Concentration gradient cell culture system for research on the control of osteoclast precursor cell differentiation by propionic acid-GPR43.

Abstract

This invention provides a concentration gradient cell culture system that solves the problem of large errors in manual drug addition and the difficulty in constructing a stable, continuous concentration gradient in studies on the control of osteoclast progenitor cell differentiation using propionic acid-GPR43. [Solution] The system comprises a housing assembly with an inner container, a first liquid storage cylinder 2, a second liquid storage cylinder 3, a tuned crossbeam 4, an adjustable screw rod 5, a flow-dividing branch pipe array frame, a spiral mixing unit, a porous diffusion plate, and a culture tray 9. The tuned crossbeam is driven by the adjustable screw rod to synchronously supply liquid from both liquid storage cylinders. After gradient distribution by the flow-dividing branch pipe array frame and mixing by the spiral mixing unit, the porous diffusion plate forms a continuous and stable propionic acid concentration gradient field on the culture tray. This avoids manual errors, provides accurate concentration gradients to RAW264.7 and BMMs cells in a single experiment, and supports the calculation of dose-effect curves and IC50, as well as the elucidation of the mechanism of osteoclast differentiation control by propionic acid-GPR43 signaling.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture technology, and specifically, it is a concentration gradient cell culture device used for research on the regulation of osteoclast precursor cell differentiation by propionic acid - GPR43.

Background Art

[0002] Osteoporosis is a systemic disease characterized by bone loss and imbalance in bone remodeling. Deep exploration of the abnormal differentiation mechanism of osteoclasts has important reference value for the clinical intervention of bone metabolic diseases. Osteoclasts are multinucleated giant cells derived from monocyte / macrophage lineage precursor cells, and their abnormal differentiation and hyperfunction are the core pathological processes in which bone resorption exceeds bone formation and bone mass decreases net. Therefore, targeting the suppression of excessive osteoclast formation is an important strategy for the prevention and treatment of osteoporosis.

[0003] In recent years, the regulatory role of short-chain fatty acids (SCFAs), which are metabolites of the gut microbiota, in the "gut-bone axis" has attracted wide attention. In particular, the process by which propionic acid affects the differentiation of osteoclast precursor cells via its specific receptor GPR43 is regarded as a key factor in elucidating the homeostasis of the bone microenvironment. Propionic acid is one of the main SCFAs, mainly produced by the fermentation of dietary fiber by gut bacteria, and functions as a signaling molecule that interacts with the G protein-coupled receptor 43 (GPR43, also known as free fatty acid receptor 2: FFAR2) present on the surface of immune cells and bone tissue cells. Existing studies have shown that SCFAs such as butyric acid and propionic acid may suppress osteoclast formation and improve bone loss in osteoporosis animal models, but these findings are mainly based on the observation of the phenotypes of the whole animals, and the precise cellular and molecular mechanisms by which a specific SCFA component (such as propionic acid) directly regulates the differentiation of osteoclast precursor cells via its specific receptor are still lacking detailed verification by in vitro tests.

[0004] When conducting such biological research, it is usually necessary to simulate the in vitro environment of cell growth using a cell culture system and combine it with a specific dosing plan. To precisely evaluate the inhibitory effect of propionic acid on osteoclast formation, researchers often have to construct multi-group concentration gradient experimental environments and observe the trajectory of cell differentiation under different doses to screen for the optimal effective concentration that combines safety and efficacy, which is extremely important for subsequent pharmacological research and clinical applications. In particular, in studies to elucidate the mechanism of osteoclast differentiation regulation by the propionic acid-GPR43 signaling axis, the biological effect of propionic acid shows a significant dose-dependence, and its effective window is often narrow, for example, within the physiological and pharmacologically relevant concentration range of 0.1 mM to 10 mM. Therefore, whether or not a stable, continuous, and slowly increasing propionic acid concentration gradient can be constructed in a unified culture space is directly related to the precise plotting of dose-effect curves and the accurate calculation of the median inhibitory concentration (IC50).

[0005] However, most existing cell culture equipment achieves concentration changes by manually adding culture media of pre-set concentrations to different wells of a multi-well plate, thus achieving dose control of metabolites through manual dispensing and addition. However, with such discrete addition methods, random errors are likely to occur during the operation, making it difficult to create a continuous and stable concentration gradient within a unified culture space. Due to the lack of an integrated concentration control component, existing equipment generally makes it inconvenient to make small, precise concentration changes for active substances such as propionic acid, and there is still room for improvement in the accuracy of experimental data when it comes to finding accurate effective concentration limits. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention provides a concentration gradient cell culture device for use in research on the control of osteoclast precursor cell differentiation using propionic acid-GPR43. In the process of exploring the inhibitory effect of propionic acid on osteoclast formation, it can solve the problems of large concentration errors caused by manual drug addition and the difficulty in constructing a stable, continuous concentration gradient within a unified space. [Means for solving the problem]

[0007] The technical means employed in this invention are as follows:

[0008] A concentration gradient cell culture device for research on the control of osteoclast precursor cell differentiation by propionic acid-GPR43, A housing assembly comprising a housing assembly having an inner container, A first liquid storage cylinder and a second liquid storage cylinder are both fixedly installed above the top of the inner container, the first liquid storage cylinder having a first outlet at its bottom, the second liquid storage cylinder having a second outlet at its bottom, and a sealing piston is slidably installed inside both the first and second liquid storage cylinders. A tuning crossbeam is installed above the first and second liquid storage cylinders, wherein both ends of the tuning crossbeam are fixedly connected to the top ends of the piston rods of the two sealing pistons, and a screw hole is drilled in the center of the tuning crossbeam. An adjustment screw rod having a screw hole and a screw thread, wherein the bottom end of the adjustment screw rod is rotatably supported on the top of the housing assembly, A flow-diverting pipe array frame having a first main channel and a second main channel that do not communicate with each other, wherein the first outlet is in communication with the first main channel, the second outlet is in communication with the second main channel, and at least two pairs of branch pipes are led out in parallel along the longitudinal direction of the flow-diverting pipe array frame, and each pair of branch pipes includes a first branch pipe that communicates with the first main channel and a second branch pipe that communicates with the second main channel, A helical mixing unit comprising at least two helical mixing units, the inlet of each helical mixing unit being connected to the first branch end and the second branch end of a pair of branch pipes, A porous diffusion plate fixed to the lower part of the top wall of the inner container, wherein the upper surface of the porous diffusion plate has the same number of independent liquid inlets as the number of helical mixing units, the bottom outlet of each helical mixing unit is connected to one independent liquid inlet in a one-to-one correspondence, and the lower surface of the porous diffusion plate has multiple rows of diffusion micropores, The invention is characterized by comprising a culture tray slidably installed inside the inner container and directly below the porous diffusion plate.

[0009] Furthermore, the housing assembly includes an outer casing, the inner container is fixed inside the outer casing, a thermal insulation intermediate layer is filled between the outer casing and the inner container, a waste liquid collection tank is provided in the bottom wall of the inner container, and a drain pipe that penetrates the outer casing is connected to the bottom of the waste liquid collection tank.

[0010] Furthermore, the edges of the culture tray extend upward to form a surrounding frame.

[0011] Furthermore, the helical mixing unit is made of stainless steel 316L and is covered with a constant temperature heating sleeve on its outside. [Effects of the Invention]

[0012] This invention provides the following beneficial effects.

[0013] This invention involves installing a first liquid storage cylinder, a second liquid storage cylinder, a synchronous crossbeam, and an adjustment screw rod. Both ends of the synchronous crossbeam are fixed to the top ends of two pressurized push rods. By rotating a graduated dial, the adjustment screw rod is driven to rotate, causing the synchronous crossbeam to move downward in sync. This results in two different liquids being injected at exactly the same speed into two independent main channels of the branching pipe array frame. This avoids concentration fluctuations caused by separate manual additions or asynchronous supply pressures of the two systems, and ensures that the initial flow velocity and pressure conditions of the two original liquid flows entering the subsequent mixing unit are matched.

[0014] This invention utilizes two independent main channels that do not communicate with each other within a flow-dividing and branching pipe array frame, and multiple pairs of branching pipes that are led out in parallel along their longitudinal direction. Furthermore, the ratio of the inner diameter or inlet cross-sectional area of ​​each pair of branching pipes changes in a predetermined gradient manner, resulting in different initial volumetric flow rates of propionic acid culture solution and basal medium entering each helical mixing unit. As a result, after thorough mixing by six sets of helical disturbance plates arranged at 90-degree angles to each other within each helical mixing unit, a series of mixed solutions with different concentrations can be formed along the longitudinal direction of the porous diffusion plate. The structure itself enables the physical coding and stable construction of spatial concentration gradients.

[0015] This invention involves fixing a porous diffusion plate to the lower part of the top wall of the inner container, maintaining a 20 mm gap between it and the culture tray, and providing a matrix of diffusion micropores on the surface of the porous diffusion plate. As a result, the mixed liquid from each spiral mixing unit is uniformly dripped onto the corresponding position on the culture tray below through the diffusion micropores. This provides a spatially continuous propionic acid concentration gradient field to osteoclast precursor cells seeded in the culture tray within the same culture experiment. This makes it possible to synchronously observe cell differentiation responses under different concentrations without repeatedly opening the container, significantly improving experimental efficiency and the reliability of data comparison.

[0016] The stable, spatially continuous propionic acid concentration gradient field constructed by this invention greatly facilitates the development of downstream mechanism studies and quantitative analysis of data. Specifically, within the corresponding concentration range of the same culture tray, researchers can synchronously acquire osteoclast precursor cell samples under different concentrations of propionic acid intervention, thereby enabling direct comparative analysis of differences in the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, changes in the expression levels of osteoclast-specific genes (e.g., Nfatc1, Acp5, Ctsk), and the phosphorylation status of important signaling pathway molecules such as nuclear factor κB (NF-κB) and mitogenic factor-activated protein kinase (MAPK). This not only helps in the precise plotting of dose-effect curves and calculation of IC50 values ​​for propionic acid's suppression of osteoclast differentiation, but also provides a direct and high-resolution hardware foundation for clarifying the effective concentration window in which propionic acid exerts specific regulatory effects via the GPR43 receptor, avoiding the oversight of important phenotypes due to sloppiness in concentration setting or dispensing errors. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 is a schematic diagram showing the overall structure of the present invention. [Figure 2] Figure 2 is a schematic diagram showing the connection structure between the liquid storage pressurization component and the concentration gradient mixing and flow distribution component. [Figure 3] Figure 3 is a schematic diagram showing the structure of the concentration gradient diffusion component. [Modes for carrying out the invention]

[0018] The embodiments of the present invention will be described below with reference to the drawings. These embodiments are provided for the purpose of clearly and completely illustrating the present invention and do not limit its scope.

[0019] Referring to FIGS. 1 to 3, an embodiment of the present invention provides a concentration gradient cell incubator for studying the regulation of osteoclast precursor cell differentiation by propionic acid - GPR43. This incubator is applicable to the scenario of culturing osteoclast precursor cells in vitro and constructing a drug concentration gradient in the field of biomedical research, and includes a housing assembly 1, a first liquid storage cylinder 2, a second liquid storage cylinder 3, a synchronous cross beam 4, an adjustment screw rod 5, a dial with scale 6, a plurality of parallel spiral mixing units 7, a porous diffusion plate 8, and a culture tray 9. Here, The housing assembly 1 includes an outer housing, an inner container, a heat - insulating intermediate layer, and a sealed door. The inner container is fixedly installed inside the outer housing, and a gap of 5 cm to 8 cm is ensured between the inner container and the outer housing. The heat - insulating intermediate layer is filled in the gap between the outer housing and the inner container, and the material of the heat - insulating intermediate layer is polyurethane foam. The sealed door is movably connected to the front - side edge of the outer housing through three stainless - steel hinges. The internal space of the inner container constitutes a constant - temperature environment for cell culture. Horizontally sliding rails are symmetrically provided on both inner - wall sides of the inner container, and a culture tray 9 is slidably connected above the horizontally sliding rails.

[0020] The first liquid storage cylinder 2 and the second liquid storage cylinder 3 constitute a liquid - storage pressurization structure. The first liquid storage cylinder 2 and the second liquid storage cylinder 3 are fixedly installed above the top of the inner container, and the axes of the first liquid storage cylinder 2 and the second liquid storage cylinder 3 are parallel to each other. The first liquid storage cylinder 2 is a container for storing a high - concentration propionic acid culture solution, and the second liquid storage cylinder 3 is a container for storing a basal medium. The materials of the first liquid storage cylinder 2 and the second liquid storage cylinder 3 are both transparent organic glass, and volume scale lines in mL units are engraved on their outer walls. A first liquid outlet is provided at the bottom end of the first liquid storage cylinder 2, and a second liquid outlet is provided at the bottom end of the second liquid storage cylinder 3. Sealed pistons are provided inside both the first liquid storage cylinder 2 and the second liquid storage cylinder 3. Double nitrile rubber sealed rings are installed on the outer peripheral edge of the sealed piston, and the sealed piston is in close contact with the inner wall of the liquid storage cylinder. A pressurization push rod is fixedly connected to the central position of the sealed piston, and the pressurization push rod extends upward through the top cover of the liquid storage cylinder.

[0021] The synchronization crossbeam 4, adjustment screw rod 5, and graduated dial 6 constitute the fluid supply pressurized drive structure. The left and right ends of the synchronization crossbeam 4 are fixedly connected to the top ends of two pressurized push rods. A through hole with a female thread is provided in the center of the synchronization crossbeam 4. The adjustment screw rod 5 passes through the through hole and is rotatably mounted to the through hole, and fine threads are machined on the surface of the adjustment screw rod 5. The bottom end of the adjustment screw rod 5 is rotatably connected to a support frame fixed to the top of the housing via a deep groove ball bearing seat. A transmission gear is fixedly connected to the top end of the adjustment screw rod 5. The graduated dial 6 is installed on the outside of the top of the outer housing, and the central axis of the graduated dial 6 passes through the top of the outer housing, and a driving gear that meshes with the transmission gear is fixed to it. The circumferential surface of the graduated dial 6 displays scale values ​​from 0 to 100, and the scale value represents the piston displacement (i.e., the total stroke of the synchronization pressurized gear).

[0022] The plurality of parallel spiral mixing units 7 and the porous diffusion plate 8 together constitute a concentration gradient diffusion structure. The first liquid outlet and the second liquid outlet are respectively connected to the two liquid inlet ends of a shunt branch pipe arrangement frame via polytetrafluoroethylene conduits. Inside the shunt branch pipe arrangement frame, two independent main flow paths that do not communicate with each other are provided, and are respectively used for transporting the high-concentration propionic acid culture solution and the basal medium. Along the longitudinal direction of the shunt branch pipe arrangement frame, a plurality of sets of branch pipe pairs are derived in parallel, and each set of branch pipe pairs includes a propionic acid branch pipe communicating with the first main flow path and a basal medium branch pipe communicating with the second main flow path. The ends of the two branch pipes within the same set are both connected to the top inlet of one independent spiral mixing unit 7. Importantly, the ratio of the inner diameter of the pipe or the inlet cross-sectional area of each set of branch pipes changes in a preset gradient shape along the longitudinal direction of the shunt branch pipe arrangement frame. For example, in the leftmost set, the ratio of the flow cross-sectional areas of the propionic acid branch pipe and the medium branch pipe is the largest, so that the highest-concentration mixing precursor liquid is introduced, and in the rightmost set, the cross-sectional area ratio is the smallest, so that the lowest-concentration mixing precursor liquid is introduced. Inside each spiral mixing unit 7, six sets of spiral turbulator plates arranged at 90-degree intersections with each other are provided. The turbulator plates divide and recombine the two flowing liquids multiple times to complete sufficient independent mixing inside, and form a propionic acid culture solution with a specific concentration. The bottom outlets of all the spiral mixing units 7 are correspondingly connected one-to-one to a plurality of independent liquid inlets opened on the upper surface of the porous diffusion plate 8.

[0023] The surface of the porous diffusion plate 8 has diffusion micropores arranged in a matrix. These diffusion micropores are divided into multiple diffusion regions according to the specific concentration discharged by the helical mixing unit 7, and the diffusion micropores in each region uniformly drop the mixed culture medium of that specific concentration. Since the propionic acid concentrations discharged from different helical mixing units 7 are different and their positions are arranged sequentially along the longitudinal direction of the porous diffusion plate 8, a concentration gradient field that changes continuously along the longitudinal direction is formed on the culture tray 9 below. When conducting research on the control of osteoclast precursor cell differentiation by propionic acid-GPR43, the concentration gradient field can cover a wide concentration range, for example, from 0.1 mM to 10 mM. To achieve precise degradation within the effective inhibitory interval, the branching tube sequence frame can be pre-configured with numerous branching tube pairs during manufacturing. This forms a high-resolution propionic acid concentration gradient sequence on the culture tray, providing RAW264.7 cells and primary myeloid macrophages (BMMs) with a co-stimulatory environment of different concentrations of propionic acid and NF-κB receptor activator ligand (RANKL).

[0024] In this embodiment, a controlled culture environment is established by installing the housing assembly 1. The heat-insulating intermediate layer blocks heat exchange between the inside and outside, and the material of the inner container is mirror-finished stainless steel with a surface roughness of less than 0.4 μm. A magnetic adsorption type sealing strip is provided on the edge of the sealed door, and the sealing strip generates a sealing pressure at the contact surface with the outer housing. The observation window is embedded in the center of the sealed door, and the material of the observation window is double-layered tempered glass.

[0025] The installation of the first liquid storage cylinder 2 and the second liquid storage cylinder 3 enables the independent storage of two liquids of different concentrations. The sealing piston slides downward along the inner wall of the cylinder driven by the pressurizing push rod, pressurizing the liquid inside the cylinder. Since the diameters of the first liquid storage cylinder 2 and the second liquid storage cylinder 3 are the same, the synchronized crossbeam 4 can move the two pistons downward in sync, ensuring that the pressure and initial flow velocity of the two original liquid flows entering the flow division branch pipe array frame can be controlled synchronously.

[0026] The synchronized crossbeam 4, adjustment screw rod 5, and graduated dial 6 work together to achieve mechanized precision control for the synchronous supply of two types of liquids. When the operator rotates the graduated dial 6, the drive gear drives the transmission gear, which in turn rotates the adjustment screw rod 5 around its axis. Due to the screw configuration between the adjustment screw rod 5 and the synchronized crossbeam 4, the rotational motion of the adjustment screw rod 5 is converted into a vertical linear displacement of the synchronized crossbeam 4. The synchronized crossbeam 4 moves two pressurizing push rods downward at exactly the same speed, thereby pressurizing the propionic acid culture medium in the first reservoir 2 and the basal culture medium in the second reservoir 3 into the two independent main channels of the branching pipe array frame, respectively, under equivalent pressure conditions. The fine-threaded design provides high-precision displacement compensation, with one rotation of the graduated dial 6 corresponding to a 1 mm displacement of the synchronized crossbeam 4.

[0027] The installation of a flow-dividing and branching pipe array frame and multiple parallel helical mixing units 7 enables the generation of a concentration gradient and physical mixing. Since the cross-sectional area ratio of each set of branching pipes within the flow-dividing and branching pipe array frame is pre-set to change in a gradient, the initial volumetric flow rates of the propionic acid culture medium and basal medium entering each helical mixing unit 7 differ. After mixing within each helical mixing unit 7, a series of mixed culture solutions of specific concentrations that continuously change along the longitudinal direction are formed. As the two fluids flow through the helical flow-dispersing plate within each unit, the flow path is altered, generating a swirling flow, ensuring that liquids of different concentrations come into sufficient contact within the pipe and are uniformly mixed. The outside of all helical mixing units 7 is covered with a common constant-temperature heating sleeve, which maintains the mixed liquid at 37°C.

[0028] The installation of the porous diffusion plate 8 enables the spatially stable dropping and application of a concentration gradient. Mixtures of specific concentrations from different helical mixing units 7 each enter corresponding independent diffusion regions on the porous diffusion plate 8 and are uniformly dropped onto corresponding positions on the culture tray 9 via the diffusion micropores below. Since the liquid concentrations within each diffusion region are predetermined and arranged in a gradient along the longitudinal direction, different positions on the culture tray 9 below receive propionic acid culture solutions of different concentrations, thereby forming a predetermined, continuously changing concentration field on the surface of the culture tray 9.

[0029] In this embodiment, a waste liquid collection tank is provided in the bottom wall of the inner container, and the cross-section of the waste liquid collection tank is inverted trapezoidal. A drain pipe is connected to the bottom of the waste liquid collection tank, and the drain pipe extends to the outside through the outer casing. A manual ball valve is provided at the end of the drain pipe. The waste liquid collection tank collects excess liquid overflowing from the culture tray 9 and discharges it outside the casing via the drain pipe. After the experiment is completed, the culture tray can be removed, and in-situ fixation, TRAP staining, and microscopic observation can be performed directly on the cells in the culture dish or well plate according to the concentration divisions pre-marked on the porous diffusion plate. This ensures a one-to-one correspondence between the spatial position of the data and the concentration gradient, avoiding contamination risks and batch-to-batch differences associated with multiple opening operations.

[0030] In this embodiment, a Teflon® lubricating layer is applied to the outer surface of the pressurized push rod, a guide hole is provided in the support frame for the pressurized push rod to pass through, and a linear bearing is installed on the inner wall of the guide hole. The linear bearing limits the radial runout of the pressurized push rod and maintains its vertical motion.

[0031] In this embodiment, the porous diffusion plate 8 is fixed to the lower part of the top wall of the inner container by four support columns 50 mm in length. A 20 mm gap is maintained between the porous diffusion plate 8 and the culture tray 9. This gap provides physical space for droplet formation and fall.

[0032] In this embodiment, four support legs are fixed to the bottom of the outer casing, and rubber anti-slip pads are provided at the bottom ends of the support legs. The rubber anti-slip pads increase the frictional force between the equipment and the ground, and also act as a buffer against external vibrations.

[0033] In this embodiment, the first liquid storage cylinder 2 and the second liquid storage cylinder 3 are fixed to the support frame via stainless steel clamp rings. The clamp rings are fastened with bolts, and the liquid storage cylinders can be easily disassembled and cleaned. A one-way exhaust valve is provided at the top of the sealing piston, and the one-way exhaust valve discharges air from below the piston when the liquid is filled.

[0034] In this embodiment, the material of the helical mixing unit 7 and its connecting conduit is stainless steel 316L. A platinum resistance thermometer is embedded inside the constant temperature heating sleeve.

[0035] The operating procedure for this invention is as follows.

[0036] 1. A high-concentration propionic acid culture medium of a predetermined concentration is loaded into the first reservoir 2, the basal culture medium is loaded into the second reservoir 3, a sealing piston is attached, the pressurized push rod is connected to the synchronized crossbeam 4, and then the culture dish seeded with osteoclast precursor cells is placed on the culture tray 9, and the culture tray 9 is pushed into the predetermined position in the inner container by the horizontal sliding rail.

[0037] 2. The operator rotates the graduated dial 6, which rotates the adjustment screw rod 5 via the meshing of the drive gear and the transmission gear. The adjustment screw rod 5 drives the synchronization crossbeam 4 downward, and the sealing piston pressurizes the two original fluids into the two independent main channels of the branching pipe array frame.

[0038] 3. The two liquids are distributed within the branching pipe array frame to each helical mixing unit 7 according to a predetermined cross-sectional area ratio for each set of branching pipes. They are thoroughly mixed within each unit, forming a series of mixed solutions with different concentrations. These solutions are then dropped onto corresponding culture dishes on the culture tray 9 via the diffusion micropores of the porous diffusion plate 8, thereby creating a stable and continuous propionic acid concentration gradient within the culture space. At this time, osteoclast precursor cells located in different regions of the culture tray are exposed to propionic acid environments ranging from low to high concentrations. Researchers can collect cells from each region at the end of the experiment and directly compare changes in phosphorylation dynamics of the NF-κB and MAPK signaling pathways downstream of the GPR43 receptor, as well as differences in nuclear translocation of the core transcription factor NFATc1. This allows for a preliminary analysis of the dose-response relationship and molecular mechanism of propionic acid in a single experiment. After the experiment, the waste liquid is treated using a waste liquid collection tank and drainage pipe. [Explanation of Symbols]

[0039] 1. Enclosure Assembly 2 First liquid storage cylinder 3 Second liquid storage cylinder 4. Synchronized Crossbeam 5. Adjustable screw rod 6-point dial with scale 7. Helical mixing unit 8. Porous Diffuser 9 Culture trays

Claims

1. A concentration gradient cell culture device for research on the control of osteoclast precursor cell differentiation by propionic acid-GPR43, A housing assembly having an inner container, A first liquid storage cylinder and a second liquid storage cylinder are both fixedly installed above the top of the inner container, the first liquid storage cylinder having a first outlet at its bottom, the second liquid storage cylinder having a second outlet at its bottom, and a sealing piston is slidably installed inside both the first and second liquid storage cylinders. A tuning crossbeam is installed above the first and second liquid storage cylinders, wherein both ends of the tuning crossbeam are fixedly connected to the top ends of the piston rods of the two sealing pistons, and a screw hole is drilled in the center of the tuning crossbeam. An adjustment screw rod having a screw hole and a screw thread, wherein the bottom end of the adjustment screw rod is rotatably supported on the top of the housing assembly, A flow-diverting pipe array frame having a first main channel and a second main channel that do not communicate with each other, wherein the first outlet is in communication with the first main channel, the second outlet is in communication with the second main channel, and at least two pairs of branch pipes are led out in parallel along the longitudinal direction of the flow-diverting pipe array frame, and each pair of branch pipes includes a first branch pipe that communicates with the first main channel and a second branch pipe that communicates with the second main channel, A helical mixing unit comprising at least two helical mixing units, the inlet of each helical mixing unit being connected to the first branch end and the second branch end of a pair of branch pipes, A porous diffusion plate fixed to the lower part of the top wall of the inner container, wherein the upper surface of the porous diffusion plate has the same number of independent liquid inlets as the number of helical mixing units, the bottom outlet of each helical mixing unit is connected to one independent liquid inlet in a one-to-one correspondence, and the lower surface of the porous diffusion plate has multiple rows of diffusion micropores, A culture tray is slidably installed inside the inner container and directly below the porous diffusion plate, A concentration gradient cell culture device characterized by being equipped with the following features.

2. The concentration gradient cell culture apparatus according to claim 1, characterized in that the housing assembly comprises an outer casing, the inner container is fixed inside the outer casing, a heat-insulating intermediate layer is filled between the outer casing and the inner container, a waste liquid collection tank is provided in the bottom wall of the inner container, and a drain pipe that penetrates the outer casing is connected to the bottom of the waste liquid collection tank.

3. The concentration gradient cell culture apparatus according to claim 2, characterized in that the edge of the culture tray extends upward to form a surrounding frame.

4. The concentration gradient cell culture apparatus according to claim 3, characterized in that the helical mixing unit is made of stainless steel 316L and is covered with a constant temperature heating sleeve on its outside.

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