CN116609313A

CN116609313A - Cell high throughput testing method, system and apparatus

Info

Publication number: CN116609313A
Application number: CN202310875411.4A
Authority: CN
Inventors: 梁俊强; 李梦浩; 王海涛
Original assignee: Beijing Xinlian Photoelectric Technology Co ltd
Current assignee: Beijing Xinlian Photoelectric Technology Co ltd
Priority date: 2023-07-18
Filing date: 2023-07-18
Publication date: 2023-08-18
Anticipated expiration: 2043-07-18
Also published as: CN116609313B

Abstract

The invention relates to the technical field of cell testing, and particularly discloses a method, a system and equipment for testing high throughput of cells, wherein the method comprises the following steps: firstly, setting and identifying a positioning mark on a culture container; numbering the wells on the culture vessel based on the positioning marks, and positioning the cells to be tested in the wells; based on the serial number and the positioning information, performing a plurality of test flows on the cells to be tested to obtain test results of different test periods; and finally, generating a test report based on the test result. Therefore, the test data of a large number of cells can be obtained at one time because each hole in the culture container comprises a plurality of cells to be tested, meanwhile, the test data of the single cells with specificity can be obtained by testing the single cells, and the cells to be measured can be rapidly determined when the cells are required to be measured for a plurality of times because the serial numbers and the positioning are carried out, so that a plurality of groups of comparison data and change ratios are obtained, and the change of the cells to be tested is more visual.

Description

Cell high throughput testing method, system and apparatus

Technical Field

The invention relates to the technical field of cell testing, in particular to a method, a system and equipment for testing high throughput of cells.

Background

The cell is a basic unit of life activities, and the basic characteristics, functions and change rules of the cell can be known and mastered to better understand the mechanism of macroscopic life phenomena, so that the cell has important significance for testing the cell including shape, size, activity, mechanics, movement, intracellular ion concentration and the like.

Traditional "whole body" cell analysis methods can mask the signal of intercellular differences, thereby losing the cell heterogeneity of the test results, resulting in a departure from true biological phenomena; the single cell isolation test method can provide data support for cell research to a certain extent because the single cell isolation test method can be used for carrying out detailed test on single cells, but the single cell isolation method has very high requirements on cell selection, and complicated cell selection is needed in the early stage, so that representative cells are obtained, representative data are obtained, a test result has certain unilaterality, the cell characteristics in various states cannot be comprehensively reflected, and in the prior art, the cells cannot be accurately measured for multiple times to obtain the data of the same cells in different times or states.

In summary, there is a need for a method that can perform detailed testing of cells, and that can fully test the characteristics of cells in various states.

Disclosure of Invention

In view of the above, the present application is directed to a method, a system and a device for testing cells with high throughput, so as to overcome the problem of poor cell testing effect.

In order to achieve the above purpose, the application adopts the following technical scheme:

in a first aspect, embodiments of the present application provide a method for high throughput testing of cells, comprising:

setting and identifying a positioning mark on a culture container to be tested;

numbering holes on the culture container to be tested based on the positioning marks, and positioning cells to be tested in the holes; wherein the culture container comprises at least one hole, and at least one cell to be tested is contained in the hole;

based on the hole number and the positioning information of the cells to be tested, the cells to be tested enter a test flow to obtain a test result of a first test period; the positioning information of the cells to be tested comprises coordinate information of the positions of the cells to be tested, and the cells to be tested can apply preset stimulus in the testing process;

after a preset time interval and/or a preset stimulus is applied, the cells to be tested reenter a test flow based on the serial numbers of the holes and the positioning information of the cells to be tested, so as to obtain a test result of a new test period;

And generating a test report based on the test result of the first test period and/or the test result of the new test period.

Optionally, the numbering the wells on the culture vessel to be tested based on the positioning mark, and positioning the cells to be tested in the wells, includes:

numbering holes on the culture container to be tested based on the positioning mark;

positioning the cells to be tested in the wells based on the number of wells on the culture vessel.

Optionally, the test procedure includes:

determining the number of the current hole and the positioning information of the cell to be tested in the current hole, and performing preset test and recording on the current cell to be tested;

based on the serial number of the hole, the positioning information of the cell to be tested in the hole corresponding to the next serial number is obtained, the cell to be tested is subjected to preset test and recorded until the serial number of the last hole to be tested.

Optionally, the preset test includes:

transmitting a target beam to the cell to be tested through an illumination system; the target light beam comprises at least one of visible light, first excitation light and second excitation light;

recording the state of the cell to be tested under the irradiation of the target beam, and obtaining the test result of the first test period.

Optionally, the emitting, by an illumination system, a target beam to the cell to be tested includes:

emitting the visible light beam to the cell to be tested through an illumination system;

emitting the first excitation light and/or the second excitation light to the cells to be tested by an illumination system.

Optionally, before the step of testing all the cells to be tested to obtain the test result of the first test period, the method further includes:

performing global photographing on the hole to obtain a first global image;

before the cell to be tested reenters the testing process based on the serial number of the hole and the positioning information of the cell to be tested, the method further comprises the following steps:

performing global photographing on the hole to obtain a second global image;

and comparing the similarity between the first global image and the second global image, and if the similarity is larger than a preset value, re-entering the test flow by the cells to be tested based on the serial numbers of the holes and the positioning information of the cells to be tested.

Optionally, the method further comprises:

receiving a manual instruction;

based on the manual instruction, determining the number of the hole on the culture container to be tested, and determining the positioning information of the cell to be tested in the hole.

In a second aspect, embodiments of the present application also provide a cell high throughput test system, comprising: a microscopic imaging subsystem;

the microscopic imaging subsystem is used to implement the cell high throughput test method described above.

Optionally, the microscopic imaging subsystem comprises:

the device comprises an objective table, a lens, a circulator, a first light splitting element, a second light splitting element, a visible light source, a first excitation light source, a second excitation light source and a camera;

the visible light emitted by the visible light source irradiates the cells to be tested in the culture container to be tested fixed on the objective table, and the formed transmitted light is transmitted into the camera through the lens, the circulator and the second light splitting element to form visible light signals;

the first excitation light source and/or the excitation light emitted by the second excitation light source irradiates the cells to be tested in the culture container to be tested fixed on the objective table through the first light splitting element, the circulator and the lens to form excitation fluorescence, and the excitation fluorescence is transmitted into the camera through the lens, the circulator and the second light splitting element to form excitation fluorescence signals.

In a third aspect, an embodiment of the present application further provides a cell high-throughput testing apparatus, including a processor and a memory, where the processor is connected to the memory:

the processor is used for calling and executing the program stored in the memory;

the memory is used for storing the program, and the program is at least used for executing the cell high-throughput testing method.

The application provides a cell high-throughput test method, which comprises the steps of firstly setting and identifying a positioning mark on a culture container to be tested; then numbering holes on the culture container to be tested based on the positioning marks, and positioning cells to be tested in the holes; wherein the culture container comprises at least one hole, and at least one cell to be tested is contained in the hole; then, based on the hole number and positioning information of the cells to be tested, carrying out a test flow on the cells to be tested to obtain a test result of a first test period; and re-entering the test flow of the cells to be tested based on the serial numbers of the holes and the positioning information of the cells to be tested after the preset time interval and/or the preset stimulus is applied, so as to obtain the test result of a new test period; and finally, generating a test report based on the test result of the first test period and/or the test result of the new test period. Thus, since a plurality of wells can be provided in the culture vessel, and a plurality of cells to be tested can be included in each well, a large amount of cell test data can be obtained at a time, and simultaneously, since individual cells to be tested can be individually tested, test data of individual cells having specificity can be obtained, and since numbering and positioning are performed, when a plurality of times of measurement are required for the cells, the cells to be measured before can be rapidly determined, thereby conveniently obtaining a plurality of sets of comparison data and change ratios, and thus, the change of the cells to be tested is more visual.

Drawings

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic flow chart of a method for high throughput testing of cells according to an embodiment of the present application;

FIG. 2 is a schematic flow chart of a test in a method for high throughput testing of cells according to an embodiment of the present application;

FIG. 3 is a schematic flow chart of a method for high throughput testing of cells according to another embodiment of the present application;

FIG. 4 is a schematic flow chart of a method for high throughput testing of cells according to another embodiment of the present application;

FIG. 5 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 6 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 7 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 8 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 9 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 10 is a schematic flow chart of a method for high throughput testing of cells according to still another embodiment of the present application;

FIG. 11 is a schematic flow chart of a method for high throughput testing of cells according to yet another embodiment of the present application;

FIG. 12 is a schematic block diagram of a cellular high throughput test system provided in an embodiment of the present application;

FIG. 13 is a schematic diagram of a microscopic imaging subsystem in a cellular high throughput test system according to an embodiment of the present application;

fig. 14 is a schematic structural diagram of a cell high-throughput testing apparatus according to an embodiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, based on the examples herein, which are within the scope of the application as defined by the claims, will be within the scope of the application as defined by the claims.

Summary of the application:

as mentioned in the background of the application, cells are the basic unit of life activities, and understanding and mastering the basic functions and change laws of cells can better understand the mechanism of macroscopic life phenomena. Traditional "whole body" cell analysis methods can mask the information of intercellular differences, thereby losing cellular heterogeneity, resulting in a departure from true biological phenomena. The single cell isolation method can effectively solve the defects of the traditional cell analysis method, and disperses the whole cells into single cells, and the common single cell isolation method and technology comprise the following steps: limiting dilution, micromanipulation, excitation light capture, flow fluorescence sorting, patch clamp technology, microfluidic technology, electric cage technology, natural sedimentation based on picoliter microplates, and the like. Studies of the shape, size, activity, mechanics, movement, intracellular ion concentration, etc. of individual cells are critical to understanding the size, survival, differentiation, proliferation, movement, ion distribution, concentration changes, etc. of cells.

In recent years, expert scholars at home and abroad have made a great deal of researches on biomechanics of cells, ion concentration of cells and the like. The research on the mechanical property and ion concentration of single cells can help human beings to understand the pathogenesis of diseases, and provide theoretical basis and method for early screening and diagnosis of diseases and the like.

The high flux is an important means for drug screening, and has the characteristics of high efficiency, high speed, trace quantity and the like in the research and discovery process of innovative lead drugs. The high throughput drug screening is to evaluate the biological activity of a large number of new drug samples from which new drug samples with activity against specific targets can be found. With the continuous discovery of potential drug targets, the appearance of new drug targets provides a wide development prospect for the development of innovative drugs, and simultaneously provides higher requirements on the efficiency of high-flux drug screening.

Then, various single cell isolation methods in the prior art have certain defects, such as high experimental skill, time consumption and low cell flux required by limiting dilution, micromanipulation and excitation light capture; the flow type fluorescence sorting does not need higher experimental skill, but can not sort rare cell samples, and an expensive sorting type flow cytometer is needed, so that a large amount of fluorescent antibodies are consumed in the experimental process; the patch clamp technology can obtain electrophysiological signals of cells while separating single cells, and the single-hand patch clamp technology has high technical requirements for users, and the automatic patch clamp technology can only test suspended cells; the micro-fluidic technology can sort cells by size, observe cell morphology under a microscope after sorting, but is limited by the physical space of a chip, and the number of the separated cells is limited; the electric cage technology is a novel single-cell separation technology, is a variant of the microfluidic technology, and is still essentially limited by the physical space of the chip.

Therefore, a high-flux cell testing system capable of simultaneously testing multiple cell biological parameters is developed, the problem of single function of equipment can be solved, and higher comprehensive application benefit is realized.

In view of the above technical problems, fig. 1 is a flow chart of a method for testing high throughput of cells according to an embodiment of the present application, referring to fig. 1, the method may include the following steps:

s101, setting and identifying positioning marks on the culture container to be tested.

S102, numbering holes on the culture container to be tested based on the positioning marks, and positioning cells to be tested in the holes.

Wherein the culture container comprises at least one hole, and at least one cell to be tested is contained in the hole.

Specifically, in the present application, cells to be tested may be cultured in wells on a culture vessel, which is then mounted as a whole on a stage of a cell high throughput testing system, such as a microscopy imaging subsystem, for measurement to effect testing of the cells to be tested.

For example, in some embodiments, a plurality of chemical reagents can be added to a cell culture vessel to be tested to prepare a base gel, fluorescent microbeads are added to the gel, and the surface of the gel to which the fluorescent microbeads are added is treated to form a pattern on the surface of the gel that can accommodate individual cells. Inoculating the cells to be tested to the surface of a gel substrate with patterns, culturing for a period of time, adding fluorescent dye into a culture container to incubate the cells, and cleaning after incubation to obtain the cells to be tested.

Wherein the cell to be tested may comprise: cells that are autonomously contractile, cells that are capable of producing contractions upon stimulation, cells that are autonomously movable, cells that are incapable of producing any contractions and are not free to move, and the like.

In some embodiments, the culture vessel is accompanied by a plate having a thickness tailored to the arrangement of the pattern, and the gel surface mentioned above may form a pattern capable of accommodating individual cells to be tested, wherein the arrangement of the pattern includes both regular arrangement and irregular arrangement, and the shape of the pattern includes rectangular, square, oval, circular, and irregular shapes.

In addition, in some embodiments, the cell culture vessel may be made of transparent material, on the basis of which the light beam emitted from the high throughput cell testing system can penetrate the culture vessel to irradiate or focus on the cells to be tested.

After obtaining the cells to be tested, the relevant staff can place the culture vessel carrying the cells to be tested on a testing position of the testing system, such as a stage, for subsequent procedures.

In some embodiments, the positioning mark of the culture container can be directly aligned to the target position by setting a preset mark or shape on the objective table, and when the relevant staff is placed, the positioning mark on the culture container can be directly identified by the cell high-throughput test system; in other embodiments of the present application, the positioning identification module in the high throughput cell test system may automatically identify the positioning mark of the culture container without excessively setting the stage, so as to position the culture container. For example, for single-well culture vessels and irregularly shaped multi-well culture vessels, a specific stage can be used by being fixed in a specific fixture; the multi-hole culture plate with regular shape can be directly fixed on the common objective table through a clamp.

After the culture container is positioned, the zero point coordinate position is determined based on the positioning mark so as to position the culture container, and the serial number of the hole and the positioning information of the cells to be tested are determined on the basis.

S103, entering a test flow by the cells to be tested based on the hole numbers and positioning information of the cells to be tested to obtain a test result of a first test period, wherein the positioning information of the cells to be tested comprises coordinate information of the positions of the cells to be tested, and the cells to be tested can apply preset stimulus in the test process.

Depending on the cell type assay or experimental condition settings, stimulation may be applied to the cells during both the first and subsequent test cycles.

S104, re-entering the test flow of the cells to be tested based on the serial numbers of the holes and the positioning information of the cells to be tested after the preset time interval and/or the preset stimulus is applied, and obtaining the test result of the new test period.

Specifically, after the serial number of the wells of the culture vessel is determined, the cells to be tested in the wells are positioned. It should be noted that, the numbering and positioning may be performed based on the positioning marks of the culture container, for example, numbering Kong Zhuyi based on the zero coordinate positions of the holes and the positioning marks, and performing coordinate positioning on the cells to be tested based on the coordinates of the positioning marks, so as to obtain the positioning marks of the cells to be tested.

In some embodiments, other distinguishing manners besides numbering may be performed on the holes, for example, the holes are named one by one according to preset logic, and the purpose of the distinguishing manners is the same, that is, the holes are distinguished, which is not described herein.

After numbering and positioning are completed, the cells to be tested enter a test flow, the cells to be tested are irradiated through a target light beam, and after a preset time, the states of the irradiated cells are recorded to obtain a test result.

In the application, the test flow can be carried out for a plurality of times, namely, the cells to be tested are tested for a plurality of times, a plurality of test results are correspondingly obtained, and record files corresponding to the test flows are recorded and generated, so that the application is convenient for subsequent research and use.

It should be noted that the above-mentioned multiple test procedures may be based on time and/or various stimuli, for example, the first test is a test under a conventional state, and after a preset time and/or after adding a certain stimulus and adding a certain drug, the test is performed, so as to obtain characteristics of cells under various conditions.

S105, generating a test report based on the test result of the first test period and/or the test result of the new test period.

Specifically, after the serial number and the positioning information are determined, the cells to be tested in the culture container can be measured for multiple times based on the same test flow, and the test result of each test can be used as a group of data for subsequent study; and moreover, a plurality of test results obtained by multiple tests are correspondingly used for generating a comparison test report, so that comparison data and data change ratios of cells under different conditions and states can be provided, and further subsequent researches are facilitated.

The invention provides a cell high-throughput test method, which comprises the steps of firstly setting and identifying a positioning mark on a culture container to be tested; then numbering holes on the culture container to be tested based on the positioning marks, and positioning cells to be tested in the holes; wherein the culture container comprises at least one hole, and at least one cell to be tested is contained in the hole; then, based on the hole number and positioning information of the cells to be tested, the cells to be tested enter a test flow to obtain a test result of a first test period; and re-entering the test flow of the cells to be tested based on the serial numbers of the holes and the positioning information of the cells to be tested after the preset time interval and/or the preset stimulus is applied, so as to obtain the test result of a new test period; and finally, generating a test report based on the test result of the first test period and/or the test result of the new test period. Thus, since a plurality of holes can be formed in the culture container, and a plurality of cells to be tested can be included in each hole, a large amount of cell test data can be obtained at one time, meanwhile, since a single cell to be tested can be tested, test data of a single cell with specificity can be obtained, and since numbering and positioning are performed, when a plurality of times of measurement are required to be performed on the cells, the cells to be measured before can be rapidly determined, so that a plurality of groups of comparison data and change ratios can be conveniently obtained, and the change of the cells to be tested is more visual.

Further, in some embodiments of the present application, different positioning marks may be set for the single-well culture container and the multi-well culture container, or no positioning mark may be set for the single-well culture container, for example, the center position of the single-well culture container is directly set as the zero coordinate position, and then subsequent positioning of the cells to be tested is performed; and setting marks at fixed positions at the bottoms of the porous culture containers, detecting the marks under the lens of the test system, moving the marks to the center of the visual field of the microscopic imaging subsystem, marking the marks as coordinate zero points, and then carrying out subsequent positioning of cells to be tested.

Fig. 2 is a schematic flow chart of a test in the cell high-throughput test method provided by the embodiment of the application, and as shown in fig. 2, in the cell high-throughput test method provided by the application, holes on a culture container to be tested are numbered based on positioning marks, and cells to be tested in the holes are positioned and tested, which specifically may include:

s201, numbering holes on the culture container to be tested based on the positioning marks.

S202, positioning cells to be tested in the holes in sequence based on the numbers of the holes on the culture container until all the cells to be tested are tested.

S203, judging whether the re-measurement is needed.

If yes, repeating S201 and S202 until the times reach the requirement, and ending the test; if not, directly ending the test.

Specifically, after numbering all the holes, positioning and testing the cells to be tested in the holes according to the numbers, and after positioning and testing the cells to be tested in one hole, continuing to perform positioning and testing on the cells to be tested in the hole with the next number until the last hole to be tested is numbered, namely, positioning and testing on the cells to be tested in all the holes are completed. If the number of the current hole and the positioning information of the cells to be tested in the current hole are determined, carrying out preset test on the cells to be tested and recording; and then based on the serial number of the hole, acquiring the positioning information of the cell to be tested in the hole corresponding to the next serial number, and carrying out preset test and recording on the cell to be tested until the serial number of the last hole to be tested.

For example, when the culture vessel has 6 wells, the 6 wells are divided into two rows, the up numbers are 1, 2 and 3, and the down numbers are 4, 5 and 6, the cells to be tested in the well 1 are first positioned, detected and recorded, and then the cells to be tested in the well 2 are positioned, detected and recorded … … until the positioning, detection and recording of the cells to be tested in the well with the number 6 are completed.

In other embodiments of the present application, after numbering is completed, positioning information of cells to be tested in all the numbered holes can be obtained by positioning, and then the cells to be tested are tested one by one according to the numbers of the holes.

Based on the above embodiments, in the method for testing cells with high throughput provided by the present application, the testing of the cells, that is, the cells to be tested, may specifically include recording the states and characteristics of the cells under irradiation of different light beams.

Specifically, based on the type of the test environment light, the whole test flow can be divided into a bright field test and a dark field test, and the bright field test can obtain a bright field test result under the irradiation of visible light; the dark field test refers to obtaining a dark field test result under irradiation of excitation light with a specific wavelength, and recording the test result in a time after the irradiation is started, wherein the test result can be recorded in the form of photos and videos.

For example, in some embodiments, the bright field test may be by illuminating the cell with visible light and the dark field test may be focused onto the cell under test by one or more excitation lights. The information in the recorded test results may include, among others, cell shape data, living cell number, cell mechanics data, cell movement data, and intracellular ion concentration data, and the rate of change that can be formed, and the like.

It should be noted that, the cell shape may be obtained based on photographs and videos, and the data such as the number of living cells, the data of cell mechanics, the data of ion concentration in the cells, etc. may be obtained based on the change of the optical signal through a preset module.

In some embodiments, the cell high-throughput test system may pre-store test information or input by a worker in the system, and then control each subsystem to perform a test corresponding to the test information, such as the number of living cells, the concentration of cellular ions, the cell force, etc., on the cells to be tested, and record corresponding data, such as one or more of cell shape data, the number of living cells, the data of cell mechanics, the data of cell movement, and the data of intracellular ion concentration, so as to implement a personalized test, thereby meeting various detection requirements.

Based on the above embodiments, the method for testing cells with high throughput provided by the present application further includes re-measuring and recording cells in the culture container that have been tested based on the numbering and positioning information, that is, testing the cells to be tested in the same culture container multiple times.

Specifically, before the first test is performed on the culture container, global detection is performed on the cells to be tested in each hole according to the determined hole numbers, so as to obtain a global photograph, namely a first global image, of the cells to be tested in the hole. The method for obtaining the global image comprises the following steps: and photographing the field of view in the hole by a camera in a microscopic imaging subsystem in the cell high-throughput test system, dividing the hole area into a plurality of small areas, photographing each small area, performing similarity comparison on the edges of two adjacent photos, ensuring that the field of view is continuous and non-missing, and splicing the obtained adjacent photos to finally obtain the global image of the cell to be tested in the hole.

When the cell to be tested, which needs to be tested for multiple times, is measured again, firstly, a culture container such as a culture plate is positioned, and based on the serial number of the upper hole of the culture container and the positioning information of the cell to be tested, which are measured for the first time or are manually introduced, different positions in the hole are shot under a lens, so that a global detection photo in the hole during retesting, namely a second global image, is obtained.

And then, comparing the second global image with the first global image in similarity, and if the similarity of the second global image and the first global image is greater than a preset threshold value, such as 95%, performing a subsequent testing process, namely performing subsequent detection on the cells to be tested.

The principle is the same as that of the first test, in this process, after the similarity of one hole is determined, and after the test and recording of the cells to be tested in the hole are completed, the same operation is performed on the hole with the next number, so that the whole test process is ensured to be orderly performed.

In some embodiments, more than three measurements may be made on the cells to be tested in the same culture vessel with reference to the re-measurement procedure described above. The image for global detection contrast in the current test flow can be a first global image obtained in the first test flow or a global image obtained in the last measurement flow of the current test flow.

It should be noted that, the re-measurement of the cells to be tested in the same culture container may be performed after a preset time interval without any treatment after the first measurement, so as to obtain development data for researching the cells to be tested in a natural state; or after the first measurement is completed, the test is performed again after the study drug is added or the study stimulus is applied, so that the data for studying the influence of the specific drug or stimulus on the cells is obtained, and thus the requirements of various cell researches are met.

In addition, to meet the need of comparison with the first-time test data, the data recorded in the subsequent test may include cell shape data, living cell number, cell mechanics data, cell movement data, intracellular ion concentration data, and graphs, heat maps, fitted curves, residual analysis, etc. of cell death rate, percentage change in cell mechanics, percentage change in intracellular ion concentration, and the respective data over time, drug addition amount, etc.

The data calculation method of the nth cell death rate, the cell mechanical change percentage and the intracellular ion concentration change percentage obtained by the nth test comprises the following steps:

in the above formula, M is the number of dead cells, the cell strength and the intracellular ion concentration, N is the number of dead cells, the cell strength and the cell ion concentration of the nth test, mn is the number of dead cells, the cell strength and the cell ion concentration of the nth test, M (N-1) is the number of dead cells, the cell strength and the cell ion concentration of the nth test, N is the number of living cells, nn is the number of living cells in the nth test, and N (N-1) is the number of living cells in the nth test.

It should be noted that, for the recording of the test results, the content may include photographs, videos, electronic reports and paper reports, the video recording time may be adjusted, the electronic reports and paper reports may output data, graphs and correlation analysis, the data and graphs include graphs of cell shape data (including length, width, aspect ratio, cell area, deformation area), cell activity (including living cell number, mortality), mechanical data (including cell force magnitude, cell force distribution, cell beating frequency, power, moment), cell movement data (including movement direction, speed), ion concentration data (including intracellular ion concentration magnitude, ion concentration change speed, ion distribution) and various data over time, drug addition amount, etc., heat maps, fitting curves, residual analysis, etc.

Of course, in the present application, the data to be recorded at last is not completely fixed, and in particular, the test information input by the user can be received by the system before the test, and various conditions in the test process and the recorded data information can be determined based on the test information.

In the process of carrying out multiple tests on cells to be tested in a culture container, besides the first test, in the subsequent test, a manual instruction is received, the zero coordinate position is determined based on the manual instruction, the repositioning of the culture container is realized, or the system is automatically triggered and positioned, then the serial number of a hole obtained by the first test in a preset system and the positioning information of the cells to be tested are called, and after the global verification is completed, a retesting flow is carried out; of course, in other embodiments, the serial number of the hole and the positioning information of the cells to be tested can be received by the culture container manually introduced by the relevant staff, so as to manually identify a plurality of culture containers needing to be measured for many times; or receiving the serial number of the hole which is manually guided by the relevant staff and is determined by the staff and the positioning information of the cells to be tested. Thus, the full-automatic semi-automatic manual test device meets various test requirements of full automation, semi-automation and pure manual operation.

In addition, in the above embodiment of the present application, the chemical agent added to the cell culture vessel may be changed according to actual requirements, and the present application is not limited to the type of chemical agent added;

when the cells to be tested are cultivated through the culture container, the hardness range of the prepared gel can be between 1Pa and 10000 kPa; the mass ratio of the fluorescent microspheres added into the gel can be 0.001% -10%, and the diameter range of the fluorescent microspheres can be 100nm-10 mu m; the gel surface forms a pattern capable of containing single cells, the application does not limit the manufacturing method of the pattern, and the length, width, depth and specific pattern shape of the pattern can be determined by practical test schemes and requirements; when the cells to be tested are inoculated to the surface of the gel with the pattern for culturing, the environment and the time for culturing the cells are determined according to the cells to be tested and a test scheme; the fluorescent dye can comprise Flou-3AM, flou-4AM, SBFI AM, mag-Fluo-4 AM and the like, and the specific fluorescent dye type used in the experiment needs to be determined according to the ion type to be tested; when adding fluorescent dye into the cultured cell culture container to incubate the cells, the incubation time is determined by the ions to be tested and the cells to be tested; whether the fluorescent dye is added for incubation for multiple times or not in the multiple test process, and the times of adding the fluorescent dye for incubation can be determined by the time from the first time to the last time of test, the ions to be tested, the types of the fluorescent dye and the types of the cells to be tested; cell culture containers may include single Kong Peiyang plates, multi-well plates, culture containers, etc., cell culture containers may include disposable culture containers and reusable culture containers, and cell culture containers may include round culture containers and square culture containers; the cell culture container is attached with a plate with a thickness customized according to the pattern arrangement state, wherein the plate is made of PVC, organic glass (PMMA), chromium plates, stainless steel plates and the like, and the thickness of the plate is not limited; the names of the finally formed report comprise automatic names and manual names, wherein the automatic names are named according to the sequence of the test time, the serial number or name of the cell culture container to be tested, the cell coordinates and the detection times; the test information may include: the name or number of the testers, the test name, the number of the cell culture containers to be tested, the number or name of the cell culture containers to be tested currently, the number or name of the cell culture container holes to be tested currently, the test mode and the like; in the application, in a full-automatic mode and a semi-automatic mode, according to the input current cell culture container hole number to be tested, the hole number automatically extends to the next hole after the current cell hole to be tested is tested, until the last current cell culture container hole number to be tested; the number of the current cell culture container to be tested is automatically and smoothly changed into the next number according to the number of the culture containers to be tested after all holes in the current cell culture container to be tested are tested, and the number is up to the last culture container to be tested; the video recording time is determined by the shooting frequency of a camera, the cell beating period and the requirement of a tester; in the drug screening process, the drugs can be not added into each hole of the porous culture container for blank test, the same drugs with different concentrations can be added for screening, and the drugs with different types can also be added for screening.

The following description will be made with respect to various test embodiments, including various numbers and types of cells to be tested, different test requirements, and different manual, automatic, and semi-automatic test modes of the test procedures, respectively, to illustrate the above-mentioned cell high throughput test methods of the present application.

First, as shown in fig. 3, in an embodiment of the present application, it includes:

the cells to be tested are first obtained, and as mentioned in the above-described embodiment, the cells to be tested may be obtained by a cell culture vessel to be tested such as a culture vessel.

At this time, the relevant staff can input test information including various test conditions and data to be recorded on a preset system such as a cell high-throughput test system.

The test system automatically recognizes or receives the manual command, and sets the zero point coordinate position of the cell culture container to be tested, such as the culture container, based on the positioning mark on the culture container, so as to position the cell culture container to be tested.

Then the test system numbers the cell culture container to be tested, such as the holes in the culture container, namely the culture container, according to the zero point coordinate position;

the test system sequentially carries out global detection on the cells to be tested in each hole based on the holes to obtain a global photo, namely a global image.

After global detection, the test system acquires the coordinates of a single cell to be tested in the hole according to the zero coordinate position, and then enters a cell test process, namely a cell test stage, and the method specifically comprises the following steps: the cells to be tested mentioned in the above embodiments are irradiated with visible light or excitation light or the like, the cells are recorded, a first test result is obtained, and a first record file is obtained.

The test system or manually then compares the cells to be tested, which need to be measured multiple times, with the cells to be tested first, as in the global test mentioned in the examples above, and after confirming that there is no error, the cell entry test phase is again performed, and a re-measurement is performed (again, the second measurement and the third measurement may be performed, etc.).

After each measurement is completed, the test results of corresponding times are recorded, and the record files of corresponding times are obtained.

Based on this embodiment, corresponding adjustments are made for different types and numbers of cells and different test requirements (which may be determined based on the test information entered by the user and the test information). The following describes various cases in various embodiments.

Test example 1: for a fully automated process for detecting cell force and intracellular ion concentration of autonomously contractile cells, as shown in FIG. 3, the test procedure may specifically include:

Firstly, obtaining cells to be tested through a culture container, which can specifically comprise: adding polyethylene glycol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like with the same volume into each well of a six-well culture plate to prepare gel with the hardness of 500kPa, adding fluorescent microbeads with the mass ratio of 1% and the diameter of 10 mu m into the gel, treating the surface of the gel added with the fluorescent microbeads, placing a customized chromium plate on the hydrogel, irradiating the surface of the gel by an ultraviolet lamp, and forming an elliptic regular array pattern capable of containing single cells after the non-shielded part of the gel is deactivated. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ Culturing for 7 days under the condition, adding a fluorescent probe Flou-4 AM into a cell culture container after 7 days for incubation, and washing after incubation for 30min to obtain the cells to be tested.

After the high-throughput test system is started, the user may enter verification information and test information, such as a tester name or number, e.g., input number 03511, test name: human cardiomyocytes, number of cell culture vessels to be tested: 5, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, test mode: and (5) full-automatic testing.

For the first test, i.e. the first test, it may comprise:

the six-hole culture plate (same as the function of the culture container to be tested mentioned in the embodiment, the same as the function of the culture container to be tested mentioned in the embodiment) with the number 001 is fixed on the objective table by using a clamp, the central position of the six-hole culture plate is marked by using a black dot, the control system is operated to move the objective table to enable the six-hole culture plate to be placed at the position between the ocular lens and the objective lens, the position marked with the black dot is set as a zero point coordinate position, the six-hole culture plate with the number 001 is positioned, after the zero point coordinate position is recorded, the numbers of all holes in the six-hole culture plate with the number 001 are respectively 01, 02, 03, 04, 05 and 06, and the hole numbers are edited to finish the test process. The method comprises the steps of irradiating visible light to 01 holes under a bright field test condition, globally detecting cells to be tested in the 01 holes to obtain coordinates of all the cells to be tested, recording the cells to be tested under the bright field condition, taking pictures and videos of the cells to be tested, converting the bright field into a dark field, focusing two excitation lights to the 01 holes at the same time, recording the cells to be tested in the form of pictures and videos, recording the video time for 20s, automatically naming a record file according to the test time, the cell culture container number, the cell coordinate position and the test times after the test is finished, and outputting a test report, wherein the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force size, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the intracellular calcium ion concentration. After the test of the holes 01 is completed, the control system is operated to move the objective table to enable the ocular lens and the objective lens to be aligned with the holes 02, 03, 04, 05 and 06 in sequence, global detection is carried out on the holes 02, 03, 04, 05 and 06 respectively, the coordinates of all cells to be tested in each hole are obtained, and the test process of 01 Kong Mingchang and a dark field is repeated.

The six-well plates for cells to be tested, numbered 002, 003, 004 and 005, were sequentially placed on the stage, and the test procedure for the six-well plates for cells to be tested, numbered 001, was repeated.

A second test was performed:

the method comprises the steps of placing a six-hole culture plate of cells to be tested with the number 001 on an objective table, fixing the six-hole culture plate by using a clamp, controlling a control system to adjust the position of the six-hole plate, finding the zero coordinate position of the six-hole culture plate, positioning the six-hole culture plate, introducing the numbers of all holes and the coordinates of all cells of the six-hole culture plate of cells to be tested with the number 001 in the first test, performing global detection on the cells to be tested with the number 01 hole, obtaining a global detection photo of the second test, performing similarity comparison with the global detection photo of the first test, and entering a test process after confirming that the similarity of the two global detection photos is more than 95%. Taking a photograph and video of 01 Kong Daice test cells under bright field conditions, wherein the video time is 10s, converting the bright field into a dark field, focusing two beams of excitation light to 01 holes at the same time, recording the time to be tested of the 01 holes in the form of the photograph and the video, wherein the video time is 20s, automatically naming a record file according to the test time, the serial number of the cell culture container to be tested, the cell coordinate position and the test times after the test is finished, outputting a test report, and the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the intracellular calcium ion concentration, the change percentage of each item of data and the cell death rate. After the test of the holes 01 is finished, the control system is operated to move the objective table to enable the ocular lens and the objective lens to be aligned with the holes 02, 03, 04, 05 and 06 in sequence, global detection is carried out on all cells to be tested in the holes 02, 03, 04, 05 and 06 respectively, similarity comparison is carried out on the cells to be tested in the holes with the corresponding numbers obtained by the first test, and after the similarity is confirmed to be more than 95%, the test process of the holes 01 and Kong Mingchang and the dark field is repeated.

The six well plates for cells to be tested, numbered 002, 003, 004 and 005, were sequentially placed on the stage, and the second test procedure of the six well plates for cells to be tested, numbered 001, was repeated.

Third test, fourth test and fifth test:

and sequentially placing six pore plates of five cells to be tested on an objective table and fixing the six pore plates by using a clamp, controlling a control system to adjust the positions of the six pore plates, finding the zero coordinate positions of the six pore plates, positioning the six pore plates, wherein the cell coordinates imported by each test are the cell coordinates generated during the first test, comparing the similarity of the global detection photos generated during the current test with the similarity of the global detection photos generated during the previous test, and repeating the second test process after confirming that the similarity is more than 95%.

The total test time from the first test to the fifth test was 3 hours, during which incubation without the addition of Fluo-4 AM again was not required.

Test example 2: for a fully automated test procedure that can generate electrical stimulation, cellular forces and intracellular ion concentrations of contracted cells upon stimulation, as shown in fig. 4, the test procedure may specifically include:

First for the cellular force test:

adding polyvinyl alcohol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like into a disposable single-hole culture container to prepare gel with the hardness of 800Pa, adding fluorescent microbeads with the mass ratio of 0.001% and the diameter of 100nm into the gel, treating the surface of the gel added with the fluorescent microbeads, pressing a mold with a protruding pattern on the surface of the gel, enabling the contact distance between the mold and the surface of the gel to be about 0.5mm under the action of buoyancy, drying the gel to form cell grooves with the depth of about 0.2mm, and enabling the surface of the gel to form an irregular array pattern capable of accommodating single cells. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ 、1%N ₂ Culturing for 3 days under the condition to obtain the cells to be tested.

Entering the tester name or number after the high throughput test system is started: 03512, test name: human muscle cells, number of cell culture vessels to be tested: 1, current cell culture vessel name to be tested: disposable single well culture vessel, current cell culture vessel well name to be tested: 1, test mode: and (5) full-automatic testing.

For the first test:

The disposable single well culture vessel was fixed to the stage using a specific jig. And a cross mark is used at any position of the bottom of the disposable single-hole culture container, and the crossing position in the middle of the cross mark is a zero coordinate position, so that the disposable single-hole culture container is positioned. After the zero position recording is completed, the name of the editing hole is cell hole. Under the open field test condition, visible light irradiates the disposable single-hole culture container, a control system is operated to adjust the distance between the single-hole culture container and the ocular lens and the objective lens, and global detection is carried out on cells to be tested in the disposable single-hole culture container, so that coordinates of all cells to be tested in a hole named as a cell hole are obtained; placing electric stimulators on two sides of the disposable single-hole culture container, and introducing current to make the cell beating frequency the same; recording cells to be tested under the condition of bright field, taking photos and videos of the cells to be tested, changing the bright field into dark field, focusing first excitation light into a disposable single-hole culture container, recording the cells to be tested in the form of photos and videos, wherein the video time is 5s, automatically naming a record file according to the test time, the disposable single-hole culture container, the cell coordinate position and the test times after the test is finished, outputting a test report, and the report content comprises cell length, width, length-width ratio, cell area, number of living cells, cell force size, cell force distribution, cell beating frequency, power, moment and the like.

For the second test:

fixing a disposable single-hole culture container on an objective table by using a specific clamp, controlling a control system to adjust the distance between the disposable single-hole culture container and an ocular lens, finding the zero coordinate position of the disposable single-hole culture container, positioning the disposable single-hole culture container, introducing the coordinates of all cells to be tested in a first test time single Kong Peiyang container, performing global detection on the disposable single-hole culture container, taking global photos of all the cells to be tested in the disposable single-hole culture container, performing similarity comparison with the global photos obtained by the first test, entering a test process after confirming that the similarity is more than 95%, placing electric stimulators on two sides of the disposable single-hole culture container, and introducing current to make the cell beating frequency the same; recording cells to be tested under the condition of bright field, taking photos and videos of the cells to be tested, changing the bright field into dark field, focusing first excitation light into a disposable single-hole culture container, recording the cells to be tested in the form of photos and videos, wherein the video time is 5s, automatically naming a record file according to the test time, the disposable single-hole culture container, the cell coordinate position and the test times after the test is finished, outputting a test report, and the report content comprises cell length, width, length-width ratio, cell area, number of living cells, cell force size, cell force distribution, cell beating frequency, power, moment, cell death rate, cell force size change percentage and the like.

Third test, fourth test … … Nth test

The second test procedure was repeated.

The testing for intracellular ion concentration may include:

adding polyvinyl alcohol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like into a disposable single-hole culture container to prepare gel with the hardness range of 800Pa, adding fluorescent microbeads with the mass ratio of 0.001% and the diameter of 100nm into the gel, treating the surface of the gel added with the fluorescent microbeads, pressing a mold with a protruding pattern on the surface of the gel, enabling the contact distance between the mold and the surface of the gel to be about 0.5mm under the action of buoyancy, drying the gel to form cell grooves with the depth of about 0.2mm, and enabling the surface of the gel to form an irregular array pattern capable of accommodating single cells. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ 、1%N ₂ Culturing for 3 days under the condition, adding fluorescent dye SBFI AM into a cell culture container after 3 days for incubation, and washing after incubation for 10min to obtain the cells to be tested.

The name of the tester is input after the high-flux test system is started: x, test name: human muscle cells, number of cell culture vessels to be tested: 1, current cell culture vessel name to be tested: single well culture vessel, current cell culture vessel well name to be tested: cell well, test mode: and (5) full-automatic testing.

First test:

the disposable single-well culture vessel was fixed on the stage. And a cross mark is used at any position of the bottom of the disposable single-hole culture container, and the crossing position in the middle of the cross mark is a zero coordinate position, so that the disposable single-hole culture container is positioned. After the zero position recording is completed, the name of the editing hole is cell hole. Under the open field test condition, visible light irradiates the disposable single-hole culture container, a control system is operated to adjust the distance between the disposable single-hole culture container and the ocular lens and the objective lens, and the cells in the disposable single-hole culture container are subjected to global detection to obtain the coordinates of all cells to be tested in the holes named as cell holes; recording cells to be tested under the condition of bright field, taking photos and videos of the cells to be tested, changing the bright field into dark field, focusing second excitation light into a disposable single-hole culture container, recording the cells to be tested in the form of photos and videos, wherein the video time is 5s, automatically naming a record file according to the test time, the single-hole cell culture container, the cell coordinate position and the test times after the test is finished, and outputting a test report, wherein the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the average fluorescence intensity in the cells, the concentration of sodium ions in the cells and the like.

Second test:

the fluorescent dye SBFI AM is added into a disposable single-well culture container for incubation, and the culture container is washed after incubation for 10 min. After cleaning, the disposable single-hole culture container is fixed on the objective table by using a specific clamp, the control system is operated to adjust the distance between the disposable single-hole culture container and the ocular lens and the distance between the disposable single-hole culture container and the objective lens, the zero point coordinate position of the disposable single-hole culture container is found, and the disposable single-hole culture container is positioned. Importing the coordinates of all cells to be tested in a first test time sheet Kong Peiyang container, performing global detection on a disposable single-hole culture container, taking global photos of all the cells to be tested in the disposable single-hole culture container, performing similarity comparison with the global photos obtained by the first test, entering a test process after confirming that the similarity is more than 95%, placing electric stimulators on two sides of the disposable single-hole culture container, and introducing current to enable the cell beating frequencies to be the same; recording the cells to be tested under the condition of a bright field, taking a photo and a video of the cells to be tested, changing the bright field into a dark field, focusing second excitation light into a disposable single-hole culture container, recording the cells to be tested in the form of the photo and the video, wherein the video time is 5s, automatically naming a record file according to the test time, the disposable single-hole culture container, the cell coordinate position and the test times after the test is finished, outputting a test report, and the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the cell death rate, the average fluorescence intensity change percentage in the cell force, the sodium ion concentration change percentage in the cell and the like.

Third test, fourth test … … Nth test

The second test procedure was repeated.

Test example 3: for a fully automated test procedure that does not produce any shrinkage and does not allow free movement of cells, the test procedure may specifically include, as shown in fig. 5:

the gel with the hardness of 10000KPa is prepared by adding sodium carboxymethylcellulose, silk fibroin, tannic acid and the like into each hole of a four-hole culture plate, fluorescent microbeads with the mass ratio of 0.01% and the diameter of 200nm are added into the gel, the surface of the gel added with the fluorescent microbeads is treated, and an irregular array pattern is marked on the surface of the gel by using an extremely fine needle. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ 、2%N ₂ For 5 days, adding fluorescent dye Mag-Fluo-4 AM to each control of the four-hole culture plate for incubation after 5 days, and clearly obtaining the cells to be tested after incubation for 15 minutes.

The tester number is input after the high-throughput test system is started: 03513, test name: mouse stomach wall cells, number of cell culture vessels to be tested: 3, number of cell culture vessel to be tested currently: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, test mode selection full automatic test.

The method comprises the steps of placing a cell four-hole culture plate to be tested with the number of 001 on an objective table, fixing the cell four-hole culture plate by using a clamp, marking the center position of the six-hole culture plate by using a triangle, operating a control system to move the objective table, enabling the four-hole culture plate to be placed at a position between an ocular lens and an objective lens, setting the position of the marked triangle as a zero coordinate position, and positioning the four-hole culture plate. After the zero point coordinate position record is completed, the serial numbers of all holes in the cell four-hole culture plate to be tested with the serial number of 001 are respectively 01, 02, 03 and 04, and the hole serial numbers are completed to enter the test process. The method comprises the steps of irradiating visible light to 01 holes under the condition of bright field test, globally detecting cells to be tested in the 01 holes to obtain coordinates of all the cells to be tested, recording the cells to be tested under the condition of bright field, taking pictures and videos of the cells to be tested, changing the bright field into dark field, focusing two excitation light beams to the 01 holes at the same time, recording the cells to be tested in the form of pictures and videos, and recording the video time to be 20s. And (3) after the recording is finished, digesting the cells to be tested by using trypsin, photographing a dark field photo and a video of the cells to be tested in the digestion process and after digestion by using trypsin, automatically naming a record file according to the test time, the number of the cell culture container to be tested currently, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force size, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the concentration of intracellular magnesium ions. After the 01 hole test is completed, the control system is operated to move the objective table to enable the ocular lens and the objective lens to be aligned to the 02 hole, the 03 hole and the 04 hole in sequence, and the 01 hole test process is repeated.

The cell four-well culture plates to be tested with the numbers 002 and 003 are sequentially placed on the objective table, and the test process of the cell four-well culture plate to be tested with the number 001 is repeated.

Test example 4: for a fully automated process of detecting cell force, cell movement and intracellular ion concentration that can move cells freely, as shown in fig. 6, the test process may specifically include:

the gel with the hardness of 100kPa is prepared by adding lignocellulose, sephades G-25 cross-linked dextran, agar and the like into each hole of a twelve-hole culture plate, the custom-made chromium plate is placed on the hydrogel with the mass ratio of 0.5%, the ultraviolet light lamp irradiates the surface of the gel, a circular regular array pattern which can contain single cells is formed after the non-shielded part of the gel is deactivated, and a PVC baffle with the thickness of 0.5cm is used for forming independent spaces between the single patterns. Inoculating the cells to be tested onto the gel surface at 37deg.C with 5% CO ₂ Culturing for 7 days under the condition, adding fluorescent dye SBFI AM into a cell culture container after 7 days for incubation, and washing after 20 minutes of incubation to obtain the cells to be tested.

The tester number is input after the high-throughput test system is started: 03514, input test name: neutrophils, number of cell culture vessels to be tested: 1, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, the test mode selects full automatic testing.

The twelve-hole culture plate of the cells to be tested, which is numbered 001, is placed on an objective table and fixed by using a clamp, the central position of the twelve-hole culture plate is marked by using a black dot, a control system is operated to move the objective table, the twelve-hole culture plate is placed between an ocular lens and an objective lens, the position of the black dot marked by the position is set as a zero coordinate position, and the twelve-hole culture plate is positioned. After the zero point coordinate position record is completed, the serial numbers of all holes of cells to be tested in the twelve-hole culture plate with the edit serial number 001 are respectively 01, 02, 03, 04, 05 and 06, and the hole serial numbers are completed to enter the test process. Under the condition that a baffle is not taken, illumination field test visible light irradiates to the 01 hole, global detection is carried out on cells to be tested of the 01 hole, coordinates of all the cells to be tested are obtained, a photo and a video of the cells to be tested are taken, the video time is 10s, the illumination field is converted into a dark field, two bundles of excitation light are focused to the 01 hole at the same time, the cells to be tested are recorded in the form of the photo and the video, and the video time is 20s. Under the condition that the baffle is taken out, visible light is irradiated to the 01 hole in a bright field test, a photo and a video of the cell to be tested are shot, the video time is 10s, the bright field is converted into a dark field, two excitation lights are focused to the 01 hole at the same time, the cell to be tested is recorded in the form of the photo and the video, and the video time is 20s. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the concentration of intracellular sodium ions, the cell moving distance, the cell moving speed and the like. After the 01 hole test is completed, the control system is operated to move the objective table to enable the ocular lens and the objective lens to be aligned to the 02 hole, the 03 hole, the 04 hole, the 05 hole and the 06 hole in sequence, and the 01 hole test process is repeated.

Test example 5: for a semi-automatic test procedure for electrical stimulation, cellular forces and intracellular ion concentration of cells capable of producing contractions upon stimulation, as shown in fig. 7, the test procedure may specifically include:

adding polyvinyl alcohol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like into each hole of a six-hole culture plate to prepare gel with the hardness of 1Pa, adding fluorescent microbeads with the mass ratio of 0.1% and the diameter of 50 mu m into the gel, treating the surface of the gel added with the fluorescent microbeads, pressing a mold with protruding patterns on the surface of the gel, enabling the mold to have a contact distance of about 0.5mm with the surface of the gel under the action of buoyancy, drying the gel to form cell grooves with the depth of about 0.2mm, and enabling the surface of the gel to form an irregular array pattern capable of containing single cells. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ Culturing for 3 days under the condition, adding fluorescent dye Flou-3 AM into a cell culture container for incubation, and washing after incubation for 30min to obtain the cells to be tested.

The tester number is input after the high-throughput test system is started: 03515, test name: rat muscle cells, number of cell culture vessels to be tested: 2, current cell culture vessel number to be tested: 001, current cell culture vessel well name to be tested: 01. 02, 03, 04, 05, 06, test mode selection semi-automatic test.

First test:

the six-hole culture plate with the number of 001 is placed on the objective table and fixed by using a clamp, the center position of the six-hole culture plate is marked by using a black dot, the control system is operated to move the objective table, the six-hole culture plate is placed between the ocular and the objective lens, the position of the black dot marked is set as a zero coordinate position, and the six-hole culture plate is positioned. After the zero point coordinate position record is completed, the serial numbers of all holes in the six-hole culture plate of the cell to be tested with the serial number of 001 are 01, 02, 03, 04, 05 and 06 respectively, and the hole serial numbers are completed to enter the test process. And (3) manually searching a cell area under the bright field condition, and sequentially adding cell coordinates after the cell area is found. Placing electric stimulators on two sides of the hole with the number of 01 to make the cell contraction frequency the same; recording cells to be tested under the condition of bright field, taking photos and videos of the cells to be tested, changing the bright field into dark field, focusing two beams of exciting light to 01 holes at the same time, recording the cells to be tested in the form of photos and videos, wherein the video time is 10s, deriving a cell coordinate information file after the test, automatically naming the recorded file according to the test time, the serial number of a cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the intracellular calcium ion concentration. After the test of the holes 01 is completed, the control system is operated to move the objective table to enable the ocular lens and the objective lens to be aligned with the holes 02, 03, 04, 05 and 06 in sequence, the cell areas of the holes 02, 03, 04, 05 and 06 are respectively searched, the coordinates of all cells to be tested in each hole are obtained, and the test process of the holes 01 and Kong Mingchang and the dark field is repeated.

The six-well culture plates for the cells to be tested, numbered 002, were sequentially placed on the stage, and the test procedure for the six-well culture plates for the cells to be tested, numbered 001, was repeated.

Second test:

placing a six-hole culture plate with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, controlling a control system to adjust the position of the six-hole plate, finding the zero point coordinate position of the six-hole culture plate, positioning the six-hole culture plate, introducing the serial numbers of all cells and the serial numbers of all cells of the six-hole culture plate with the number of 001 to be tested in the first test, and placing an electric stimulator above the serial number 01 of the holes to ensure that the cell beating frequencies are the same; taking a photograph and video of a 01 Kong Daice test cell under a bright field condition, converting the bright field into a dark field with the video time of 5s, focusing two beams of excitation light to 01 holes at the same time, recording the cell to be tested in the form of the photograph and the video with the video time of 10s, automatically naming a record file according to the test time, the cell culture container number, the cell coordinate position and the test times after the test is finished, outputting a test report, wherein the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the intracellular calcium ion concentration, the change percentage of each item of data and the cell death rate. After the 01 hole test is completed, the control system is operated to move the objective table to align the ocular and the objective lens to the 02, 03, 04, 05 and 06 holes in sequence, and the test process of 01 Kong Mingchang and the dark field is repeated.

Third test, fourth test … … Nth test

And sequentially placing the six pore plates of the two cells to be tested on the object stage, fixing the six pore plates by using a clamp, controlling a control system to adjust the positions of the six pore plates, finding the zero coordinate positions of the six pore culture plates, and repeating the second test process after the introduced cell coordinates are the cell coordinates generated during the first test.

Test example 6: for a process of manually detecting cell force and intracellular ion concentration of autonomously contractile cells, as shown in fig. 8, the test process may specifically include:

adding polyethylene glycol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like with the same volume into each well of a six-well culture plate to prepare gel with the hardness of 500kPa, adding fluorescent microbeads with the mass ratio of 1% and the diameter of 100 mu m into the gel, treating the surface of the gel added with the fluorescent microbeads, placing a customized chromium plate on the hydrogel, irradiating the surface of the gel by an ultraviolet lamp, and forming a regular array pattern capable of containing single cells after the non-shielded part of the gel is deactivated. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ Culturing for 7 days under the condition, adding fluorescent dye Flou-4 AM into a cell culture container after 7 days for incubation, and washing after incubation for 30min to obtain the cells to be tested.

The tester number is input after the high-throughput test system is started: 03516, test name: number of cells cultured in the mouse heart muscle cells to be tested: 1, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, the test mode selects full automatic testing.

First test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, marking the central position of the six-hole culture plate by using a black dot, operating a control system to move the objective table, enabling the six-hole culture plate to be placed at the position between an ocular lens and an objective lens, setting the position of the black dot marked as a zero coordinate position, and positioning the six-hole culture plate. After the zero point coordinate position record is completed, the serial numbers of all holes in the six-hole culture plate of the cell to be tested with the serial number of 001 are 01, 02, 03, 04, 05 and 06 respectively, and the hole serial numbers are completed to enter the test process. The control system is operated to align the ocular lens and the objective lens with the 01 hole, the cell area is manually searched under the bright field condition, and the cell coordinates are sequentially added after the cell area is found. The manual regulation and control system is in a bright field mode, records the cells to be tested under the bright field condition, takes a photo and video of the cells to be tested, the video time is 10s, manually converts the bright field into a dark field, simultaneously focuses two excitation lights to 01 holes, records the cells to be tested in the form of the photo and video, and the video time is 15s. The control system is operated to align the ocular lens and the objective lens with the holes 02, 03, 04, 05 and 06 in sequence, manually find the cell area of each hole, sequentially add the cell coordinates after finding the cell area, repeat the test process of the bright field and dark field of the hole 01 until the test of the hole 06 is finished, derive the cell coordinate information, analyze the data leading-in system, automatically name the record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times after the test is finished, output the test report, and the report content comprises the cell length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the intracellular calcium ion concentration.

Second test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, finding the zero point coordinate position of the six-hole culture plate, positioning the six-hole culture plate, introducing the coordinates of the cells to be tested obtained by the first test, respectively aligning an ocular lens and an objective lens with 01 holes by a control system, manually searching a cell area under a bright field condition, manually regulating and controlling the system to be in a bright field mode after the cells are found, recording the cells to be tested under the bright field condition, taking photos and videos of the cells to be tested, changing the bright field into a dark field by manually, focusing two excitation lights to the 01 holes simultaneously, recording the cells to be tested in the form of the photos and videos, and recording the cells to be tested for 15s. The control system is operated to aim the ocular lens and the objective lens at the holes 02, 03, 04, 05 and 06 in sequence, the cell coordinates to be tested of each hole are led in, the bright field and dark field testing process of the hole 01 is repeated until the hole 06 is tested, the test is finished, the recorded file is automatically named according to the test time, the serial number of the cell culture container, the position of the cell coordinates and the test times, and a test report is output, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force size, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the intracellular calcium ion concentration, the change percentage of each item of data and the cell death rate.

Third test, fourth test … … Nth test

And sequentially placing six pore plates of cells to be tested on an objective table, fixing the six pore plates by using a clamp, controlling a control system to adjust the positions of the six pore plates, finding the zero coordinate positions of the six pore plates, positioning the six pore plates, wherein the cell coordinates led in each test are the cell coordinates generated in the first test, and repeating the second test process.

Test example 7: for the process of automatically shrinking cell force and intracellular ion concentration of cells with fully automatic changing the light and dark field test sequence, as shown in fig. 9, the test process may specifically include:

adding polyethylene glycol, trimethylolpropane tri (3-aziridinyl propionate), polybutylene succinate and the like with the same volume into each well of a six-well culture plate to prepare gel with the hardness of 500kPa, adding fluorescent microbeads with the mass ratio of 1% and the diameter of 100 mu m into the gel, treating the surface of the gel added with the fluorescent microbeads, placing a customized chromium plate on the hydrogel, irradiating the surface of the gel by an ultraviolet lamp, and inactivating the non-shielded part of the gel to form an elliptic regular array pattern capable of containing single cells. Inoculating the cells to be tested onto the surface of gel with patterns, culturing at 37deg.C and 5% CO ₂ Culturing for 7 days under the condition, adding fluorescent dye Flou-4 AM into a cell culture container after 7 days for incubation, and washing after incubation for 30min to obtain the cells to be tested.

The tester number is input after the high-throughput test system is started: 03517, test name: human cardiomyocytes, number of cell culture vessels to be tested: 1, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, the test mode selects full automatic testing.

First test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, marking the central position of the six-hole culture plate by using a black dot, operating a control system to move the objective table, enabling the six-hole culture plate to be placed at the position between an ocular lens and an objective lens, setting the position of the black dot marked as a zero coordinate position, and positioning the six-hole culture plate. After the zero point coordinate position record is completed, the serial numbers of all holes in the six-hole culture plate of the cell to be tested with the serial number of 001 are 01, 02, 03, 04, 05 and 06 respectively, and the hole serial numbers are completed to enter the test process. And (3) irradiating visible light to the 01 holes under the bright field test condition, performing global detection on the cells to be tested in the 01 holes to obtain coordinates of all the cells to be tested, recording the cells to be tested under the bright field condition, and taking photos and videos of the cells to be tested, wherein the video time is 10s. After the 01-hole open field test is completed, a control system is operated, an ocular lens and an objective lens are respectively aligned to the 02 holes, the 03 holes, the 04 holes, the 05 holes and the 06 holes, the cells to be tested in each hole are subjected to global detection, the coordinates of all the cells to be tested are obtained, and the 01-hole open field test process is repeated. After the 06-hole bright field test is completed, converting the bright field into a dark field, focusing two excitation lights to 01 holes at the same time, and recording the cells to be tested in the form of pictures and videos, wherein the video time is 20s. After the 01-hole dark field test is finished, the control system is operated to align the ocular and the objective lens with the 02, 03, 04, 05 and 06 holes respectively, and the 01-hole dark field test process is repeated until the 06-hole dark field test is finished. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the concentration of intracellular calcium ions.

Second test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number 001 on an objective table, fixing the six-hole culture plate by using a clamp, controlling a control system to adjust the position of the six-hole plate, finding the zero coordinate position of the six-hole culture plate, positioning the six-hole culture plate, introducing the numbers of all holes and the coordinates of all cells of the six-hole culture plate of cells to be tested with the number 001 in the first test, radiating visible light to 01 holes under the bright field test condition, performing global detection on the cells to be tested with the 01 holes, obtaining a global detection photo of the second test, performing similarity comparison with the global detection photo of the first test, and entering a test stage after confirming that the similarity of the two global detection photos is more than 95%. Recording the cells to be tested under the bright field condition, taking photos and videos of the cells to be tested, and taking the video for 10s. After the 01-hole bright field test is finished, a control system is operated, an ocular lens and an objective lens are respectively aligned to 02 holes, 03 holes, 04 holes, 05 holes and 06 holes, global detection is carried out on cells to be tested in each hole, a global detection photo of a second test of each hole is obtained, similarity comparison is carried out on the global detection photo of the first test of each hole, after the similarity of the global detection photo is confirmed to be greater than 95%, the 01 Kong Mingchang test process is repeated until the 06-hole bright field test is finished, the bright field is converted into a dark field, two excitation lights are focused to the 01 holes at the same time, the cells to be tested are recorded in the form of photos and videos, and the video time is 20s. After the 01-hole dark field test is finished, the control system is operated to align the ocular and the objective lens with the 02, 03, 04, 05 and 06 holes respectively, and the 01-hole dark field test process is repeated. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the intracellular calcium ion concentration, the change percentage of each item of data and the cell death rate.

Third test, fourth test … … Nth test

And sequentially placing six pore plates of cells to be tested on an objective table, fixing the six pore plates by using a clamp, controlling a control system to adjust the positions of the six pore plates, finding the zero coordinate positions of the six pore plates, positioning the six pore plates, wherein the cell coordinates imported by each test are the cell coordinates generated during the first test, comparing the similarity of the two global detection photos to the similarity of the global detection photo generated during the current test and the global detection photo generated during the previous test, and repeating the second test process after confirming that the similarity is more than 95%.

Test example 8: for the process of fully automated-manual testing of cell forces and intracellular ion concentrations of individual autonomously contractile cells, the details are shown in fig. 10:

The tester number is input after the high-throughput test system is started: 03518, test name: human cardiomyocytes, number of cell culture vessels to be tested: 1, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, the test mode selects full automatic testing.

First test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, marking the central position of the six-hole culture plate by using a black dot, operating a control system to move the objective table, enabling the six-hole culture plate to be placed at the position between an ocular lens and an objective lens, setting the position of the black dot marked as a zero coordinate position, and positioning the six-hole culture plate. After the zero point coordinate position record is completed, the serial numbers of all holes in the six-hole culture plate of the cell to be tested with the serial number of 001 are 01, 02, 03, 04, 05 and 06 respectively, and the hole serial numbers are completed to enter the test process. And under the bright field test condition, visible light irradiates to the 01 hole, a control system is controlled to adjust the distance between the six-hole plate and the ocular lens and the distance between the six-hole plate and the objective lens, global detection is carried out on cells to be tested in the 01 hole, coordinates of all the cells to be tested are obtained, the cells to be tested under the bright field condition are recorded, photographs and videos of the cells to be tested are taken, and the video time is 10s. After the 01 hole bright field test is finished, converting the bright field into a dark field, focusing two excitation lights to 01 holes at the same time, recording cells to be tested in the form of pictures and videos, wherein the video time is 20s, and operating a control system after the 01 hole dark field test is finished. And respectively aligning the ocular lens and the objective lens with holes 02, 03, 04, 05 and 06, performing global detection on the cells to be tested in each hole to obtain the coordinates of all the cells to be tested, and repeating the 01 Kong Mingchang and dark field testing processes. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the concentration of intracellular calcium ions.

Second test

After the high-flux test system is started, other information is unchanged, and the test mode is selected for manual test.

The six-hole culture plate for the cells to be tested, which is numbered 001, is placed on an objective table and fixed by using a clamp, a control system is operated to adjust the position of the six-hole plate, the zero coordinate position of the six-hole culture plate is found, the six-hole culture plate is positioned, and the coordinates of the cells to be tested, which are required to be tested, are manually added. And (3) irradiating visible light to the cells to be tested with the coordinates under the bright field test condition, taking pictures and videos of the cells to be tested, and taking the video for 10s. The bright field is manually converted into the dark field, two beams of excitation light are simultaneously focused on the cells to be tested with the coordinates positioned, the cells to be tested are recorded, and the video time is 20s. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions, the intracellular calcium ion concentration and the change percentage of each item of data.

Third test, fourth test … … Nth test

The procedure of the second test was repeated.

Test example 9: for the process of performing a drug screening test of fully automatic autonomously contractile cells to obtain cell force and intracellular ion concentration of the cells to be tested, the process is shown in fig. 11:

The tester number is input after the high-throughput test system is started: 03519, test name: human cardiomyocytes, number of cell culture vessels to be tested: 1, current cell culture vessel number to be tested: 001, current cell culture vessel well number to be tested: 01. 02, 03, 04, 05, 06, the test mode selects full automatic testing.

First test

The method comprises the steps of placing a six-hole culture plate of cells to be tested with the number of 001 on an objective table, fixing the six-hole culture plate by using a clamp, marking the central position of the six-hole culture plate by using a black dot, operating a control system to move the objective table, enabling the six-hole culture plate to be placed at the position between an ocular lens and an objective lens, setting the position of the black dot marked as a zero coordinate position, and positioning the six-hole culture plate. After the zero point coordinate position record is completed, the serial numbers of all holes in the six-hole culture plate of the cell to be tested with the serial number of 001 are 01, 02, 03, 04, 05 and 06 respectively, and the hole serial numbers are completed to enter the test process. And (3) irradiating visible light to the 01 holes under the bright field test condition, performing global detection on the cells to be tested in the 01 holes to obtain coordinates of all the cells to be tested, recording the cells to be tested under the bright field condition, and taking photos and videos of the cells to be tested, wherein the video time is 10s. After the 01 hole bright field test is finished, converting the bright field into a dark field, focusing two excitation lights to the 01 hole at the same time, recording the cells to be tested in the form of pictures and videos, and the video time is 20s. After the 01-hole dark field test is completed, a control system is operated, an ocular lens and an objective lens are respectively aligned to the 02 holes, the 03 holes, the 04 holes, the 05 holes and the 06 holes, the cells to be tested in each hole are subjected to global detection, the coordinates of all the cells to be tested in each hole are obtained, and the 01 Kong Mingchang and dark field test process is repeated. And after the test is finished, automatically naming a record file according to the test time, the serial number of the cell culture container, the cell coordinate position and the test times, and outputting a test report, wherein the report content comprises the length, the width, the length-width ratio, the cell area, the number of living cells, the cell force distribution, the cell beating frequency, the power, the moment, the average fluorescence intensity of intracellular ions and the concentration of intracellular calcium ions.

After the first test was completed, no drug was added to the well numbered 01, drug A at 0.1% was added to the well numbered 02, drug A at 0.2% was added to the well numbered 03, drug A at 0.3% was added to the well numbered 04, drug B at 0.2% was added to the well numbered 05, drug C at 0.2% was added to the well numbered 06, and the second test was performed after 5 minutes of drug addition.

Second test

The six-hole culture plate of the cells to be tested, which is numbered 001, is placed on an objective table and fixed by using a clamp, a control system is operated to adjust the position of the six-hole plate, the zero coordinate position of the six-hole culture plate is found, the six-hole culture plate is positioned, and the numbers of all holes and the coordinates of all cells of the six-hole culture plate of the cells to be tested, which are numbered 001 in the first test, are imported. Taking a photo and video of 01 Kong Daice test cells under the bright field condition, converting the bright field into a dark field, focusing two beams of excitation light to the 01 holes at the same time, recording the time to be tested of the 01 holes in the form of the photo and video, automatically naming a record file according to the test time, the serial number of the cell culture container to be tested, the cell coordinate position and the test times after the test is finished, outputting a test report, wherein the report content comprises graphs, heat maps, fitting curves, residual analysis and the like of cell length, width, length-width ratio, cell area, number of living cells, cell force distribution, cell beating frequency, power, moment, average fluorescence intensity of intracellular ions, the concentration of intracellular calcium ions, the change percentage of each item of data, the cell death rate, the drug addition and the like. After the 01 hole test is completed, the control system is operated to move the objective table to align the ocular and the objective lens to the 02, 03, 04, 05 and 06 holes in sequence, and the test process of 01 Kong Mingchang and the dark field is repeated.

And after the second test is completed for 5min, performing a third test, after the third test is completed for 10min, performing a fourth test, after the fourth test is completed for 10min, performing a fifth test … …, and repeating the process of the second test from the third test.

If the test time is longer than 8 hours, the first test after 8 hours needs to use the fluorescent dye Fluo-4 AM to incubate the cells again, and the cells can be tested after incubation for 30 minutes and cleaning.

According to the cell high-throughput test method provided by the application, firstly, a culture container provided with cells to be tested is obtained, then after relevant staff inputs test information on a cell high-throughput test system, the zero point coordinate position of the culture container is set based on a positioning mark on the culture container, the culture container is positioned, holes on the culture container are numbered or named according to the zero point coordinate position, the cells to be tested in each hole are sequentially subjected to global detection according to the hole numbers or names, a global picture of the cells to be tested in the holes is obtained, the coordinates of a single cell to be tested are obtained according to the zero point coordinate position after the global detection, the coordinates of the cells to be tested are determined, then the cells enter a cell test stage, and the first test result is recorded after the test is completed and a first record file is generated; and comparing the cells to be tested which need to be tested for multiple times with the cells to be tested for the previous time, entering a cell testing stage after confirming that the cells are correct, and recording the test results of corresponding times and generating a record file of corresponding times after the tests are completed. Thus, by the method provided by the application, a large number of biological parameter test data of cells to be tested can be obtained simultaneously, meanwhile, single cells can be tested to obtain test data of the single cells with specificity, and based on serial number and positioning information, multiple measurements can be rapidly and accurately carried out to obtain comparison data, and detailed research data is provided for various stimulus or drug screening researches.

In addition, in the scheme provided by the application, the position coordinates of all cells to be tested are obtained in an automatic or manual mode according to the zero coordinate position by setting the zero coordinate position of the culture container, and the uniqueness corresponding to the cell position to be tested and the quick finding of the cells to be tested can be ensured by determining the position coordinates of the cells to be tested; the global photos of the cells to be tested are obtained, and all the tests after the second test need to compare the global photos of the cells to be tested, which are currently tested, with the global photos of the cells to be tested, which are obtained by the first test, so that a guarantee is provided for finding accurate cells to be tested; and different test modes and test types can be selected for different types of cells so as to meet various test requirements; and providing photos and videos of cells to be tested, testing results of various cell parameters and variation trend of the cell parameters, and realizing visualization, imaging and datamation of analysis results.

Based on the same inventive concept, the present application also provides a cell high throughput testing system, as shown in fig. 12, which may include a microscopic imaging subsystem.

The microscopic imaging subsystem 121 is used to perform the cell high throughput test method provided by the method embodiments described above.

Further, in other embodiments of the present application, the test system further comprises a cell culture subsystem 122 and a cell stimulator subsystem 123. Wherein the cell culture subsystem 121 comprises a cell temperature control incubator and a cell culture vessel for culturing cells to be tested; the cell stimulating subsystem 124 is used to stimulate the cells to be tested to produce changes in the cells to be tested.

In some embodiments, the microscopic imaging subsystem may include a lens (e.g., including an eyepiece and an objective lens), a stage, a clamp, etc., and the test system may further include a control subsystem for controlling movement of the stage in the microscopic imaging subsystem. Comprises the steps of placing the cell culture container to be tested on the objective table between the ocular lens and the objective lens, adjusting the distance between the cell culture container to be tested and the ocular lens and the objective lens, and the like.

FIG. 13 is a schematic diagram of a microscopic imaging subsystem in a cellular high throughput test system according to an embodiment of the present application, where the microscopic imaging subsystem may include: the lens, stage, fixture, bright field test subsystem and dark field test subsystem (i.e., illumination system) are specifically shown in fig. 13:

The bright field test subsystem comprises a visible light source 1, a lens 6, a circulator 5, a second light splitting element 7, a first camera 8, a second camera 9 and a computer 10, and the dark field test subsystem comprises a first excitation light source 2, a second excitation light source 3, a first light splitting element 4, the circulator 5, the lens 6, the second light splitting element 7, the first camera 8, the second camera 9 and the computer 10. It should be noted that, because the lens 6, the circulator 5, the first light splitting element 6, the second light splitting element 7, the first camera 8 and the second camera 9 play similar roles and functions in the field test subsystem and the dark field test subsystem, in some embodiments, the devices in the two subsystems may be shared, for example, one circulator 5 may be disposed at a preset position, so that the field test subsystem and the dark field test subsystem are shared, thereby achieving the effect of saving the devices.

It should be noted that the above-mentioned lens may include an eyepiece and an objective lens, wherein the eyepiece is used for the relevant staff to observe and find the cell area or the cell to be tested; the objective table is used for bearing a cell culture container of cells to be tested; the clamp is used for fixing a culture container of the cells to be tested which are placed on the objective table; the open field test subsystem is used for providing an open field test environment to test the cells to be tested in an open field state and obtain photos, videos and data of the cells to be tested in the open field state; the dark field test subsystem is used for providing a dark field test environment to test the cells to be tested in a dark field state and obtaining photos, videos and data of the cells to be tested in the dark field state; the visible light source 1 is used for emitting visible light to irradiate the cells to be tested on the object stage; the first excitation light source 2 is used for emitting excitation light with a specific wavelength for enabling the fluorescent microbeads to generate fluorescence, and the excitation light is focused on cells in the culture container on the object stage through the light splitting element, the circulator and the lens; the second excitation light source 3 is configured to emit excitation light of a specific wavelength (excitation light of a wavelength different from that of the first excitation light source 2) that causes intracellular ions to generate fluorescence, and the excitation light is focused on cells in the culture vessel by the spectroscopic element, the circulator, and the lens.

The lens also comprises an objective lens which is externally displayed by the microscopic imaging subsystem and is used for receiving transmitted light and excitation light which are formed after visible light irradiates the cells to be tested, fluorescent microbeads and fluorescence generated after intracellular ions are irradiated by the excitation light.

The first light-splitting element is used for combining the excitation light emitted by the first excitation light source and the excitation light emitted by the second excitation light source into new excitation light based on the test requirement, and then transmitting the new excitation light to the circulator.

The second light splitting element is used for receiving light transmitted by the circulator, splitting the transmitted light into a plurality of transmitted lights, splitting the fluorescent light into a plurality of fluorescent lights, and respectively transmitting the fluorescent lights to different cameras, wherein the first camera and the second camera are included.

The camera is used for receiving the transmitted light or fluorescence and converting the light signal into a digital signal and transmitting the digital signal to the computer.

The computer is used for generating photos and videos according to the received digital signals, obtaining corresponding test data according to the received signal information and forming a report; and also used for receiving the test information input by the related staff; the test information includes selection of a test mode, such as including a full-automatic mode, a semi-automatic mode, a manual mode, and the like.

The following describes in detail the process of delivering and converting object beams (including visible light beams, first excitation beams, and second excitation beams) from various light sources in a microscopic imaging subsystem in a few complete implementations, including:

First application: the visible light source emits visible light to perform bright field test.

The method specifically comprises the following steps: the visible light beam emitted by the visible light source 1 can emit visible light to the sample 13 to form third transmission light, the third transmission light is received by the lens 6, the lens 6 enters the circulator 5 through the second channel B, the transmission light entering the circulator 5 enters the third channel C and then irradiates the second light splitting element 7 through the tail end of the third channel C optical fiber, the second light splitting element 7 splits the third transmission light into first transmission light and second transmission light, the first transmission light is captured by the first camera 8, the first transmission light signal is converted into a first digital signal and then transmitted to the computer 10, the second transmission light is captured by the second camera 9, and the second transmission light signal is converted into a second digital signal and then transmitted to the computer 10.

A second application: the first excitation light source and the second excitation light source respectively emit excitation light, and dark field test is carried out:

the method specifically comprises the following steps: the first excitation light source 2 or the second excitation light source 3 emits first excitation light or second excitation light with specific wavelength, the first excitation light enters the first light splitting element 4 through the fourth channel D, the second excitation light enters the first light splitting element 4 through the fifth channel E, the sixth channel F of the first light splitting element 4 is connected with the first channel A of the circulator 5, the first excitation light or the second excitation light enters the circulator 5 through the sixth channel F of the first light splitting element 4 and the first channel A of the circulator 5, the first excitation light or the second excitation light irradiates the lens 6 through the second channel B of the circulator 5, the first excitation light or the second excitation light is focused on the sample 13 through the lens 6 to generate first fluorescence or second fluorescence, the first fluorescence or the second fluorescence enters the circulator 5 through the lens 6 and the second channel B and irradiates the second light splitting element 7 through the tail end of the third channel C, the optical signal of the first fluorescence is captured by the first camera 8, the optical signal of the first fluorescence is converted into a digital signal by the second camera 10, the optical signal of the second camera is converted into a digital signal by the second camera 9, and the optical signal of the second fluorescence is captured by the second camera 9 is converted into a digital signal by the second camera 9.

Third application: the first excitation light source and the second excitation light source emit excitation light simultaneously, and dark field test is performed:

the method specifically comprises the following steps: the first excitation light source 2 and the second excitation light source 3 emit first excitation light and second excitation light at the same time, the first excitation light and the second excitation light are combined into third excitation light in the light splitting element 4 through the fourth channel D and the fifth channel E, the third excitation light enters the circulator 5 through the sixth channel F and the first channel A of the second light splitting element 4 and then irradiates the lens 6 through the second channel B, the third excitation light is focused on the sample 13 through the lens 6, the sample 13 generates third fluorescence, the third fluorescence is transmitted to the circulator 5 through the lens 6 and the second channel B, the third fluorescence is irradiated to the second light splitting element 7 from the tail end of the third channel C of the circulator 5, the second light splitting element 7 splits the third fluorescence into first fluorescence and second fluorescence, the optical signal of the first fluorescence is captured by the first camera 8, the optical signal of the first fluorescence is converted into a digital signal and then transmitted to the computer 10, the optical signal of the second fluorescence is captured by the second camera 9, and the optical signal of the second fluorescence is converted into the digital signal by the second camera 9 and then transmitted to the computer 10.

Based on the same inventive concept, the present application also provides a cell high-throughput testing apparatus, as shown in fig. 14, which includes a processor 141 and a memory 142, where the processor 141 is connected to the memory 142: wherein, the processor 141 is used for calling and executing the program stored in the memory 142; the memory 141 is configured to store a program at least for executing the cell high throughput test method in the above-described method embodiment.

Specific embodiments of the cell high-throughput test apparatus provided in the embodiments of the present application may refer to the embodiments of the cell high-throughput test method in any of the above embodiments, and will not be described herein.

It is to be understood that the same or similar parts in the above embodiments may be referred to each other, and that in some embodiments, the same or similar parts in other embodiments may be referred to.

It should be noted that in the description of the present application, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. Furthermore, in the description of the present application, unless otherwise indicated, the meaning of "plurality" means at least two.

Any process or method descriptions in flow charts or otherwise described herein may be understood as representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps of the process, and further implementations are included within the scope of the preferred embodiment of the present application in which functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those reasonably skilled in the art of the present application.

It is to be understood that portions of the present invention may be implemented in hardware, software, firmware, or a combination thereof. In the above-described embodiments, the various steps or methods may be implemented in software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented in hardware, as in another embodiment, may be implemented using any one or combination of the following techniques, as is well known in the art: discrete logic circuits having logic gates for implementing logic functions on data signals, application specific integrated circuits having suitable combinational logic gates, programmable Gate Arrays (PGAs), field Programmable Gate Arrays (FPGAs), and the like.

Those of ordinary skill in the art will appreciate that all or a portion of the steps carried out in the method of the above-described embodiments may be implemented by a program to instruct related hardware, where the program may be stored in a computer readable storage medium, and where the program, when executed, includes one or a combination of the steps of the method embodiments.

In addition, each functional unit in the embodiments of the present invention may be integrated in one processing module, or each unit may exist alone physically, or two or more units may be integrated in one module. The integrated modules may be implemented in hardware or in software functional modules. The integrated modules may also be stored in a computer readable storage medium if implemented in the form of software functional modules and sold or used as a stand-alone product.

The above-mentioned storage medium may be a read-only memory, a magnetic disk or an optical disk, or the like.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A method of high throughput testing of cells comprising:

setting and identifying a positioning mark on a culture container to be tested;

2. The method of claim 1, wherein the numbering of wells on the test culture vessel based on the positioning markers and positioning test cells within the wells comprises:

3. The method of claim 2, wherein the test procedure comprises:

4. A method of high throughput testing of cells according to claim 3, wherein said predetermined test comprises:

5. The method according to claim 4, wherein the emitting the target beam to the cell to be tested by the illumination system comprises:

6. The method according to claim 1, wherein all the cells to be tested are tested to obtain a test result of a first test cycle, further comprising:

performing global photographing on the hole to obtain a first global image;

performing global photographing on the hole to obtain a second global image;

7. The method of claim 1, further comprising:

receiving a manual instruction;

8. A cellular high throughput testing system, comprising: a microscopic imaging subsystem;

the microscopic imaging subsystem is used to implement the cell high throughput test method of any one of claims 1-7.

9. The cellular high throughput testing system of claim 8, wherein the microscopic imaging subsystem comprises:

10. A cell high throughput testing apparatus comprising a processor and a memory, the processor being coupled to the memory:

the memory for storing the program at least for performing the cell high throughput test method of any one of claims 1-7.