Plant cell culture device capable of regulating and controlling fluid shear force

Abstract

The invention discloses a plant cell culture device capable of regulating and controlling fluid shear force, and relates to the technical field of plant cell culture, and the plant cell culture device comprises a space positioning module used for obtaining three-dimensional coordinate data of plant cells attached to the surface of a microcarrier through an optical sensor array arranged in a closed culture cavity; the flow field construction module is used for calculating streamline distribution of the culture solution in the culture cavity based on coordinates of a cell dense region in the cell space distribution map; the shear stress calculation module is used for obtaining a shear stress value of each point on the surface of the cell through velocity gradient tensor operation and generating a shear stress distribution matrix; the dynamic regulation and control module is used for generating a flow channel sectional area adjustment instruction and a circulation flow adjustment instruction; and the execution response module is used for driving the variable aperture baffle group to change the local flow channel drift diameter based on the flow channel sectional area adjustment instruction and controlling the variable frequency pump to adjust the culture solution circulation rate based on the circulation flow adjustment instruction. And the success rate of plant cell culture is improved.

Description

Technical Field

[0001] This invention relates to the field of plant cell culture technology, and more particularly to a device for plant cell culture with adjustable fluid shear force. Background Technology

[0002] Plant cell culture technology has significant applications in biopharmaceuticals, functional foods, and the production of plant secondary metabolites. Existing plant cell culture devices generally employ fixed stirring rates or constant circulation flow rates, which cannot dynamically adjust the fluid environment based on changes in cell growth status and spatial distribution. This results in uneven shear stress distribution during culture, with some areas experiencing excessively high or low stress, severely impacting cell culture efficiency and product yield.

[0003] In existing technologies, operators can only adjust stirring parameters based on experience, lacking a scientific feedback mechanism and unable to dynamically optimize shear stress requirements in response to factors such as changes in cell density and metabolic state transitions during culture. This extensive control method not only limits the large-scale application of plant cell culture but also restricts the industrial production efficiency of high-value-added plant secondary metabolites.

[0004] Therefore, there is an urgent need for a device for plant cell culture that can regulate fluid shear force. Summary of the Invention

[0005] This invention provides a device for plant cell culture with adjustable fluid shear force to solve the above-mentioned problems in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A device for plant cell culture with adjustable fluid shear force, comprising: The spatial positioning module is used to acquire the three-dimensional coordinate data of plant cells attached to the surface of the microcarrier in real time through an optical sensor array set in a closed culture chamber, and generate a cell spatial distribution map. The flow field construction module is used to calculate the streamline distribution of the culture medium in the culture chamber based on the coordinates of the densely populated areas in the cell spatial distribution map, and generate the initial flow velocity vector field. The shear stress calculation module is used to obtain the shear stress values ​​at various points on the cell surface based on the initial flow velocity vector field and the viscosity coefficient of the culture medium through velocity gradient tensor calculation, and generate a shear stress distribution matrix. The dynamic control module is used to compare the shear stress distribution matrix with the preset suitable stress range for cell growth, identify the spatial location where the stress deviation exceeds the threshold, and generate flow channel cross-sectional area adjustment instructions and circulation flow rate adjustment instructions. The execution response module is used to drive the variable aperture baffle group to change the local flow channel diameter based on the flow channel cross-sectional area adjustment command, control the variable frequency pump to adjust the culture medium circulation rate based on the circulation flow rate adjustment command, update the flow velocity vector field and feed it back to the shear stress calculation module.

[0007] Furthermore, the spatial positioning module includes: The image acquisition unit is used to simultaneously capture orthogonal projection images of the microcarrier group using multi-angle CCD cameras arranged on the transparent sidewall of the culture chamber. The three-dimensional reconstruction unit is used to calculate the center coordinates of each microcarrier based on the contour feature points of the microcarrier in the orthogonal projection image using a stereo vision algorithm. The cell recognition unit is used to perform fluorescence staining imaging on the surface of the microcarrier corresponding to the central coordinates to identify the number and distribution density of attached cells. The map generation unit is used to integrate the center coordinates of the microcarrier with the corresponding cell distribution density into a cell spatial distribution map containing spatial location and density information.

[0008] Furthermore, the flow field construction module includes: The dense region extraction unit is used to perform density clustering analysis on the spatial distribution map of cells and extract the boundary coordinates of spatial regions where the cell density exceeds a preset density threshold. The streamline planning unit is used to calculate the mainstream path of the culture medium from the inlet to the outlet based on the spatial region boundary coordinates and the positions of the inlet and outlet of the culture chamber through computational fluid dynamics simulation. The velocity field calculation unit is used to solve the velocity magnitude and flow direction of each spatial grid point in the culture chamber based on the main flow path and the set circulation flow rate, and generate an initial velocity vector field containing velocity vectors.

[0009] Furthermore, the shear stress calculation module includes: The viscosity acquisition unit is used to measure the dynamic viscosity coefficient of the current culture medium via an online viscometer; The gradient calculation unit is used to perform spatial partial derivative calculations on the velocity vector of each grid point in the initial velocity vector field to obtain the velocity gradient tensor. The stress transformation element is used to perform tensor multiplication between the velocity gradient tensor and the dynamic viscosity coefficient to obtain the shear stress tensor at each grid point. The surface mapping unit is used to extract the shear stress tensor components corresponding to the cell surface coordinates in the cell spatial distribution map and construct a shear stress distribution matrix indexed by the cell surface position.

[0010] Furthermore, the dynamic control module includes: The threshold comparison unit is used to obtain the upper and lower limits of the preset suitable stress range for cell growth, and compares the stress value at each position in the shear stress distribution matrix with the upper and lower limits. The deviation location unit is used to mark high stress locations where the stress value exceeds the upper limit and low stress locations where the stress value is below the lower limit, and to generate a set of stress deviation locations containing the coordinates of the abnormal locations. The flow channel adjustment strategy unit is used to generate adjustment commands to increase the flow channel cross-sectional area for high stress locations and to generate adjustment commands to decrease the flow channel cross-sectional area for low stress locations. The flow rate adjustment strategy unit generates an adjustment command to increase the circulation flow rate when the spatial volume covered by the stress deviation location set exceeds a preset proportion of the total volume of the culture chamber.

[0011] Furthermore, the flow channel adjustment strategy unit includes: The radial distance calculation sub-unit is used to calculate the radial distance between each abnormal location in the stress deviation location set and the nearest flow channel wall. The baffle positioning subunit is used to identify the position of the flow channel wall where the radial distance is less than the preset critical distance, and mark the position as the baffle adjustment target position. The aperture adjustment calculation subunit is used to calculate the required aperture change based on the stress deviation amplitude at the baffle adjustment target position using a stress-flow velocity correlation model. The instruction encoding subunit is used to encode the baffle adjustment target position and the change in aperture into a flow channel cross-sectional area adjustment instruction that includes the stepper motor rotation angle.

[0012] Furthermore, the execution response module includes: The baffle drive unit is used to drive the inner ring of the concentric ring variable aperture baffle group to rotate relative to the outer ring based on the stepper motor rotation angle in the flow channel cross-sectional area adjustment command, thereby changing the flow aperture of the baffle group. The flow control unit is used to adjust the speed of the variable frequency pump based on the target flow value in the circulating flow adjustment command through a PID control algorithm. The flow field update unit is used to remeasure the flow velocity distribution in the culture chamber and generate an updated flow velocity vector field after the flow orifice diameter of the baffle group is changed and the speed of the variable frequency pump is adjusted. The closed-loop feedback unit is used to transmit the updated velocity vector field to the shear stress calculation module, triggering a new round of shear stress distribution matrix calculation.

[0013] Furthermore, it also includes: The environmental parameter acquisition module is used to acquire the temperature, pH and dissolved oxygen concentration of the culture medium in real time through a distributed sensor network, and generate a time series of environmental parameters. The metabolic status assessment module is used to calculate the oxygen consumption rate of plant cells based on the rate of decrease of dissolved oxygen concentration in the time series of environmental parameters, and to determine the level of cell metabolic activity based on the oxygen consumption rate. The appropriate stress range adjustment module is used to retrieve a new stress range corresponding to the metabolic activity level from the pre-stored stress range database when the cell metabolic activity level changes, and update the appropriate stress range for cell growth in the dynamic regulation module.

[0014] Furthermore, the metabolic state assessment module includes: The concentration difference unit is used to extract the dissolved oxygen concentration values ​​at adjacent time points in the time series of environmental parameters and to calculate the rate of change of dissolved oxygen concentration over time. The consumption rate normalization unit is used to divide the dissolved oxygen concentration change rate by the current total number of cells to obtain the oxygen consumption rate per unit cell. The activity grading unit is used to compare the oxygen consumption rate per unit cell with a preset activity grading threshold sequence to determine the cell metabolic activity level. The stress demand mapping unit is used to query a pre-stored stress demand mapping table based on the cell metabolic activity level to obtain the upper and lower limits of shear stress for the corresponding level.

[0015] Compared with the prior art, the present invention has the following advantages: This invention integrates modules such as spatial positioning, flow field construction, stress calculation, and dynamic control to achieve precise monitoring and real-time adjustment of fluid shear force during plant cell culture. It solves the technical problems of inaccurate shear stress control and inability to dynamically adapt to cell growth needs in existing technologies, and significantly improves the success rate and product yield of plant cell culture.

[0016] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a structural diagram of a plant cell culture device with adjustable fluid shear force according to an embodiment of the present invention; Figure 2 This is a simplified diagram of the plant cell culture apparatus in an embodiment of the present invention; Figure 3This is a structural diagram of the spatial positioning module in an embodiment of the present invention. Detailed Implementation

[0019] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0020] The embodiments of the present invention provide, as follows Figure 1 As shown, a device for plant cell culture with adjustable fluid shear force includes: The spatial positioning module is used to acquire the three-dimensional coordinate data of plant cells attached to the surface of the microcarrier in real time through an optical sensor array set in a closed culture chamber, and generate a cell spatial distribution map. The flow field construction module is used to calculate the streamline distribution of the culture medium in the culture chamber based on the coordinates of the densely populated areas in the cell spatial distribution map, and generate the initial flow velocity vector field. The shear stress calculation module is used to obtain the shear stress values ​​at various points on the cell surface based on the initial flow velocity vector field and the viscosity coefficient of the culture medium through velocity gradient tensor calculation, and generate a shear stress distribution matrix. The dynamic control module is used to compare the shear stress distribution matrix with the preset suitable stress range for cell growth, identify the spatial location where the stress deviation exceeds the threshold, and generate flow channel cross-sectional area adjustment instructions and circulation flow rate adjustment instructions. The execution response module is used to drive the variable aperture baffle group to change the local flow channel diameter based on the flow channel cross-sectional area adjustment command, control the variable frequency pump to adjust the culture medium circulation rate based on the circulation flow rate adjustment command, update the flow velocity vector field and feed it back to the shear stress calculation module.

[0021] The working principle and beneficial effects of the above technical solution are as follows: Figure 2 As shown, the optical sensor array 1 is distributed on the inner wall of the cavity, the flow field construction module 2 is connected to the cavity, the shear stress calculation module 3 is the processing unit, the dynamic control module 4 is the control unit, the variable aperture baffle group 5 is installed in the flow channel, the frequency converter pump 6 is connected to the culture medium circulation pipeline 7, and the microcarrier 8 is suspended in the culture medium.

[0022] This device achieves precise control of fluid shear force during plant cell culture through multi-module collaboration. During operation, the spatial positioning module first activates the optical sensor array to scan the microcarriers within the sealed culture chamber. The sensors capture images of cell attachment from different angles, and the three-dimensional spatial coordinates of each microcarrier and its surface plant cells are extracted through image processing algorithms. These coordinate data are combined with cell density information to form a spatial map that reflects the cell distribution state within the culture chamber.

[0023] After receiving the spatial map, the flow field construction module identifies the specific locations of densely celled regions. Using these regions as key control points and combining the geometric parameters of the culture chamber, it calculates the complete flow path of the culture medium from the inlet to the outlet using fluid dynamics equations. For the three-dimensional grid divided within the chamber, it solves the magnitude and direction of the flow velocity point by point, and finally establishes an initial flow velocity vector field covering the entire culture space.

[0024] After obtaining the initial velocity vector field, the shear stress calculation module extracts the real-time viscosity parameters of the culture medium, performs partial derivative calculations on each spatial point in the velocity vector field to obtain the rate of change of the velocity in each direction at that point, forming a velocity gradient tensor. This tensor is then mathematically calculated with the viscosity coefficient of the culture medium to convert it into a shear stress tensor at that point. Subsequently, the shear stress values ​​corresponding to each position on the cell surface are extracted to construct a distribution matrix reflecting the stress state of the cell.

[0025] The dynamic control module compares the shear stress distribution matrix with the pre-set suitable stress range for cell growth point by point. When it finds that the stress values ​​at certain locations exceed the suitable range, it records the coordinates of these abnormal locations. For locations with excessively high stress, it generates control commands to expand the flow channel, and for locations with excessively low stress, it generates control commands to shrink the flow channel. At the same time, it evaluates the distribution range of abnormal locations. If the abnormal area occupies a large proportion of the space, it generates additional commands to adjust the circulation flow rate.

[0026] After receiving the adjustment command, the execution response module drives the drive mechanism of the variable aperture baffle group according to the flow channel cross-sectional area adjustment command, so that the flow aperture of the baffle changes accordingly, thereby changing the flow capacity of the local flow channel. At the same time, according to the circulation flow adjustment command, the variable frequency pump is controlled to change the operating frequency, thereby adjusting the overall circulation speed of the culture medium. After these adjustment actions are completed, the flow velocity distribution in the culture chamber changes. The system remeasures the updated flow velocity vector field and feeds it back to the shear stress calculation module, forming a closed-loop control cycle to continuously optimize the shear stress environment on the cell surface.

[0027] In another embodiment, such as Figure 3 As shown, the spatial positioning module includes: The image acquisition unit is used to simultaneously capture orthogonal projection images of the microcarrier group using multi-angle CCD cameras arranged on the transparent sidewall of the culture chamber. The three-dimensional reconstruction unit is used to calculate the center coordinates of each microcarrier based on the contour feature points of the microcarrier in the orthogonal projection image using a stereo vision algorithm. The cell recognition unit is used to perform fluorescence staining imaging on the surface of the microcarrier corresponding to the central coordinates to identify the number and distribution density of attached cells. The map generation unit is used to integrate the center coordinates of the microcarrier with the corresponding cell distribution density into a cell spatial distribution map containing spatial location and density information.

[0028] The working principle and beneficial effects of the above technical solution are as follows: The spatial positioning module activates the CCD cameras deployed on the transparent sidewalls of the culture chamber at different angles through the image acquisition unit. Multiple cameras are triggered to capture images simultaneously, obtaining projection images of the microcarrier group from different perspectives. These images record the position information of the microcarriers in various observation directions.

[0029] After receiving the orthogonal projection image, the 3D reconstruction unit uses an image segmentation algorithm to extract the edge contour of each microcarrier, identify the feature points on the contour, and, based on the principle of stereo vision geometry, uses the positional differences of the same microcarrier in different images taken by different cameras to infer the true coordinates of the microcarrier in the 3D space of the culture chamber through a triangulation algorithm, and calculates the center point position of each microcarrier.

[0030] After the cell recognition unit obtains the center coordinates of the microcarrier, it controls the fluorescent excitation light source to irradiate the surface of the microcarrier at the corresponding position. Since the plant cells attached to the microcarrier have been treated with fluorescent staining, the cells emit fluorescence of a specific wavelength under the excitation light. These fluorescence signals are captured by the fluorescence imaging system, and the number and intensity distribution of fluorescence spots on the surface of each microcarrier are statistically analyzed using image processing technology, thereby determining the number of cells attached to the microcarrier and their distribution density on the surface of the microcarrier.

[0031] The atlas generation unit integrates the center coordinates of the microcarriers provided by the 3D reconstruction unit with the cell distribution density data obtained by the cell recognition unit. It establishes a data record for each microcarrier containing spatial location information and cell density information. By summarizing the data records of all microcarriers, a complete cell spatial distribution atlas is formed. This atlas can reflect the spatial distribution characteristics of cells in the culture chamber and show the degree of cell density at each location.

[0032] In another embodiment, the flow field construction module includes: The dense region extraction unit is used to perform density clustering analysis on the spatial distribution map of cells and extract the boundary coordinates of spatial regions where the cell density exceeds a preset density threshold. The streamline planning unit is used to calculate the mainstream path of the culture medium from the inlet to the outlet based on the spatial region boundary coordinates and the positions of the inlet and outlet of the culture chamber through computational fluid dynamics simulation. The velocity field calculation unit is used to solve the velocity magnitude and flow direction of each spatial grid point in the culture chamber based on the main flow path and the set circulation flow rate, and generate an initial velocity vector field containing velocity vectors.

[0033] The working principle and beneficial effects of the above technical solution are as follows: After receiving the spatial distribution map of cells, the dense area extraction unit in the flow field construction module performs density clustering operation on the map data, classifies spatially adjacent and density-similar positions into the same cluster, calculates the average cell density of each cluster, identifies clusters with density exceeding a preset threshold as dense cell regions, extracts the spatial boundary coordinates of these dense regions, and determines the flow field control areas that need to be focused on.

[0034] After obtaining the boundary coordinates of the dense region, the streamline planning unit, combined with the fixed position information of the inlet and outlet of the culture chamber, establishes a three-dimensional geometric model of the culture chamber. The dense region is taken as the key area for flow field calculation. The boundary conditions are set using computational fluid dynamics software to simulate the flow process of the culture medium after entering the chamber from the inlet. The movement trajectory of the culture medium particles is tracked to generate the main flow path from the inlet to the outlet. These streamlines describe the main flow direction of the culture medium in the chamber.

[0035] The velocity field calculation unit, based on the main flow path and user-defined circulation flow parameters, applies the fluid dynamics control equations to divide the three-dimensional space of the culture chamber into a computational grid. For each grid node, a velocity solution equation is established. Considering the continuity constraints of the culture medium and the momentum conservation relationship, the velocity magnitude and flow direction vector at each grid point are solved through numerical iteration. The velocity vectors of all grid points are organized into structured data to generate an initial velocity vector field covering the entire culture space. This vector field completely describes the flow state of the culture medium at each location in the chamber.

[0036] In another embodiment, the shear stress calculation module includes: The viscosity acquisition unit is used to measure the dynamic viscosity coefficient of the current culture medium via an online viscometer; The gradient calculation unit is used to perform spatial partial derivative calculations on the velocity vector of each grid point in the initial velocity vector field to obtain the velocity gradient tensor. The stress transformation element is used to perform tensor multiplication between the velocity gradient tensor and the dynamic viscosity coefficient to obtain the shear stress tensor at each grid point. The surface mapping unit is used to extract the shear stress tensor components corresponding to the cell surface coordinates in the cell spatial distribution map and construct a shear stress distribution matrix indexed by the cell surface position.

[0037] The working principle and beneficial effects of the above technical solution are as follows: The shear stress calculation module first starts the online viscometer through the viscosity acquisition unit to measure the culture medium in real time. The probe of the viscometer is immersed in the culture medium and the resistance characteristics of the culture medium to external forces are determined by vibration or rotation method. Based on the measurement results, the dynamic viscosity coefficient of the culture medium under the current temperature and composition conditions is calculated. This coefficient reflects the frictional resistance between molecules when the culture medium flows inside.

[0038] After receiving the initial velocity vector field data, the gradient calculation unit extracts the velocity vector components of each grid point and its neighboring points for each grid point in the vector field. It performs partial derivative operations on each component of the velocity vector in three spatial directions to calculate the rate of change of the velocity in the x, y, and z directions. These nine partial derivative components are organized into a third-order tensor to obtain the velocity gradient tensor of that point. This tensor completely describes the changes in velocity in all directions near that point.

[0039] After obtaining the velocity gradient tensor and dynamic viscosity coefficient from the stress transformation unit, the velocity gradient tensor and viscosity coefficient are multiplied using a tensor multiplication operation based on the constitutive relationship between shear stress and velocity gradient in fluid mechanics, and the calculation is performed according to the following formula: Where τ represents the shear stress tensor and μ represents the dynamic viscosity coefficient. This represents the velocity gradient tensor. This operation converts the velocity field information into stress field information, resulting in the shear stress tensor for each grid point.

[0040] The surface mapping unit extracts the spatial coordinates of each point on the cell surface from the cell spatial distribution map, finds the grid points corresponding to these coordinates in the shear stress tensor field, and extracts the components of the shear stress tensor at the grid points that are related to the cell surface normal vector. These components represent the actual shear stress acting on the cell surface. The extracted stress values ​​are indexed and organized according to the cell surface position to construct a distribution matrix with cell surface coordinates as the index and shear stress values ​​as the content. This matrix intuitively reflects the shear stress condition at each position on the cell surface.

[0041] In another embodiment, the dynamic control module includes: The threshold comparison unit is used to obtain the upper and lower limits of the preset suitable stress range for cell growth, and compares the stress value at each position in the shear stress distribution matrix with the upper and lower limits. The deviation location unit is used to mark high stress locations where the stress value exceeds the upper limit and low stress locations where the stress value is below the lower limit, and to generate a set of stress deviation locations containing the coordinates of the abnormal locations. The flow channel adjustment strategy unit is used to generate adjustment commands to increase the flow channel cross-sectional area for high stress locations and to generate adjustment commands to decrease the flow channel cross-sectional area for low stress locations. The flow rate adjustment strategy unit generates an adjustment command to increase the circulation flow rate when the spatial volume covered by the stress deviation location set exceeds a preset proportion of the total volume of the culture chamber.

[0042] The working principle and beneficial effects of the above technical solution are as follows: The threshold comparison unit in the dynamic control module first reads the preset suitable stress range for cell growth from the parameter library stored in the system. This range is defined by the upper limit and the lower limit, which represent the range of shear stress that plant cells can withstand for normal growth. The unit extracts the stress value at each position in the shear stress distribution matrix one by one, compares the value with the upper limit and the lower limit, and determines whether the stress at that position is within the suitable range.

[0043] The deviation localization unit classifies and marks abnormal locations based on the comparison results. Locations with stress values ​​greater than the upper limit are marked as high-stress locations, where excessive shear force may damage cells. Locations with stress values ​​less than the lower limit are marked as low-stress locations, where insufficient shear force affects nutrient exchange in cells. All marked abnormal locations and their coordinate information are summarized to generate a set of stress deviation locations containing location coordinates and deviation types.

[0044] The flow channel adjustment strategy unit analyzes the deviation type of each abnormal location in the stress deviation location set, formulates a strategy to reduce the flow rate for high stress locations, and generates an adjustment command to expand the cross-sectional area of ​​the flow channel near the location, so that the flow rate of the culture medium at that location is reduced, thereby reducing shear stress. For low stress locations, formulate a strategy to increase the flow rate, and generate an adjustment command to reduce the cross-sectional area of ​​the flow channel near the location, so that the flow of the culture medium at that location is accelerated, thereby increasing shear stress.

[0045] The flow adjustment strategy unit statistically analyzes the spatial range covered by the set of stress deviation locations and calculates the ratio of the volume occupied by the abnormal location to the total volume of the culture chamber. When this ratio exceeds the preset critical ratio, it indicates that the problem cannot be solved by adjusting the local flow channel alone, and the overall circulation flow needs to be controlled. At this time, the unit generates a global adjustment command to increase or decrease the circulation flow, thereby optimizing the stress environment by changing the overall circulation speed of the culture medium.

[0046] In another embodiment, the flow channel adjustment strategy unit includes: The radial distance calculation sub-unit is used to calculate the radial distance between each abnormal location in the stress deviation location set and the nearest flow channel wall. The baffle positioning subunit is used to identify the position of the flow channel wall where the radial distance is less than the preset critical distance, and mark the position as the baffle adjustment target position. The aperture adjustment calculation subunit is used to calculate the required aperture change based on the stress deviation amplitude at the baffle adjustment target position using a stress-flow velocity correlation model. The instruction encoding subunit is used to encode the baffle adjustment target position and the change in aperture into a flow channel cross-sectional area adjustment instruction that includes the stepper motor rotation angle.

[0047] The working principle and beneficial effects of the above technical solution are as follows: After the radial distance calculation subunit in the flow channel adjustment strategy unit obtains the set of stress deviation locations, it extracts the three-dimensional coordinates of the abnormal locations one by one, identifies the flow channel wall closest to the coordinates in the geometric model of the culture chamber, and calculates the vertical distance from the abnormal location point to the wall. This distance reflects the spatial correlation between the abnormal location and the adjustable flow channel.

[0048] The baffle positioning subunit compares the calculated radial distance with the preset critical distance. When the radial distance is less than the critical distance, it indicates that the abnormal position is close to the flow channel wall and is significantly affected by the flow velocity of the flow channel. The flow channel wall position corresponding to the abnormal position is marked as the baffle adjustment target position, and it is determined that a variable aperture baffle needs to be installed or adjusted at this position.

[0049] The aperture adjustment calculation subunit, for each baffle adjustment target position, extracts the stress deviation amplitude at that position, i.e., the difference between the actual stress and the boundary of the suitable stress range, and calls the pre-stored stress-flow velocity correlation model. This model uses the formula... Describing the amount of stress change With aperture change The mapping relationship between them, where The nonlinear function is established based on the principles of fluid mechanics. The stress deviation amplitude is substituted into the model for inverse solution to calculate the amount of aperture change that needs to be adjusted. This amount of change indicates the specific degree to which the baffle needs to be opened or closed.

[0050] After obtaining the target position and aperture change of the baffle, the instruction encoding subunit converts the aperture change into the rotation angle required to drive the stepper motor according to the mechanical structure parameters of the baffle group. The target position number and the calculated rotation angle are encoded according to the communication protocol to generate a flow channel cross-sectional area adjustment instruction that can be directly sent to the actuator.

[0051] In another embodiment, the execution response module includes: The baffle drive unit is used to drive the inner ring of the concentric ring variable aperture baffle group to rotate relative to the outer ring based on the stepper motor rotation angle in the flow channel cross-sectional area adjustment command, thereby changing the flow aperture of the baffle group. The flow control unit is used to adjust the speed of the variable frequency pump based on the target flow value in the circulating flow adjustment command through a PID control algorithm. The flow field update unit is used to remeasure the flow velocity distribution in the culture chamber and generate an updated flow velocity vector field after the flow orifice diameter of the baffle group is changed and the speed of the variable frequency pump is adjusted. The closed-loop feedback unit is used to transmit the updated velocity vector field to the shear stress calculation module, triggering a new round of shear stress distribution matrix calculation.

[0052] The working principle and beneficial effects of the above technical solution are as follows: During the execution response phase, the baffle drive unit analyzes the stepper motor rotation angle parameter in the flow channel cross-sectional area adjustment command, drives the inner ring of the concentric ring baffle group to rotate relative to the outer ring by a specific angle, and the misalignment of the inner and outer rings changes the flow orifice diameter. The flow control unit reads the flow target value in the circulating flow adjustment command, calculates the deviation between the current speed and the target speed of the variable frequency pump through the PID control algorithm, and outputs a control signal to adjust the pump speed until the actual flow reaches the target value. After the baffle group and variable frequency pump have completed their execution, the flow field update unit uses a flow velocity sensor to remeasure the flow velocity at various locations in the culture chamber, and re-solves the flow velocity vector field based on the new flow channel geometry and circulating flow, generating an updated flow velocity vector field. The closed-loop feedback unit transmits the updated flow velocity vector field to the shear stress calculation module, triggering a new round of velocity gradient calculation and shear stress distribution matrix generation, forming a continuously optimized closed-loop control.

[0053] In another embodiment, it further includes: The environmental parameter acquisition module is used to acquire the temperature, pH and dissolved oxygen concentration of the culture medium in real time through a distributed sensor network, and generate a time series of environmental parameters. The metabolic status assessment module is used to calculate the oxygen consumption rate of plant cells based on the rate of decrease of dissolved oxygen concentration in the time series of environmental parameters, and to determine the level of cell metabolic activity based on the oxygen consumption rate. The appropriate stress range adjustment module is used to retrieve a new stress range corresponding to the metabolic activity level from the pre-stored stress range database when the cell metabolic activity level changes, and update the appropriate stress range for cell growth in the dynamic regulation module.

[0054] The working principle and beneficial effects of the above technical solution are as follows: The environmental parameter acquisition module continuously collects temperature, pH, and dissolved oxygen concentration data of the culture medium through temperature sensors, pH sensors, and dissolved oxygen sensors distributed at different locations in the culture chamber, and records them in chronological order to form an environmental parameter time series. The metabolic state assessment module extracts dissolved oxygen concentration data from the time series, calculates the rate of decrease in dissolved oxygen concentration per unit time, and this rate of decrease reflects the oxygen consumption rate of plant cells. The consumption rate is compared with a preset metabolic activity grading standard to determine whether the cell is currently in a vigorous, normal, or slow metabolic state. The suitable stress range adjustment module monitors changes in the cell's metabolic activity level. When a change in level is detected, it retrieves the appropriate upper and lower limits of the stress range corresponding to the new metabolic state from the pre-stored stress range database, updates the new range values ​​to the dynamic control module, and ensures that subsequent shear stress regulation is based on the cell's current metabolic needs.

[0055] In another embodiment, the metabolic state assessment module includes: The concentration difference unit is used to extract the dissolved oxygen concentration values ​​at adjacent time points in the time series of environmental parameters and to calculate the rate of change of dissolved oxygen concentration over time. The consumption rate normalization unit is used to divide the dissolved oxygen concentration change rate by the current total number of cells to obtain the oxygen consumption rate per unit cell. The activity grading unit is used to compare the oxygen consumption rate per unit cell with a preset activity grading threshold sequence to determine the cell metabolic activity level. The stress demand mapping unit is used to query a pre-stored stress demand mapping table based on the cell metabolic activity level to obtain the upper and lower limits of shear stress for the corresponding level.

[0056] The working principle and beneficial effects of the above technical solution are as follows: During the metabolic state assessment, the concentration difference unit extracts dissolved oxygen concentration values ​​from adjacent sampling times in the environmental parameter time series, calculates the concentration difference, and divides it by the time interval to obtain the rate of change of dissolved oxygen concentration over time. The consumption rate normalization unit obtains the total number of cells in the current culture chamber, divides the rate of change of dissolved oxygen concentration by the total number of cells to eliminate the influence of cell number differences, and obtains the average oxygen consumption rate of a single cell. The activity grading unit compares the oxygen consumption rate per unit cell with a preset activity grading threshold sequence (such as low activity threshold, normal activity threshold, and high activity threshold), and determines the cell metabolic activity level based on the range of the value. The stress demand mapping unit queries a pre-stored stress demand mapping table, which records the suitable shear stress range for cells under different metabolic activity levels, extracts the upper and lower limits of the shear stress for the corresponding level, and provides this information to the suitable stress range adjustment module.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of this invention.

Claims

1. A device for plant cell culture with adjustable fluid shear force, characterized in that, The method comprises the following steps: A spatial positioning module is used to obtain three-dimensional coordinate data of plant cells attached to the surface of microcarriers in real time through an optical sensor array arranged in a closed culture cavity, and generate a cell spatial distribution map; A flow field construction module is used to calculate the streamline distribution of the culture solution in the culture cavity based on the cell dense area coordinates in the cell spatial distribution map, and generate an initial flow velocity vector field; A shear stress calculation module is used to obtain the shear stress values of each point on the cell surface by velocity gradient tensor operation according to the initial flow velocity vector field and the viscosity coefficient of the culture solution, and generate a shear stress distribution matrix; A dynamic regulation module is used to compare the shear stress distribution matrix with a preset cell growth suitable stress interval, identify the spatial positions with stress deviation exceeding the threshold, and generate flow passage cross-sectional area adjustment instructions and circulation flow rate adjustment instructions; An execution response module is used to drive the variable-aperture baffle group to change the local flow passage diameter based on the flow passage cross-sectional area adjustment instructions, control the variable frequency pump to adjust the culture solution circulation rate based on the circulation flow rate adjustment instructions, update the flow velocity vector field and feed back to the shear stress calculation module.

2. The device for the cultivation of plant cells with controllable fluid shear according to claim 1, characterized in that The spatial positioning module comprises: An image acquisition unit is used to synchronously capture orthogonal projection images of the microcarrier group through multi-angle CCD cameras arranged on the transparent side wall of the culture cavity; A three-dimensional reconstruction unit is used to calculate the center coordinates of each microcarrier through a stereo vision algorithm based on the profile feature points of the microcarriers in the orthogonal projection images; A cell recognition unit is used to perform fluorescence staining imaging on the surface of the microcarriers corresponding to the center coordinates, and identify the number and distribution density of the attached cells; A map generation unit is used to integrate the microcarrier center coordinates and the corresponding cell distribution density into a cell spatial distribution map containing spatial position and density information.

3. The device for the controlled fluid shear of plant cell cultures of claim 1, wherein, The flow field construction module comprises: A dense area extraction unit is used to perform density clustering analysis on the cell spatial distribution map, and extract the boundary coordinates of the spatial area with a cell density exceeding a preset density threshold; A streamline planning unit is used to calculate the main streamline path of the culture solution from the liquid inlet to the liquid outlet of the culture cavity based on the spatial area boundary coordinates and the positions of the liquid inlet and the liquid outlet of the culture cavity through computational fluid dynamics simulation; A velocity field solving unit is used to solve the flow velocity and flow direction of each spatial grid point in the culture cavity based on the main streamline path and the set circulation flow rate, and generate an initial flow velocity vector field containing velocity vectors.

4. The device for the controlled fluid shear of plant cell cultures of claim 1, wherein, The shear stress calculation module comprises: A viscosity acquisition unit is used to measure the dynamic viscosity coefficient of the current culture solution through an online viscometer; A gradient calculation unit is used to perform spatial partial derivative operation on the velocity vectors of each grid point in the initial flow velocity vector field, and obtain the velocity gradient tensor; A stress conversion unit is used to perform tensor multiplication operation on the velocity gradient tensor and the dynamic viscosity coefficient to obtain the shear stress tensor of each grid point; A surface mapping unit is used to extract the shear stress tensor components corresponding to the cell surface coordinates in the cell spatial distribution map, and construct a shear stress distribution matrix indexed by the cell surface positions.

5. The device for the controlled fluid shear of plant cell cultures of claim 1, wherein, The dynamic regulation module comprises: a threshold comparison unit configured to obtain an upper limit value and a lower limit value of a preset suitable stress interval for cell growth, and compare stress values at each position in the shear stress distribution matrix with the upper limit value and the lower limit value; a deviation positioning unit configured to mark high-stress positions with stress values exceeding the upper limit value and low-stress positions with stress values lower than the lower limit value, and generate a stress deviation position set containing coordinates of abnormal positions; a flow channel adjustment strategy unit configured to generate an adjustment instruction for expanding the cross-sectional area of the flow channel for the high-stress positions, and generate an adjustment instruction for reducing the cross-sectional area of the flow channel for the low-stress positions; a flow rate adjustment strategy unit configured to generate an adjustment instruction for increasing the circulating flow rate when the spatial volume covered by the stress deviation position set exceeds a preset proportion of the total volume of the culture cavity.

6. The device for the controlled fluid shear force plant cell culture of claim 5, wherein, The flow channel adjustment strategy unit comprises: a radial distance calculation subunit configured to calculate the radial distance of each abnormal position in the stress deviation position set from the nearest flow channel wall surface; a baffle positioning subunit configured to identify the position of the flow channel wall surface with a radial distance less than a preset critical distance, and mark the position as a baffle adjustment target position; a pore size adjustment amount calculation subunit configured to calculate the required pore size change amount based on the stress deviation amplitude of the baffle adjustment target position through a stress-flow rate correlation model; an instruction encoding subunit configured to encode the baffle adjustment target position and the pore size change amount into a flow channel cross-sectional area adjustment instruction containing the rotation angle of the stepper motor.

7. The device for the controlled fluid shear of plant cell cultures of claim 1, wherein, The execution response module comprises: a baffle driving unit configured to drive the inner ring of the concentric ring variable pore size baffle set to rotate relative to the outer ring based on the rotation angle of the stepper motor in the flow channel cross-sectional area adjustment instruction, and change the flow-through pore size of the baffle set; a flow rate control unit configured to adjust the rotation speed of the variable frequency pump through a PID control algorithm based on the flow rate target value in the circulating flow rate adjustment instruction; a flow field updating unit configured to re-measure the flow rate distribution in the culture cavity after the flow-through pore size of the baffle set is changed and the rotation speed of the variable frequency pump is adjusted, and generate an updated flow rate vector field; a closed-loop feedback unit configured to transmit the updated flow rate vector field to the shear stress calculation module to trigger a new round of shear stress distribution matrix calculation.

8. The device for the controlled fluid shear of plant cell cultures of claim 1, wherein, Further comprising: an environmental parameter acquisition module configured to obtain temperature values, pH values, and dissolved oxygen concentration values of the culture solution in real time through a distributed sensor network, and generate an environmental parameter time series; a metabolic state evaluation module configured to calculate the oxygen consumption rate of plant cells based on the decline rate of the dissolved oxygen concentration in the environmental parameter time series, and determine the cell metabolic activity level according to the oxygen consumption rate; a suitable stress interval adjustment module configured to retrieve a new stress interval corresponding to the metabolic activity level from a pre-stored stress interval database when the cell metabolic activity level changes, and update the cell growth suitable stress interval in the dynamic regulation module.

9. The device for the controlled fluid shear of plant cell cultures according to claim 8, characterized in that The metabolic state evaluation module comprises: a concentration difference unit configured to extract the dissolved oxygen concentration values at adjacent time points in the environmental parameter time series, and calculate the change rate of the dissolved oxygen concentration over time; a consumption rate normalization unit configured to divide the change rate of the dissolved oxygen concentration by the current total number of cells to obtain the oxygen consumption rate per cell; An activity grading unit is configured to compare the oxygen consumption rate of the unit cell with a preset activity grading threshold sequence to determine a cell metabolic activity grade. A stress demand mapping unit is configured to query a pre-stored stress demand mapping table according to the cell metabolic activity grade to obtain an upper limit value and a lower limit value of shear stress corresponding to the grade.

Images