Cell and microorganism perforated plate culture method and system

Abstract

The invention relates to the field of cell and microorganism culture, and discloses a cell and microorganism perforated plate culture method, which comprises the following steps: acquiring the drug concentration of each culture unit of a culture dish, the drug viscosity of a drug inlet pipe and the image data of the culture dish; judging whether the number of the culture units of which the drug concentrations are not within a preset interval is greater than a preset threshold value, and if so, constructing a layered color concentration diagram of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit; and outputting a dosing list according to the layered color setting concentration diagram. Each culture unit is subjected to independent drug concentration detection and drug delivery, so that the balance of the drug concentration of the whole culture dish is increased, and the drug concentration can be conveniently adjusted with higher quality.

Description

Technical Field

[0001] The invention relates to the field of cell and microorganism cultivation, in particular to a multi-well plate cultivation method for cells and microorganisms. Background Art

[0002] In the culture of cells and microorganisms, it is necessary to regularly add, drain and add drugs to the culture dishes.

[0003] In the traditional culture method, an upstream water inlet and a downstream water outlet are added to the culture dish to complete the operation of liquid inlet and liquid outlet. When the liquid is inlet, the culture liquid is mixed with the drug to complete the operation of drug inlet.

[0004] However, this mode of drug administration is often accompanied by excessively high local drug concentrations, which is not conducive to the cultivation of cells and microorganisms that are sensitive to drug concentrations.

[0005] Therefore, the more advanced ones in the industry are porous plate-type medicine feeding or porous plate-type liquid feeding or multi-hole plate-type liquid discharge. This porous plate does not take into account the three functions of liquid feeding, liquid discharge and medicine feeding at the same time.

[0006] In addition, since conventional multi-well plates simply pump drugs evenly to all wells of the multi-well plate, this inevitably leads to high drug consumption and insufficient drug concentration in locations where cells and microorganisms are densely populated, while drug concentrations in other locations may be too high, making it impossible to evenly transport drugs to cells and microorganisms through the multi-well plate, which is not conducive to the cultivation of cells and microorganisms.

[0007] Therefore, there is a need for a cell and microorganism multi-well plate culture method that can better culture cells and microorganisms through a multi-well plate. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a cell and microorganism multi-well plate culture method for better culturing cells and microorganisms through a multi-well plate.

[0009] In a first aspect, the present invention provides a method for culturing cells and microorganisms in a multi-well plate, comprising:

[0010] The multi-well plate is arranged on the culture dish, wherein the multi-well plate is arranged with a plurality of culture wells, and every three adjacent culture wells form a culture unit, and each culture unit is arranged with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole;

[0011] The liquid inlet pipe, the liquid discharge pipe and the drug inlet pipe are respectively arranged in the liquid inlet hole, the liquid discharge hole and the drug inlet hole, wherein the liquid inlet port of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe and the drug inlet port of the drug inlet pipe are bent or inclined in a clockwise direction;

[0012] Collect the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, and determine whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold. If so, construct a layered color concentration map of each culture unit in the culture dish based on the image data of the culture dish and the drug concentration of each culture unit; output a drug addition list based on the layered color concentration map;

[0013] Output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

[0014] The present invention provides a cell and microorganism multi-well plate culture method, wherein the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0015] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0016] Each culture unit is classified according to the concentration level corresponding to the drug concentration of each culture unit; the concentration level of the culture unit with the largest number of the same concentration level in each first area is configured as the concentration level of this first area, and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

[0017] The present invention provides a cell and microorganism multi-well plate culture method, wherein the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0018] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0019] Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level;

[0020] The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

[0021] The present invention provides a cell and microorganism multi-well plate culture method, wherein the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0022] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0023] The average drug concentration of the first area is output according to the average drug concentration of each culture unit in each first area, and the concentration level of the first area is configured according to the concentration level corresponding to the average drug concentration; and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

[0024] The present invention provides a cell and microorganism multi-well plate culture method, wherein the output of the drug addition list according to the layered color concentration map includes:

[0025] Draw a target point according to the image centroid or geometric center of each first area of ​​the layered color density map; output the modulus of the pre-stored vector corresponding to the density level of the first area;

[0026] Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit;

[0027] outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located;

[0028] Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

[0029] The present invention provides a method for culturing cells and microorganisms in a multi-well plate, wherein the control strategy for outputting the culturing unit according to the first region where the end point of the first vector is located comprises:

[0030] Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map;

[0031] If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes;

[0032] Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

[0033] In a second aspect, the present invention provides a cell and microorganism multi-well plate culture system, comprising:

[0034] A culture module, which is used to configure a multi-well plate on a culture dish, wherein the multi-well plate is configured with a plurality of culture holes, and every three adjacent culture holes form a culture unit, and each culture unit is configured with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole; a liquid inlet pipe, a liquid discharge pipe, and a drug inlet pipe are respectively configured in the liquid inlet hole, the liquid discharge hole, and the drug inlet hole, wherein the liquid inlet port of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe, and the drug inlet port of the drug inlet pipe are bent or inclined in a clockwise direction;

[0035] The drug adding module is used to collect the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, and determine whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold. If so, a layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit; and a drug adding list is output according to the layered color concentration map;

[0036] The processing module is used to output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

[0037] The present invention provides a cell and microorganism multi-well plate culture system, wherein the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0038] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0039] Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level;

[0040] The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

[0041] The present invention provides a cell and microorganism multi-well plate culture system, wherein the output of the drug addition list according to the layered color concentration map includes:

[0042] Draw a target point according to the image centroid or geometric center of each first area of ​​the layered color density map; output the modulus of the pre-stored vector corresponding to the density level of the first area;

[0043] Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit;

[0044] outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located;

[0045] Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

[0046] The present invention provides a cell and microorganism multi-well plate culture system, wherein the control strategy for outputting the culture unit according to the first region where the end point of the first vector is located includes:

[0047] Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map;

[0048] If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes;

[0049] Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

[0050] The difference between the cell and microorganism multi-well plate culture method of the present invention and the prior art is that the cell and microorganism multi-well plate culture method of the present invention can split a larger culture dish into smaller culture modules by constructing multiple culture units in the culture dish, and in each culture module, due to the different densities of cells and microorganisms, their consumption of drugs is different, resulting in the problem that the drug may be larger or smaller in part of the culture dish. The present invention performs separate drug concentration detection and drug delivery for each culture unit, thereby increasing the balance of the drug concentration of the entire culture dish, and facilitating the adjustment of the drug concentration with higher quality.

[0051] The cell and microorganism multi-well plate culture method of the present invention will be further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a schematic diagram of the structure of a multi-well plate culture method for cells and microorganisms;

[0053] Figure 2 It is the parts diagram of the perforated plate;

[0054] Figure 3 It is a partial enlarged view of the porous plate;

[0055] Figure 4 is the first contour map;

[0056] Figure 5 It is a layered color concentration map;

[0057] Figure 6 It is a method flow chart of a multi-well plate culture method for cells and microorganisms. DETAILED DESCRIPTION

[0058] like Figures 1 to 6 As shown, see Figure 1 , 6 The present invention provides a method for culturing cells and microorganisms in a multi-well plate, comprising:

[0059] See also Figure 2 , 3 , placing a multi-well plate on a culture dish, wherein the multi-well plate is provided with a plurality of culture wells, and every three adjacent culture wells form a culture unit, and each culture unit is provided with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole;

[0060] The liquid inlet pipe, the liquid discharge pipe, and the drug inlet pipe are respectively arranged in the liquid inlet hole, the liquid discharge hole, and the drug inlet hole, wherein the liquid inlet of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe, and the drug inlet of the drug inlet pipe are bent or tilted in the clockwise direction, and the clockwise and counterclockwise directions are the clockwise and counterclockwise directions when looking down at the culture dish. The bent liquid inlet of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe, and the drug inlet of the drug inlet pipe are more convenient for forming vortices or turbulences of the fluid in the culture dish, thereby increasing the uniformity of liquid inlet, drug feeding, and liquid discharge, making it easier for drugs and culture fluids to diffuse in the culture dish, and improving the culture effect;

[0061] See also Figure 6 , collecting the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, judging whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold, and if so, constructing a layered color concentration map of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit; outputting a drug addition list according to the layered color concentration map;

[0062] Output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

[0063] The present invention constructs multiple culture units in a culture dish, so that a larger culture dish can be split into smaller culture modules. In each culture module, due to the different densities of cells and microorganisms, their consumption of drugs is different, resulting in the problem that the drug may be larger or smaller in a part of the culture dish. The present invention performs separate drug concentration detection and drug delivery for each culture unit, thereby increasing the balance of the drug concentration of the entire culture dish and facilitating the adjustment of the drug concentration with higher quality.

[0064] Among them, the liquid inlet pipe and the drug inlet pipe are both arranged at the bottom of the culture dish, and the lowest point of the drainage pipe is arranged at the same level as the liquid surface of the culture dish, that is, the drainage pipe cannot completely absorb all the culture liquid, and the drug inlet pipe and the liquid inlet pipe are injected from the bottom, so they are farther away from the drainage pipe and more difficult to be directly absorbed.

[0065] The culture dish may be an independent circular culture dish configured on each culture unit. Of course, multiple culture units may also culture one culture dish at the same time.

[0066] Among them, the multi-well plate can be directly placed on the upper edge opening of the culture dish;

[0067] Among them, every three culture wells can be Figure 1 , 2 The circular frame shown in ,3 forms a culture unit.

[0068] The liquid inlet hole, the liquid discharge hole and the drug inlet hole are respectively fixed near the ports of the liquid inlet pipe, the liquid discharge pipe and the drug inlet pipe.

[0069] The porous plate is provided with a concentration detection module at the center of each culture unit, that is, at the center of the circle, for detecting the drug concentration of the culture unit.

[0070] When capturing image data, the multi-well plate can be opened to capture the culture dish from a bird's-eye view. Of course, a camera can also be arranged under the multi-well plate to capture image data of the culture dish.

[0071] The preset interval may be a normal drug solution concentration interval, for example, 25-50 mg / mL. If the concentration is not within this interval, it means that the density of cells and microorganisms in the area is too high.

[0072] The preset threshold is 1 to 1000, preferably 20. For example, if the multi-well plate has 100 culture units and 300 culture wells, and the drug concentration of 20 culture units is not within the normal preset range of 25 to 50 mg / mL, they should no longer be cultured in a uniform dosing manner, but each culture unit should adopt a different dosing dose to implement a drug control strategy and dosing list. Finally, the pump or the solenoid valve of each drug inlet pipe is controlled according to the dosing list to implement different drug inlet schemes for different drug inlet pipes, so that the drug concentration is relatively balanced within a certain period of time, and the culture effect is better.

[0073] In some embodiments, see Figure 4 , 5 , the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0074] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0075] Each culture unit is classified according to the concentration level corresponding to the drug concentration of each culture unit; the concentration level of the culture unit with the largest number of the same concentration level in each first area is configured as the concentration level of this first area, and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

[0076] The present invention identifies the approximate aggregation contours of cells and microorganisms through image data, thereby finding the areas that should be focused on cultivation and drug administration. Specifically, the image data can reflect contour lines, and such contour lines often represent the contours of aggregation. Multiple first areas are divided according to the contours, and the concentration levels of multiple culture units in each first area are compared. The concentration level with the highest proportion is used as the concentration level of the first area, and the colors corresponding to the concentration levels are configured to generate a layered color concentration map.

[0077] Among them, in the step of identifying the first contour map having at least two first contour lines based on the image data of the culture dish, the first contour lines and the first contour map can be obtained by using a layered color topographic map drawn by a radar camera overlooking the mountain range.

[0078] Specifically, different intervals may be defined according to color changes of the image data, and the first contour line and the first contour graph may be generated by drawing adjacent intervals based on the image data.

[0079] The conversion of the closed figure formed between two adjacent first contour lines into the first region can be understood as: the annular first region formed by the two first contour lines in the middle region.

[0080] The closed figure formed by the first contour lines without other first contour lines inside is converted into the first area, which can be understood as: a relatively central area, the last roughly circular first area.

[0081] Among them, converting a closed figure formed by a first contour line with no other first contour lines outside into a first area can be understood as: a relatively marginal area, whose shape comes partly from the border of the first contour figure and partly from the first contour line, which is roughly similar to a square with a circle cut off, or a roughly circle with straight edges.

[0082] Among them, each culture unit is classified according to the concentration level corresponding to the drug concentration of each culture unit, which can be understood as follows: the concentration level can be set to 5 levels, for example, when the preset interval is 25-50 mg / mL, the first level is 0-15 mg / mL, the second level is 15-25 mg / mL, the third level is 25-50 mg / mL, the fourth level is 50-75 mg / mL, and the fifth level is above 75 mg / mL. By classifying the concentration levels and displaying them in layers and colors, the observability and analyzability of the culture dish concentration can be increased. For example, the drug concentration of a culture unit of 5 mg / mL can be configured as the first level, which appears roughly at the edge. Among them, the colors of the 5 levels gradually turn red, that is, the first level is the reddest and the fifth level is the least red, which is convenient for users to intuitively observe that the center is relatively red and the drug should be added in particular, thereby drawing a layered color concentration map.

[0083] In some embodiments, see Figure 4 , 5 , the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0084] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0085] Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level;

[0086] The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

[0087] The present invention no longer uses the coordinate points collected from a certain culture unit as the evaluation standard for the concentration level of the first area, but uses the connecting line of the coordinate points of the collected concentrations of adjacent culture units of the same concentration level as the evaluation standard; thereby taking into account the trend change of concentration more, and using the connecting line to represent more virtual points, and using the connecting line in the form of more points as the evaluation standard and basis, the concentration level of the first area can be obtained more accurately.

[0088] Among them, the first stratified connection line is drawn according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first stratified connection line is a connection line of the coordinate points of the drug concentration collection of adjacent culture units of the same concentration level, which can be understood as: assuming that among the 5 concentration levels, the concentration level of a part of the culture units is in the third level, then the coordinates of the collection points connecting the adjacent third-level culture units can roughly surround a closed square or circle or a similar figure, so as to virtualize more points of the same concentration in the form of lines, and configure the concentration level of the first area by considering the proportion of these lines as points and the first area, which is more reasonable and closer to the real concentration of the first area, and effectively eliminates noise data.

[0089] In some embodiments, see Figure 4 , 5, the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0090] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0091] The average drug concentration of the first area is output according to the average drug concentration of each culture unit in each first area, and the concentration level of the first area is configured according to the concentration level corresponding to the average drug concentration; and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

[0092] The present invention configures the average drug concentration of each first area as the average concentration of the first area, and then generates the corresponding first area concentration level and color, so as to fairly refer to the concentration level of each culture unit for the first area of ​​the entire contour, and to eliminate the collected noise. That is, the output is based on the first contour map and the average drug concentration of each culture unit.

[0093] Among them, taking the average value is more conducive to data organization and statistics.

[0094] In some embodiments, see Figure 4 , 5 The output of the dosing list according to the layered color concentration map includes

[0095] Draw a target point according to the image centroid or geometric center of each first area of ​​the layered color density map; output the modulus of the pre-stored vector corresponding to the density level of the first area;

[0096] Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit;

[0097] outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located;

[0098] Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

[0099] The present invention uses the centroid and geometric center of each of the first regions as the target point to indicate the direction in which the concentration of the first region of the layer of the layered color concentration map should be increased theoretically; with this as the direction, the length is constructed with its own concentration level, and the culture unit is used as the starting point. The constructed vector has a good reference significance, and the first region where the end point of this vector is located can reflect whether this position is relatively close or far from the first region with a higher concentration level. If it is close, a higher drug administration control strategy must be configured, that is, the dosage of the drug should be increased, and the frequency of liquid injection and discharge should be increased. If it is far, the opposite is true. The drug addition list drawn in this way can be more in line with whether the cells and microorganisms are dense or not, and different control strategies for each culture unit can be customized and output in the form of a drug addition list.

[0100] Among them, each drug inlet pipe, liquid inlet pipe, and liquid discharge pipe should be connected to a pump and a sink, and each pump connected to the pipeline is equipped with a corresponding solenoid valve to adapt to different outward and inward pumping times and flows.

[0101] Usually, a solenoid valve is provided at the liquid outlet of the pump, and of course, a solenoid valve may also be provided at the liquid inlet.

[0102] The flow rate regulation function can be achieved by adjusting the power of the solenoid valve or the pump.

[0103] Among them, the liquid and drug injection needles are deep and directly inserted into the bottom of the culture dish, and the liquid aspiration needle is shallow and flush with the liquid surface to avoid sucking away all of the liquid.

[0104] Among them, the control strategy can be to sometimes not administer the drug first, culture first, and then administer the drug after culturing for a period of time; or it can be to administer the drug directly.

[0105] Among them, there are several ways of drug administration: continuous administration, intermittent administration, and phased administration; when multiple drugs are administered, more than two drugs are administered at the same time;

[0106] The drug administration flow rate is 1 to 5 ul / min.

[0107] When administering drugs, whether intermittently or continuously, even if the flow rate of the replacement solution is small, it will affect the current concentration of the drug in the liquid, thus affecting the final experimental results. Therefore, the corresponding drug concentration can be converted when the solution is replaced, so as to ensure that the drug concentration after the solution change is the same as before the solution change.

[0108] Among them, the target point is drawn according to the image centroid or geometric center of each first area of ​​the layered color density map, which can be understood as: the image centroid represents the center of gravity of this figure. In other words, assuming that the weight of each unit area of ​​the first annular area is the same, then the target point of the first annular area is its equivalent center of gravity. The geometric center of gravity can be understood as the center point of the equivalent area of ​​its image. That is, the center of mass in the traditional sense is the center of gravity and the center of the set is the center.

[0109] By constructing this target point, we can clearly know the relative center of each first area, and thus determine the direction of the changing trend. The first vector of the component in this direction is more meaningful for reference.

[0110] Among them, outputting the modulus of the vector corresponding to the concentration level of the first region pre-stored can be understood as: the modulus of the vector of each first region is pre-stored in the database, for example, taking three adjacent culture units, and calculating the maximum distance between the three adjacent culture units is the modulus of the vector. It can also be adjusted and set according to the actual concentration and activity of the drug. The length of the modulus of the vector of the first region of each concentration level is different.

[0111] Among them, the control strategy of the culture unit is output according to the first area where the end point of the first vector is located. It can be understood that: the first area where the end point of the vector falls can represent the control strategy of this culture unit. For example, when the vector length is constant and they all point toward the center, assuming that the change is relatively gentle, then the interval between the first areas is large, and the end point of the first vector is not easy to fall into another first area. If the end point is in the original first area, less medication should be given, less liquid should be added, and less liquid should be sucked. If it falls into another first area, it may be that the adjacent areas are close, or the concentration at this position may vary greatly, then more medication should be given, more liquid should be added, and more liquid should be sucked, so as to maintain a relatively balanced drug concentration within a certain period of time, which is more conducive to the culture of cells and microorganisms. The above control strategy can be preset in the database or adjusted by the user.

[0112] That is to say, the control strategy for how the drug should be administered stored in the database is actually the control strategy for the third concentration level. The drug administration strategies for the first and second concentration levels are minimum and less administration; the drug administration strategies for the fourth and fifth concentration levels are more and maximum administration, thereby achieving better drug administration and culture effects. That is, assuming that there is no consumption of cells and microorganisms in the above-mentioned culture dish, the final concentration after a period of rest is roughly equal to the drug concentration of the control strategy for how the drug should be administered that was previously stored in the database. Among them, the maximum and minimum administration should meet the reasonable range of drug concentration tolerance of cells and microorganisms.

[0113] In some embodiments, see Figure 4 , 5 The control strategy of outputting the culture unit according to the first region where the end point of the first vector is located includes

[0114] Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map;

[0115] If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes;

[0116] Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

[0117] In the present invention, when there are fewer first vectors not in this area, the first areas of other concentration levels bypassed by the length of the conventional first vector can be regarded as noise and can be ignored, so that the noise area is merged with the other first areas, and after the first contour map is regenerated, the layered color concentration map is regenerated according to the concentration of each culture unit, and the subsequent steps. If it is true that too many first vectors pass through the first areas of other peak or trough concentration levels, the length of the vector should be appropriately reduced so that the end point of the vector stays at the highest or lowest point, so as to facilitate the control of the culture unit according to the position adjacent to the highest or lowest first area. The configuration of the control strategy and the dosing list is not to be eliminated. For example, a smaller first area is a small ring-shaped first area with only one first area inside the second to last. With the first vector configuration of the conventional length, it is likely that the end point of this first vector is configured in the third to last first area. Then, the end point of this first vector should fall into the last first area, and the corresponding control strategy and dosing list should be adapted.

[0118] In other words, it is indicated that the present region is a focal region and it should be considered whether the first region having a larger concentration level or a smaller concentration level is bypassed by the first vector of a regular length.

[0119] It should be noted that the modulus of the first vector is reduced to fall into the first region, based on the length of the modulus that is reduced the least. That is, the first region is a region, and the first vector is no longer reduced as long as it falls into the highest or lowest first region.

[0120] Among them, the first threshold value can be 1% to 99%, preferably 50%, that is, assuming that the end points of less than half of the first vectors are not in the original first area, that is, not higher than the first threshold value; then the first areas other than the first area of ​​the starting point and the first area of ​​the end point that the first vector passes through can be considered as noise first areas. For example, assuming that the end points of less than half of the first vectors are not in the original first area, one of the first vectors covers three first areas, and except for the first areas of the starting point and the end point, the middle first area is the noise area. Then, this noise area should be deleted, that is, by merging the noise area with other areas, that is, after merging, the starting point and the end point of this first vector are located in the same first area. Since the definition of the first area has changed, it is necessary to jump back to the above step of "identifying a first contour map with at least two first contour lines based on the image data of the culture dish," and re-perform the step polling.

[0121] Among them, it is judged whether there is a first area with a higher concentration level than the first area of ​​the end point and the starting point configured between the end point and the starting point of the first vector. It can be understood that: the same overly long first vector covers at least three first areas, and the drug concentration in the middle area is the highest. Then, the length of the first vector should be reduced, so that the middle is not considered to be a noise area, but a guiding area. For example, the middle area in the layered color concentration map has the lowest concentration level, which should be the most able to guide the direction of the first vector. Then, it is not a noise area that should be removed, but should be referenced, so the length of the first vector is adjusted. Conversely, the processing method for lower levels is the same.

[0122] like Figures 1 to 6 As shown, see Figure 1 , 6 , a cell and microorganism multi-well plate culture system, comprising

[0123] A culture module, which is used to configure a multi-well plate on a culture dish, wherein the multi-well plate is configured with a plurality of culture holes, and every three adjacent culture holes form a culture unit, and each culture unit is configured with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole; a liquid inlet pipe, a liquid discharge pipe, and a drug inlet pipe are respectively configured in the liquid inlet hole, the liquid discharge hole, and the drug inlet hole, wherein the liquid inlet port of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe, and the drug inlet port of the drug inlet pipe are bent or inclined in a clockwise direction;

[0124] The drug adding module is used to collect the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, and determine whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold. If so, a layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit; and a drug adding list is output according to the layered color concentration map;

[0125] The processing module is used to output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

[0126] The present invention constructs multiple culture units in a culture dish, so that a larger culture dish can be split into smaller culture modules. In each culture module, due to the different densities of cells and microorganisms, their consumption of drugs is different, resulting in the problem that the drug may be larger or smaller in a part of the culture dish. The present invention performs separate drug concentration detection and drug delivery for each culture unit, thereby increasing the balance of the drug concentration of the entire culture dish and facilitating the adjustment of the drug concentration with higher quality.

[0127] Among them, the liquid inlet pipe and the drug inlet pipe are both arranged at the bottom of the culture dish, and the lowest point of the drainage pipe is arranged at the same level as the liquid surface of the culture dish, that is, the drainage pipe cannot completely absorb all the culture liquid, and the drug inlet pipe and the liquid inlet pipe are injected from the bottom, so they are farther away from the drainage pipe and more difficult to be directly absorbed.

[0128] In some embodiments, see Figure 4 , 5 , the layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit, including

[0129] A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region;

[0130] Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level;

[0131] The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

[0132] The present invention no longer uses the coordinate points collected from a certain culture unit as the evaluation standard for the concentration level of the first area, but uses the connecting line of the coordinate points of the collected concentrations of adjacent culture units of the same concentration level as the evaluation standard; thereby taking into account the trend change of concentration more, and using the connecting line to represent more virtual points, and using the connecting line in the form of more points as the evaluation standard and basis, the concentration level of the first area can be obtained more accurately.

[0133] Among them, the first stratified connection line is drawn according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first stratified connection line is a connection line of the coordinate points of the drug concentration collection of adjacent culture units of the same concentration level, which can be understood as: assuming that among the 5 concentration levels, the concentration level of a part of the culture units is in the third level, then the coordinates of the collection points connecting the adjacent third-level culture units can roughly surround a closed square or circle or a similar figure, so as to virtualize more points of the same concentration in the form of lines, and configure the concentration level of the first area by considering the proportion of these lines as points and the first area, which is more reasonable and closer to the real concentration of the first area, and effectively eliminates noise data.

[0134] In some embodiments, see Figure 4 , 5 The output of the dosing list according to the layered color concentration map includes

[0135] Draw a target point according to the image centroid or geometric center of each first area of ​​the layered color density map; output the modulus of the pre-stored vector corresponding to the density level of the first area;

[0136] Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit;

[0137] outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located;

[0138] Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

[0139] The present invention uses the centroid and geometric center of each of the first regions as the target point to indicate the direction in which the concentration of the first region of the layer of the layered color concentration map should be increased theoretically; with this as the direction, the length is constructed with its own concentration level, and the culture unit is used as the starting point. The constructed vector has a good reference significance, and the first region where the end point of this vector is located can reflect whether this position is relatively close or far from the first region with a higher concentration level. If it is close, a higher drug administration control strategy must be configured, that is, the dosage of the drug should be increased, and the frequency of liquid injection and discharge should be increased. If it is far, the opposite is true. The drug addition list drawn in this way can be more in line with whether the cells and microorganisms are dense or not, and different control strategies for each culture unit can be customized and output in the form of a drug addition list.

[0140] In some embodiments, see Figure 4 , 5 The control strategy of outputting the culture unit according to the first region where the end point of the first vector is located includes

[0141] Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map;

[0142] If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes;

[0143] Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

[0144] In the present invention, when there are fewer first vectors not in this area, the first areas of other concentration levels bypassed by the length of the conventional first vector can be regarded as noise and can be ignored, so that the noise area is merged with the other first areas, and after the first contour map is regenerated, the layered color concentration map is regenerated according to the concentration of each culture unit, and the subsequent steps. If it is true that too many first vectors pass through the first areas of other peak or trough concentration levels, the length of the vector should be appropriately reduced so that the end point of the vector stays at the highest or lowest point, so as to facilitate the control of the culture unit according to the position adjacent to the highest or lowest first area. The configuration of the control strategy and the dosing list is not to be eliminated. For example, a smaller first area is a small ring-shaped first area with only one first area inside the second to last. With the first vector configuration of the conventional length, it is likely that the end point of this first vector is configured in the third to last first area. Then, the end point of this first vector should fall into the last first area, and the corresponding control strategy and dosing list should be adapted.

[0145] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for culturing cells and microorganisms in a multi-well plate, characterized in that: include The multi-well plate is arranged on the culture dish, wherein the multi-well plate is arranged with a plurality of culture wells, and every three adjacent culture wells form a culture unit, and each culture unit is arranged with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole; The liquid inlet pipe, the liquid discharge pipe and the drug inlet pipe are respectively arranged in the liquid inlet hole, the liquid discharge hole and the drug inlet hole, wherein the liquid inlet port of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe and the drug inlet port of the drug inlet pipe are bent or inclined in a clockwise direction; Collect the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, and determine whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold. If so, construct a layered color concentration map of each culture unit in the culture dish based on the image data of the culture dish and the drug concentration of each culture unit; output a drug addition list based on the layered color concentration map; Output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

2. A cell and microorganism multi-well plate culture method according to claim 1, characterized in that: The method constructs a layered color concentration map of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit, including A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region; Each culture unit is classified according to the concentration level corresponding to the drug concentration of each culture unit; the concentration level of the culture unit with the largest number of the same concentration level in each first area is configured as the concentration level of this first area, and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

3. A cell and microorganism multi-well plate culture method according to claim 1, characterized in that: The method constructs a layered color concentration map of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit, including A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region; Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level; The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

4. A cell and microorganism multi-well plate culture method according to claim 1, characterized in that: The method constructs a layered color concentration map of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit, including A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region; The average drug concentration of the first area is output according to the average drug concentration of each culture unit in each first area, and the concentration level of the first area is configured according to the concentration level corresponding to the average drug concentration; and a layered color concentration map is drawn according to the color corresponding to the concentration level of the first area.

5. A cell and microorganism multi-well plate culture method according to any one of claims 2, 3 or 4, characterized in that: The output of the dosing list according to the layered color concentration map includes: Draw a target point according to the image centroid or geometric center of each first area of ​​the layered tint density map; Outputting the magnitude of the pre-stored vector corresponding to the concentration level of the first area; Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit; outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located; Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

6. A cell and microorganism multi-well plate culture method according to claim 5, characterized in that: The control strategy of outputting the culture unit according to the first region where the end point of the first vector is located includes Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map; If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes; Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

7. A cell and microorganism multi-well plate culture system, characterized in that: include A culture module, which is used to configure a multi-well plate on a culture dish, wherein the multi-well plate is configured with a plurality of culture holes, and every three adjacent culture holes form a culture unit, and each culture unit is configured with a liquid inlet hole, a liquid discharge hole, and a drug inlet hole; a liquid inlet pipe, a liquid discharge pipe, and a drug inlet pipe are respectively configured in the liquid inlet hole, the liquid discharge hole, and the drug inlet hole, wherein the liquid inlet port of the liquid inlet pipe, the liquid discharge port of the liquid discharge pipe, and the drug inlet port of the drug inlet pipe are bent or inclined in a clockwise direction; The drug adding module is used to collect the drug concentration of each culture unit of the culture dish, the drug viscosity of the drug inlet pipe, and the image data of the culture dish, and determine whether the number of culture units whose drug concentration is not within the preset interval is greater than a preset threshold. If so, a layered color concentration map of each culture unit in the culture dish is constructed according to the image data of the culture dish and the drug concentration of each culture unit; and a drug adding list is output according to the layered color concentration map; The processing module is used to output the drug feeding data of the drug feeding pipe of each culture unit according to the drug feeding list.

8. A cell and microorganism multi-well plate culture system according to claim 7, characterized in that: The method constructs a layered color concentration map of each culture unit in the culture dish according to the image data of the culture dish and the drug concentration of each culture unit, including A first contour figure having at least two first contour lines is identified according to the image data of the culture dish, and a closed figure formed between two adjacent first contour lines is converted into a first region; a closed figure formed by first contour lines without other first contour lines inside is converted into a first region; and a closed figure formed by first contour lines without other first contour lines outside is converted into a first region; Draw a first layered connection line according to the concentration level corresponding to the drug concentration of each culture unit, wherein the first layered connection line is a line connecting the coordinate points collected from the drug concentrations of adjacent culture units of the same concentration level; The concentration level corresponding to the first layered connection line with the highest proportion in the first region is configured as the first region concentration level, and a layered coloring concentration map is drawn according to the color corresponding to the concentration level of the first region.

9. A cell and microorganism multi-well plate culture system according to claim 8, characterized in that: The output of the dosing list according to the layered color concentration map includes: Draw a target point according to the image centroid or geometric center of each first area of ​​the layered tint density map; Outputting the magnitude of the pre-stored vector corresponding to the concentration level of the first area; Taking the coordinate point of the collected concentration of each culture unit as the starting point of the vector, and heading toward the target point as the direction of the vector, a first vector is drawn with the modulus of the vector generated by the first area of ​​the culture unit; outputting a control strategy of the culture unit according to a first region where the end point of the first vector is located; Output the corresponding dosing list according to each culture unit and the corresponding control strategy.

10. A cell and microorganism multi-well plate culture system according to claim 9, characterized in that: The control strategy of outputting the culture unit according to the first region where the end point of the first vector is located includes Determine whether a first percentage of a first number of first vectors in the first region where the end point and the starting point of the first vector of the first region are not in the same first region to a first percentage of a total number of all first vectors in the first region as the starting point is higher than a first threshold; if not, define the first region excluding the end point and the starting point through which the first vector of the first region where the end point and the starting point are not in the same first region as a noise region, delete the first contour line of the noise region, and regenerate the first contour map; If yes, determine whether a first region with a higher concentration level than the first regions of the end point and the start point is configured between the end point and the start point of the first vector, and if yes, reduce the vector modulus of the first vector until the end point of the first vector falls within the first region with the highest concentration level through which the first vector passes; Alternatively, if so, determine whether there is a first area between the end point and the starting point of the first vector, and if so, reduce the vector modulus of the first vector until the end point of the first vector falls within the first area with the lowest concentration level through which the first vector passes.

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