Controllable multi-channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment

Abstract

The invention belongs to the technical field of biology, and discloses a controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, the controllable multi-channel three-dimensional dynamic culture system comprises a driving platform, a flexible driver and a cell culture box, the flexible driver is provided with a planar substrate and an outer wall provided with a periodic protrusion structure, and is provided with a hollow structure serving as an air chamber; one end is connected with the air path module through an air hole channel, and the other end is sealed; a groove is also formed in the outer surface of the planar substrate and is used for accommodating cells to be cultured; the flexible driver air chamber can be pressurized or depressurized, so that the bending degree is enhanced or reduced according to the preset frequency, and a mechanical microenvironment is generated. The three-dimensional dynamic culture system constructed by the invention can simulate a cell mechanical microenvironment, realizes dynamic culture of cells, and promotes cell proliferation and expression of specific proteins. In addition, the system can be conveniently designed into multi-channel culture, and has a very good practical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a controllable multi-channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment. Background Art

[0002] In modern biomedicine, tissue organoids and drug screening play a key role in clinical applications. As an indispensable step, in vitro cell culture has become an important research method in biomedical engineering. Mechanical factors in the cell microenvironment, including cell-cell interaction forces, the forces of the surrounding fluid on cells, and the mechanical stimulation of organ movement on tissues, have a decisive influence on biological processes such as cell proliferation, differentiation, migration, and matrix remodeling. Therefore, accurately simulating the mechanical microenvironment in the cell body is crucial for a deep understanding of cell behavior and function and for the development of effective tissue engineering and drug screening models.

[0003] However, current cell culture methods still rely mainly on traditional culture flasks, a static culture environment that cannot fully simulate the complex mechanical microenvironment in vivo. Cells in culture flasks are usually in a two-dimensional static environment, lacking the true three-dimensional mechanical stimulation in vivo, including fluid shear force, tensile force, and compression force. This leads to significant differences in the behavior of cells cultured in vitro and their performance in vivo, which limits the credibility and applicability of research results and makes it difficult to provide sufficient scientific basis for tissue engineering and drug screening.

[0004] In order to overcome the limitations of traditional culture methods, a variety of bioreactor systems have been developed, mainly including rotary and perfusion reactors. These systems, to a certain extent, simulate part of the in vivo microenvironment by providing sufficient mixing of oxygen and nutrients to enable cells to grow in a controlled fluid environment. For example, the rotary bioreactor uses the low shear force generated by the rotational motion to facilitate the three-dimensional aggregation and differentiation of cells; the perfusion bioreactor provides cells with stable oxygen and nutrient support through continuous perfusion of nutrient solution. However, these bioreactors still have many shortcomings in practical applications. First, they are usually large in size, complex in structure, expensive to manufacture and maintain, and have high requirements for the experimental environment, which limits their application in conventional laboratories and large-scale cell culture. Secondly, these systems usually require a large amount of culture medium, resulting in increased operating costs. In addition, existing bioreactors generally lack multi-channel design, making it difficult to achieve dynamic simulation culture of cells of different cell types or under different culture conditions, which limits the development of high-throughput and diversified research.

[0005] Therefore, there is an urgent need for a new, multi-channel, three-dimensional dynamic cell culture system that can accurately simulate the mechanical microenvironment of cells in vivo and provide more physiologically relevant culture conditions. At the same time, the system should have the characteristics of compact structure, low cost, easy operation and strong adaptability to meet different cell culture needs and promote the development of fields such as cell biology, tissue engineering and drug screening. Summary of the invention

[0006] In view of the above defects or improvement needs of the prior art, the purpose of the present invention is to provide a controllable multi-channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment. A three-dimensional dynamic culture system is constructed with the participation of a cell culture box by using a specially designed driving platform and a flexible driver, which can simulate the cell mechanical microenvironment, realize dynamic culture of cells, and promote cell proliferation and expression of specific proteins. In addition, the system can be conveniently designed for multi-channel culture, and has very good practical application prospects.

[0007] To achieve the above object, according to one aspect of the present invention, a controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment is provided, comprising a driving platform, a flexible driver and a cell culture box, wherein:

[0008] The driving platform includes a power supply module, a signal control module and an air circuit module, wherein the power supply module is used to convert the mains power into a DC power supply with a preset amplitude; the signal control module is provided with electric energy by the DC power supply and is used to control the on and off of the electric control switch at a preset frequency; the air circuit module is connected to the flexible actuator, and includes an air pump, a pressure regulating valve and an electromagnetic reversing valve; wherein the air pump is used to provide air circuit power, and the electric energy is provided by the DC power supply or the mains power; the pressure regulating valve is used to adjust the air flow of the air circuit; the electromagnetic reversing valve is used to adjust the air flow direction under the influence of the on and off of the electric control switch;

[0009] The flexible actuator is made of a flexible material, and has a planar base and an outer wall provided with a periodic protrusion structure; the flexible actuator has a hollow structure as an air chamber, one end of the hollow structure is connected to the air path module through an air hole channel, and the other end remains sealed; a groove is also provided on the outer surface of the planar base for accommodating cells to be cultured;

[0010] The cell culture box comprises an open box and a cover, wherein the interior of the open box is used to simultaneously contain the culture medium and the flexible driver;

[0011] The driving platform can control the electromagnetic reversing valve by controlling the electric control switch, and utilize the air pump to input gas for pressurization or exhaust gas for depressurization through the air hole channel of the flexible driver, so that the flexible driver can strengthen or reduce the bending degree according to the preset frequency, thereby generating a mechanical microenvironment; the bending degree of the flexible driver is affected by the pressure in the air chamber of the flexible driver.

[0012] As a further preferred embodiment of the present invention, there are multiple flexible actuators, each of which corresponds to one signal control module and one gas path module in the driving platform, and can independently generate a mechanical microenvironment to achieve multi-channel culture;

[0013] The power supply module also includes a junction box for providing a DC power supply as electrical energy to the multi-channel signal control module and the multi-channel gas path module;

[0014] Preferably, a plurality of the flexible actuators are accommodated in the inner space of one or more cell culture boxes.

[0015] As a further preferred embodiment of the present invention, for any one of the flexible actuators, the length of the flexible actuator is 20-36 mm, the height is 5-10 mm, the width is 6-10 mm, and the wall thickness is 1-2 mm;

[0016] The pore channel has a diameter of 1 to 3 mm and a length of 2 to 8 mm;

[0017] The number of the periodic protrusion structures is 4 to 8;

[0018] The number of the grooves is 1 to 2; the length of any one of the grooves is 8 to 15 mm, the width is 2 to 4 mm, and the depth is 0.5 to 2 mm.

[0019] As a further preferred embodiment of the present invention, a support frame is further contained in the internal space of each cell culture box, and a flexible actuator is placed at each placement position on the support frame to avoid mutual interference between the flexible actuators contained in the internal space of the same cell culture box;

[0020] A plurality of the flexible actuators can be placed side by side on the same support frame, the width of the support frame can accommodate these side-by-side flexible actuators, and the interval between two adjacent flexible actuators is 2 to 6 mm; the length of the support frame is greater than or equal to the length of the flexible actuator; the length of the cell culture box is greater than the length of the support frame, and the width is greater than the width of the support frame; the height is 60 to 80 mm.

[0021] As a further preferred embodiment of the present invention, the flexible actuator is prepared by pouring, curing and demoulding silicone in a mold with a predetermined shape to form a planar base and an outer wall provided with a periodic protrusion structure, respectively, and then bonding the planar base and the outer wall provided with the periodic protrusion structure, thereby;

[0022] Preferably, the silica gel is one or more of AB glue, ecoflex, and PDMS.

[0023] As a further preferred embodiment of the present invention, the cell culture box is made of PMMA, PLA or silica gel;

[0024] Preferably, the silica gel is selected from: AB glue, ecoflex, PDMS.

[0025] As a further preferred embodiment of the present invention, the electronically controlled switch includes a relay, a thyristor, a field effect transistor (MOSFET), a solid-state relay (SSR), a photocoupler or an insulated gate bipolar transistor (IGBT);

[0026] The electromagnetic reversing valve is selected from a two-way electromagnetic reversing valve, a three-way electromagnetic reversing valve, a four-way electromagnetic reversing valve and a five-way electromagnetic reversing valve; preferably a two-position three-way electromagnetic reversing valve;

[0027] The power supply module is specifically an AC to DC power adapter.

[0028] As a further preferred embodiment of the present invention, the air pump includes both a positive pressure pump and a negative pressure pump, and the pressure regulating valve includes both a positive pressure regulating valve and a negative pressure regulating valve, wherein the positive pressure regulating valve is connected to the positive pressure pump, and the negative pressure regulating valve is connected to the negative pressure pump;

[0029] Preferably, the rated voltage of the positive pressure pump is 6V, and the rated voltage of the negative pressure pump is 12V.

[0030] According to another aspect of the present invention, the present invention provides the use of the above-mentioned controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment in dynamic cell culture.

[0031] According to another aspect of the present invention, the present invention provides the use of the above-mentioned controllable channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment in the dynamic culture of cells for the purpose of in vitro tissue organoid preparation and drug screening.

[0032] Compared with the prior art, the above technical solution conceived by the present invention uses a specially designed driving platform and a flexible driver to construct a three-dimensional dynamic culture system with the participation of a cell culture box. The driving platform controls the bending of the flexible driver located in the cell culture box, converts the macroscopic bending into mechanical stimulation of the cells, and realizes three-dimensional dynamic culture to simulate the cell mechanical microenvironment. The flexible driver is placed in the cell culture box, made of flexible material, and has a planar base and an outer wall with a periodic protrusion structure; the outer surface of the planar base is also provided with grooves for accommodating the cells to be cultured. The flexible actuator is a hollow structure, which serves as an air chamber; one end of the hollow structure is connected to the air path module of the driving platform through the air hole channel, and the other end remains sealed; the driving platform controls the electromagnetic reversing valve (the electromagnetic reversing valve realizes the output control of the air path) by controlling the electric control switch, and uses the air pump to input and / or discharge the gas in the air chamber of the flexible actuator through the air hole channel of the flexible actuator, and finally controls the pressure in the air chamber of the flexible actuator. Due to the design of the outer wall of the periodic protrusion structure, the flexible actuator as a whole can strengthen the bending degree and reduce the bending degree according to the preset frequency (such as bending-restoration, the bending degree of the flexible actuator is affected by the pressure in the air chamber of the flexible actuator). By controlling the bending and deformation of the flexible actuator, it is possible to provide adjustable mechanical stimulation for the cultured cells or tissues, simulating the mechanical stress in the microenvironment in the body. This mechanical stimulation has an important influence on the proliferation, differentiation and functional expression of cells. The design of the cell culture box matches the flexible actuator to ensure that the cells are in an ideal growth environment during the dynamic culture process, while reducing the consumption of culture medium and improving the culture efficiency.

[0033] Moreover, the driving platform in the present invention can flexibly design the number of channels according to experimental requirements to meet the requirements of cell culture of different scales and complexities. The increase in the number of channels not only improves the experimental throughput, but also significantly improves the cost performance of the driving platform. The system can realize multi-channel operation at the same time, so as to carry out cell culture experiments under various conditions, greatly improving research efficiency and data reliability.

[0034] In the present invention, the bending degree and frequency of the flexible actuator are controllable, and the cell culture box can be flexibly adjusted according to the size of the flexible actuator and the number of channels. The flexible actuator can disturb the culture medium when working, promoting the uniform distribution of oxygen and nutrients. The system provides an excellent and controllable in vitro culture platform for cells, cell-hydrogel, cell-scaffold structures, and cell-hydrogel-scaffold structures, etc., which can simulate the in vivo environment and realize the dynamic culture of cells, and has important application value in the research of cell culture related models.

[0035] The present invention may preferably adopt a small-sized flexible actuator design. The length of the flexible actuator may preferably be 20-36 mm, the height may preferably be 5-10 mm, the width may preferably be 6-10 mm, and the wall thickness may preferably be 1-2 mm. The small-sized flexible actuator is more sensitive to deformation when bending and requires a lower amount of cells.

[0036] Specifically, the controllable multi-channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment in the present invention can achieve the following beneficial effects:

[0037] (1) The number of channels and flexible actuator parameters of the system of the present invention can be flexibly customized according to actual needs to meet the special requirements of various cell culture experiments. Whether it is for the precise culture of a single cell type or complex experiments involving multiple cell types, the system can provide customized solutions. At the same time, by designing grooves on the planar substrate of the flexible actuator, the system can perform various types of in vitro culture, including cell-hydrogel, cell-scaffold structure and cell-hydrogel-scaffold structure, etc., to adapt to different practical application scenarios.

[0038] (2) The present invention can design driving platforms with different numbers of channels according to needs, flexibly adjust the number, style and size of flexible actuators (each flexible actuator corresponds to one channel), and the size of the cell culture box, to achieve space and cost optimization and meet different scientific research needs.

[0039] The culture device in the prior art is bulky, and the number of channels often requires several instruments, which takes up a lot of space. In addition, each instrument is expensive. The more channels there are, the more expensive the whole unit is, and the cost performance is low. The multi-channel design in the present invention is small in size and low in cost. In addition, the multi-channel design allows experiments of different frequencies to be cultured in one cell culture box, reducing the influence of non-variable factors.

[0040] (3) During the operation of the system of the present invention, the flexible driver strengthens the bending degree and reduces the bending degree so that the oxygen and nutrients in the culture medium in the cell culture box are evenly mixed, thereby promoting the material exchange of cells and improving the culture efficiency.

[0041] (4) Compared with traditional rotary and perfusion bioreactors, the cell culture box of this system is smaller in size. In addition to being able to accommodate the support frame (that is, the bottom surface of the open box can completely cover the projection of the support frame on the bottom surface; taking 4 channels as an example, the width of the support frame can enable 4 flexible actuators to be fixed side by side on the support frame, that is, the width of the support frame is greater than or equal to the width of the 4 flexible actuators plus the distance between the two flexible actuators and the reserved width at the edge, and one side of the bottom surface of the cell culture box is larger than the width of the support frame and the other side of the bottom surface is larger than the length of the support frame), the depth is 60 to 80 mm; the amount of culture medium added can be determined according to the bending requirements of the flexible actuator, and it is only necessary to ensure that the culture medium contained in the open box can cover the highest height that the flexible actuator can reach during the bending process (that is, when in use, especially when the flexible actuator is bent, the culture medium can still cover the flexible actuator), so a smaller amount of culture medium can be used, greatly reducing the culture cost.

[0042] (5) This system supports multi-channel dynamic simulation culture of cells. The dynamic culture method helps to improve the physiological relevance of cells, promote cell proliferation, differentiation and functional expression, and make the experimental results closer to the actual situation in vivo.

[0043] (6) The controllable multi-channel three-dimensional dynamic culture system for simulating the cell mechanical microenvironment in the present invention can be preferably designed as a multi-channel three-dimensional dynamic culture system for simulating the cell mechanical microenvironment, wherein the signal control module and the gas path module in the driving platform are multi-channel, and there are multiple flexible drivers, and each flexible driver corresponds to one signal control module and one gas path module in the driving platform, and can independently generate a mechanical microenvironment. The multi-channel design allows experiments under multiple conditions to be carried out simultaneously in the same system, greatly improving the data output efficiency. The design of the system effectively reduces costs while maintaining high performance, making it an ideal choice for laboratory research and industrial applications, taking into account both experimental efficiency and economic benefits.

[0044] (7) The culture system of the present invention can be constructed of different materials according to actual needs (such as polycaprolactone and acrylic types that can be sterilized by ethylene oxide, and silicone types that can be sterilized by high-temperature and high-pressure autoclaves, as shown in the examples below), so that researchers can use products that are suitable for their own laboratories.

[0045] (8) This system is simple in design and easy to operate, suitable for ordinary laboratory environments. Its structure is stable and reliable, which reduces maintenance requirements and user workload, further reducing operating costs and providing researchers with a convenient and efficient experimental platform.

[0046] In summary, the present invention has the advantages of simple structure, low cost, convenient operation and stable operation, and is easy to build and use in various research sites. The number of channels of the driving platform can be adjusted according to actual needs, the size and style of the flexible driver can be customized, and can be prepared into miniaturization, thereby saving raw materials and facilitating batch experiments to study dynamic cell culture. The controllable multi-channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment proposed in the present invention shows significant advantages in batch cell culture and single-factor variable control research, and can provide reliable data support for in-depth understanding of the behavior of cells in the mechanical microenvironment. Its high-throughput, multi-channel design not only improves the experimental efficiency, but also ensures the accuracy and repeatability of the experimental results, and provides strong technical support for fields such as tissue engineering, drug screening and regenerative medicine, which is of great significance to the construction and improvement of in vitro dynamic cell culture models. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram and physical diagram of the eight-channel driving platform of the controllable multi-channel three-dimensional dynamic culture system simulating the cell mechanical microenvironment in Example 3; wherein, Figure 1 (a) in the figure corresponds to the schematic diagram, Figure 1 (b) in the figure corresponds to the actual picture.

[0048] Figure 2 These are three views of the flexible actuator of the controllable multi-channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment in Example 5.

[0049] Figure 3 These are diagrams showing different bending angles driven by the flexible actuator of the controllable multi-channel three-dimensional dynamic culture system simulating the cell mechanical microenvironment in Example 5.

[0050] Figure 4 This is a dimensional diagram of the open box and lid of the controllable multi-channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment in Example 8.

[0051] Figure 5 This is a physical picture of the open box and lid parts obtained by laser cutting in the controllable multi-channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment in Example 8.

[0052] Figure 6 It is a three-dimensional diagram of the support frame of the controllable multi-channel three-dimensional dynamic culture system that simulates the cell mechanical microenvironment in Example 6 and Example 9.

[0053] Figure 7 This is a diagram of a PLA cell culture box loaded with a flexible actuator and culture medium obtained in Example 9.

[0054] Figure 8 This is a diagram of a silicone cell culture box loaded with a flexible actuator and culture medium obtained in Example 10.

[0055] Fig. 9 This is a diagram of a PMMA cell culture box loaded with a flexible actuator and culture medium obtained in Example 11.

[0056] Fig.10 It is the expression of extracellular matrix in the simulated microtissue in Example 13 under the simulated mechanical microenvironment (i.e., driving stimulation according to Low Frequency, Middle Frequency and High Frequency and 60° angle amplitude, driving stimulation for 30 minutes per day, driving stimulation for 7 days, and the control group means that the same conditions as the experimental group are maintained except for driving stimulation). Fig.10 a in the figure is the immunofluorescence staining of type I collagen CoL-I, chondroitin sulfate CS-56, and elastin Elastin; Fig.10 b in the figure is the semi-quantitative fluorescence statistics of CoL-I; Fig.10 c in the figure is the semi-quantitative fluorescence statistics of CS-56; Fig.10 The d in the figure is the semi-quantitative fluorescence statistics of Elastin.

[0057] Figure 1 The meanings of the various figures are as follows: step-down and voltage stabilizing module 1; DC power supply 2; development board 3; junction box 4; electronically controlled switch 5; positive pressure pump 6; positive pressure regulating valve 7; solenoid reversing valve 8; negative pressure regulating valve 9; negative pressure pump 10; and, thin solid lines represent circuit connections, dotted lines represent signal control, and thick solid lines represent gas path connections. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0059] The connectors in the following embodiments are not described in detail. The connectors include common wires, trachea and quick-plug connectors / pagoda connectors. The wires are used to connect all electronic devices. The trachea and quick-plug connectors / pagoda connectors are used to connect the gas circuit system and connect the cell culture box to achieve electrical conduction and gas conduction. For example, the trachea can be a PU tube, a FEP / PTFE tube, a PA tube, a PVC tube, and a silicone tube. The air pump is the power source of the entire air circuit and can include a positive pressure pump and a negative pressure pump.

[0060] Example 1

[0061] A controllable channel three-dimensional dynamic culture system simulating a cell mechanical microenvironment, wherein the construction of a single channel driving platform comprises the following steps:

[0062] Power supply system: The switching power supply is an AC 220V to DC 12V power supply, which is used to power the Arduino uno development board, a single-channel solid-state relay and a negative pressure pump through a 1-input 5-output junction box. At the same time, the power cord is connected to a two-position three-way electromagnetic reversing valve through the output end of the electromagnetic relay, and the positive pressure pump is powered by a 6V power adapter DC.

[0063] Signal system: The single-channel running code is written into the Arduino uno development board. Through the program running, the control signal is sent to the solid-state relay. The solid-state relay affects the air port conversion of the two-position three-way electromagnetic reversing valve by turning on and off, changing the direction of the airflow, thereby affecting the bending of the flexible actuator.

[0064] Air circuit system: the positive pressure pump is connected to the air inlet of the positive pressure regulating valve, the air outlet of the positive pressure regulating valve is connected to the air inlet of the two-position three-way solenoid reversing valve, the negative pressure pump is connected to the air outlet of the negative pressure regulating valve, the air inlet of the negative pressure regulating valve is connected to the exhaust port of the two-position three-way solenoid reversing valve, and the air outlet of the two-position three-way solenoid reversing valve is reserved for the flexible driver.

[0065] Example 2

[0066] A controllable multi-channel three-dimensional dynamic culture system simulating a cell mechanical microenvironment, the construction of a four-channel driving platform includes the following steps:

[0067] Power supply system: The switching power supply is an AC 220V to DC 12V power supply, which powers the Arduino uno development board and four-channel MOSFET through a 1-input and 8-output junction box. The four power lines are connected to the four two-position three-way solenoid reversing valves through the output end of the MOSFET, and the air compressor and vacuum pump are powered separately.

[0068] Signal system: The four-channel running code is written into the Arduino nano development board. Through the program running, the control signal is sent to the four-channel MOSFET. The MOSFET affects the air port conversion of each two-position three-way electromagnetic reversing valve by turning on and off, changing the direction of the airflow, thereby affecting the working state of the flexible driver (there are 4 flexible drivers, which can be controlled independently).

[0069] Air circuit system: The air compressor is connected to the air inlets of four positive pressure regulating valves through a 5-hole quick-connect connector, the air outlet of the positive pressure regulating valve is connected to the air inlet of the two-position three-way solenoid reversing valve, the vacuum pump is connected to the air outlet of the negative pressure regulating valve through a 5-hole quick-connect connector, the air inlet of the negative pressure regulating valve is connected to the exhaust port of the two-position three-way solenoid reversing valve, and the air outlet of the two-position three-way solenoid reversing valve is reserved for the flexible driver.

[0070] Example 3

[0071] A controllable multi-channel three-dimensional dynamic culture system simulating a cell mechanical microenvironment, the construction of an eight-channel driving platform includes the following steps:

[0072] Power supply system: The power supply converts 220V AC into 12V DC, and supplies power to the ESP 32 development board, eight-channel relays and eight negative pressure pumps through a 1-input 20-output junction box. The power line is connected to eight two-position three-way solenoid reversing valves through relays. At the same time, a DC step-down voltage regulator module is used to convert the 12V voltage into 6V and supply power to the eight positive pressure pumps through a 1-input 8-output junction box. Figure 1 shown.

[0073] Signal system: The eight-channel running program is written into the ESP 32 development board. Through the program running, the control signal is sent to the eight-channel electromagnetic relay. The electromagnetic relay affects the air port conversion of each two-position three-way electromagnetic reversing valve by switching on and off, changing the direction of the airflow, thereby affecting the bending of the flexible actuator (there are 8 flexible actuators, which can be controlled independently). Figure 1 shown.

[0074] Air circuit system: The positive pressure pump is connected to the air inlet of the positive pressure regulating valve, the air outlet of the positive pressure regulating valve is connected to the air inlet of the two-position three-way electromagnetic reversing valve, the negative pressure pump is connected to the air outlet of the negative pressure regulating valve, the air inlet of the negative pressure regulating valve is connected to the exhaust port of the two-position three-way electromagnetic reversing valve, and the air outlet of the two-position three-way electromagnetic reversing valve is reserved for the flexible driver, as shown in the following figure. Figure 1 shown.

[0075] Example 4

[0076] A controllable multi-channel three-dimensional dynamic culture system simulating a cell mechanical microenvironment, the construction of a twenty-four-channel driving platform includes the following steps:

[0077] Power supply system: The switching power supply is an AC 220V to DC 12V power supply, which powers the Arduino uno development board and the twenty-four-channel MOSFET through a 1-input and 28-output junction box. The twenty-four power lines are connected to the twenty-four two-position three-way solenoid reversing valves through the output ends of the MOSFET. The air compressor and vacuum pump are powered separately.

[0078] Signal system: The twenty-four-channel running code is written into the Arduino nano development board. The Arduino nano development board is connected to two I / O expansion chips MCP23017 to expand 32 pins. Through the program running, the control signal is sent to the twenty-four-channel MOSFET. The MOSFET affects the air port conversion of each two-position three-way solenoid reversing valve by turning on and off, changing the direction of the airflow, thereby affecting the working state of the flexible actuator (there are 24 flexible actuators, which can be controlled independently).

[0079] Air circuit system: The air compressor is connected to the air inlets of twenty-four positive pressure regulating valves through 24-hole quick-connect connectors, the air outlets of the positive pressure regulating valves are connected to the air inlets of the two-position three-way solenoid reversing valves, the vacuum pump is connected to the air outlets of the negative pressure regulating valves through 24-hole quick-connect connectors, the air inlet of the negative pressure regulating valves is connected to the exhaust port of the two-position three-way solenoid reversing valves, and the air outlets of the two-position three-way solenoid reversing valves are reserved for the flexible driver.

[0080] Example 5

[0081] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, wherein the flexible actuator preparation comprises the following steps:

[0082] (1) Solidworks software was used to design a flexible actuator serrated outer wall mold master (after demolding, a serrated flexible actuator component was obtained, which, after bonding with the substrate, formed an air chamber with a single inlet) and a substrate mold master (after demolding, the flexible actuator component was a substrate with one side smooth and the other side containing a groove structure).

[0083] (2) Export the stl file, slice it with Snapmaker and complete the mold master printing.

[0084] (3) The ecoflex is mixed evenly and injected into the mold master. After it is completely cured, the mold is opened to remove the serrated outer wall and the substrate. The groove design on the substrate facilitates the fixation of the cell-hydrogel / cell-scaffold structure / cell-hydrogel-scaffold structure on the flexible actuator.

[0085] (4) The sawtooth outer wall and the substrate are bonded with a silicone adhesive to obtain a flexible actuator with a length of 26 mm, a height of 6 mm, a width of 8 mm, and a wall thickness of 1 mm. The diameter of the pore channel is 1.5 mm and the length is 4 mm. The number of periodic protrusion structures is 6; there are two grooves on the substrate (the length, width and height of each groove are 12 mm, 2 mm and 1 mm respectively), and the thickness of the substrate itself is 2 mm. The three-view drawing is as follows Figure 2 As shown, the flexible actuator is driven to bend using a driving platform, as shown in Figure 3 As shown, it was found that the flexible actuator can be bent at least 180°, wherein one protrusion structure is located inside the fixture shown in the figure, thereby forming a relatively fixed effect on the actuator.

[0086] (5) Multi-channel actuators can be used to prepare multiple flexible actuators as needed.

[0087] Example 6

[0088] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, wherein the flexible actuator preparation comprises the following steps:

[0089] (1) 3D MAX software was used to design a flexible actuator serrated outer wall mold master (after demolding, a serrated flexible actuator component was obtained, which, after bonding with the substrate, formed an air chamber with a single inlet) and a substrate mold master (after demolding, the flexible actuator component was a substrate with one side smooth and the other side containing a groove structure).

[0090] (2) Export the stl file, slice it using Allcct Printer and complete the mold master printing.

[0091] (3) Mix the AB glue evenly and inject it into the mold master. After it is completely cured, open the mold and take out the serrated outer wall and base.

[0092] (4) The serrated outer wall and the substrate are bonded with silicone adhesive to obtain a flexible actuator with a length of 20 mm, a height of 5 mm, a width of 6 mm, and a wall thickness of 1 mm. The pore channel has a diameter of 1 mm and a length of 2 mm. There are 4 periodic protrusion structures, a groove on the substrate (whose length, width, and height are 8 mm, 2 mm, and 1 mm, respectively), and the thickness of the substrate itself is 2 mm.

[0093] (5) Multi-channel actuators can be used to prepare multiple flexible actuators as needed.

[0094] Example 7

[0095] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, wherein the flexible actuator preparation comprises the following steps:

[0096] (1) Autodesk CAD software was used to design a flexible actuator serrated outer wall mold master (after demolding, a serrated flexible actuator component was obtained, which, after bonding with a substrate, formed an air chamber with a single inlet) and a substrate mold master (after demolding, the flexible actuator component was a substrate with one side smooth and the other side containing a groove structure).

[0097] (2) Export the stl file, slice it with Snapmaker and complete the mold master printing.

[0098] (3) The PDMS sample is mixed with the curing agent and injected into the mold master. After complete curing, the mold is opened and the serrated outer wall and base are removed.

[0099] (4) The serrated outer wall and the substrate are bonded with silicone adhesive to obtain a flexible actuator with a length of 36 mm, a height of 10 mm, a width of 10 mm, and a wall thickness of 2 mm. The pore channel has a diameter of 3 mm and a length of 8 mm. There are 8 periodic protrusion structures, two grooves on the substrate (the length, width, and height of each groove are 15 mm, 4 mm, and 2 mm, respectively), and the thickness of the substrate itself is 3 mm.

[0100] (5) Multi-channel actuators can be used to prepare multiple flexible actuators as needed.

[0101] Example 8

[0102] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, the preparation of a four-channel cell culture box comprises the following steps:

[0103] (1) Draw the dimensions of the four-channel open box and cover in Autodesk CAD. The specific dimensions are as follows: Figure 4 As shown (i.e., the length, width and height of the open box are 70mm, 70mm and 55mm respectively; the length, width and height of the lid are 75mm, 75mm and 5mm respectively).

[0104] (2) Import the pattern into the laser cutting machine and cut the 2 mm thick PMMA to obtain a block of PMMA, such as Figure 5 shown.

[0105] (3) Use shadowless glue to bond the PMMA blocks to obtain an open box and a lid. The cell culture box is a relatively closed space used to accommodate a support frame, culture medium, and four flexible actuators (in actual use, these four flexible actuators can be placed side by side on the same support frame, and the internal space of the open box can accommodate the support frame). When placed in a cell culture incubator, it can meet basic gas exchange and prevent cells, culture medium, etc. from being contaminated by the outside world.

[0106] (4) Use silicone to coat and cure the joint gaps.

[0107] (5) Install the quick-connect connector through the reserved hole in the open box.

[0108] (6) Draw a model of the support frame in 3D Max so that the spacing between the flexible actuators is 5 mm, as shown below: Figure 6 As shown, the support frame serves as a movable support between the flexible actuator and the cell culture box, so as to realize the orderly arrangement of the flexible actuator in the cell culture box and avoid mutual interference between the actuators during operation.

[0109] (7) Export the stl file, slice it using Snapmaker and complete the 3D mold printing.

[0110] (8) Use a PU air tube with an outer diameter of 2 mm to connect one end to the quick-connect interface inside the open box, and the other end to connect to the air hole channel of the flexible actuator in Example 5.

[0111] (9) Fix the flexible actuator on the support frame.

[0112] (10) The assembled cell culture box, the flexible actuator, and the connectors therein are sterilized with ethylene oxide. The volume of the cell culture box is about 350 cubic centimeters.

[0113] Example 9

[0114] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, the preparation of a four-channel cell culture box comprises the following steps:

[0115] (1) Draw the four-channel open box, cover and support frame in 3D Max (see the sample picture). Figure 6 shown) model.

[0116] (2) Export the stl file, slice it with Snapmaker and use PLA to complete the three-dimensional model printing. The size of the obtained four-channel cell culture box is similar to that of Example 8.

[0117] (3) Install the quick-connect connector through the reserved hole in the open box.

[0118] (4) One end of a PTFE air tube with an outer diameter of 2 mm is connected to the quick-connect interface inside the open box, and the other end is connected to the air hole channel of the flexible actuator obtained in Example 6.

[0119] (5) Fix the flexible actuator on the support frame.

[0120] (6) The assembled cell culture box, the flexible actuator and the connectors therein are sterilized with ethylene oxide, and then loaded with the flexible actuator and the culture medium. Figure 7 As shown, the volume of the cell culture box is approximately 350 cubic centimeters.

[0121] Example 10

[0122] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, the preparation of a four-channel cell culture box comprises the following steps:

[0123] (1) Draw the four-channel open box mold master, the lid mold master, and the support frame mold master in 3D Max.

[0124] (2) Export the stl file, slice it with Snapmaker and complete the 3D mold master printing.

[0125] (3) Injecting silica gel into the master mold and demoulding after solidification, the obtained four-channel cell culture box has a size similar to that of Example 8.

[0126] (4) Install the pagoda joint through the reserved hole of the open box.

[0127] (5) Use one end of the silicone air tube to connect to the internal pagoda interface of the open box, and the other end to connect to the air hole channel of the flexible driver through the L-shaped pagoda interface.

[0128] (6) Fix the flexible actuator on the support frame.

[0129] (7) The assembled cell culture box, the flexible actuator and the connectors therein are sterilized by high temperature and high pressure. After loading the flexible actuator and the culture medium, Figure 8 As shown, the volume of the cell culture box is approximately 366 cubic centimeters.

[0130] Embodiment 11

[0131] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, the preparation of a twenty-four-channel cell culture box comprises the following steps:

[0132] (1) Draw the dimensions of the twenty-four-channel open box and cover in Autodesk CAD.

[0133] (2) Import the pattern into a laser cutting machine and cut the PMMA to obtain a block of PMMA.

[0134] (3) Use shadowless adhesive to bond the PMMA blocks together to obtain an open box and a lid.

[0135] (4) Use silicone to coat and cure the joint gaps.

[0136] (5) Install the quick-connect connector through the reserved hole in the open box.

[0137] (6) Draw a model of the support frame in 3D Max so that the spacing between the flexible actuators is 4 mm.

[0138] (7) Export the stl file, slice it using Snapmaker and complete the 3D mold printing.

[0139] (8) Use a PU air tube with an outer diameter of 2 mm to connect one end to the quick-connect interface inside the open box, and the other end to connect to the air hole channel of the flexible actuator obtained in Example 5.

[0140] (9) Fix the flexible actuator on the support frame.

[0141] (10) The assembled cell culture box, the flexible actuator, and the connectors therein are sterilized by ethylene oxide. The volume of the cell culture box is about 2000 cubic centimeters.

[0142] (11) The cell culture box can realize simultaneous stimulation experiments of actuators under multiple conditions, such as Fig. 9In one of the cases shown, the angle amplitude (denoted as α) can be divided into large amplitude (Large Amplitude, 90°≤α≤180°. At this time, in order to ensure that the cells to be cultured are always located in the groove of the flexible actuator, the hydrogel loaded with cells can be added to the groove of the flexible actuator, and the groove can be simply sutured with thread) and small angle amplitude (Small Amplitude, 0°≤α<90°. At this time, due to the small angle amplitude, the cells to be cultured can always be located in the groove of the flexible actuator without suturing. Each angle amplitude zone can achieve driving stimulation of different frequencies such as Low Frequency (such as 1 / 3Hz, i.e., bending and recovering 20 times within 1 minute, simulating bradycardia), Middle Frequency (such as 1Hz, i.e., bending and recovering 60 times within 1 minute, simulating normal heart rate) and High Frequency (such as 3Hz, i.e., bending and recovering 180 times within 1 minute, simulating tachycardia).

[0143] Example 12

[0144] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, wherein the dynamic cell culture comprises the following steps:

[0145] (1) Fresh pig hearts were obtained from a local slaughterhouse. After washing, cutting, digestion, and filtration, the 0th generation of porcine valvular interstitial cells (VICs) were obtained. The 3rd generation of VICs were obtained by culture and amplification. After trypsin digestion and counting, the cells were counted at a rate of 1×10 6 pcs / mL were inoculated into 10% sterilized GelMA solution to obtain bio-ink.

[0146] (2) The bio-ink is subjected to bio-printing and photocuring treatment with a 10W wavelength 405nm ultraviolet light at a distance of 10cm for 15s to obtain simulated microtissues, which are then migrated into the groove of the flexible actuator in the sterilized open box, culture medium is added, and the lid is closed.

[0147] (3) The cell culture box was placed in a cell culture incubator, and the cells were driven and stimulated at a Low Frequency (i.e., 1 / 3 Hz), Middle Frequency (i.e., 1 Hz), and High Frequency (i.e., 3 Hz) at an angle of 60° for 30 min per day for 7 days. Meanwhile, the control group was cultured at an angle of 0° (i.e., no drive stimulation) as a blank control.

[0148] Embodiment 13

[0149] A controllable multi-channel three-dimensional dynamic culture system for simulating a cell mechanical microenvironment, wherein the cell staining experiment comprises the following steps:

[0150] (1) Example 12 After the cells have completed the predetermined culture, the simulated microtissue is taken out.

[0151] (2) The simulated microtissue was washed three times with PBS solution, fixed with 0.04 g / mL paraformaldehyde solution for 15 min, and then permeabilized with 5 mg / mL Triton / PBS solution at room temperature for 8 min. After washing with PBS three times, type I collagen (CoL-I), chondroitin sulfate (CS-56) and elastin (Elastin) antibodies were used to stain type I collagen, polysaccharide and elastin expressed by VIC cells in the simulated microtissue, and photographed with a laser confocal microscope. The results are shown in Figure 2. Fig.10 As shown in the figure, it can be seen that the cells in the simulated microtissue with driving stimulation are superior to the cells without mechanical stimulation in proliferation and matrix remodeling, and when the driving stimulation frequency is 1Hz, the cells' secretion expression of CoL-1, CS-56 and Elastin is much higher than that of the driving frequencies of 1 / 3Hz and 3Hz, indicating that the three-dimensional dynamic culture system is reliable in simulating the in vivo cell mechanical microenvironment.

[0152] The above embodiments are only examples. For example, there are many types of development boards, including Arduino series, STM32 series, ESP32 / ESP8266 series, Particle series and Xilinx series, etc., which can realize the controllable opening and closing of the electric switch through program programming and signal interaction; the specifications of the junction box are selected according to the actual number of channels of the driving platform, which is used to transmit the power of the switching power supply to various electronic devices; AC to DC power adapters include AC 220V to DC 24V, AC 220V to DC 12V, AC220V to DC 6V, ... and other specifications (the actual selection should unify the voltage of the electronic devices as much as possible, and is determined by the power and number of the electronic devices. The buck regulator module assists the switch power supply to complete the construction of the drive platform); the pressure regulating valve uses a high-precision positive pressure gas pressure regulating valve and a high-precision negative pressure gas pressure regulating valve; for example, the positive pressure pump selects the rated voltage of 6V, and the negative pressure pump selects the rated voltage of 12V; if the positive pressure pump selects a large-flow air compressor and the negative pressure pump selects a large-flow vacuum pump, both are connected to 220V AC (i.e., AC power). The electric control switch realizes the instant reversal of the electromagnetic reversing valve through the upstream signal. The electromagnetic reversing valve is rich in types. According to the passage, it is divided into two-way electromagnetic reversing valve, three-way electromagnetic reversing valve, four-way electromagnetic reversing valve and five-way electromagnetic reversing valve, etc., and according to the working position of the valve core, it is divided into two-position electromagnetic reversing valve, three-position electromagnetic reversing valve, etc., receiving the upstream current signal, and finally realizing the rapid conversion of the airflow direction in the gas path. The size of the flexible actuator can be flexibly designed according to demand; the size of the cell culture box can be designed and manufactured according to the size of the flexible actuator and the number of channels (for example, the side-by-side direction of the flexible actuator is the width direction, the length of the open box can be longer than the length of the support frame (for example, it can be longer by more than 10 mm), the width can be longer than the width of the support frame (for example, it can be longer by 1 to 5 mm), the height is 60 to 80 mm, and the lid size is sufficient to cover the open box).

[0153] The above embodiments are only examples. In addition to the sawtooth protrusions, other periodic protrusion structures such as semicircular protrusions and rectangular protrusions can also be used. In this way, a flexible actuator is formed in conjunction with a planar substrate, which can bend when pressurized and recover when depressurized. For another example, the support frame is an optional component. When a cell culture box accommodates multiple flexible actuators and the space inside the cell culture box is relatively compact, there is a possibility that the two actuators will be entangled with each other during operation. Therefore, the support frame can avoid this effect.

[0154] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A controllable channel three-dimensional dynamic culture system simulating a cell mechanical microenvironment, characterized in that: It includes a driving platform, a flexible driver and a cell culture box, wherein: The driving platform includes a power supply module, a signal control module and an air circuit module, wherein the power supply module is used to convert the mains power into a DC power supply with a preset amplitude; the signal control module is provided with electric energy by the DC power supply and is used to control the on and off of the electric control switch at a preset frequency; the air circuit module is connected to the flexible actuator, and includes an air pump, a pressure regulating valve and an electromagnetic reversing valve; wherein the air pump is used to provide air circuit power, and the electric energy is provided by the DC power supply or the mains power; the pressure regulating valve is used to adjust the air flow of the air circuit; the electromagnetic reversing valve is used to adjust the air flow direction under the influence of the on and off of the electric control switch; The flexible actuator is made of a flexible material, and has a planar base and an outer wall provided with a periodic protrusion structure; the flexible actuator has a hollow structure as an air chamber, one end of the hollow structure is connected to the air path module through an air hole channel, and the other end remains sealed; a groove is also provided on the outer surface of the planar base for accommodating cells to be cultured; The cell culture box comprises an open box and a cover, wherein the interior of the open box is used to simultaneously contain the culture medium and the flexible driver; The driving platform can control the electromagnetic reversing valve by controlling the electric control switch, and utilize the air pump to input gas for pressurization or exhaust gas for depressurization through the air hole channel of the flexible driver, so that the flexible driver can strengthen or reduce the bending degree according to the preset frequency, thereby generating a mechanical microenvironment; the bending degree of the flexible driver is affected by the pressure in the air chamber of the flexible driver.

2. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment as claimed in claim 1, characterized in that: There are multiple flexible actuators, each of which corresponds to one signal control module and one gas path module in the driving platform, and can independently generate a mechanical microenvironment to achieve multi-channel culture; The power supply module also includes a junction box for providing a DC power supply as electrical energy to the multi-channel signal control module and the multi-channel gas path module; Preferably, a plurality of the flexible actuators are accommodated in the inner space of one or more cell culture boxes.

3. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment according to claim 1, characterized in that: For any one of the flexible actuators, the length of the flexible actuator is 20-36 mm, the height is 5-10 mm, the width is 6-10 mm, and the wall thickness is 1-2 mm; The pore channel has a diameter of 1 to 3 mm and a length of 2 to 8 mm; The number of the periodic protrusion structures is 4 to 8; The number of the grooves is 1 to 2; the length of any one of the grooves is 8 to 15 mm, the width is 2 to 4 mm, and the depth is 0.5 to 2 mm.

4. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment as claimed in claim 2, characterized in that: A support frame is also contained in the internal space of each cell culture box, and a flexible actuator is placed at each placement position on the support frame to avoid mutual interference between the flexible actuators contained in the internal space of the same cell culture box; A plurality of the flexible actuators can be placed side by side on the same support frame, the width of the support frame can accommodate the side-by-side flexible actuators, and the interval between two adjacent flexible actuators is 2 to 6 mm; the length of the support frame is greater than or equal to the length of the flexible actuator; The length of the cell culture box is greater than the length of the support frame, and the width is greater than the width of the support frame; The height is 60~80mm.

5. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment according to claim 1, characterized in that: The flexible actuator is prepared by pouring, curing and demoulding silicone in a mold with a predetermined shape to form a planar base and an outer wall with a periodic protrusion structure, respectively, and then bonding the planar base and the outer wall with the periodic protrusion structure. Preferably, the silica gel is one or more of AB glue, ecoflex, and PDMS.

6. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment according to claim 1, characterized in that: The cell culture box is made of PMMA, PLA or silica gel; Preferably, the silica gel is selected from: AB glue, ecoflex, PDMS.

7. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment according to claim 1, characterized in that: The electronically controlled switch includes a relay, a thyristor, a field effect transistor (MOSFET), a solid-state relay (SSR), a photocoupler or an insulated gate bipolar transistor (IGBT); The electromagnetic reversing valve is selected from a two-way electromagnetic reversing valve, a three-way electromagnetic reversing valve, a four-way electromagnetic reversing valve and a five-way electromagnetic reversing valve; preferably a two-position three-way electromagnetic reversing valve; The power supply module is specifically an AC to DC power adapter.

8. The controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment according to claim 1, characterized in that: The air pump includes both a positive pressure pump and a negative pressure pump, and the pressure regulating valve includes both a positive pressure regulating valve and a negative pressure regulating valve, wherein the positive pressure regulating valve is connected to the positive pressure pump, and the negative pressure regulating valve is connected to the negative pressure pump; Preferably, the rated voltage of the positive pressure pump is 6V, and the rated voltage of the negative pressure pump is 12V.

9. Use of the controllable channel three-dimensional dynamic culture system for simulating cell mechanical microenvironment as claimed in any one of claims 1 to 8 in dynamic cell culture.

10. Use of the controllable channel three-dimensional dynamic culture system simulating cell mechanical microenvironment as claimed in any one of claims 1 to 8 in dynamic cell culture for the purpose of in vitro tissue organoid preparation and drug screening.

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