Cell growth state real-time monitoring method and system

Abstract

The invention provides a cell growth state real-time monitoring method which comprises the following steps: S1, putting cells to be detected into a culture device for culturing, and measuring an impedance value of the culture device in a culture process; s2, converting the impedance value into a cell index of the to-be-detected cell in a culture process; and S3, processing the cell index in the culture process to obtain the growth state of the to-be-detected cell. The change of the impedance value of the culture device in the culture process of the to-be-detected cells is detected, the cell indexes of the to-be-detected cells in the culture process are calculated, the cell indexes are drawn into the growth curve, the growth state of the to-be-detected cells can be intuitively reflected through the growth curve, the experiment steps are few, the operation is simple, and the personal error is small.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method and system for real-time monitoring of cell growth status. Background Art

[0002] At present, drug therapy is one of the main ways to fight cancer. In recent years, new drugs for cell therapy have continued to emerge. Detection methods for cytotoxicity, such as the tetrazolium salt method (MTT method), use the activity of enzymes inside mitochondria to convert specific tetrazolium salts, and then detect them by microplate reader; lactate dehydrogenase method (LDH method), detect cytotoxicity by detecting the enzyme activity of lactate dehydrogenase in cell culture supernatant; luciferin luminescence method, cell viability detection, adenylate kinase (AK) exists in the cytoplasm of all eukaryotic and prokaryotic cells, adenylate kinase has the ability to activate adenosine diphosphate (ADP) to generate adenosine triphosphate (ATP). When cells are damaged, the cell membrane is damaged, and adenylate kinase will be released into the culture supernatant. This kit uses luciferase and luciferin to emit light under the action of adenosine triphosphate, and can be quantitatively detected by a chemiluminometer.

[0003] However, the existing conventional methods for testing drug efficacy require the use of a combination of multiple technologies to collect data at multiple time points. The experiments are complex, labor-intensive, time-consuming, and the products produced during the experiment will affect the accuracy of the results. Therefore, it is necessary to improve them. Summary of the invention

[0004] The purpose of the present invention is to provide a method for real-time monitoring of cell growth status to solve the technical problems that the existing conventional methods for detecting drug efficacy need to use a combination of multiple technologies to collect data at multiple time points, the experiments are complex, the workload is large, time-consuming and labor-intensive, and the products of the experimental process will affect the accuracy of the results.

[0005] To achieve the above object, the present invention provides a method for real-time monitoring of cell growth status, the method comprising:

[0006] S1: placing the cells to be tested in a culture device for culture, and measuring the impedance value of the culture device during the culture process;

[0007] S2: converting the impedance value into a cell index of the cells to be tested during the culture process;

[0008] S3: Processing the cell index during the culture process to obtain the growth status of the cells to be tested.

[0009] In a preferred embodiment, S2 is specifically:

[0010] The impedance value is substituted into a calculation formula to obtain the cell index of the cells to be tested during the culture process.

[0011] In a preferred embodiment, the calculation formula is:

[0012]

[0013] Among them, Z norm is the cell index value at time point t after normalization, Z t is the impedance value at time point t, Z1 is the impedance value of the selected normalized point, and Z0 is the impedance value at the initial point.

[0014] In a preferred embodiment, S3 includes:

[0015] S31: drawing a growth curve of the cells to be tested according to the cell index during the culture process;

[0016] S32: Obtaining the growth status of the cells to be tested through the growth curve.

[0017] In a preferred embodiment, the S1 includes:

[0018] S11: preparing the adherent cells of the cells to be tested into a cell suspension to be tested;

[0019] S12: placing the cell suspension to be tested into a culture device and culturing it at a temperature of 37° C. and a CO2 concentration of 5%;

[0020] S13: Measure the impedance value of the culture device during the culture process.

[0021] In a preferred embodiment, the S3 is S3';

[0022] S3': Processing the cell index during the culture process to obtain at least one parameter of the adhesion, spread, death and morphological change of the cells to be tested.

[0023] In a preferred embodiment, the cells to be tested are any one of glioma cells, human brain astrocytoblastoma cells, breast cancer cells, liver cancer cells, ileocecal cancer cells, colorectal cancer cells, ovarian cancer cells, breast ductal cancer cells, human non-small cell lung cancer cells, gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, renal cancer cells, bladder cancer cells, prostate cancer cells, cervical cancer cells and osteosarcoma cells.

[0024] In a preferred embodiment, the culture device is a cell culture plate.

[0025] The present invention also provides a real-time monitoring system for cell growth status, and the real-time monitoring system for cell growth status can use the above-mentioned real-time monitoring method for cell growth status to perform real-time monitoring on the cells to be tested.

[0026] In a preferred embodiment, the cell growth status real-time monitoring system comprises:

[0027] A culture device, wherein the culture device is used to culture the cells to be tested;

[0028] A detection device, the detection device is connected to the culture device by signal, and the detection device is used to measure the impedance value of the culture device during the culture process;

[0029] An output device is signal-connected to the detection device, and is used to convert the impedance value into the growth state of the cell to be detected.

[0030] In a preferred embodiment, the real-time monitoring method of cell growth status or the real-time monitoring system of cell growth status is applied to new drug screening, new drug efficacy evaluation, cytotoxicity analysis, stem cell research, cell signal transduction research, cell adhesion research or cell barrier function analysis.

[0031] The beneficial effects of the present invention are:

[0032] (1) The present invention detects the change in the impedance value of the culture device during the culture process of the cells to be tested, calculates the cell index of the cells to be tested during the culture process, and plots the cell index into a growth curve. The growth curve can intuitively reflect the growth status of the cells to be tested, has few experimental steps, is simple to operate, and has a small human error.

[0033] (2) The method and system for real-time monitoring of cell growth status of the present invention have high sensitivity and accuracy. Once the cells to be tested are added to the culture device, the detection device and the output device can begin to detect parameters such as cell status, cell number, cell proliferation rate, cell size, cell morphology, and cell-matrix adhesion strength in real time.

[0034] (3) The method for real-time monitoring of cell growth status of the present invention does not use labeling. Compared with traditional fluorescent labeling or radioactive labeling, which will affect the physiological state of cells, the present invention can monitor cells in real time without interfering with the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of a real-time monitoring system for cell growth status of the present invention;

[0036] Figure 2 This is a flow chart of the method for real-time monitoring of cell growth status of the present invention;

[0037] Figure 3 This is a flow chart of step S1 in the method for real-time monitoring of cell growth status of the present invention;

[0038] Figure 4 This is a flow chart of step S3 in the method for real-time monitoring of cell growth status of the present invention;

[0039] Figure 5 The killing curve diagram of human ovarian cancer cell SK-OV-3 of Examples 1 to 5 of the present invention;

[0040] Figure 6 The killing curves of ES-2 cells of Comparative Examples 1 to 5 of the present invention are shown;

[0041] Figure 7 The growth curves of ovarian cancer cells Caov-3 of Examples 6 to 9 of the present invention;

[0042] Figure 8 It is a growth curve diagram of breast cancer cell HCC1143 of Examples 10 to 13 of the present invention;

[0043] Fig. 9 It is a growth curve diagram of ileocecal cancer cell HCT-8 of Examples 14 to 17 of the present invention;

[0044] Explanation of reference numerals: culture device 10, detection device 20, output device 30. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the usual meanings understood by people with general skills in the field to which the present invention belongs. "Including" and similar words used in this article mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Unless otherwise specified, the "connection" described in this article can be a direct connection or an indirect connection, that is, connected through an intermediate.

[0046] In this example, if Figure 1As shown, the present invention provides a real-time monitoring system for cell growth status, including a culture device 10, a detection device 20 and an output device 30. Among them, the culture device 10 is used to culture the cells to be tested; the detection device 20 is connected to the culture device 10 by signal, so that the detection device 20 detects the impedance value of the culture device 10 during the culture process; the output device 30 is connected to the detection device 20 by signal, so that the output device 30 receives the impedance value data of the culture device 10 detected by the detection device 20. After obtaining the impedance value data, the output device 30 converts the impedance value into a cell index, and then plots the cell index into a growth curve of the cells to be tested. Through the growth curve, the growth state of the cells to be tested can be intuitively reflected. The real-time monitoring system for cell growth status of the present invention has high sensitivity and accuracy. It only needs to add the cells to be tested to the culture device 10, and the detection device 20 and the output device 30 can start to detect parameters such as cell growth status, cell number, cell proliferation rate, cell size, cell morphology, and cell-matrix adhesion strength in real time.

[0047] In this example, the culture device 10 includes an incubator and a cell culture plate. The incubator is used to provide culture conditions for the cells to be tested; the cell culture plate is placed in the incubator for culturing the cells to be tested; the detection device 20 is connected to the cell culture plate signal so that an electrical circuit is formed between the detection device 20 and the cell culture plate. When detection is required, the cells to be tested are attached to the cell culture plate, and then the cell culture plate is immersed in a conductive solution. After voltage is applied, the electrons in the conductive solution will leave the negative electrode, pass through the cells to be tested, and finally return to the positive electrode to form a complete electrical circuit, while the cells to be tested will hinder the flow of electrons. Under different physiological conditions, the degree to which the cells to be tested hinder the flow of electrons varies. Therefore, the detection device 20 will measure the impedance value data of the cell culture plate.

[0048] In this example, the detection device 20 includes a probe and a microelectronic biosensor chip. The microelectronic biosensor chip forms an electrical circuit with the cell culture plate through the probe, that is, the microelectronic biosensor chip is connected to the cell culture plate signal through the probe, and the microelectronic biosensor chip is connected to the output device 30 signal. When cell monitoring is performed, the cells to be tested hinder the flow of electrons. The microelectronic biosensor chip measures the impedance value data of the cell culture plate, and after storing the data, sends it to the output device 30 for processing. The output device 30 converts the impedance value into a cell index, and then plots the cell index into a growth curve of the cells to be tested. The growth curve can intuitively reflect the growth state of the cells to be tested. In a more preferred embodiment, the microelectronic biosensor chip includes a storage chip and a processing chip. The storage chip and the processing chip form an electrical circuit with the cell culture plate through the probe, and the storage chip and the processing chip are electrically connected. The processing chip is used to measure the impedance value of the cell culture plate, and the storage chip is used to store the measured impedance value data.

[0049] In a preferred example, the microelectronic biosensor chip is a micro-gold electrode embedded in the bottom surface of a cell culture plate. When immersed in a conductive solution (such as a buffer or a standard tissue culture medium), applying a voltage causes the electrons in the conductive solution to leave the negative electrode, and the electrons flow through the cells or culture medium, and then return to the positive electrode to form a complete electrical circuit. The adherent cells present at the junction of the electrode and the solution will hinder the flow of electrons. The strength of the impedance depends on the number, size, shape and adhesion strength of the cells-matrix, and the surface of the micro-gold electrode and the potential used will not affect cell viability and cell behavior. The cells to be tested interact with the micro-gold electrodes of the cell culture plate to produce changes in electrical impedance. The real-time monitoring method for cell growth status of the present invention is used to convert the signals of these changes in electrical impedance into cell indexes. The cell index is a good measure of the state of the cell, such as growth, diffusion, shape change, death, stress state, etc. The cell index is a dimensionless parameter, which is obtained by calculating the measured impedance value through a calculation formula. The change in the cell index can reflect the comprehensive changes in the number, morphology, adhesion and size of the cells to be tested. Due to the different distribution positions of the detection holes on the cell culture plate, there are certain differences in the detection of impedance values. The cell index calculated from the impedance value can remove the background difference of impedance and ensure that the cell index reflects the activity and state of the cells to be tested.

[0050] In a preferred example, the detection device 20 also includes a voltage conversion chip, which is electrically connected to the microelectronic biosensor chip, and the voltage conversion chip is also connected to the cell culture plate signal through a probe. The voltage conversion chip can perform signal filtering, amplification, compensation and correction, and can detect and eliminate noise, drift and nonlinearity in the voltage signal, and improve signal quality and accuracy. It is a chip for converting voltage signals into other forms. It can convert the input voltage signal into different voltage ranges, current ranges or output characteristics. The main function of the chip is to collect, process and convert voltage signals. It usually has multiple input channels and output channels, and can process multiple signals at the same time. These input channels can receive voltage signals from external sensors or other devices and convert them into digital signals or other voltage ranges that meet the requirements.

[0051] In this example, the output device 30 includes a computer and an analog-to-digital conversion chip. Wherein, the analog-to-digital conversion chip is electrically connected to the detection device 20 and the cell culture plate. The analog-to-digital conversion chip is an integrated circuit that converts an analog signal into a digital signal, and converts a continuous analog quantity (such as voltage, current, etc.) into a discrete digital quantity by sampling. The main function is to convert the input analog signal into a corresponding digital signal so that the digital system can process and analyze it. Therefore, the analog-to-digital conversion chip is used to convert the impedance value into a cell index, specifically by bringing the measured impedance value into a calculation formula, calculating the cell index, and then sending the obtained cell index to the computer, and the computer draws the growth curve of the cells to be tested, so as to intuitively reflect the growth state of the cells to be tested.

[0052] In a preferred example, after the output device 30 obtains the impedance value data, the output device 30 converts the impedance value into a cell index, and then plots the cell index into other physiological state curves of the cells to be tested to reflect other parameters of the cells to be tested, such as at least one parameter of adhesion, development, death and morphological changes of the cells to be tested.

[0053] In a further example, after the real-time monitoring system for cell growth status is finished running, the real-time growth data and information of the cells to be tested can be exported in TXT format through a computer. The exported data can be directly copied and processed in an EXECL spreadsheet, or the curve of the current experimental data curve interface can be directly copied, or the current experimental data and information can be directly printed out to a connected printer.

[0054] In this example, if Figure 2 As shown, the present invention provides a method for real-time monitoring of cell growth status, the method comprising:

[0055] S1: placing the cells to be tested in the culture device 10 for culture, and measuring the impedance value of the culture device 10 during the culture process;

[0056] S2: converting the obtained impedance value into a cell index of the cells to be tested during the culture process;

[0057] S3: Processing the cell index obtained during the culture process to obtain the growth status of the cells to be tested.

[0058] The real-time monitoring method of the cell growth state of the present invention detects the change in the impedance value of the culture device 10 during the culture process of the cells to be tested, calculates the cell index of the cells to be tested during the culture process, and plots the cell index into a growth curve. The growth curve can intuitively reflect the growth state of the cells to be tested, with fewer experimental steps, simple operation, and less human error. In addition, the real-time monitoring method of the cell growth state of the present invention does not use labels. Compared with traditional fluorescent labels or radioactive labels that affect the physiological state of cells, the present invention can monitor cells in real time without interfering with the cells.

[0059] In a preferred example, Figure 3 As shown, S1 includes:

[0060] S11: preparing the adherent cells of the cells to be tested into a cell suspension to be tested;

[0061] S12: placing the cell suspension to be tested into the culture device 10, and culturing at a temperature of 37° C. and a CO 2 concentration of 5%;

[0062] S13: Measure the impedance value of the culture device 10 during the culture process.

[0063] In a preferred example, S2 specifically includes: substituting the obtained impedance value into a calculation formula to obtain a cell index of the cells to be tested during the culture process.

[0064] In a preferred example, the calculation formula is:

[0065]

[0066] Among them, Z norm is the cell index value at time point t after normalization, Z t is the impedance value at time point t, Z1 is the impedance value of the selected normalized point, and Z0 is the impedance value of the initial point.

[0067] The impedance value is put into the calculation formula to calculate the cell index of the cells to be tested.

[0068] In a preferred example, Figure 4 As shown, S3 includes:

[0069] S31: Draw the growth curve of the cells to be tested according to the cell index during the culture process;

[0070] S32: Obtain the growth status of the cells to be tested through the growth curve.

[0071] In another preferred example, the S3 is S3'; S3' is: processing the cell index in the culture process to obtain at least one parameter of adhesion, expansion, death and morphological change of the cells to be tested. That is to say, the real-time monitoring method of cell growth status of the present invention is not limited to obtaining the growth status of the cells to be tested. By processing the cell index, other state parameters such as adhesion, expansion, death and morphological change of the cells to be tested can also be obtained.

[0072] In a preferred example, the cells to be tested are any one of glioma cells, human brain astrocytoblastoma cells, breast cancer cells, liver cancer cells, ileocecal cancer cells, colorectal cancer cells, ovarian cancer cells, breast ductal cancer cells, human non-small cell lung cancer cells, gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, renal cancer cells, bladder cancer cells, prostate cancer cells, cervical cancer cells and osteosarcoma cells.

[0073] In this example, the real-time monitoring system and method for cell growth status of the present invention can be applied to new drug screening, new drug efficacy evaluation, cytotoxicity analysis, stem cell research, cell signal transduction research, cell adhesion research or cell barrier function analysis.

[0074] The specific principle of the real-time monitoring method of cell growth status of the present invention is: a conductive solution is added to the cell culture plate, and after voltage is applied, electrons leave the negative electrode and return to the positive electrode after passing through the culture medium to form a loop. When the cells to be tested in the cell culture plate grow by adhering to the wall, if the number of cells to be tested increases, the obstacle to the flow of electrons increases, so the impedance value increases and the cell index increases; if the number of cells to be tested decreases, the obstacle to the flow of electrons decreases, so the impedance value decreases and the cell index decreases. Therefore, when the cells to be tested are amplified, the number of cells increases, and the cell index increases with the increase in the number of cells. When the cells to be tested die, the number of cells decreases and the cell index decreases. Different cell sizes are different, so the impedance values ​​of different cells are also different, and the cell indexes obtained are also different. Taking human ovarian cancer cells SK-OV-3 and human ovarian cancer cells ES-2 as examples, the cell size of human ovarian cancer cells SK-OV-3 is larger than that of human ovarian cancer cells ES-2. Therefore, when monitoring cell growth, the cell index of human ovarian cancer cells SK-OV-3 with the same number of cells at the same time is much higher than that of ES-2 cells. The same is true for other parameters of the cells to be tested.

[0075] In addition, the different curves obtained by monitoring represent the unique adhesion, extension or proliferation states of different cells to be tested, or the changes in the unique response states of a certain cell to be tested under different stimulation conditions. Different time periods of the same curve represent the special states of the cells to be tested in different time periods. For example, in the early stage of the experiment after adding the cells to be tested, it is a special curve when the cells are attached to the wall and extended, while in the later stage of the experiment, it is a special curve when the cells to be tested are in the logarithmic growth phase. The curve after adding a certain stimulus is a special curve that reflects the stress changes of the cells to be tested to the stimulus.

[0076] Examples 1 to 5 and comparative examples 1 to 5 are real-time cell killing detection experiments, which are as follows:

[0077] Example 1

[0078] The cells to be tested are human ovarian cancer cells SK-OV-3, which are MSLN target-positive cells.

[0079] The experimental steps are as follows:

[0080] S1:

[0081] S11: Human ovarian cancer cells SK-OV-3 were digested and prepared into a cell suspension. The cells were counted after pipetting and mixing. After counting, the cell suspension was diluted to 2×10 4 cells / mL concentration to obtain a diluted cell suspension, and then place the diluted cell suspension under low temperature conditions for later use;

[0082] S12: Add 100 μL of the diluted cell suspension to a 16-well cell culture plate, place the 16-well cell culture plate in an incubator, and culture human ovarian cancer cells SK-OV-3 for 24 h at a temperature of 37°C and a CO2 concentration of 5%;

[0083] S13: the detection device 20 measures the impedance value of the culture device 10 during the 24-hour culture process;

[0084] S2: Substitute the obtained impedance value into the calculation formula to obtain the cell index of human ovarian cancer cell SK-OV-3 during 24h culture;

[0085] S3:

[0086] S31: operate a computer to draw a growth curve of human ovarian cancer cells SK-OV-3 during the 24-hour culture process according to the cell index of human ovarian cancer cells SK-OV-3 during the 24-hour culture process;

[0087] S32: The growth status of human ovarian cancer cells SK-OV-3 during 24h culture was obtained through the growth curve of human ovarian cancer cells SK-OV-3;

[0088] S4: Observe the growth curve of human ovarian cancer cells SK-OV-3. When the human ovarian cancer cells SK-OV-3 are in the logarithmic growth phase, add 100 μL of effector T cells (CAR-T) to the 16-well cell culture plate and continue to monitor the cell state (repeat S13 to S32) to obtain the growth curve of real-time cell killing detection of human ovarian cancer cells SK-OV-3. The real-time cell killing detection result of human ovarian cancer cells SK-OV-3 is obtained by the obtained growth curve of real-time cell killing detection of human ovarian cancer cells SK-OV-3.

[0089] Example 2

[0090] The ratio of effector T cells (CAR-T) to diluted cell suspension is 2.5 to 1, that is, 250 μL effector T cells (CAR-T) and 100 μL diluted cell suspension.

[0091] Other experimental steps and parameters are the same as in Example 1.

[0092] Example 3

[0093] The effector T cells (CAR-T) were replaced with uninfected T cells (NC-T), and the ratio of uninfected T cells (NC-T) to the diluted cell suspension was 1:1, that is, 100 μL of uninfected T cells (NC-T) and 100 μL of diluted cell suspension.

[0094] Other experimental steps and parameters are the same as in Example 1.

[0095] Example 4

[0096] The effector T cells (CAR-T) were replaced with uninfected T cells (NC-T), and the ratio of uninfected T cells (NC-T) to the diluted cell suspension was 2.5 to 1, that is, 250 μL of uninfected T cells (NC-T) and 100 μL of diluted cell suspension.

[0097] Other experimental steps and parameters are the same as in Example 1.

[0098] Example 5

[0099] No T cells were added to S4, only human ovarian cancer cells SK-OV-3 were added.

[0100] Other experimental steps and parameters are the same as in Example 1.

[0101] Comparative Examples 1 to 5

[0102] The cells to be tested in Comparative Examples 1 to 5 all used MSLN target negative cells ES-2 as controls;

[0103] The other experimental steps and parameters of Comparative Example 1 are the same as those of Example 1;

[0104] The other experimental steps and parameters of Comparative Example 2 are the same as those of Example 2;

[0105] The other experimental steps and parameters of Comparative Example 3 are the same as those of Example 3;

[0106] The other experimental steps and parameters of Comparative Example 4 are the same as those of Example 4;

[0107] The other experimental steps and parameters of Comparative Example 5 are the same as those of Example 5.

[0108] Examples 6 to 17 are real-time cell growth detection experiments, which are as follows:

[0109] Example 6

[0110] The cells to be tested are human ovarian cancer cells Caov-3.

[0111] The specific experimental steps are as follows:

[0112] S1:

[0113] S11: Human ovarian cancer cells Caov-3 were digested and prepared into a cell suspension. The cells were counted after pipetting and mixing. After counting, the cell suspension was diluted to 0.5×10 4 cells / mL concentration to obtain a diluted cell suspension, and then place the diluted cell suspension under low temperature conditions for later use;

[0114] S12: Add 100 μL of the diluted cell suspension to a 16-well cell culture plate, place the 16-well cell culture plate in an incubator, and culture human ovarian cancer cells Caov-3 at 37° C. and 5% CO2 (for more than 40 h);

[0115] S13: the detection device 20 measures the impedance value of the culture device 10 during the culture process;

[0116] S2: Substitute the obtained impedance value into the calculation formula to obtain the cell index of human ovarian cancer cell Caov-3 during the culture process;

[0117] S3:

[0118] S31: operating a computer to draw a growth curve of the human ovarian cancer cell Caov-3 during the culture process according to the cell index of the human ovarian cancer cell Caov-3 during the culture process;

[0119] S32: Obtaining the growth status of human ovarian cancer cell Caov-3 during the culture process through the growth curve of human ovarian cancer cell Caov-3.

[0120] Embodiments 7 to 9

[0121] In Example 7, the cell suspension was diluted to 1×10 4 cells / mL concentration;

[0122] In Example 8, the cell suspension was diluted to 2×10 4 cells / mL concentration;

[0123] In Example 9, the cell suspension was diluted to 4×10 4 cells / mL concentration;

[0124] The other experimental steps and parameters in Examples 7 to 9 are the same as those in Example 6.

[0125] Example 10

[0126] The cells to be tested were human breast cancer cells HCC1143;

[0127] Other experimental steps and parameters are the same as in Example 6.

[0128] Examples 11 to 13

[0129] In Example 11, the cell suspension was diluted to 1×10 4 cells / mL concentration;

[0130] In Example 12, the cell suspension was diluted to 2×10 4 cells / mL concentration;

[0131] In Example 13, the cell suspension was diluted to 4×10 4 cells / mL concentration;

[0132] The other experimental steps and parameters in Examples 11 to 13 are the same as those in Example 10.

[0133] Embodiment 14

[0134] The cells to be tested are human breast cancer cells, human ileocecal cancer cells HCT-8;

[0135] Other experimental steps and parameters are the same as in Example 6.

[0136] Embodiments 15 to 17

[0137] In Example 15, the cell suspension was diluted to 1×10 4 cells / mL concentration;

[0138] In Example 16, the cell suspension was diluted to 2×10 4 cells / mL concentration;

[0139] In Example 17, the cell suspension was diluted to 4×10 4 cells / mL concentration;

[0140] The other experimental steps and parameters in Examples 15 to 17 are the same as those in Example 14.

[0141] Experimental data results:

[0142] The experimental results of Examples 1 to 5 are as follows Figure 5 As shown;

[0143] The experimental results of Comparative Examples 1 to 5 are as follows Figure 6 As shown;

[0144] The experimental results of Examples 6 to 9 are as follows Figure 7 As shown;

[0145] The experimental results of Examples 10 to 13 are as follows Figure 8 As shown;

[0146] The experimental results of Examples 14 to 17 are as follows Fig. 9 shown.

[0147] Experimental conclusion:

[0148] Figure 5 and Figure 6 The “medium” in the table means that there are no effector cells but only tumor target cells. Figure 5 and Figure 6 By comparison, it can be seen that CAR-T cells specifically kill human ovarian cancer cells SK-OV-3 (tumor cells with positive MSLN target), but have no killing effect on ES-2 tumor cells that do not express the MSLN target. This reflects the high accuracy of the real-time monitoring method and system for cell growth status of the present invention. Moreover, from Figure 5 It can be seen that after adding CAR-T cells at 24h, the cell index of human ovarian cancer cell SK-OV-3 dropped sharply after a short proliferation, which shows that the number of human ovarian cancer cell SK-OV-3 cells is decreasing sharply, and the degree of reduction in the number of human ovarian cancer cell SK-OV-3 cells in Example 2 (the ratio of CAR-T to SK-OV-3 is 2.5 to 1) is significantly greater than that in Example 1 (the ratio of CAR-T to SK-OV-3 is 1 to 1). This further illustrates that the real-time monitoring method and system for cell growth status of the present invention have high sensitivity and high accuracy.

[0149] Depend on Figure 7 , Figure 8 as well as Fig. 9It can be seen that the higher the concentration of the cell suspension of the cells to be tested, the more the cell exponential growth increases over time, and the growth curve conforms to the law of cell proliferation, that is, the slope of the growth curve changes from large to small, and the growth curves of the cells to be tested at different gradients are clear. It can be seen that the real-time monitoring method and system for cell growth status of the present invention can realize real-time monitoring of the growth status of cells with high accuracy.

[0150] The above is only a specific implementation of the embodiment of the present application, but the protection scope of the embodiment of the present application is not limited thereto, and any changes or replacements within the technical scope disclosed in the embodiment of the present application should be included in the protection scope of the embodiment of the present application. Therefore, the protection scope of the embodiment of the present application should be based on the protection scope of the claims.

Claims

1. A method for real-time monitoring of cell growth status, characterized in that: The method comprises: S1: placing the cells to be tested in a culture device for culture, and measuring the impedance value of the culture device during the culture process; S2: converting the impedance value into a cell index of the cells to be tested during the culture process; S3: Processing the cell index during the culture process to obtain the growth status of the cells to be tested.

2. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The S2 is specifically: The impedance value is substituted into a calculation formula to obtain the cell index of the cells to be tested during the culture process.

3. The method for real-time monitoring of cell growth status according to claim 2, characterized in that: The calculation formula is: Among them, Z norm is the cell index at time point t after normalization, Z t is the impedance value at time point t, Z1 is the impedance value of the selected normalized point, and Z0 is the impedance value at the initial point.

4. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The S3 includes: S31: drawing a growth curve of the cells to be tested according to the cell index during the culture process; S32: Obtaining the growth status of the cells to be tested through the growth curve.

5. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The S1 includes: S11: preparing the adherent cells of the cells to be tested into a cell suspension to be tested; S12: placing the cell suspension to be tested into the culture device, and culturing at a temperature of 37° C. and a CO 2 concentration of 5%; S13: Measure the impedance value of the culture device during the culture process.

6. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The S3 is S3'; S3': Processing the cell index during the culture process to obtain at least one parameter of the adhesion, spread, death and morphological change of the cells to be tested.

7. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The cells to be tested are any one of glioma cells, human brain astrocytoblastoma cells, breast cancer cells, liver cancer cells, ileocecal cancer cells, colorectal cancer cells, ovarian cancer cells, breast ductal cancer cells, human non-small cell lung cancer cells, gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, renal cancer cells, bladder cancer cells, prostate cancer cells, cervical cancer cells and osteosarcoma cells.

8. The method for real-time monitoring of cell growth status according to claim 1, characterized in that: The culture device is a cell culture plate.

9. A real-time monitoring system for cell growth status, characterized in that: The cell growth status real-time monitoring system can use the cell growth status real-time monitoring method according to any one of claims 1 to 8 to perform real-time monitoring on the cells to be tested.

10. The real-time monitoring system for cell growth status according to claim 9, characterized in that: The cell growth status real-time monitoring system comprises: A culture device, wherein the culture device is used to culture the cells to be tested; A detection device, the detection device is connected to the culture device by signal, and the detection device is used to measure the impedance value of the culture device during the culture process; An output device is signal-connected to the detection device, and is used to convert the impedance value into the growth state of the cell to be detected.

11. Use of the method for real-time monitoring of cell growth status according to any one of claims 1 to 8 or the system for real-time monitoring of cell growth status according to any one of claims 9 to 10, characterized in that: The real-time monitoring method of cell growth status or the real-time monitoring system of cell growth status is applied to new drug screening, new drug efficacy evaluation, cytotoxicity analysis, stem cell research, cell signal transduction research, cell adhesion research or cell barrier function analysis.

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