction mode is the combination of radial contraction and torsional contraction of the ventricle structure, the contraction of the cell muscle tissue makes the overall volume of the chamber smaller, so as to squeeze the liquid in the chamber and pump it out from one port, when the cells relax, the liquid flows into the chamber from another port, fills the chamber and is pumped out again in the next cycle, so as to generate a net flow and present a pumping effect.

[0057] The application further discloses a manufacturing method of the self-assembled ventricle structure based on stress-induced coiled membrane.

[0058] PDMS is spin-coated on a silicon mold to prepare the patterned area in the top layer 4, and the spin-coated and solidified PDMS is separated and stretched to a certain proportion on a stretching clamp after the preparation of the patterned area is completed; then, PDMS is spin-coated on the middle layer 5, the patterned area in the top layer 4 after stretching is bonded to one side of the middle layer 5, and after solidification and bonding, the middle layer 5 is turned over, PDMS is spin-coated on the other side of the middle layer 5, and the bottom layer 6 after stretching is bonded thereto.

[0059] Myocardial cells are planted on the fibronectin layer 2 to obtain the myocardial cell layer 1, and then the pipeline 7 and the end face flat plug 8 are connected respectively to obtain the self-assembled ventricle structure based on stress-induced coiled membrane.

[0060] The application will be further described in detail in combination with the drawings:

[0061] As shown in the drawings, Figure 1 the self-assembled ventricle structure based on stress-induced coiled membrane disclosed in the application comprises a life-like unit and a mechanical unit, wherein the life-like unit comprises a scaffold layer 3, a fibronectin layer 2 and a myocardial cell layer 1; and the mechanical unit is composed of a pipeline 7 and an end face flat plug 8.

[0062] As shown in the drawings, Figure 2 the scaffold layer 3 is composed of three layers, namely a top layer 4, a middle layer 5 and a bottom layer 6, the top layer 4 is composed of a patterned area and a connecting “hinge” layer, microgrooves are arranged on the patterned area and the middle layer 5, the fibronectin layer 2 is in the form of a film and covers the upper surface of the scaffold layer 3 containing the microgrooves, myocardial cells in the myocardial cell layer 1 grow directionally above the fibronectin layer 2 along the direction of the microgrooves, and the myocardial tissue in the myocardial cell layer contains myofibrils; the overall thickness of the scaffold layer 3 is about 30 μm, and the material is polydimethylsiloxane (PDMS), the PDMS material has excellent characteristics of high biocompatibility, stable and reliable physical and chemical properties, good formability, adjustable rigidity, low processing difficulty and easy-to-obtain high-fidelity micron-level structure; the rigidity of the entire ventricle structure can be adjusted by adjusting the process parameters such as the composition and thickness of the substrate;

[0063] As shown in Figures 2-3 , the micro-groove has a protrusion height of 5 μm, a protrusion width of 4 μm, and a spacing between protrusions of 25 μm; the micro-groove guides the primary cardiomyocytes in the cardiomyocyte layer 1 to grow along the directional clue, thereby generating an anisotropic driving force;

[0064] As shown in Figures 2-3 , the fibronectin layer 2 has a thickness of 10-20 μm and is made of fibronectin; fibronectin is a macromolecular extracellular membrane protein existing on the surface of various animal cells and is a main non-collagenous glycoprotein in the extracellular matrix and basement membrane; fibronectin plays a central role in cell adhesion and can regulate cell polarity, differentiation and growth;

[0065] The cardiomyocyte layer 1 has a thickness of about 10 μm and is made of primary neonatal rat cardiomyocytes; the cardiomyocytes can spontaneously contract, and their contraction behavior can be adjusted by applying an electrical signal; compared with other contractile active biological tissues (skeletal muscle, insect dorsal blood vessels, etc.), the cardiomyocytes have stronger contraction ability;

[0066] The top layer 4 is first released, and under the action of prestress, the layer is bent to the cell side, thereby constructing a 3D helical right-handed inner layer structure; then the middle layer 5 and the bottom layer 6 are released, and under the action of tensile prestress, the bottom layer 6 is bent to the inner layer structure formed to form a 3D helical left-handed outer layer structure; due to the hydrophobic property of PDMS, the two layers are closely attached after being wrapped.

[0067] As shown in Figures 2-3 , the 2D unfolded ventricle-like structure based on stress-induced coiled membrane self-assembly ventricle-like structure has the following dimensions: the main part is a 135-degree arc surface composed of a pair of R15 and R1, and the size of the middle connecting "hinge" is 14 mm in length and 2 mm in width; it should be noted that the reference dimensions shown in the figure and mentioned below are part of an embodiment of the present application, and all other reference dimensions given by a person skilled in the art without creative work are within the scope of the present application.

[0068] As shown in Figure 4 , the stretching mechanism 10 is 3D printed by photosensitive resin and is provided with different openings; the PDMS film with an initial length of 20 mm can be clamped and stretched by 10%-100%, and other initial length films can also be stretched.

[0069] As shown in Figure 5As shown, the self-assembled ventricle structure based on stress-induced coiled membrane of the present application has a life-like part manufacturing process mainly including: spin-coating PDMS on a silicon mold, preparing a patterned area in the top layer 4, separating the spin-coated and solidified PDMS after the preparation of the patterned area, and stretching the PDMS to a certain proportion on a stretching clamp; then spin-coating PDMS on the middle layer 5, bonding the patterned area in the top layer 4 and one side of the middle layer 5, and after solidification and bonding, turning over the middle layer 5, spin-coating PDMS on the other side of the middle layer 5, and bonding the bottom layer 6 with the middle layer 5.

[0070] Planting myocardial cells on the fibronectin layer 2 to obtain a myocardial cell layer 1; then connecting with the pipeline 7 and the end face flat plug 8 respectively to obtain a self-assembled ventricle structure based on stress-induced coiled membrane.

[0071] For the silicon mold shown in the figure, the manufacturing scheme is as follows: spin-coating photoresist on a silicon wafer, placing a photo mask, and irradiating under the ultraviolet light of a photoetching machine for 90 seconds; the photoresist under the white part of the mask is exposed, and the photoresist under the black part of the mask is not affected; taking away the mask, and placing the silicon wafer in a 5‰ sodium hydroxide solution; since EPG535 is a positive photoresist, carboxylic acid is generated after exposure, and can be dissolved in the sodium hydroxide solution; finally, only the photoresist under the black part of the mask which is not exposed is left; after cleaning, the silicon wafer is subjected to plasma etching, and after cleaning with acetone, the silicon mold is obtained. A thin layer containing C and F atoms is grown on the surface of the silicon mold to make the surface of the silicon mold hydrophobic, and the mold can be turned over multiple times.

[0072] For the preparation scheme of the patterned area in the top layer 4, about 8g of PDMS prepolymer is weighed, mixed with a curing agent in a mass ratio of 10:1, stirred uniformly, and vacuumized to remove bubbles; the PDMS mixture is poured on the silicon mold to cover all the patterned areas. Vacuumize again to remove air, and make the PDMS fill the microstructure of the silicon mold. The silicon wafer is adsorbed in the center of a table-type spin coater, and the PDMS is spin-coated at 4000 rpm for 1 minute. The PDMS is solidified at room temperature for 48 hours or at 65°C for 3 hours; the PDMS middle layer 5 is spin-coated on both sides in turns at 4000 rpm, and the PDMS top layer 4 and the PDMS bottom layer 6 are bonded on the upper and lower layers of the PDMS middle layer 5 when the PDMS is not solidified; the PDMS is solidified and bonded at room temperature for 48 hours or at 65°C for 3 hours; the PD MS thin film is cut according to the width, and is transferred to a glass slide. A laser scribing machine is used to process the outer contour of the life-like part of the ventricle structure.

[0073] For the myocardial cell layer 1, the manufacturing scheme is as follows: primary rat neonatal myocardial cells are cultured for 48 hours, and are inoculated on a directional scaffold at 4E6 cells per scaffold.

[0074] AsFigure 6 As shown, the self-assembled ventricular-like structure based on stress-induced curling membrane described in this invention primarily moves through... Figure 3 This is accomplished through the contraction of the myocardial cell layer 1 shown, specifically as follows:

[0075] Because the myocardial tissue in the myocardial cell layer 1 contains contractile myofibrils, as the cells mature, the contractile force of the myofibrils increases, and the myocardial tissue generates passive stress. This stress works synergistically with the pre-stretching stress of the scaffold layer 3. Therefore, the ventricular-like structure and the life-like part still have a certain curvature even without a driving electrical signal, and aggregate into a stable cone composed of cells and PDMS.

[0076] When myocardial tissue spontaneously beats or is activated under control, the tissue contracts, increasing the tensile stress on scaffold layer 3 and fibronectin layer 2. Both undergo further elastic deformation, resulting in a further increase in the curvature of the ventricular-like, life-like structure.

[0077] After myocardial tissue contracts, it naturally relaxes, reducing the tensile stress on scaffold layer 3 and fibronectin layer 2, resulting in the restoration of the curvature of the ventricular-like, life-like structure.

[0078] like Figure 7 As shown, the stimulator 11 is a Rigol DG812 pulse frequency generator or a similar stimulator 11 with adjustable output voltage and frequency; the solution environment 13 is a culture medium or a solution that can maintain cell activity, provide nutrients for cell movement, and has a certain degree of conductivity; the electrode 12 is made of graphite, which has a better effect on driving and maintaining the activity of cardiomyocytes, but it should be noted that other common types of electrodes such as gold, platinum, and silver can also achieve the driving function and are also within the scope of this invention. The driving control part functions to control the contraction frequency and amplitude of the ventricular-like structure, and to activate cardiomyocytes when the spontaneous pulsation of the cardiomyocyte layer 1 is weak; its working mechanism is as follows: the output terminal of the stimulator 11 provides an electrical stimulation signal with a specific voltage and frequency required to control the contraction of the ventricular-like structure. The signal passes through the paired electrodes 12 to form an electric field in the solution environment 13, and the electric field provides an electrical signal to the cardiomyocyte layer 1, thereby controlling the contraction behavior of the myocardium, and thus controlling the overall contraction behavior of the ventricular-like structure.

[0079] like Figure 7 As shown, the self-assembled ventricular-like structure based on stress-induced curling membrane described in this invention can have its contractile behavior adjusted by changing the electrical stimulation parameters. Specifically, the driving voltage of the ventricular-like structure described in this invention is 4-10V. Too low a driving voltage cannot drive the structure, while too high a driving voltage will affect cell activity.

[0080] Driving frequency: the driving frequency of the ventricle-like structure is 0.5-2Hz, and too high driving frequency will cause the myocardial cell to contract strongly and cannot relax, so that the contraction process cannot be completed. When the driving frequency is about 1Hz, the ventricle-like structure has the maximum pumping efficiency.

[0081] Flow control strategy: the ventricle-like structure of the present application, the life part of the ventricle-like structure is related to the driving frequency under the appropriate voltage and driving frequency, so only the driving voltage and frequency need to be adjusted to change the contraction frequency of the ventricle-like structure, and then change the pumping flow rate of the ventricle-like structure.

[0082] The self-assembled ventricle-like structure based on stress-induced coiled membrane of the present application can realize dynamic culture of tissues, and can be used in cell muscle tissues in complex terrain environment. Specifically,

[0083] As shown in Figure 8 , the cultured tissue 9 and the self-assembled ventricle-like structure based on stress-induced coiled membrane are connected together through the pipeline 7, and with the contraction of the life part of the self-assembled ventricle-like structure based on stress-induced coiled membrane, the culture solution is accurately pumped and delivered to the cultured tissue, realizing dynamic culture of the tissue.

[0084] The above content is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application, and any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. A self-assembled ventricular-like structure based on a stress-induced curled membrane, characterized in that, include: A life-like unit and a mechanical unit; the life-like unit and the mechanical unit are connected; the life-like unit includes a scaffold layer (3), a fibronectin layer (2) and a cardiomyocyte layer (1); the life-like unit is rolled up to form a three-dimensional conical ventricular structure; The mechanical unit includes a pipe (7) and an end face plug (8); One end of the pipe (7) is connected to the small end of the three-dimensional conical ventricular structure; the end face plug (8) is connected to the large end of the three-dimensional conical ventricular structure. The support layer (3) includes a top layer (4), a middle layer (5) and a bottom layer (6) that are sequentially attached to each other; The top layer (4) is composed of patterned areas; the patterned areas and the upper surface of the intermediate layer (5) are provided with microgrooves; The fibronectin layer (2) covers the upper surface of the scaffold layer (3) containing microgrooves in the form of a thin film. The cardiomyocyte layer (1) is obtained by directional growth of cardiomyocytes on the surface of the fibronectin layer (2) along the direction of the microgrooves on the upper surface of the top layer (4); The protrusion height of the microgroove is 5~10μm, the protrusion width is 4~6μm, and the interval between the protrusions is 20~50μm; The material of the bottom layer (6) is polydimethylsiloxane; the material of the fibronectin layer (2) is fibronectin; The cardiomyocytes were primary neonatal rat cardiomyocytes; The support layer (3) and the end face flat plug (8) are both made of PDMS material; the diameter of the end face flat plug (8) is 10~15mm, the thickness is 1~1.5mm, and the diameter of the central coaxial hole is 2mm; The tensile strengths of the top layer (4), the middle layer (5), and the bottom layer (6) are 8%~10%, 0%, and 12%~15%, respectively. The top layer (4) and the middle layer (5) are bonded together to form a 3D right-handed spiral inner structure, and the bottom layer (6) is bonded together with the middle layer (5) to form a 3D left-handed spiral outer structure.

2. The self-assembled ventricular-like structure based on a stress-induced curled membrane according to claim 1, characterized in that, The material of the pipe (7) is PEEK material; the inner diameter of the pipe (7) is 1~1.9mm; The diameter of the end face flat plug and the diameter of the central coaxial hole change proportionally with the size of the support layer (3).

3. A self-assembling ventricular-like structure driving system based on a stress-induced curling membrane, characterized in that, It includes a stimulator (11), an electrode (12), and a solution environment (13); one end of the electrode (12) is connected to the stimulator (11), and the other end of the electrode (12) is placed in the solution environment (13); The self-assembled ventricular structure based on a stress-induced curled membrane as described in claim 1 or 2 is disposed in a solution environment (13).

4. The self-assembly ventricular-like structure driving system based on stress-induced curling membrane according to claim 3, characterized in that, The solution environment (13) is a culture medium; the material of the electrode (12) is any one of graphite, gold, platinum and silver; the electric field driving voltage of the stimulator (11) is 4~10V and the driving frequency is 0.5~2Hz.

5. A method for preparing a self-assembled ventricular-like structure based on a stress-induced curled membrane according to claim 1 or 2, characterized in that, Includes the following steps: The scaffold layer (3) was prepared by spin coating and curing bonding, and then fibronectin was coated on the scaffold layer (3) to form a fibronectin layer (2). Cardiac cells were seeded on the fibronectin layer (2) to obtain a cardiac cell layer (1); then connected to the conduit (7) and the end face flat plug (8) respectively to obtain a self-assembled ventricular-like structure based on stress-induced curled membrane; The method for preparing the scaffold layer (3) is as follows: PDMS is spin-coated onto a silicon mold to prepare the patterned area in the top layer (4). After the patterned area is prepared, the spin-coated and cured PDMS is separated and placed on a stretching fixture and stretched to a certain proportion by a stretching mechanism (10). Then, PDMS is spin-coated onto the upper layer of the intermediate layer (5) and the patterned area in the stretched top layer (4) is bonded to one side of the intermediate layer (5). After the bonding is cured, the intermediate layer (5) is flipped over and PDMS is spin-coated onto the other side of the intermediate layer (5) and the stretched unpatterned bottom layer (6) is bonded to it.

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