Biological cell stimulation system based on pneumatic annular mold and control method thereof

Abstract

The present invention is specifically a biological cell stimulation system based on a pneumatic annular mold and a control method thereof, comprising: an annular culture mold, an air pump device; the annular culture mold is arranged in a culture dish and connected to the air pump device through an air duct; the annular culture mold comprises: an upper mold, a lower mold and a mold base; the upper mold and the lower mold, and the lower mold and the mold base are sealed and connected to form an annular culture mold with a variable volume; the mold base is arranged in a culture dish; a through hole is provided on the upper mold, and the air pump device is connected to the lower mold through the air duct through the through hole. Since the present invention has no special requirements in the application of promoting differentiation of cell tissue culture, there is no need for complex processing of cell samples, and the integration of culture and stimulation can be realized, which effectively reduces the damage and infection probability of cell tissues during the culture and stimulation process, and is used in skeletal muscle tissue engineering, drug screening, and the research of life-like robots with muscle cells as driving units.

Description

Technical Field

[0001] The present invention relates to a biological cell culture system that can generate mechanical stimulation of any variable period and multiple modes, specifically, mechanical strain stimulation of any frequency, duration and amplitude is achieved by combining the uniform stress of the annular culture mold and the controllability of any gas output, retention and release of the air pump device. It is mainly used for electrical stimulation of biological cells to promote growth and differentiation, and has important potential applications in the field of life sciences, such as skeletal muscle tissue engineering, drug screening, and the research of life-like robots with muscle cells as driving units. Background Art

[0002] The life-like robot system formed by the organic deep integration of life system and electromechanical system may have the advantages of both organisms and machines, such as the unique multi-degree of freedom, high energy conversion efficiency, high energy density, biocompatibility, and self-repair characteristics of life, as well as the high precision, high strength, high repeatability, and good controllability of electromechanical system. Therefore, the research on life-like robots driven by living muscle cells has attracted a lot of attention. Now, micro-robots driven by active molecular motors, microorganisms, muscle cells, insect dorsal vascular tissue, etc. have been realized. Among them, skeletal muscle, as the main power source of mammals, has the advantages of large contraction force and good controllability, and is expected to become the main driving unit of the new generation of life-like robots. However, myoblasts need to differentiate into myotubes to produce contraction movement under external stimulation (such as electrical stimulation), and the low differentiation rate limits the development of skeletal muscle cell-driven life-like robot research. Studies have shown that periodic mechanical stimulation can promote muscle cell growth, such as myoblast proliferation, differentiation, myotube maturity, arrangement, and contraction characteristics of myotubes. In the current research on mechanical stimulation to promote cell growth and differentiation, the parallel plate stretching stimulation method is mostly used, but there are certain limitations in its application. For example, when using a parallel stretching stimulation system for muscle cell stimulation training, the cell tissue culture must first be formed, and then the formed tissue is transferred to the mechanical stimulation training device, and the two ends of the cell tissue are fixed to the stretching structure to conduct cell tissue training. Although similar mechanical stimulation methods can promote cell growth and differentiation, since cell tissue culture and stimulation need to be completed on two systems separately, the complexity of the process and the probability of contamination are increased. Therefore, from a physical principle, the development of a biological cell stimulation system based on a pneumatic annular mold plays an important role in realizing the in vitro culture and promotion research of biological cells / tissues. Summary of the invention

[0003] The purpose of the present invention is to provide an intelligent lithium-ion battery management system for electric vehicles with stable performance, small size, low cost and low power consumption, so as to overcome the defects of the above-mentioned battery management system.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0005] A biological cell stimulation system based on a pneumatic annular mold, comprising: an annular culture mold, an air pump device;

[0006] Wherein, the annular culture mold is arranged in a culture dish;

[0007] The air pump device is used to output pulsed air pressure and is connected to the annular culture mold (1) through a conduit, thereby causing the annular mold to produce periodic deformation, thereby achieving variable mechanical stimulation of biological cells.

[0008] The annular culture mold comprises: an upper mold, a lower mold and a mold base; the upper mold and the lower mold, and the lower mold and the mold base are sealed to form an annular culture mold with a variable volume; the mold base is arranged in a culture dish;

[0009] The upper mold is provided with a through hole, and the air pump device is connected with the lower mold through an air guide pipe through the through hole.

[0010] The upper mold is a disc-shaped mold, and the through hole is opened at the center of the mold.

[0011] The lower mold is a hollow cylindrical mold, which is used to change the diameter of the hollow cylindrical body in the center of the mold under the action of the gas input through the conduit;

[0012] The cross-sectional diameter of the lower mold is the same as the cross-sectional diameter of the upper mold.

[0013] The mold base is a disc-shaped base, and a circular groove is formed on the disc-shaped base. The circular groove is used to culture a biological cell mixture to form an annular microtissue.

[0014] The lower mold is installed in the circular groove, and the axis of the lower mold is concentrically arranged with the center of the circular groove; the height of the lower mold is greater than the groove height of the circular groove.

[0015] The height of the annular culture mold is smaller than the height of the culture dish.

[0016] The lower mold is a round tube made of polydimethylsiloxane.

[0017] The air guide tube is any one of a PP air guide tube, a PE air guide tube and a PVC air guide tube.

[0018] The control method of the biological cell stimulation system based on the pneumatic annular mold is characterized by comprising the following steps:

[0019] 1) Fix the annular culture mold with the biogel on the bottom of the culture dish and connect the airway to the air pump device;

[0020] 2) Changing the driving parameters of the air pump device, starting the air pump device, and making the air pump device realize periodic pulse gas output, thereby achieving mechanical stress stimulation on the cell tissue.

[0021] The air pump device realizes periodic pulse gas output, comprising the following steps:

[0022] The frequency of pneumatic pulse stimulation is N Hz, within a stimulation cycle T, T=1 / N, the pressure rise time of the variable volume annular culture mold (1) is t1, the pressure holding time is t2, the pressure drop time is t3, and the pressure-free time is t4, then a stimulation cycle T=t1+t2+t3+t4; the air pump is enabled to perform periodic pressure output, holding, and release, thereby achieving periodic deformation of the annular culture mold and promoting cell tissue growth and differentiation.

[0023] The present invention has the following beneficial effects and advantages:

[0024] 1. The present invention utilizes a ring-shaped culture mold to perform uniform stress stimulation on the cultured cell tissues, thereby improving the efficiency of in vitro differentiation of the tissues.

[0025] 2. The present invention utilizes an annular culture mold and a controllable air pump device to generate mechanical stimulation with controllable inflation rates, final pressures, deflation rates, and duty cycles, which can be used for optimization and analysis of in vitro cell tissue growth and differentiation conditions.

[0026] 3. Since the present invention has no special requirements in the application of promoting cell tissue culture differentiation, there is no need for complicated processing of cell samples, and the integration of culture and stimulation can be achieved, which can effectively reduce the damage and contamination rate of cell tissues during the culture and stimulation process. Therefore, it has important potential applications in the field of life sciences, such as skeletal muscle tissue engineering, drug screening, and the research of life-like robots with muscle cells as driving units. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1a It is a schematic diagram of the system structure principle of the present invention;

[0028] Figure 1b This is a schematic diagram of the pneumatic annular culture mold in the present invention;

[0029] Among them, 1 is a pneumatic annular culture mold, 2 is a culture dish, 3 is a culture box, 4 is an air guide tube, 5 is an air pump device, 6 is an upper mold, and 7 is a lower mold;

[0030] Figure 2 This is a picture of the results of promoting cell tissue growth and differentiation using the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] like Figure 1a As shown, the present invention relates to a biological cell stimulation system based on a pneumatic annular mold and a control method thereof, including a volume-variable annular culture mold and an air pump device, which can realize mechanical stress stimulation to promote cell growth and differentiation, and can be used in conjunction with electric pulse stimulation to achieve further differentiation of skeletal muscle microtissues.

[0033] The present invention comprises: a volume-variable annular culture mold 1 and an air pump device 5 connected thereto;

[0034] The annular culture mold 1 is arranged in the culture dish 2; the air pump device 5 is connected to the annular culture mold 1 through the air guide tube 4, and is used to output pulsed air pressure. It is connected to the annular culture mold 1 with variable volume through the air guide tube 4, so that the annular culture mold 1 produces periodic deformation, thereby realizing variable mechanical stimulation of biological cells.

[0035] The volume-variable annular culture mold 1 is hollow inside, and is used to change the diameter of the hollow column in the center of the mold under the action of gas input through the air guide tube 4.

[0036] The volume-variable annular culture mold 1 has an annular groove on its outside for culturing a biological cell mixture to eventually form an annular microtissue.

[0037] The volume-variable annular culture mold 1 is fixed to the bottom of the culture dish 2 .

[0038] The height of the variable-volume annular culture mold 1 is smaller than the height of the culture dish 2 .

[0039] The air pump device 5 is used to output pulsed air pressure and is connected to the variable-volume annular culture mold through a conduit, thereby causing the annular culture mold 1 to produce periodic deformation, thereby achieving variable mechanical stimulation of biological cells.

[0040] In the biological cell stimulation system based on the pneumatic annular mold 1, the annular mold can be assembled on different types of biological culture dishes 2, fixed at the bottom of the culture dish 2, and connected to the output end of the air pump device 5 through the air guide tube 4. By changing the driving parameters of the air pump device 5, including the output inflation rate, final pressure, deflation rate, duty cycle, etc., the deformation mode of the annular culture mold 1 can be adjusted, thereby performing multi-mode controllable mechanical stimulation on the cultured cell tissue. When the system is applied to promote the growth and differentiation of cell tissues (such as differentiation of myoblasts into myotubes), the annular culture mold 1 is fixed to the bottom of the culture dish 2 and placed in the incubator 3, and connected to the biological culture device, that is, the air pump device 5 outside the incubator, through the air guide tube 4 to achieve mechanical stimulation.

[0041] The annular culture mold 1 can be assembled on different types of culture dishes 2, and the annular culture mold 1 is connected to the output port of the air pump device 5. During use, the culture dish 2 equipped with the annular culture mold 1 is placed in the incubator 3 for in vitro culture and stimulation of cell tissues.

[0042] like Figure 1b As shown, the annular culture mold 1 comprises an upper and lower part, a lower part and a mold base, the mold base is fixed to the culture dish, and the upper part is connected to the gas guide tube 4. When in use, the upper and lower parts are bonded to form a hollow shape. The total height of the annular culture mold 1 is lower than the height of the culture dish 2, and thus it can be completely submerged in the culture dish 2 during use. Driven by the air pump device 5, the diameter of the culture mold 1 can produce a controllable periodic deformation of less than 10 Hz and less than 10%, thereby mechanically stimulating the cell tissue therein to promote the growth and differentiation of the cell tissue.

[0043] The control method of the present invention is as follows:

[0044] 1) Fixing the annular culture mold 1 with the biogel on the bottom of the culture dish 2, and connecting the air guide tube 4 to the air pump device 5;

[0045] 2) Changing the driving parameters of the air pump device 5, starting the air pump device 5, and making the air pump device 5 realize periodic pulse gas output, thereby realizing mechanical stress stimulation on the cell tissue.

[0046] The air pump device 5 realizes periodic pulse gas output, including the following steps:

[0047] The frequency of pneumatic pulse stimulation is N Hz, within a stimulation cycle T, T=1 / N, the pressure rise time of the variable volume annular culture mold (1) is t1, the pressure holding time is t2, the pressure drop time is t3, and the pressure-free time is t4, then a stimulation cycle T=t1+t2+t3+t4; the air pump is enabled to perform periodic pressure output, holding, and release, thereby achieving periodic deformation of the annular culture mold and promoting cell tissue growth and differentiation.

[0048] Embodiment 1

[0049] 1) Myoblasts (C2C12), matrix gel, fibrinogen, thrombin, and culture medium biological mixture are added to the PDMS circular culture mold 1. After the mixture solidifies, 10 ml of cell proliferation medium (89% volume of high-glucose DMEM medium + 10% volume of fetal bovine serum + 1% volume of penicillin-streptomycin mixture) is added to submerge the biological gel in the cell culture mold, and cultured in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide;

[0050] 2) Replace the cell proliferation medium every day until 24 hours after culture, then aspirate the cell proliferation medium in the culture dish, wash the cells three times with sterile phosphate buffered saline (PBS), 3 minutes each time, add cell differentiation medium (97% volume of high-glucose DMEM medium + 2% volume of horse serum + 1% volume of penicillin-streptomycin mixture), and continue to culture in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide;

[0051] 3) After 24 hours of culture, aspirate the differentiation medium in the culture dish, wash the cells three times with sterile PBS for 3 minutes each time, and add new cell differentiation medium;

[0052] 4) Then, the air pump device 5 is started to apply mechanical stress stimulation to the cell tissue, and the output parameters of the air pump device 5 are adjusted, such as: the output inflation rate is 1 ml / s, the final pressure is 110 kPa, the deflation rate is 1 ml / s, the duty cycle is 30%, the stimulation time is 1 hour per stimulation, 1 hour rest, and the duration is 12 hours;

[0053] 5) After 12 hours of mechanical stimulation, the cells were cultured for another 4 days without stimulation and the cell differentiation medium was replaced every day;

[0054] Take out a cell tissue that has been cultured after mechanical stimulation training, observe the differentiation of myotubes and perform immunofluorescence staining, label myotube-specific proteins (such as myosin heavy chain), and perform immunofluorescence imaging, recording and analysis of the staining results, such as Figure 2 .

Claims

1. A biological cell stimulation system based on a pneumatic annular mold, characterized in that: include: Ring-shaped culture mold (1), air pump equipment (5); Wherein, the annular culture mold (1) is arranged in a culture dish (2); The air pump device (5) is used to output pulsed air pressure and is connected to the annular culture mold (1) through a conduit, thereby causing the annular mold to produce periodic deformation, thereby achieving variable mechanical stimulation of biological cells; The annular culture mold (1) comprises: an upper mold, a lower mold and a mold base; the upper mold and the lower mold, and the lower mold and the mold base are sealed to form an annular culture mold with a variable volume; the mold base is arranged in a culture dish (2); The upper mold is provided with a through hole, and the air pump device (5) is connected to the lower mold through the air guide pipe (4) through the through hole; The upper mold is a disc-shaped mold, and the through hole is opened at the center of the mold; The lower mold is a hollow cylindrical mold, which is used to change the diameter of the hollow cylindrical body in the center of the mold under the action of the gas input through the conduit; The cross-sectional diameter of the lower mold is the same as the cross-sectional diameter of the upper mold; The mold base is a disc-shaped base, and a circular groove is formed on the disc-shaped base. The circular groove is used to culture a biological cell mixture to form an annular microtissue.

2. The biological cell stimulation system based on pneumatic annular mold according to claim 1, characterized in that: The lower mold is installed in the circular groove, and the axis of the lower mold is concentrically arranged with the center of the circular groove; the height of the lower mold is greater than the groove height of the circular groove.

3. The biological cell stimulation system based on pneumatic annular mold according to claim 1, characterized in that: The height of the annular culture mold (1) is smaller than the height of the culture dish (2).

4. The biological cell stimulation system based on pneumatic annular mold according to claim 1, characterized in that: The lower mold is a round tube made of polydimethylsiloxane.

5. The biological cell stimulation system based on pneumatic annular mold according to claim 1, characterized in that: The air guide tube (4) is any one of a PP air guide tube, a PE air guide tube and a PVC air guide tube.

6. The control method of the biological cell stimulation system based on the pneumatic annular mold according to claim 1, characterized in that: The following steps are involved: 1) fixing the annular culture mold (1) with the biogel on the bottom of the culture dish (2), and connecting the air guide tube (4) to the air pump device (5); 2) changing the driving parameters of the air pump device (5), starting the air pump device (5), and making the air pump device (5) realize periodic pulse gas output, thereby realizing mechanical stress stimulation on the cell tissue; The air pump device (5) realizes periodic pulse gas output, comprising the following steps: The frequency of pneumatic pulse stimulation is N Hz, within a stimulation cycle T, T=1 / N, the pressure rise time of the variable volume annular culture mold (1) is t1, the pressure holding time is t2, the pressure drop time is t3, and the pressure-free time is t4, then a stimulation cycle T=t1+t2+t3+t4; the air pump is enabled to perform periodic pressure output, holding, and release, thereby achieving periodic deformation of the annular culture mold and promoting cell tissue growth and differentiation.

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