Self-assembly ventricle-like structure, system and method based on stress-induced curly membrane

Abstract

The invention discloses a self-assembly ventricle-like structure, system and method based on a stress-induced curly membrane, and belongs to the technical field of heart-like research. The ventricle-like structure is composed of a life-like body unit and a mechanical body unit, the life-like body unit is composed of a support layer, a fibronectin layer and a myocardial cell layer and used for providing driving force for the whole structure, and the mechanical body unit is composed of a pipeline and an end face flat plug and used for achieving efficient operation of one-way pumping. The structure can spontaneously generate movement and can also show different contractile capacities in different electrical stimulation environments, and pump-out flow velocity control of the ventricle-like structure can be simply achieved by changing the external electrical stimulation environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of heart-like research, and specifically relates to a self-assembled ventricle-like structure, system and method based on a stress-induced curling membrane. Background Art

[0002] Organoids are a new in vitro cell model with a complex three-dimensional cell structure that can simulate various aspects of organ development, composition and function. They can be randomly or directed differentiated into different cell types and assembled from embryonic stem cells, human induced pluripotent stem cells, adult stem cells and even tumor cells in a 3D culture system. 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 for basic research and clinical applications. First, organoids can be used to simulate and study organ development and related diseases. Second, they also have a wide range of applications in drug experiments and disease treatment. Although corresponding organoids have been developed in various organs, the progress in the field of cardiac organoids has lagged behind significantly, partly due to the complexity of early heart development. The heart is both the first functional organ formed in embryonic development and the commanding heights of the in vitro organoid culture problem.

[0003] In the heart, the left ventricle is a major component of the heart. It 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 the development of the heart. In current heart-like research, in vitro myocardial models with chambers have been generated through tissue engineering technologies such as geometric constraints, stents, and molds. The contraction of muscles is used to drive the unidirectional movement of fluids by designing and applying valves, diaphragms or asymmetric structures. However, the current research on heart-like research is a multi-cellular heart model obtained by stem cell differentiation, lacking a simple assembly method. In addition, there are few studies on the heart's blood circulation and delivery capacity, and the performance shown in existing studies is far from the real natural heart capacity. Therefore, it is of great significance to design and manufacture a life-like heart that is closer to the delivery efficiency of a real heart, and to achieve dynamic tissue culture on this basis, laying the foundation for the development of new organoids and culture. Summary of the invention

[0004] The purpose of the present invention is to provide a self-assembled ventricle-like structure, system and method based on a stress-induced curling membrane, which is used to solve the technical problem that the existing life-like heart is far from the real natural heart.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a self-assembled ventricle-like structure based on a stress-induced curling film, comprising:

[0007] A life-like unit and a mechanical unit; the life-like unit and the mechanical unit are connected; the life-like unit comprises a support layer, a fibronectin layer and a myocardial cell layer; the fibronectin layer is arranged on the surface of the support 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 includes 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 surface flat plug is connected to the large end of the three-dimensional conical ventricular structure.

[0010] Furthermore, the support layer includes a top layer, a middle layer and a bottom layer which are sequentially bonded and arranged;

[0011] The top layer is composed of a patterned area; micro grooves are arranged on the upper surfaces of the patterned area and the middle layer.

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

[0013] The myocardial cell layer is obtained by directional growth of 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 micro groove has a protrusion height of 5 to 10 μm, a protrusion width of 4 to 6 μm, and an interval between protrusions of 20 to 50 μm.

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

[0016] The cardiomyocytes are primary neonatal rat cardiomyocytes;

[0017] The support layer and the end surface flat plug are both made of PDMS material; the diameter of the end surface 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] Furthermore, the material of the pipeline is PEEK material; the inner diameter of the pipeline is 1 to 1.9 mm;

[0019] The diameter of the end face flat plug and the diameter of the central coaxial hole change in proportion to the size of the support layer.

[0020] Furthermore, the stretching rates of the top layer, the middle layer and the bottom layer are 8% to 10%, 0% and 12% to 15% respectively.

[0021] The present invention also discloses a self-assembled ventricular structure driving system based on stress-induced curling membrane, comprising a stimulator, an electrode and a solution environment; one end of the electrode is connected to the stimulator, and the other end of the electrode is placed in the solution environment;

[0022] The above-mentioned self-assembled ventricular-like structure based on stress-induced curling membrane is set in a solution environment.

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

[0024] The present invention also discloses a method for preparing the above-mentioned self-assembled ventricular-like structure based on the stress-induced curling film, comprising the following steps:

[0025] The scaffold layer is prepared by a spin coating solidification bonding method, and then fibronectin is coated on the scaffold layer to form a fibronectin layer;

[0026] Cardiomyocytes are planted on the fibronectin layer to obtain a cardiomyocyte layer; then the cardiomyocyte layer is connected to a pipe and an end-face flat plug, respectively, to obtain a self-assembled ventricular-like structure based on a stress-induced curling membrane.

[0027] Furthermore, the support layer includes a top layer, a middle layer and a bottom layer, and the materials are all PDMS materials; the top layer is composed of a patterned area;

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

[0029] PDMS is spin-coated on the silicon mold to prepare the patterned area in the top layer. After the preparation of the patterned area is completed, the spin-coated and cured PDMS is separated and placed on a stretching fixture to be stretched to a certain proportion by a stretching mechanism; then PDMS is spin-coated on the upper layer of the middle layer, and the patterned area in the stretched top layer is bonded to one side of the middle layer. After solidification and bonding, the middle layer is turned over, PDMS is spin-coated on the other side of the middle layer, and the stretched bottom layer without pattern is bonded to it.

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

[0031] The present invention discloses a self-assembled ventricle-like structure based on a stress-induced curling membrane, which is composed of a life-like unit and a mechanical unit. The life-like unit is composed of a stent layer, a fibronectin layer and a myocardial cell layer, and is used to provide a driving force for the overall structure. The mechanical unit is composed of a pipeline and an end-face flat plug, and is used to achieve efficient operation of unidirectional pumping. The above structure can spontaneously generate movement, and can also show different contraction capabilities in different electrical stimulation environments. The pumping flow rate control of the ventricle-like structure can be simply achieved by changing the external electrical stimulation environment.

[0032] Furthermore, the scaffold layer realizes the construction of a three-dimensional morphology by setting pre-stretched and unstretched PDMS films and releasing stress after bonding, thereby achieving self-assembly of a ventricular-like structure; microgrooves are set on the surface as cell-oriented topographic cues, and myocardial cells grow along the directional cues, thereby generating anisotropic driving force, which also helps to improve the contractile performance of the ventricular-like structure. The structure is composed of only a macroscopic layer of myocardial cells, a layer of fibronectin and a layer of PDMS substrate to form the living moving part of the ventricular-like structure, and has a simple structure and is easy to manufacture.

[0033] The present invention also discloses a driving system for the above-mentioned self-assembled ventricular-like structure based on stress-induced curling membrane, including a stimulator, electrodes and a solution environment. An electrical stimulation signal is given through the output end of the stimulator, and the electrical stimulation signal forms an electric field in the solution environment through paired electrodes. The electric field provides an electrical signal to the myocardial cell layer, thereby controlling the contraction behavior of the myocardial cells in the myocardial cell layer, and then controlling the overall contraction of the ventricular-like structure; the radial contraction and torsional contraction of the ventricular-like structure in the present invention are combined. During contraction, the contraction of the cell muscle tissue makes the overall volume of the chamber smaller, thereby squeezing the liquid inside the chamber so that it is pumped out from one port. When the cell relaxes, the liquid flows in from the other port, filling the chamber, and is pumped out again in the next cycle, thereby generating a net flow and presenting a pumping effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the overall structural diagram of the self-assembled ventricular-like structure based on the stress-induced curling membrane of the present invention;

[0035] Figure 2 A detailed diagram of the self-assembled ventricle-like structure based on the stress-induced curling membrane of the present invention;

[0036] Figure 3 It is a schematic diagram of the dimensions of the self-assembled ventricle-like structure based on the stress-induced curling membrane of the present invention;

[0037] Figure 4 Schematic diagram of the stretching mechanism of the stress-induced curling film;

[0038] Figure 5 A flow chart for the preparation of a living organism with a self-assembled ventricle-like structure based on stress-induced curling membranes;

[0039] Figure 6 Diagram of the contraction mechanism of the self-assembled ventricular-like structure based on stress-induced curling membrane;

[0040] Figure 7 Schematic diagram of the driving of the self-assembled ventricular-like structure based on stress-induced curled membrane;

[0041] Figure 8Schematic diagram of the dynamic culture of the self-assembled ventricular-like structure based on stress-induced curling membrane connected with other tissues;

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

[0043] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

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

[0045] The present invention discloses a self-assembled ventricular structure based on a stress-induced curling film, comprising a life-like unit and a mechanical unit, wherein the life-like unit comprises a support layer 3, a fibronectin layer 2 and a myocardial cell layer 1; wherein the support layer 3 is composed of three layers, namely a top layer 4, a middle layer 5 and a bottom layer 6, wherein the top layer 4 is composed of a patterned area, and microgrooves are arranged on the patterned area and the middle layer 5; the fibronectin layer 2 is covered on the upper surface of the support layer 3 containing the microgrooves in the form of a film; the myocardial cells of the myocardial cell layer 1 are directionally grown above the fibronectin layer 2 along the direction of the microgrooves, and the myocardial tissue in the myocardial cell layer contains myofibrils; wherein the mechanical unit is composed of a pipe 7 and an end-face flat plug 8; one end of the pipe 7 is matched and connected with the small end of a three-dimensional conical ventricular structure; the end-face flat plug 8 is matched and connected with the large end of the three-dimensional conical ventricular structure, and in order to make the match tight, surgical sutures are selected to be used to tighten the matching position.

[0046] Preferably, the top layer 4, the middle layer 5 and the bottom layer 6 have different stretching rates, which are 10%, 0% and 15% respectively, to facilitate the formation of different degrees of bending. The top layer 4 and the middle layer 5 fit together to form a smaller spiral structure of the inner layer, and the bottom layer 6 and the middle layer 5 fit together to form a larger spiral structure of the outer layer.

[0047] The present invention also discloses a driving system for the self-assembled ventricular-like structure based on the stress-induced curled membrane, comprising a stimulator 11, an electrode 12 and a solution environment 13; an electrical stimulation signal is given through the output end of the stimulator 11, and the electrical stimulation signal forms an electric field in the solution environment 13 through the paired electrodes 12, and an electrical signal is provided to the myocardial cell layer 1 through the electric field, thereby controlling the contraction behavior of the myocardial cells in the myocardial cell layer 1, and further controlling the overall contraction of the self-assembled ventricular-like structure based on the stress-induced curled membrane;

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

[0049] Preferably, the electric field driving voltage of the ventricular-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 microgrooves have a protrusion height of 5-10 μm, a protrusion width of 4-6 μm, and a spacing between protrusions of 20-50 μm;

[0053] Preferably, in the scaffold layer 3 of the 2D combined PDMS, the direction of the microgrooves is at an angle of 45 degrees to the axis of symmetry; the rise angle of the 3D myocardial spiral fibers formed after release is 45 degrees;

[0054] Preferably, the end surface flat plug 8 is made of PDMS material, with a diameter of 11 mm, a thickness of 1 mm, and a central coaxial hole diameter of 2 mm;

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

[0056] The self-assembled ventricular-like structure based on stress-induced curling membrane disclosed in the present invention contracts in a combination of radial contraction and torsional contraction of the ventricular-like structure under the drive of a driving system. During contraction, the contraction of cell muscle tissue reduces the overall volume of the chamber, thereby squeezing the liquid inside the chamber so that it is pumped out from one port. When the cells relax, the liquid flows in from the other port, filling the chamber, and is pumped out again in the next cycle, thereby generating a net flow and presenting a pumping effect.

[0057] The present invention also discloses a method for manufacturing the self-assembled ventricular-like structure based on the stress-induced curling film, comprising the following steps:

[0058] PDMS is spin-coated on the silicon mold to prepare the patterned area in the top layer 4. After the preparation of the patterned area is completed, the spin-coated and cured PDMS is separated and placed on a stretching fixture to be stretched to a certain ratio; then PDMS is spin-coated on the upper layer of the middle layer 5, and the stretched patterned area in the top layer 4 is bonded to one side of the middle layer 5. 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 stretched bottom layer 6 is bonded to it;

[0059] Cardiomyocytes are planted on the fibronectin layer 2 to obtain a cardiomyocyte layer 1; subsequently, the cardiomyocyte layer 1 is connected to a pipe 7 and an end-face flat plug 8, respectively, to obtain a self-assembled ventricular-like structure based on a stress-induced curling membrane.

[0060] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0061] like Figure 1 As shown, the self-assembled ventricular-like structure based on stress-induced curling membrane disclosed in the present invention includes a life-like unit and a mechanical unit, wherein the life-like unit includes a support layer 3, a fibronectin layer 2 and a myocardial cell layer 1; wherein the mechanical unit is composed of a pipe 7 and an end-face flat plug 8.

[0062] like Figure 2 As shown, the support 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 covered on the upper surface of the support layer 3 containing the microgrooves in the form of a film. The myocardial cells of the myocardial cell layer 1 are directionally grown on the top of the fibronectin layer 2 along the direction of the microgrooves. The myocardial tissue in the myocardial cell layer contains myofibrils; the overall thickness of the support layer 3 is about 30 μm, and the material is polydimethylsiloxane (PDMS). The PDMS material has excellent characteristics of high biocompatibility and stable and reliable physical and chemical properties, good formability, adjustable stiffness, low processing difficulty, and easy to obtain high-fidelity micron-level structure; the stiffness of the entire ventricular-like structure can be adjusted by adjusting the substrate composition, thickness and other process parameters;

[0063] like Figure 2-Figure 3 As shown, the protrusion height of the microgroove is 5 μm, the protrusion width is 4 μm, and the interval between the protrusions is 25 μm; the microgroove guides the primary cardiomyocytes in the cardiomyocyte layer 1 to grow along the directional clues, thereby generating anisotropic driving force;

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

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

[0066] First, the top layer 4 is released. Under the action of prestress, the layer will bend toward the cell side, thereby constructing a 3D spiral right-handed inner layer structure. Then the middle layer 5 and the bottom layer 6 are released. Under the action of tensile prestress, the bottom layer 6 bends and wraps toward the already formed inner layer structure, forming a 3D spiral left-handed outer layer structure. Due to the hydrophobic properties of PDMS, the two layers fit tightly after wrapping.

[0067] like Figure 2-Figure 3 The dimensions of the 2D unfolded ventricular-like structure of the self-assembled ventricular-like structure based on the stress-induced curling membrane of the present invention are as follows: the main part is a 135-degree arc surface composed of a pair of R15 and R1, and the middle connecting "hinge" has a length of 14 mm and a width of 2 mm; it should be noted that the reference dimensions shown in the figure and mentioned below are all part of the embodiments of the present invention, and all other reference dimensions given by ordinary technicians in this field without making creative work belong to the scope of the present invention.

[0068] like Figure 4 As shown, the stretching mechanism 10 is made of photosensitive resin 3D printing and is provided with different openings. It can stretch a PDMS film with an initial length of 20 mm by 10% to 100% after clamping, and can also stretch films with other initial lengths.

[0069] like Figure 5As shown, the manufacturing process of the life-like part of the self-assembled ventricular-like structure based on the stress-induced curling membrane described in the present invention mainly includes: spin coating PDMS on the silicon mold to prepare the patterned area in the top layer 4, and after the preparation of the patterned area is completed, the spin-coated and cured PDMS is separated and placed on a stretching fixture to be stretched to a certain proportion; then, PDMS is spin-coated on the upper layer of the middle layer 5, and the patterned area in the stretched top layer 4 is bonded to one side of the middle layer 5. After solidification and bonding, the middle layer 5 is turned over, and PDMS is spin-coated on the other side of the middle layer 5 to fit the stretched bottom layer 6 to the middle layer 5.

[0070] Cardiomyocytes are planted on the fibronectin layer 2 to obtain a cardiomyocyte layer 1; subsequently, the cardiomyocyte layer 1 is connected to a pipe 7 and an end-face flat plug 8, respectively, to obtain a self-assembled ventricular-like structure based on a stress-induced curling membrane.

[0071] The manufacturing scheme for the silicon mold described in the figure is as follows: Spin-coat the photoresist on the silicon wafer, place the photomask on it, and expose it to the ultraviolet light of the photolithography machine for 90 seconds. The photoresist under the white part of the mask is exposed, and the photoresist under the black part is not affected; remove the mask, and put the silicon wafer into a 5‰ sodium hydroxide solution. Since EPG535 is a positive photoresist, it generates carboxylic acid after exposure and is soluble in the sodium hydroxide solution. Finally, only the unexposed photoresist under the black part of the mask is retained; after cleaning, the silicon wafer is plasma etched, and the silicon mold is obtained after cleaning with acetone. 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 capable of multiple mold flipping.

[0072] The preparation scheme for the patterned area in the top layer 4 is as follows: weigh about 8g of PDMS prepolymer and mix it with the curing agent in a mass ratio of 10:1, stir evenly after mixing, and vacuum to remove bubbles; pour the PDMS mixture on the silicon mold to cover all pattern areas. Vacuum to degas again and fill the silicon mold microstructure with PDMS. Adsorb the silicon wafer in the center of the desktop glue machine and spin-coat PDMS at 4000rpm for 1 minute. Place it at room temperature for 48h or at 65℃ for 3h to complete PDMS curing; spin-coat PDMS on both sides of the PDMS middle layer 5 at 4000rpm in batches. When it is not cured, attach the PDMS top layer 4 and the PDMS bottom layer 6 to the upper and lower layers of the PDMS middle layer 5, and place it at room temperature for 48h or at 65℃ for 3h to complete PDMS curing and bonding; cut the PDMS film according to the format and transfer it to the glass slide. Use a laser scriber to process the outer contour of the ventricular structure and the life body part.

[0073] The cardiomyocyte layer 1 is manufactured as follows: primary rat neonatal cardiomyocytes are statically cultured for 48 hours and then inoculated onto directional scaffolds, with 4E6 cells inoculated onto each scaffold.

[0074] like Figure 6 As shown, the self-assembled ventricular-like structure based on the stress-induced curling membrane of the present invention has a basic movement mainly through Figure 3 This is accomplished by contraction of the myocardial cell layer 1 as shown, specifically:

[0075] Since 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, so that the life-like part of the ventricle-like structure still has a certain curvature in the absence of a driving electrical signal, and aggregates into a stable cone composed of cells and PDMS;

[0076] When the myocardial tissue beats spontaneously or is activated under control, the tissue contracts, and the tensile stress on the stent layer 3 and the fibronectin layer 2 increases, and both of them undergo further elastic deformation, which is manifested as a further increase in the curvature of the ventricular structure-like living body part;

[0077] The myocardial tissue naturally relaxes after contraction, and the tensile stress on the stent layer 3 and the fibronectin layer 2 is reduced, which is manifested as the curvature of the ventricular structure and the life-like part is restored.

[0078] like Figure 7 As shown, the stimulator 11 is a DG812 pulse frequency generator of Rigol Company or a stimulator 11 with similar output voltage and frequency adjustment; the solution environment 13 is a culture solution, or a solution that can maintain cell activity, provide nutrition for cell movement, and has a certain conductivity; the material of the electrode 12 is graphite, which has a better effect on the drive and activity maintenance of myocardial cells, but it should be noted that other common types of electrodes such as gold, platinum, silver, etc. can also achieve the driving function and also belong to the scope of the present invention. The driving control part thereof has the function of controlling the contraction frequency and contraction amplitude of the ventricular-like structure, and activating the myocardial cells when the spontaneous beating of the myocardial cell layer 1 is weak; its working mechanism is: an electrical stimulation signal of a specific voltage and frequency required for controlling the contraction of the ventricular-like structure is given through the output end of the stimulator 11, and the signal forms an electric field in the solution environment 13 through the paired electrodes 12, and an electrical signal is provided to the myocardial cell layer 1 through the electric field, thereby controlling the contraction behavior of the myocardium, and then controlling the contraction behavior of the ventricular-like structure as a whole.

[0079] like Figure 7 As shown, the self-assembled ventricular-like structure based on stress-induced curling membrane described in the present invention can adjust its contraction behavior by changing the electrical stimulation parameters, which is specifically realized as follows: driving voltage. The driving voltage of the ventricular-like structure described in the present invention is 4-10V. Too low driving voltage cannot drive the structure, while too high driving voltage will affect cell activity.

[0080] Driving frequency: The driving frequency of the ventricle-like structure described in the present invention is 0.5-2Hz. Too high a driving frequency will cause myocardial cells to contract rigidly and fail to relax, thus failing to complete the contraction movement process. When the driving frequency is about 1Hz, the ventricle-like structure has the maximum pumping efficiency.

[0081] Flow control strategy: The contraction of the life-like part of the ventricle-like structure described in the present invention is related to the driving frequency under appropriate voltage and driving frequency. Therefore, it is only necessary to adjust the driving voltage and frequency to change the contraction frequency of the ventricle-like structure and thereby change the pumping flow rate of the ventricle-like structure.

[0082] The self-assembled ventricular-like structure based on stress-induced curling membrane described in the present invention can realize dynamic tissue culture and can be used in cell muscle tissue in complex terrain environments. Specifically:

[0083] like Figure 8 As shown, the cultured tissue 9 is connected to the main body of the self-assembled ventricular-like structure based on stress-induced curling membrane through a pipeline 7. As the living part of the self-assembled ventricular-like structure based on stress-induced curling membrane contracts, the culture fluid is accurately pumped out and transported to the cultured tissue to achieve dynamic culture of the tissue.

[0084] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A self-assembled ventricular-like structure based on a stress-induced curling 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 comprises a support layer (3), a fibronectin layer (2) and a myocardial cell layer (1); the fibronectin layer (2) is arranged on the surface of the support layer (3), and the myocardial cell layer (1) is directionally grown on the fibronectin layer (2); the life-like unit is curled to form a three-dimensional conical ventricular structure; The mechanical unit comprises a pipe (7) and an end-face flat plug (8); One end of the pipe (7) is connected to the small end of the three-dimensional conical ventricular structure; and the end surface flat plug (8) is connected to the large end of the three-dimensional conical ventricular structure.

2. The self-assembled ventricular-like structure based on stress-induced curling membrane according to claim 1, characterized in that: The support layer (3) comprises a top layer (4), a middle layer (5) and a bottom layer (6) which are sequentially arranged in contact with each other; The top layer (4) is composed of a patterned area; the upper surfaces of the patterned area and the middle layer (5) are provided with micro grooves.

3. The self-assembled ventricular-like structure based on stress-induced curling membrane according to claim 2, characterized in that: The fibronectin layer (2) is covered on the upper surface of the top layer (4) in the form of a thin film; The myocardial cell layer (1) is obtained by directional growth of myocardial cells on the surface of the fibronectin layer (2) along the direction of the microgrooves on the upper surface of the top layer (4); The micro groove has a protrusion height of 5 to 10 μm, a protrusion width of 4 to 6 μm, and an interval between protrusions of 20 to 50 μm.

4. The self-assembled ventricular-like structure based on stress-induced curling membrane according to claim 3, characterized in that: The material of the bottom layer (6) is polydimethylsiloxane; the material of the fibronectin layer (2) is fibronectin; The cardiomyocytes are primary neonatal rat cardiomyocytes; The support layer (3) and the end surface flat plug (8) are both made of PDMS material; the end surface flat plug (8) has a diameter of 10 to 15 mm, a thickness of 1 to 1.5 mm, and a central coaxial hole diameter of 2 mm.

5. The self-assembled ventricular-like structure based on stress-induced curling membrane according to claim 4, characterized in that: The material of the pipeline (7) is PEEK material; the inner diameter of the pipeline (7) is 1 to 1.9 mm; The diameter of the end face flat plug and the diameter of the central coaxial hole vary in proportion to the size of the support layer (3).

6. The self-assembled ventricular-like structure based on stress-induced curling membrane according to claim 4, characterized in that: The stretching rates of the top layer (4), the middle layer (5) and the bottom layer (6) are 8% to 10%, 0% and 12% to 15% respectively.

7. A self-assembled ventricular-like structure driving system based on stress-induced curling membrane, characterized in that: It comprises 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); A self-assembled ventricle-like structure based on a stress-induced curling membrane as claimed in any one of claims 1 to 6 is arranged in a solution environment (13).

8. A self-assembled ventricular-like structure driving system based on stress-induced curling membrane according to claim 7, characterized in that: The solution environment (13) is a culture solution; 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 to 10V, and the driving frequency is 0.5 to 2Hz.

9. A method for preparing a self-assembled ventricular-like structure based on a stress-induced curling membrane according to any one of claims 1 to 6, characterized in that: The following steps are involved: The scaffold layer (3) is prepared by a spin coating solidification bonding method, and then fibronectin is coated on the scaffold layer (3) to form a fibronectin layer (2); Myocardial cells are planted on a fibronectin layer (2) to obtain a myocardial cell layer (1); subsequently, the layer is connected to a pipe (7) and an end-face flat plug (8) respectively to obtain a self-assembled ventricle-like structure based on a stress-induced curling membrane.

10. The method for preparing a self-assembled ventricular-like structure based on a stress-induced curling membrane according to claim 9, characterized in that: The support layer (3) comprises a top layer (4), a middle layer (5) and a bottom layer (6), and the materials are all PDMS materials; the top layer (4) is composed of a patterned area; The preparation method of the support layer (3) is as follows: PDMS is spin-coated on a silicon mold to prepare a patterned area in the top layer (4). After the preparation of the patterned area is completed, the spin-coated and solidified PDMS is separated and placed on a stretching fixture to be stretched to a certain ratio by a stretching mechanism (10); PDMS is then spin-coated on the upper layer of the middle layer (5), and the patterned area in the stretched top layer (4) is bonded to one side of the middle layer (5). 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 stretched bottom layer (6) without a pattern is bonded thereto.

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