Bionic cell 2d / 3d culture system method and control method therefor

Abstract

Provided are a bionic cell 2D / 3D culture system and a control method therefor, wherein a control cavity and a culture cavity that are isolated from each other are formed in a main housing; the bottom of a cell culture plate is an elastic material layer, and a culture groove comprising an annular wrinkle structure is formed at the bottom of a culture chamber, wherein the annular wrinkle structure simulates a fold structure in an organism; a jacking-up mechanism drives a columnar protrusion to periodically move up and down, and squeezing the elastic material layer enables the annular wrinkle structure to periodically contracts and relaxes cells, thereby providing a mechanical tensile force for cells in the culture chamber; a static pressure mechanism communicates with the culture cavity and is used for regulating the air pressure in the culture cavity to provide a hydrostatic pressure for cells in the culture chamber. The influence of an organ structure with wrinkles on cell growth and development is simulated by means of a bionic spatial structure. Moreover, deformation can be uniformly applied to a hydrogel as a whole, and an adjustable mechanical tensile force and a hydrostatic pressure are applied to cells individually or simultaneously to recreate the real mechanical environment of cells in an organism.

Description

[Corrected according to Rule 26 09.08.2024] A bionic cell 2D / 3D culture system and its control method TECHNICAL FIELD

[0001] The present application relates to the field of in vitro cell culture, in particular to a bionic cell 2D / 3D culture system and its control method. BACKGROUND

[0002] More and more evidence shows that the physical properties of the cell culture microenvironment, such as the physical forces applied to cells and tissues, are involved in tissue development, repair, homeostasis, cell movement, proliferation, metabolism and differentiation. In the in vitro culture of cells and organoids, mechanical forces acting on stem cells participate in the growth and differentiation process of stem cells forming organoids through biochemical signal transduction. Reducing the physical microenvironment system of cells is crucial for accurately constructing normal tissues and disease models.

[0003] 3D cell culture in traditional static methods, such as organoid culture, relies on passive diffusion to provide nutrients and remove waste. However, as the organoids continue to grow and have higher requirements for metabolism, the limited diffusion of nutrients and oxygen to the inner regions of the 3D support mechanism leads to the formation of a necrotic core, a significant reduction in cell viability, and ultimately the spread through the entire hydrogel structure. Normal 2D culture cannot reflect the three-dimensional structural characteristics of cells in vivo, and tumor cells cultured in it also cannot reflect the heterogeneity of tumors, and the genome changes during unlimited passage, making it easy to lose the genetic characteristics of tumors.

[0004] Current cell stretching equipment on the market is not optimized for three-dimensional cell culture, such as organoids. In traditional dynamic cell 2D culture, cells are in a single-layer structure and adhere to the surface of an elastic material. During the periodic stretching of the elastic material, the cells are passively stretched. In three-dimensional culture, cells are in a clumped state and grow in a hydrogel. If the hydrogel containing organoids is directly planted on the surface of the elastic material, the elastic material can only stretch and apply stress to the bottom surface of the hydrogel, and cannot uniformly deform the entire hydrogel. In addition, the hydrogel and the surface of the elastic material are prone to displacement and loss of adhesion, resulting in deformation of the elastic material while the hydrogel does not deform or only deforms slightly.

[0005] In addition to cell stretching and compression forces, hydrostatic pressure is another important mechanical force that cells experience in complex in vivo environments. Hydrostatic pressure exerts force on the cell membrane, affecting the stability of microtubules within the cytoskeleton in a non-directional manner, causing changes in cell morphology and function. These physical external forces can cause changes in cell matrix properties such as elasticity and stiffness, and affect cell growth, differentiation and gene expression. However, existing cell culture protocols ignore the influence of this factor.

[0006] Many cells and organs in the body are in the environment of being subjected to the action of stretching and static pressure at the same time, such as the urogenital system (bladder, urethra, ureter, vas deferens, seminal vesicle, uterus, fallopian tube, etc.), digestive system (esophagus, intestinal tract, stomach, gallbladder and bile duct, etc.), cardiovascular system (heart, artery and vein), respiratory system (trachea, lung) and other smooth muscle, skeletal muscle tissue. Taking the bladder as an example, the bladder epithelial cells are subjected to the stretching force parallel to the bladder wall direction and the hydrostatic pressure perpendicular to the bladder wall direction during the urination cycle. With the normal urination cycle of the bladder, the urine pressure produces regular dynamic changes, ranging from 0 to 40 mm of water column, and the normal urination cycle of a normal person is 6-7 hours. However, there is no dynamic bionic culture device that can simulate the stretching force and the static pressure at the same time, it is difficult to truly simulate the physical and biological regular environment of the normal cells and tumor cells in the body, and the influence of the dynamic physical environment on the metabolism, differentiation, gene expression and related signal pathways of the organoids, thereby possibly affecting the accuracy of the experiment and drug screening.

[0007] In addition, in addition to the villi and fold structure of the intestinal tract, the bladder epithelium, vascular endothelium, ureter, fallopian tube and other tissues with expansion function have a plica structure, and the plica structure in such tissues is closely related to other mechanical factors. There is no cell culture device combining a bionic biological morphological scaffold (such as a villus and a fold structure) and a dynamic mechanical force at present, and the mechanical environment in the body cannot be accurately restored.

[0008] SUMMARY

[0009] Therefore, the present application provides a bionic cell 2D / 3D culture system and a control method thereof to solve at least one of the above technical problems. The specific scheme is as follows:

[0010] A bionic cell 2D / 3D culture system comprises a main shell, a control unit, a cell culture plate, a lifting mechanism, a driving mechanism and a static pressure mechanism, the control unit is respectively connected with the driving mechanism and the static pressure mechanism in communication;

[0011] The main shell is formed with a control cavity and a culture cavity which are isolated from each other, the cell culture plate is fixed in the culture cavity and is internally distributed with a plurality of culture chambers for culturing cells; the control unit, part of the driving mechanism and at least part of the static pressure mechanism are located in the control cavity;

[0012] The bottom of the cell culture plate is an elastic material layer, and each culture chamber forms a culture groove containing an annular fold structure at the elastic material layer, and the annular fold structure is used for simulating the plica structure in the body;

[0013] The lifting mechanism is connected with the driving mechanism, and columnar protrusions are distributed below each culture tank, for driving the columnar protrusions to move up and down periodically under the driving of the driving mechanism, realizing the periodic contraction and relaxation of the annular wrinkle structure on the cells by extruding the elastic material layer in different degrees, and further providing mechanical stretching force for the cells in the culture chamber.

[0014] The static pressure mechanism is connected with the culture cavity, for adjusting the air pressure in the culture cavity to provide hydrostatic pressure for the cells in the culture chamber.

[0015] In some specific embodiments, a detection mechanism is further included, which is connected with the control unit, for detecting environmental data including air pressure, temperature and humidity in the culture cavity, and / or detecting the stretching amplitude of the annular wrinkle structure, and / or detecting the growth state of the cells.

[0016] In some specific embodiments, the lifting mechanism includes a rotating part and a lifting part; the rotating part is connected with the driving mechanism, for rotating under the driving of the driving mechanism; the lifting part is distributed with the columnar protrusions on one side and is provided with the rotating part on the other side;

[0017] One of the rotating part and the lifting part is provided with an annular wave structure, and the other of the rotating part and the lifting part is provided with a plurality of sliding parts, which slide periodically along the surface of the annular wave structure, realizing the periodic up-and-down movement of the lifting part relative to the rotating part.

[0018] In some specific embodiments, the driving mechanism includes a driving part, a first transmission part and a second transmission part;

[0019] The first transmission part is located in the control cavity and is connected with the driving part; the second transmission part is located in the culture cavity and is connected with the lifting mechanism; the first transmission part and the second transmission part realize contactless power transmission through magnetic coupling, ensuring the independence between the culture cavity and the control cavity.

[0020] In some specific embodiments, a supporting mechanism is further included in the culture cavity, and the cell culture plate is detachably fixed to the upper side of the supporting mechanism; the supporting mechanism and the inner side wall of the main shell form a lifting cavity, and the lifting mechanism is located in the lifting cavity;

[0021] A through hole is provided through the side of the supporting mechanism facing the cell culture plate, and the columnar protrusions enter and exit the lifting cavity through the through hole to contact the elastic material layer.

[0022] In some embodiments, the lifting mechanism further comprises a position detecting member in communication with the control unit, and the control unit determines the position of the cylindrical protrusion relative to the elastic material layer according to the information fed back by the position detecting member, and further obtains the stretching amplitude of the cell by the annular wrinkle structure.

[0023] The position detecting member is located at the lifting portion to detect the contact area between the annular wave structure and the sliding portion; and / or, the position detecting member is located at the rotating portion to detect the rotating orientation of the rotating portion; and / or, the position detecting member is located at the driving mechanism to detect the driving angle of the driving mechanism to the rotating portion, so as to determine the rotating orientation of the rotating portion.

[0024] In some embodiments, the static pressure mechanism comprises a pressurizing pump, a pressurizing pipeline and a gas pressure sensing device, and the pressurizing pump and at least part of the pressurizing pipeline are located in the control cavity.

[0025] The gas pressure sensing device is located in the culture cavity and in communication with the control unit.

[0026] The pressurizing pump is in communication with the culture cavity through the pressurizing pipeline, and the control unit is in communication with the pressurizing pump to obtain the gas pressure information fed back by the gas pressure sensing device, and further to adjust the gas pressure in the culture cavity by controlling the pressurizing pump, so as to change the hydrostatic pressure applied to the cell in the culture cavity.

[0027] In some embodiments, the lifting mechanism further comprises a support member; the support member is fixed inside the main housing and penetrates through the rotating portion, and is used to support the lifting portion together with the rotating portion or to support the lifting portion alone when the lifting portion is lowered to a preset position.

[0028] The bottom of the support member is provided with a pressure sensor in communication with the control unit, and the control unit is used to determine the position of the lifting portion according to the feedback of the pressure sensor.

[0029] In some embodiments, when the liquid level of the culture medium for culturing cells in the culture chamber is lower than the maximum lifting height of the cylindrical protrusion, the lifting mechanism can be further used to periodically drive the cells in the culture chamber to enter and exit the culture medium, so as to simulate the culture of the cells at the gas-liquid interface.

[0030] A control method of a bionic cell 2D / 3D culture system, used for controlling the bionic cell 2D / 3D culture system according to any one of the above embodiments; the control method comprises:

[0031] The cells are directly placed into the bottom of the culture tank for 2D culture, and the cells adhere to the surface of the elastic material layer in the bottom; or, the uncured hydrogel containing the cells is injected for 3D cell culture; in the 3D culture, the hydrogel fills the culture tank under the action of the surface tension caused by the closure structure of the culture tank; after standing, the hydrogel is cross-linked and solidified;

[0032] The control unit controls the periodic up-and-down movement of the columnar protrusion on the jacking mechanism, and the columnar protrusion causes the relaxation or contraction of the annular wrinkle structure, thereby driving the cells on the surface of the elastic material in the 2D culture or the movement of the hydrogel and the cells in the 3D culture;

[0033] During the rising of the columnar protrusion, the jacking of the elastic material layer is gradually increased, a horizontal force is generated on the annular wrinkle structure of the elastic material layer to make the annular wrinkle structure relax, thereby simulating the influence of the tissue with the wrinkle structure in the relaxed state on the cells in the biological body;

[0034] When the gas-liquid interface culture is not needed, the depth of the culture medium is greater than the jacking height of the elastic material layer, and the cells are always immersed in the culture medium during the movement of the elastic material layer;

[0035] When the gas-liquid interface culture is needed, the depth of the culture medium is less than the jacking height of the elastic material layer; when the columnar protrusion rises to the preset height, the hydrogel can be separated from the liquid surface in the culture chamber and be in contact with the air in the culture chamber, and the gas-liquid interface culture is realized within a certain period of time;

[0036] During the descending of the columnar protrusion, the jacking degree of the elastic material layer is gradually reduced, and the elastic material layer retracts under the action of elasticity and gradually contracts together with the annular wrinkle structure, thereby simulating the influence of the tissue with the wrinkle structure in the contracted state on the cells in the biological body;

[0037] When the columnar protrusion descends to the preset height, the hydrogel re-enters the liquid surface in the culture chamber and is separated from the air in the culture chamber;

[0038] During the cell culture, the control unit controls the operation of the static pressure mechanism to adjust the air pressure in the culture cavity to provide the hydrostatic pressure for the cells in the culture chamber;

[0039] After the cell culture is completed, the cell collection plate is used to cover the culture chamber on the cell culture plate, the cell culture plate is inverted, and the cultured cells are separated by centrifugation so that the cells fall into the cell collection plate after centrifugation.

[0040] Beneficial effects: the application provides a bionic cell 2D / 3D culture system and a control method thereof, which integrates a control unit, a cell culture plate, a lifting mechanism, a driving mechanism and a static pressure mechanism and other related modules into the shell, not only reduces the volume of the bionic cell 2D / 3D culture system, but also expands the functionality of the bionic cell 2D / 3D culture system, so that it can be compatible with more modes of cell culture. The influence of the bionic space structure simulating the wrinkled organ structure on the growth and development of cells is simulated, and the deformation of the hydrogel as a whole is uniformly applied, the adjustable mechanical stretching force and the fluid static pressure are applied to the cells separately or simultaneously, and the real mechanical environment of the cells in the biological body is restored. On the basis of maintaining the three-dimensional morphology of the organoid in the hydrogel, mechanical force is applied to the cells in the hydrogel, so as to reduce the probability of three-dimensional structure collapse caused by organoid digestion. Through the design of the wrinkled morphology in the culture tank, a bionic space structure is provided to simulate the organ structure with wrinkled structure such as bladder folds and digestive tract epithelium, and the relative displacement of the hydrogel and the elastic material does not occur during stretching. By adjusting the mechanical stretching force and the stretching mode of the lifting structure, the stretching amplitude of the cells is controlled, and the interface culture is indirectly realized. Through the control unit and the sensors on the multiple mechanisms, the culture state and the mechanical state can be monitored and controlled. The modular design of the cell culture plate can be replaced and adjusted according to the requirements. The culture cavities and the control cavities are isolated from each other, so as to reduce the influence between the cavities. BRIEF DESCRIPTION OF DRAWINGS

[0041] Fig. 1 is a perspective view of the bionic cell 2D / 3D culture system according to the embodiment of the application;

[0042] Fig. 2 is a front view of the bionic cell 2D / 3D culture system according to the embodiment of the application;

[0043] Fig. 3 is an exploded view of the cell culture plate according to the embodiment of the application;

[0044] Fig. 4 is an exploded view of the lifting structure according to the embodiment of the application;

[0045] Fig. 5 is a perspective view of the lifting structure according to the embodiment of the application;

[0046] Fig. 6 is a schematic view of the ring-shaped wrinkled structure according to the embodiment of the application;

[0047] Fig. 7 is a schematic view of the connection between the cell collection plate and the culture plate according to the embodiment of the application;

[0048] Fig. 8 is a schematic view of the cell culture plate with a mounting groove according to the embodiment of the application;

[0049] Fig. 9 is a schematic view of the mounting groove on the culture plate body according to the embodiment of the application;

[0050] Figure 10 is a schematic view of a culture plate cover with a funnel structure according to an embodiment of the present application.

[0051] Reference numerals: 1 - main housing; 2 - cell culture plate; 3 - lifting mechanism; 4 - driving mechanism; 5 - static pressure mechanism; 6 - supporting mechanism; 7 - fixed plate; 21 - culture plate body; 22 - elastic material layer; 23 - culture groove; 24 - annular corrugated structure; 25 - culture chamber; 26 - culture plate cover; 27 - cell collection plate; 28 - blocking wall; 29 - mounting groove; 31 - columnar protrusion; 32 - rotating part; 33 - lifting plate; 34 - annular wave structure; 35 - sliding part; 36 - first limiting column; 37 - second limiting column; 38 - supporting plate; 39 - position detection member; 41 - driving part; 42 - first transmission part; 43 - second transmission part; 61 - spring fixing member; 71 - through hole; A - culture cavity; B - control cavity; C - lifting cavity. DETAILED DESCRIPTION

[0052] Hereinafter, various embodiments of the present disclosure will be described more fully. The present disclosure can have various embodiments, and adjustments and changes can be made therein. However, it should be understood that there is no intention to limit various embodiments of the present disclosure to the specific embodiments disclosed herein, but the present disclosure should be understood to encompass all adjustments, equivalents, and / or alternatives falling within the spirit and scope of various embodiments of the present disclosure.

[0053] Embodiment 1

[0054] The present embodiment discloses a bionic cell 2D / 3D culture system, which provides a bionic spatial structure, simulates the influence of an organ structure with corrugations on cell growth and development, and applies mechanical stretching force and hydrostatic pressure to cells, thereby restoring the mechanical environment involved by cells in vivo. The bionic cell 2D / 3D culture system and each module are shown in Figs. 1-10 of the specification. The specific scheme is as follows:

[0055] A bionic cell 2D / 3D culture system includes a control unit, a main housing 1, a cell culture plate 2, a lifting mechanism 3, a driving mechanism 4, and a static pressure mechanism 5. The control unit, the cell culture plate 2, the lifting mechanism 3, the driving mechanism 4, and the static pressure mechanism 5 are all located inside the main housing 1. The control unit, as the control core of the bionic cell 2D / 3D culture system, can be selected from a single-chip microcomputer or the like. The control unit is communicatively connected to the driving mechanism 4 and the static pressure mechanism 5, respectively, and provides mechanical stretching and hydrostatic pressure for cells in the cell culture plate 2 by controlling the operation of the driving mechanism 4 and the static pressure mechanism 5. In some embodiments, a cell collection plate 27 for collecting cells is further included.

[0056] In some embodiments, the bionic cell 2D / 3D culture system further comprises a detection mechanism connected to the control unit, for detecting environmental data in the culture cavity, including air pressure, temperature, humidity, and / or detecting the expansion range of the annular pleat structure 24, and / or detecting the growth state of the cells. The detection mechanism includes various sensors, including temperature sensors, humidity sensors, air pressure sensors, pressure sensors, position sensors, grating rotation position sensors, etc., which can monitor the system state and cell culture state in real time, facilitating the control unit to intervene.

[0057] In this embodiment, the main housing 1 has a control cavity and a culture cavity isolated from each other, which can reduce the mutual influence between the cavities. The cell culture plate 2 is fixed in the culture cavity and is internally distributed with a plurality of culture chambers 25 for culturing cells; the control unit, part of the driving mechanism 4 and at least part of the static pressure mechanism 5 are located in the control cavity. In FIG. 2, the control cavity is shown as range B, and the culture cavity is shown as range A. The culture cavity is responsible for culturing cells, which can be sampled for irradiation sterilization or ethylene oxide for disinfection. The control cavity is responsible for accommodating mechanical equipment such as thin film flat cable, motor drive board, motor, pressure pump, controller, etc. The culture cavity needs to be arranged into an environment required for cell growth and development, and the environmental parameters such as temperature and humidity require high requirements. Separating the culture cavity and the control cavity can prevent important electronic components such as motors from being damp and corroded in a humid environment, and also reduces the influence of electronic components on cell growth and development. Further, a drying agent can be placed in the control cavity to further reduce the humidity of the control cavity. In this embodiment, the control cavity and the culture cavity need to be isolated to ensure the relative independence of the two cavities, which can reduce the interference of the external environment and electronic components on the cell development environment, ensure a sterile growth environment, and also improve the durability of electronic components.

[0058] The bionic cell 2D / 3D culture system of this embodiment can be used independently for cell culture, or can be used in combination with an external cell culture box. When used independently, the culture cavity also needs to be configured with related functional modules for controlling environmental parameters such as air, temperature, humidity, etc. In some embodiments, without removing the cell culture box partition, the bionic cell 2D / 3D culture system is placed in the cell culture box, directly using the temperature, humidity and gas environment in the cell culture box, without the need to separately set up pipelines and related functional modules to provide the gas environment required for cell culture. Although the control cavity is located in the cell culture box, it is independent of the environment in the cell culture box and is not affected by the environment.

[0059] In some embodiments, the bionic cell 2D / 3D culture system further comprises a supporting mechanism 6 located in the culture cavity, and the cell culture plate 2 is detachably fixed to the upper side of the supporting mechanism 6; the supporting mechanism 6 and the inner side wall of the main shell 1 form a jacking cavity, and the jacking mechanism 3 is located in the jacking cavity; the supporting mechanism 6 is provided with a fixing plate for connecting the cell culture plate, and the side of the fixing plate 7 facing the cell culture plate 2 is provided with a through hole 71, and the columnar protrusion 31 enters and exits the jacking cavity through the through hole 71 to contact the elastic material layer 22. For example, in FIG. 2, a cavity is formed in the supporting mechanism 6, as shown in the range C, and the jacking mechanism 3 is located in the cavity. The cell culture plate 2 is fixedly connected above the supporting mechanism 6. Moreover, the cell culture plate 2 and the supporting mechanism 6 are detachably connected, so that the cell culture plate 2 can be directly detached from the supporting mechanism 6. In order to further reinforce the connection between the cell culture plate 2 and the supporting mechanism 6, a plurality of elastic fixing buckles are arranged on the supporting mechanism 6, which lock the cell culture plate 2 when the cell culture plate 2 is installed on the supporting mechanism 6.

[0060] For example, the supporting mechanism 6 is provided with a connecting opening for installing the cell culture plate 2, and the connecting opening is provided with a groove. The bottom of the cell culture plate 2 is fixedly provided with a fixing plate 7, and the fixing plate 7 is slightly larger than the cell culture plate 2, so that the fixing plate 7 protrudes relative to the cell culture plate 2. The cell culture plate 2 can be slid into the connecting opening along the groove from the side, and at this time, the part of the fixing plate 7 protruding relative to the cell culture plate 2 is embedded in the groove, so that the fixation of the cell culture plate 2 in the vertical direction is completed, and the up-down movement of the culture plate is limited. The area between the connecting opening and the fixing plate 7 is shown as area a in FIG. 1. At the same time, two spring buckles are installed on the supporting mechanism 6 as spring fixing members 61, which lock the cell culture plate 2 after the cell culture plate 2 is slid into the opening, and limit the displacement of the cell culture plate 2 in the horizontal direction. When the culture plate is removed, the spring buckle can be actuated.

[0061] In this embodiment, the cell culture plate 2 is a place for culturing cells, tissues and organoids, and is divided into a plurality of independent culture chambers 25, and the culture chambers 25 are places for cell growth and development. The bottom of the cell culture plate 2 is an elastic material layer 22, and the bottom of each culture chamber 25 is formed with a culture groove 23 containing an annular wrinkle structure 24 at the elastic material layer 22, and the annular wrinkle structure 24 is used to simulate the wrinkle structure in the living body. The jacking mechanism 3 is responsible for pulling the annular wrinkle structure 24 to generate a pulling force acting on the cells. The jacking mechanism 3 is connected to the driving mechanism 4, and columnar protrusions 31 are distributed below each culture groove 23, which are used to periodically move up and down under the driving of the driving mechanism 4, so as to realize the periodic contraction and relaxation of the annular wrinkle structure 24 by different degrees of extrusion of the elastic material layer 22, drive the movement of the hydrogel and the cells in the culture chamber 25, and further provide mechanical stretching force for the cells in the culture chamber 25.

[0062] In some embodiments, the cell culture plate 2 comprises, from top to bottom, a culture plate cover 26, a culture plate body 21, and an elastic material layer 22, the culture plate body 21 is connected to the elastic material layer 22, the cell culture plate 2 is provided with n culture chambers 25, the culture chambers 25 are used for planting cells or for planting hydrogels containing cells, the culture plate cover 26 is responsible for covering the culture chambers 25 and ensuring that each culture chamber is in communication with the external environment to provide air for cell growth. Each culture chamber 25 penetrates through the culture plate body 21 and forms a culture groove 23 on the elastic material layer 22; wherein n is an integer not less than 2. In addition, a fixing plate 7 can be arranged at the bottom of the elastic material layer 22 to fix the cell culture plate 2 to the support mechanism 6. The cell culture plate 2 is shown in FIG. 3.

[0063] The lifting mechanism 3 is provided with n columnar protrusions 31, which are distributed below each culture groove 23, and the lifting mechanism 3 is used to drive the columnar protrusions 31 to move up and down periodically, which can not only exert a traction force on the annular wrinkle structure 24 on the elastic material layer 22 to make the hydrogel deform uniformly, but also realize the switching of the hydrogel between the gas-liquid interfaces. The culture groove 23 is provided with an annular wrinkle structure 24 in contact with the hydrogel, which simulates the plica structure in the biological body, can not only increase the contact area with the hydrogel, but also uniformly drive the movement of the hydrogel and the cells in it. In addition, the morphology of the cultured cells, tissues or organoids will also be similar to the annular wrinkle structure 24, and the shape of the desired cells, tissues or organoids can be cultivated by involving the morphology of the annular wrinkle structure 24. In actual application, cells are directly placed into the bottom of the culture groove 23 for 2D culture, and the cells adhere to the surface of the elastic material layer at the bottom; or, un-solidified hydrogel containing cells is injected for 3D cell culture; during 3D culture, the hydrogel fills the culture groove under the action of the surface tension caused by the culture groove closure structure; after standing, the hydrogel is cross-linked and solidified;

[0064] Each culture chamber 25 corresponds to a culture groove 23, and each culture groove 23 is provided below with a columnar protrusion 31 that moves up and down under the drive of the lifting mechanism 3, thereby driving the contraction and relaxation of the culture groove 23 to simulate the mechanical force stretching in the body. In this embodiment, the lifting mechanism 3 can move up and down periodically according to the needs of the simulated environment, so that the annular wrinkle structure 24 on the elastic material layer 22 contracts and relaxes periodically, better simulating the biological environment. In some embodiments, sensors can be provided to monitor the up-and-down movement of the lifting mechanism 3 to sense the relaxation and stretching of the wrinkle structure. In actual application, the columnar protrusion 31 on the lifting mechanism 3 is driven to move up and down periodically, the annular wrinkle structure 24 is caused to expand or contract by the columnar protrusion 31, and the cells on the surface of the elastic material layer during 2D culture or the hydrogel and the cells in the hydrogel during 3D culture are moved; during the rising of the columnar protrusion 31, the elastic material layer 22 is gradually lifted, a horizontal force is generated on the annular wrinkle structure 24 on the elastic material layer 22 to make the annular wrinkle structure 24 expand, simulating the influence of the tissue with the wrinkle structure in the body on the cells in the relaxed state; when gas-liquid interface culture is not needed, the depth of the culture medium is greater than the lifting height of the elastic material layer 22, and the cells are always immersed in the culture medium during the movement of the elastic material layer 22; when gas-liquid interface culture is needed, the depth of the culture medium is less than the lifting height of the elastic material layer 22; when the columnar protrusion 31 rises to the preset height, the hydrogel can be separated from the liquid surface in the culture chamber 25 and contact with the air in the culture chamber 25, and the gas-liquid interface culture is realized within a certain period of time;

[0065] Specifically, the cell culture plate 2 is used to culture the water gel containing cells, tissues or organoids. The cell culture plate 2 as a whole can be sterilized by ethylene oxide. In this embodiment, the cell culture plate 2 is divided into a plurality of independent culture chambers 25, and each culture chamber 25 can culture cells. The cell culture plate 2 comprises, from top to bottom, a culture plate cover 26, a culture plate main body 21 and an elastic material layer 22, and each culture chamber 25 involves the three parts. The culture chamber 25 on the cell culture plate 2 can exert mechanical force on the organoids on the basis of maintaining the three-dimensional morphology of the organoids, thereby reducing the collapse of the three-dimensional structure caused by the digestion of the organoids. At the same time, compared with the traditional microfluidic chip, the modular design of the cell culture plate 2 can select the number of wells according to the experiment, replace the culture plate with different number of wells according to the needs, and has the characteristics of large throughput, large space and large sample size.

[0066] In some embodiments, the cell culture plate 2 is of a standard laboratory well plate design, and the layer of elastic material 22 is of a transparent material, so that the cell culture plate 2 can be directly observed under an inverted / upright microscope after the cell culture plate 2 is directly detached. In addition, the standard well plate design facilitates the loading of the culture plate on other equipment such as a microplate reader and the like for detection. For example, the cell culture plate 2 is of a size of about 127*85 mm, which is consistent with the size of common 6, 12, 24, 96 well plates on the market, and can be used in the base and support of conventional biological experimental facilities.

[0067] The plate cover 26 can be understood as an upper cover of the cell culture plate 2. Opening the plate cover 26 allows the injection of hydrogel into the culture chamber 25, and closing the plate cover 26 on the plate body 21 can relatively close the culture chamber 25 and ensure air flow between the culture chamber 25 and the outside to provide air for cell growth and development. The plate body 21 is provided with a through hole 71 extending from top to bottom, one end of the through hole 71 is communicated with the plate cover 26, and the other end is communicated with the layer of elastic material 22. Cells are initially planted in the layer of elastic material 22, and the plate body 21 provides a wider space for the growth and development of cells, and also provides a space for the mechanical force involved in simulating the environment of the organism.

[0068] The layer of elastic material 22 is provided with a plurality of culture grooves 23, in which single-layer cells, 3D cultured cells or organoids growing in hydrogel can be planted. The design of the wrinkle-shaped culture grooves 23 provides a biomimetic spatial structure to simulate the structure of organs with wrinkle structure such as bladder folds and digestive tract epithelium. The annular wrinkle structure 24 not only provides biological morphological simulation, but also increases the surface contact area and friction of the three-dimensional structure of the hydrogel, which helps to drive the movement of the hydrogel and the cells in it during the stretching process. In addition, the layer of elastic material 22 contains wrinkle-shaped culture grooves 23, which can also wrap the hydrogel containing organoids in three dimensions, so that the stress is uniform, and the hydrogel and the elastic material do not move relative to each other during stretching. Preferably, the layer of elastic material 22 is made of PDMS material. PDMS (polydimethylsiloxane) is a common organic silicon polymer material with excellent physical and chemical properties. The PDMS material has good biocompatibility and will not produce harmful chemical reactions with cells, nor will it produce toxic substances, and has no toxic effect on cells, thereby ensuring the survival and growth of cells during the culture process. In addition, the PDMS material is soft and elastic, and can be prepared into various shapes and sizes of the layer of elastic material 22 as needed, so that researchers can design suitable culture environments according to experimental needs to simulate the mechanical stimulation of biological tissues. In addition, the surface of the PDMS material is not easy to attach bacteria and other microorganisms, and is easy to clean and disinfect, which helps to maintain the sterile state of the cell culture environment and prevent cells from being contaminated and infected.

[0069] In the embodiment, the bionic cell 2D / 3D culture system is also provided with a static pressure mechanism 5, and the main shell 1 is provided with a static pressure input port, as shown in FIG. 1. The static pressure mechanism 5 is connected to the culture cavity through the static pressure input port, and is used to adjust the air pressure in the culture cavity, so as to provide the hydrostatic pressure for the cells in the culture chamber 25. By changing the air pressure in the culture cavity, the hydrostatic pressure in the culture chamber 25 is indirectly affected, and then the growth and development of the cells, tissues or organoids are affected. In some specific embodiments, the static pressure mechanism 5 includes a pressurizing pump, a pressurizing pipeline and an air pressure sensing device. The pressurizing pump and at least part of the pressurizing pipeline are located in the control cavity. The air pressure sensing device is located in the culture cavity and is communicatively connected to the control unit. The pressurizing pump is connected to the culture cavity through the pressurizing pipeline, and the control unit is communicatively connected to the pressurizing pump, used to obtain the air pressure information fed back by the air pressure sensing device, and to adjust the air pressure in the culture cavity by controlling the pressurizing pump, so as to change the hydrostatic pressure applied to the cells in the culture cavity.

[0070] For example, the control cavity contains a single-chip microcomputer, a thin film wire, a motor driving board, a desiccant container, a stepping motor / servo motor, a pressurizing pump and a pressure relief electromagnetic valve. The pressurizing pump is connected to the outside of the main shell 1 through a pipeline and is connected with a filter to prevent pollutants from being sucked in. The pressurizing pump sucks air into the culture cavity under the control of the single-chip microcomputer to pressurize the culture cavity. In addition, a one-way valve is installed in the pressurizing pump pipeline to prevent backflow of gas. The pressure relief electromagnetic valve is connected to the pipeline to connect the culture cavity with the outside of the main shell 1, and a filter is also provided at the connection position for filtration. The pressure relief electromagnetic valve is opened and closed under the control of the single-chip microcomputer, so as to depressurize the culture cavity and achieve the effect of pressure reduction. A one-way valve is also installed in the pipeline connected to the electromagnetic valve to prevent backflow of gas. After the pressure sensor in the culture cavity transmits the pressure data to the single-chip microcomputer, the single-chip microcomputer coordinates the operation of the pressurizing pump and the pressure relief electromagnetic valve to control the air pressure in the culture cavity, indirectly affect the hydrostatic pressure in the liquid culture medium, and then affect the cells in the culture chamber 25. In the embodiment, the air pressure can be controlled independently, the mechanical stretching force can also be controlled independently, or the air pressure and the mechanical stretching force can be adjusted synchronously, so that the air pressure changes with the change of the mechanical stretching force, so as to simulate the influence of the cell tension and the hydrostatic pressure of some organs (such as the bladder) in the body on the growth and development of cells.

[0071] Fig. 4 shows a schematic diagram of a lifting mechanism 3. In some embodiments, the lifting mechanism 3 comprises a rotating part 32 and a lifting part; the rotating part 32 is connected to the driving mechanism 4 and rotates under the driving of the driving mechanism 4; the rotating part 32 is located below the lifting part, and the lifting part is provided with columnar protrusions 31 on one side and the rotating part 32 on the other side; one of the rotating part 32 and the lifting part is provided with an annular wavy structure 34, and the other is provided with a plurality of sliding parts 35 that periodically move up and down along the surface of the annular wavy structure 34, realizing the periodic up-and-down movement of the lifting part relative to the rotating part 32. The annular wavy structure 34 has a wavy outer surface on which the sliding parts 35 move, and the position of the sliding parts 35 fluctuates, realizing the approach and departure of the lifting part and the rotating part 32. In Fig. 4, the sliding parts 35 are located on the rotating part 32, and the annular wavy structure 34 belongs to the lifting part. In addition, the stretching strength of the cells can be adjusted by controlling the rotation speed of the rotating part 32, and the continuous stretching of the cells can be realized by controlling the start and stop of the rotating part 32.

[0072] In some embodiments, the lifting mechanism 3 further comprises a position detection member 39 connected to the control unit, and the control unit determines the position of the columnar protrusions 31 relative to the elastic material layer 22 according to the information fed back by the position detection member 39, and further obtains the stretching amplitude of the annular wavy structure 34 on the cells; the position detection member 39 is located on the lifting part and is used to detect the contact area between the annular wavy structure 34 and the sliding parts 35; and / or, the position detection member 39 is located on the rotating part 32 and is used to detect the rotation direction of the rotating part 32; and / or, the position detection member 39 is located on the driving mechanism 4 and is used to detect the driving angle of the driving mechanism 4 on the rotating part 32, so as to determine the rotation direction of the rotating part 32.

[0073] Specifically, the annular wavy structure 34 is located on the lifting part, and the plurality of sliding parts 35 are distributed on the rotating part 32, which is a large gear structure, and the sliding parts 35 can be selected as a wheel body structure such as a universal wheel. The surface of the annular wavy structure 34 is smooth, each part is smoothly connected, and the head and tail are connected, which can continuously realize the relative displacement between the sliding parts 35 and the annular wavy structure 34. In some embodiments, the annular wavy structure 34 comprises staggered protrusions and recesses, and the surface presents a sinusoidal effect, as shown in Fig. 4. Similarly, the height and period of the protrusions and recesses of the annular wavy structure 34 can be changed to control the extrusion of the columnar protrusions 31 on the elastic material layer 22. An adjacent protrusion and recess can be defined as a wave segment, and the annular wavy structure 34 comprises a plurality of wave segments, and the structure of each wave segment can be the same or different. The greater the fluctuation of the annular wavy structure 34, the wider the range of tension that can be provided to the cells.

[0074] Further, the protrusion or the recess is provided with a marker, and the culture cavity is fixedly provided with a marker detector capable of sensing the marker; the marker detector is communicatively connected to the master control unit, and the master control unit is used to determine the stretching amplitude of the cells in the culture tank 23 according to the position information of the jacking part 3. For example, the marker is a magnetic substance located on the protrusion, and the marker detector is a Hall sensor. Once the Hall sensor detects the magnetic substance, it indicates that the current position is at the protrusion, and then it is determined whether the annular corrugated structure 24 is in a relaxation state or a contraction state.

[0075] In some embodiments, the rotating part 32 is a gear, the annular wave structure 34 constitutes a curved surface piston, and the curved surface piston is separated from other structures of the jacking part. The gear is fixedly provided with a grating structure, and a grating encoder sensor is fixed inside the culture cavity and can be located in the central region of the gear or on the periphery of the gear. The grating encoder sensor can accurately sense the grating structure, determine the rotating direction of the gear, and then obtain the stretching amplitude information of the cells. The gear is also provided with a plurality of protruding parts, and each protruding part is provided with a universal wheel at the end. As shown in FIG. 3, the side of the gear is a worm gear, and the worm gear is connected to a driving motor located in the control cavity through a shaft. The worm gear can amplify the torque and self-lock, thereby reducing the requirement for the load of the motor.

[0076] For example, the driving part 41 is a stepping motor with a position feedback function, which can know the rotating angle of the motor. The rotating angle can be used to infer the moving distance of the jacking part, and then the rotating direction information of the rotating part 32 can be determined. The control unit determines the stretching amplitude of the cells in the culture tank 23 according to the rotating direction information of the rotating part 32. Without considering the transmission ratio, the rotating accuracy of a pure stepping motor reaches 0.089°, and one traction cycle can be arbitrarily edited between 2 seconds and several hours, thereby accurately simulating the contraction cycle of an organ such as the bladder.

[0077] In FIG. 4, the gear as the rotating part 32 has a relatively large size, which can provide sustained rotating motion and fixed-range reciprocating motion, realize the periodic up-down motion of the curved surface piston as the annular wave structure 34, and then cause the periodic stretching motion of the cells. When a too strong stretching force is not needed, the single-chip microcomputer can control the gear to perform reciprocating rotating motion within a certain angle range by using the stepping motor or the servo motor with a position feedback function and the rotating position information of the gear provided by the grating encoder, thereby providing a corresponding range of stretching force. The gear can be stopped from rotating and maintained for a certain period of time at any stretching force according to the program setting, thereby providing a certain duration of sustained stretching force.

[0078] In some embodiments, the jacking mechanism 3 further comprises a support fixed inside the main housing 1 and extending through the rotating part 32, for supporting the jacking part together with the rotating part 32 or separately supporting the jacking part when the jacking part is lowered to the preset position; the support is provided with a pressure sensor in communication with a control unit, which is used to determine the position of the jacking part according to the feedback of the pressure sensor. When the jacking part is lowered to the preset position, the distance between the rotating part 32 and the jacking part is small, and the jacking part is no longer supported by the rotating part 32, but is supported by the support or the rotating part 32 and the jacking part together. The pressure feedback by the pressure sensor at the bottom of the jacking part can determine whether the support supports the jacking part, and further determine the current stretching state of the cells.

[0079] For example, three modes of the jacking mechanism 3 can be set, which are unidirectional rotation and stretching mode, maintenance waveform mode and ladder mode. In the unidirectional rotation and stretching mode, the motor continuously rotates in the same direction, the rotating part 32 continuously rotates in one direction, the curved surface piston moves periodically up and down, all curved surface strokes of the curved surface piston are used, and full motion stroke periodic stretching is achieved. In the maintenance waveform mode, the motor only rotates the rotating part 32 in the positive direction to a certain angle under the feedback of the position detection part 39, and then stops rotating, keeps for a certain time, and then reverses to the diastolic position. The rotation angle of this mode is less than half of the period of the piston surface. Taking the four convex parts of the annular wave structure 34 as an example, corresponding to four periods, the rotation angle should be less than 360 / 4 / 2=45 degrees from the diastolic position. In the ladder mode, the rotating part 32 rotates a certain angle and stops for a certain time, and then rotates a certain angle and stops for a certain time, and so on. Then stop for a certain time after reversing a certain angle, and then stop for a certain time after reversing a certain angle, and so on. In addition to the above control modes, the present application can support arbitrary mechanical waveform simulation within the piston lifting range through precise control of the stepping motor or servo motor, including static waveform, positive rotation waveform, heart waveform, triangular waveform, rectangular waveform and various special waveforms. The area under the curve can also be calculated to reflect the accumulation of mechanical stretching and hydrostatic pressure.

[0080] Exemplarily, in FIG. 5, the lifting part includes a lifting plate 33, a columnar protrusion 31 and a curved piston as an annular wavy structure 34, the support part includes a support plate 38 and a first limiting column 36 and a second limiting column 37 on the support plate 38. The second limiting column 37 is scattered on the support plate 38 and can be arranged around the central region, the lifting plate 33 is sleeved on the second limiting column 37 and moves along the second limiting column 37. The rotating part 32 and the annular wavy structure 34 are sleeved on the first limiting column 36 which is located in the central region of the support plate 38, thereby limiting the curved piston to move up and down along the first limiting column 36 and the rotating part 32 to rotate around the first limiting column 36. The bottom of the support plate 38 is provided with a pressure sensor. The first limiting column 36 alternately supports the lifting plate 33 with the rotating part 32. The curved piston and the lifting plate 33 are in a separated structure, and the sliding part 35 moves along the surface of the annular wavy structure 34. When the distance between the curved piston and the rotating part 32 is less than a preset distance, the lifting part is fixed on the first limiting column 36, and the curved piston no longer contacts the lifting part or supports the lifting part together with the first limiting column 36. At this time, the pressure borne by the first limiting column 36 becomes larger, and feedback can be obtained through the pressure sensor at the bottom. When the curved piston rises to a preset height, the lifting part is moved by the curved piston alone, and the pressure becomes smaller.

[0081] In the embodiment, the control cavity and the culture cavity are relatively independent, and the driving mechanism 4 must transmit power to the culture cavity, so the driving mechanism 4 needs to be structurally designed. In some specific embodiments, the driving mechanism 4 includes a driving part 41, a first transmission part 42 and a second transmission part 43; the first transmission part 42 is located in the control cavity and is connected to the driving part 41; the second transmission part 43 is located in the culture cavity and is connected to the lifting mechanism 3; the first transmission part 42 and the second transmission part 43 realize contactless power transmission through magnetic coupling, thereby ensuring the independence between the culture cavity and the control cavity. The use of magnetic coupling separates the driving cavity and the culture cavity, thereby ensuring the sterile environment of the culture cavity and the corrosion resistance of the driving cavity. In some specific embodiments, the first transmission part 42 and the second transmission part 43 adopt a synchronous belt + reduction gear mechanism,

[0082] In some specific embodiments, power supply and data transmission are realized through a thin film data flat cable. The thin film flat cable can lead out from the culture cavity and connect to the power supply and transmit data without affecting the sealing performance of the culture cavity.

[0083] In some embodiments, the fixing structure is a fixing plate 7. The elastic material layer 22 is sandwiched between the culture plate main body 21 and the fixing plate 7, and the three-layer structure can be fixed by screws or other fixing members. In addition, the fixing plate 7 is slightly larger than the elastic material layer 22 and the culture plate main body 21 above, so as to better fix the cell culture plate 2 to the support.

[0084] In some embodiments, the fixed plate 7 has n openings, and the lifting mechanism 3 is located below the fixed plate 7, and the columnar protrusion 31 can pass through the openings to contact the elastic material layer 22. The number of openings is equal to the number of culture chambers 25 and the number of columnar protrusions 31. For example, the support has a cell culture plate 2 fixed at the upper end and a lifting mechanism 3 movable up and down at the lower end, and the columnar protrusion 31 on the lifting mechanism 3 can pass through the support to contact the elastic material layer 22. The cell culture plate 2 is fixed to the support as a whole, and the elastic material layer 22 is extruded by the upward movement of the columnar protrusion 31 to extrude the wrinkle structure on it to the hydrogel, simulating the influence of the wrinkle structure in the body on the growth and development of cells.

[0085] In some embodiments, the culture groove 23 has a tapered structure, and the opening of the culture groove 23 is tapered relative to the inner side wall to limit the separation of the hydrogel from the opening and provide surface tension to the hydrogel. In the drawings, the side view of the culture groove 23 is a "convex" structure, and the top of the convex structure is the opening of the culture groove 23, which is obviously smaller than the main part of the culture groove 23. When the hydrogel is injected, the hydrogel is bound to the groove by surface tension; on the other hand, the "convex" structure restricts the hydrogel from the bottom, side and top surfaces together, together with the annular wrinkle structure 24, to fix the position of the hydrogel, so that the hydrogel deforms together with the elastic material layer 22 when the elastic material layer 22 deforms, and does not displace with the elastic material layer 22, which makes up for the deficiency of ordinary cell stretching equipment that is difficult to perform three-dimensional cell model stretching. When injecting the un-solidified hydrogel into the culture groove 23, the injection head of the sample injector only needs to be injected at a single position, and the hydrogel can fill the entire annular groove along the channel under the action of surface tension without spilling, reducing the difficulty of injection.

[0086] In some embodiments, the columnar protrusion 31 includes a cylindrical structure and a contact end, and the cylindrical structure is connected to the contact end. The cross section of the contact end on the tangent plane of the preset first direction is circular, the area of the cross section gradually decreases along the first direction, and a bevel is formed at the end of the contact end, so that the annular wrinkle structure 24 is located on the bevel and expands and contracts the annular wrinkle structure 24 under the action of the lifting mechanism 3. The contact end is responsible for contacting the annular wrinkle structure 24, and the annular wrinkle structure 24 adaptively contracts and dilates on the contact end as the columnar protrusion 31 rises and falls. The contact end is designed as a bevel structure, and the annular wrinkle structure 24 is sleeved on the bevel and gradually expands under the extrusion of the bevel, so that the dilatation process of the annular wrinkle structure 24 is more smooth, avoiding damage to the annular wrinkle structure 24 due to excessive expansion. At the same time, the arrangement of the bevel makes the contraction and dilation of the annular wrinkle structure 24 more consistent with the change of the stretching force in the in vivo environment.

[0087] In some embodiments, the annular wrinkle structure 24 comprises a plurality of V-shaped structures connected in sequence to form a ring, and the openings of the V-shaped structures are directed to the center of the annular wrinkle structure 24. The corners of each V-shaped structure and the connections between the V-shaped structures are arc-shaped to reduce damage to the hydrogel and cells. The annular wrinkle structure 24 is shown in FIG. 6. In FIG. 6, the inner side and the outer side of the V-shaped structure are arc-shaped, the vertex of the lower end of the V-shaped structure is arc-shaped, and the connections between the V-shaped structures are also arc-shaped to avoid sharp parts that may injure the cells in the culture medium. The annular wrinkle structure 24 is connected end to end and has a certain elasticity, and can contract and dilate with the lifting of the columnar protrusion 31. Preferably, the inclined surface is arc-shaped, and under the lifting of the inclined surface, the inner ring part of the annular wrinkle structure 24 is lifted upward under the action of friction, and the outer ring part is sleeved on the inclined surface, and the distance between the inner ring and the outer ring is increased. When the lifting mechanism 3 rises, the columnar protrusion 31 presses the elastic material layer 22, and the annular wrinkle structure 24 on the elastic material layer 22 is stretched horizontally.

[0088] In some embodiments, the culture chamber 25 contains a culture solution, and when the liquid level of the culture medium for culturing cells in the culture chamber 25 is lower than the maximum lifting height of the columnar protrusion 31, the lifting mechanism 3 can also be used to periodically drive the cells in the culture chamber 25 to enter and exit the culture medium, so as to simulate the culture of cells at the gas-liquid interface. When the columnar protrusion 31 rises to the preset height, the hydrogel is separated from the culture solution in the culture chamber 25 and contacts the air in the culture chamber 25; until the columnar protrusion 31 descends to the preset height, the hydrogel re-enters the culture solution in the culture chamber 25 and is separated from the air in the culture chamber 25. By lifting the lifting mechanism 3, the cells are switched between the gas-liquid interface to simulate the culture environment of the cells at the gas-liquid interface in vivo.

[0089] In some embodiments, each culture chamber 25 is provided with a mounting groove 29 on the periphery of the culture plate body 21, and the groove opening faces the culture plate cover 26, as shown in Figs. 8 and 9. The cell collection plate 27 is provided with a blocking wall 28 at the corresponding culture chamber 25, which can be embedded in the mounting groove 29, and the blocking wall 28 is formed with a receiving space on the culture plate cover 26, which is used to accommodate the cultured cells after centrifugation of the culture chamber 25. The cell collection plate 27 is shown in Figs. 7 and 8. The cultured cells, hydrogel or organoids need to be separated from the culture chamber 25 by centrifugation. After embedding the cell collection plate 27 in the culture plate, the two are inverted and centrifuged, and the cells in the culture chamber 25 will be collected in the receiving space. The blocking wall 28 forms a receiving space on the cell collection plate 27, and the cell culture plate 2 is inverted before centrifugation, and after centrifugation, the cultured cells, hydrogel or organoids fall into the receiving space, completing the collection work. Each culture chamber 25 is provided with a groove, and the cell collection plate 27 is provided with a blocking wall 28, and the cylindrical protrusion 31 can be embedded in the groove around the culture chamber 25, which can seal the culture chamber 25 from above, facilitate the positioning and covering of the culture chamber 25 by the cell collection plate 27, and facilitate the collection of the cultured cells.

[0090] In some embodiments, the cell collection plate 27 is provided with a conical funnel structure at the corresponding culture chamber 25, and the funnel structure and the blocking wall 28 jointly form a receiving space, and the funnel structure is used to concentrate the cultured cells at the end after centrifugation of the culture chamber 25, to facilitate the extraction of the cells from the end of the funnel structure. The end of the cell collection plate 27 is changed from a flat surface to a three-dimensional funnel structure, which facilitates the collection of the scattered cells, hydrogel or organoid structure after centrifugation at the funnel structure. The funnel structure on the culture plate cover 26 is shown in Fig. 10. In Fig. 10, the receiving space includes a lower funnel space and an upper column space.

[0091] The embodiment provides a bionic cell 2D / 3D culture system, which integrates a control unit, a cell culture plate, a jacking mechanism, a driving mechanism and a static pressure mechanism and other related modules into the shell. The bionic cell 2D / 3D culture system not only reduces the volume, but also expands the functionality, so that it can be compatible with more modes of cell culture. The influence of the bionic space structure on the growth and development of cells with wrinkled organ structure is simulated, and the whole hydrogel can be uniformly deformed. By applying adjustable mechanical tension and fluid static pressure to the cells, the real mechanical environment of the cells in the biological body is restored. On the basis of maintaining the three-dimensional morphology of the organoids in the hydrogel, mechanical force is applied to the cells in the hydrogel, so as to reduce the probability of three-dimensional structure collapse caused by organoid digestion. Through the design of the wrinkle morphology in the culture tank, a bionic space structure is provided to simulate the organ structure with wrinkle structure such as bladder folds and digestive tract epithelium. At the same time, the ring-shaped wrinkle structure binds the hydrogel, and makes the force on each part of the hydrogel uniform during the movement. On the basis of maintaining the three-dimensional morphology of the cell clusters in the hydrogel, mechanical force is applied to the cells in the hydrogel, so as to reduce the probability of three-dimensional structure collapse of the 3D cultured cells.

[0092] Embodiment 2

[0093] The embodiment discloses a control method of a bionic cell 2D / 3D culture system, which is used for controlling the bionic cell 2D / 3D culture system of embodiment 1, so as to make it more practical. The specific scheme is as follows:

[0094] The control method of the bionic cell 2D / 3D culture system is used for controlling the bionic cell 2D / 3D culture system of any one of embodiment 1. The control method comprises the following steps:

[0095] 101, directly placing cells into the bottom of the culture tank for 2D culture, and the cells adhere to the surface of the elastic material layer at the bottom; or, injecting un-solidified hydrogel containing cells for 3D cell culture; during 3D culture, the hydrogel fills the culture tank under the action of the surface tension caused by the culture tank closing structure; after standing, the hydrogel is cross-linked and solidified;

[0096] 102, the control unit controls the periodic up-down movement of the columnar protrusion on the jacking mechanism, the ring-shaped wrinkle structure is caused to dilate or contract through the columnar protrusion, and then the cells on the surface of the elastic material during 2D culture or the hydrogel and the cells in the hydrogel during 3D culture are moved;

[0097] During the rising of the columnar protrusion, the jacking of the elastic material layer is gradually increased, a horizontal force is generated on the ring-shaped wrinkle structure of the elastic material layer to make the ring-shaped wrinkle structure expand, and the influence of the tissue with the wrinkle structure in the biological body on the cells in the dilated state is simulated;

[0098] When the gas-liquid interface culture is not needed, the depth of the culture medium is greater than the height of the elastic material layer being lifted up, and the cells are always immersed in the culture medium during the movement of the elastic material layer;

[0099] When the gas-liquid interface culture is needed, the depth of the culture medium is less than the height of the elastic material layer being lifted up; when the columnar protrusion rises to the preset height, the hydrogel can be separated from the liquid surface in the culture chamber and be in contact with the air in the culture chamber, and the gas-liquid interface culture is realized within a certain period of time;

[0100] During the descending of the columnar protrusion, the lifting degree of the elastic material layer is gradually reduced, the elastic material layer is retracted under the elastic action and drives the annular wrinkle structure to gradually contract, thereby simulating the influence of the tissue with the wrinkle structure in the biological body on the cells in the contraction state;

[0101] When the columnar protrusion descends to the preset height, the hydrogel re-enters the liquid surface in the culture chamber and is separated from the air in the culture chamber;

[0102] During the cell culture, the static pressure mechanism is controlled to operate by the control unit, the air pressure in the culture cavity is adjusted, and the hydrostatic pressure is provided for the cells in the culture chamber;

[0103] 103、After the cell culture is completed, the culture chamber on the cell culture plate is covered by the cell collection plate, the cell culture plate is inverted, and the cultured cells are separated by the centrifugal method, so that the cells fall into the cell collection plate after the centrifugation.

[0104] The application provides a kind of bionic cell 2D / 3D culture system and its control method, will control unit, cell culture plate, jacking mechanism, driving mechanism and static pressure mechanism and other related modules are integrated into the shell inside, not only reduce the volume of bionic cell 2D / 3D culture system, also can expand the functionality of bionic cell 2D / 3D culture system, make it can be compatible with more mode cell culture. By simulating the influence of bionic space structure on the growth and development of cells with wrinkled organ structure, and can uniformly apply deformation to the whole hydrogel, by applying adjustable mechanical tension and fluid static pressure to cells alone or simultaneously, the real mechanical environment of cells in vivo is restored. On the basis of maintaining the three-dimensional morphology of organoids in hydrogel, mechanical force is applied to cells in hydrogel, reducing the probability of three-dimensional structure collapse caused by organoid digestion. By designing the morphology of the culture tank, a bionic space structure is provided to simulate organ structures with wrinkled structures such as bladder folds and digestive tract epithelium, and during stretching, the hydrogel and elastic material do not displace. By adjusting the degree of mechanical tension and stretching mode through the design of the jacking structure, the control of cell stretching amplitude is realized, and indirect interface culture is realized. Through the control unit and the sensors on the multiple mechanisms, the culture state and mechanical state can be monitored and controlled. The modular design of the cell culture plate can be replaced and adjusted according to the needs. The culture cavities and control cavities are isolated from each other, reducing the influence between the cavities.

[0105] Those skilled in the art can understand that the drawings are only a schematic diagram of a preferred implementation scenario, and the modules or processes in the drawings are not necessarily required to implement the present application. Those skilled in the art can understand that the modules in the devices in the implementation scenario can be distributed in the devices in the implementation scenario according to the description of the implementation scenario, or can be changed and located in one or more devices different from the implementation scenario.

Claims

1. A biomimetic cell 2D / 3D culture system, characterized in that, The main shell, the control unit, the cell culture plate, the lifting mechanism, the driving mechanism and the static pressure mechanism, the control unit is respectively connected with the driving mechanism and the static pressure mechanism; The control cavity and the culture cavity are formed in the main shell, the cell culture plate is fixed in the culture cavity, and a plurality of culture chambers for culturing cells are distributed in the culture cavity; the control unit, part of the driving mechanism and at least part of the static pressure mechanism are located in the control cavity; The bottom of the cell culture plate is an elastic material layer, and each culture chamber forms a culture groove with an annular wrinkle structure at the elastic material layer, and the annular wrinkle structure is used to simulate the wrinkle structure in the organism; The lifting mechanism is connected with the driving mechanism, and a columnar protrusion is distributed below each culture groove, which is used to drive the columnar protrusion to move up and down periodically under the driving of the driving mechanism, so as to realize the periodic contraction and relaxation of the annular wrinkle structure to the cells by extruding the elastic material layer to different degrees, and further provide mechanical stretching force for the cells in the culture chamber; The static pressure mechanism communicates with the culture cavity, which is used to adjust the air pressure in the culture cavity to provide hydrostatic pressure for the cells in the culture chamber.

2. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, It also includes a detection mechanism connected with the control unit, which is used to detect the environmental data including air pressure, temperature and humidity in the culture cavity, and / or detect the stretching amplitude of the annular wrinkle structure, and / or detect the growth state of the cells.

3. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, The lifting mechanism includes a rotating part and a lifting part; the rotating part is connected with the driving mechanism and is used to rotate under the driving of the driving mechanism; the lifting part is provided with the columnar protrusion on one side and the rotating part on the other side; One of the rotating part and the lifting part is provided with an annular wave structure, and the other of the rotating part and the lifting part is provided with a plurality of sliding parts, which slide periodically along the surface of the annular wave structure to realize the periodic up and down movement of the lifting part relative to the rotating part.

4. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, The driving mechanism includes a driving part, a first transmission part and a second transmission part; The first transmission part is located in the control cavity and connected with the driving part; the second transmission part is located in the culture cavity and connected with the lifting mechanism; the first transmission part and the second transmission part realize non-contact power transmission through magnetic coupling, which ensures the independence between the culture cavity and the control cavity.

5. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, It also includes a support mechanism located in the culture cavity, and the cell culture plate is detachably fixed to the upper side of the support mechanism; the support mechanism and the inner side wall of the main shell form a lifting cavity, and the lifting mechanism is located in the lifting cavity; The side of the support mechanism facing the cell culture plate is provided with a through hole, and the columnar protrusion enters and exits the lifting cavity through the through hole to contact the elastic material layer.

6. The biomimetic cell 2D / 3D culture system according to claim 3, wherein, The lifting mechanism also includes a position detection member connected with the control unit, and the control unit determines the position of the columnar protrusion relative to the elastic material layer according to the information fed back by the position detection member, and further obtains the stretching amplitude of the annular wrinkle structure to the cells. The position detecting member is located on the lifting part, and is used for detecting the contact area between the annular wave structure and the sliding part; And / or, the position detecting member is located on the rotating part, and is used for detecting the rotating direction of the rotating part; And / or, the position detecting member is located on the driving mechanism, and is used for detecting the driving angle of the driving mechanism to the rotating part, so as to determine the rotating direction of the rotating part.

7. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, The static pressure mechanism comprises a pressurizing pump, a pressurizing pipeline and a gas pressure sensing device, the pressurizing pump and at least part of the pressurizing pipeline are located in the control cavity; The gas pressure sensing device is located in the culture cavity and is in communication connection with the control unit; The pressurizing pump is in communication with the culture cavity through the pressurizing pipeline, and the control unit is in communication connection with the pressurizing pump, so as to acquire the gas pressure information fed back by the gas pressure sensing device, and to adjust the gas pressure in the culture cavity by controlling the pressurizing pump, thereby changing the hydrostatic pressure applied to the cells in the culture cavity.

8. The biomimetic cell 2D / 3D culture system according to claim 3, wherein, The lifting mechanism further comprises a supporting member, which is fixed inside the main shell and penetrates through the rotating part, and is used for supporting the lifting part together with the rotating part or supporting the lifting part alone when the lifting part is lowered to a preset position. The bottom of the supporting member is provided with a pressure sensor in communication connection with the control unit, and the control unit is used for judging the position of the lifting part according to the feedback of the pressure sensor.

9. The biomimetic cell 2D / 3D culture system according to claim 1, wherein, When the liquid level of the culture medium for culturing cells in the culture chamber is lower than the maximum lifting height of the columnar protrusion, the lifting mechanism can further be used to drive the cells in the culture chamber to periodically enter and exit the culture medium, so as to simulate the culture of cells at the gas-liquid interface. 10.A method for controlling a biomimetic cell 2D / 3D culture system, characterized in that, The control method is used for controlling the bionic cell 2D / 3D culture system of any one of claims 1-9, and the control method comprises the following steps: The cells are directly placed into the bottom of the culture tank for 2D culture, and the cells adhere to the surface of the elastic material layer at the bottom; or, the non-solidified hydrogel containing cells is injected for 3D cell culture; in the 3D culture, the hydrogel fills the culture tank under the action of the surface tension caused by the converging structure of the culture tank; after standing, the hydrogel is cross-linked and solidified; The control unit controls the columnar protrusion on the lifting mechanism to periodically move up and down, and the annular wrinkle structure is relaxed or contracted through the columnar protrusion, so as to drive the cells on the surface of the elastic material in the 2D culture or the movement of the hydrogel and the cells in the hydrogel in the 3D culture; In the lifting process of the columnar protrusion, the lifting of the elastic material layer is gradually increased, a horizontal force is generated on the annular wrinkle structure of the elastic material layer to relax the annular wrinkle structure, and the influence of the tissue with the wrinkle structure in the living body on the cells in the relaxed state is simulated; When the gas-liquid interface culture is not needed, the depth of the culture medium is greater than the lifting height of the elastic material layer, and the cells are always immersed in the culture medium during the movement of the elastic material layer; When gas-liquid interface culture is needed, the depth of the culture medium is less than the height of the elastic material layer being pushed up; when the columnar protrusion rises to a preset height, the hydrogel can be separated from the liquid surface in the culture chamber and contact with the air in the culture chamber, realizing gas-liquid interface culture in a certain period of time; During the descending of the columnar protrusion, the pushing up degree of the elastic material layer is gradually reduced, the elastic material layer is retracted under the action of elasticity and drives the annular wrinkle structure to gradually contract, simulating the influence of the tissue with the wrinkle structure in the body on cells in the contraction state; When the columnar protrusion descends to a preset height, the hydrogel re-enters the liquid surface in the culture chamber and is separated from the air in the culture chamber; During the cell culture, the control unit controls the operation of the static pressure mechanism to adjust the air pressure in the culture cavity to provide hydrostatic pressure for the cells in the culture chamber; After the cell culture is completed, the cell collection plate covers the culture chamber on the cell culture plate, the cell culture plate is inverted, and the cultured cells are separated by centrifugation, so that the cells fall into the cell collection plate after centrifugation.

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