Cell migration ability testing and evaluating method based on cell cells
By using serum-free DMEM culture medium and image processing technology in the cell chamber, the problem of low detection accuracy in the existing technology is solved, efficient and reliable cell migration ability detection is achieved, and the accuracy and repeatability of the test results are ensured.
Patent Information
- Application Number
- CN202510801715.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-16
AI Technical Summary
Existing methods for detecting cell migration ability have the following disadvantages: low detection accuracy, inability to effectively compare the migration effects of different brands of chambers, random migration directions and large calculation deviations, and difficulty in obtaining an accurate number of migrating cells.
A cell chamber-based testing method was used. Cells were pretreated with serum-free DMEM medium, and the culture environment was set at 37°C and 5% CO2. The culture medium volume ratio and staining time were precisely controlled. Combined with image processing technology, the migration rate was calculated and the chamber membrane curvature was corrected to establish a standardized process system.
The accuracy and repeatability of the test are improved, errors are reduced, the comparison of migration effects between chambers of different brands is ensured, and efficient and reliable cell migration ability detection is achieved.
Smart Images
Figure CN120648772A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a biological cell testing technology, and more particularly to a cell chamber-based cell migration ability testing and evaluation method. Background Art
[0002] Cell migration refers to the ability of cells to move in a certain direction under the guidance of chemical or physical signals. It is the core mechanism of many physiological and pathological processes. Research on cell migration ability is of great significance for understanding disease mechanisms, drug screening and regenerative medicine. At the same time, cell migration is closely related to the occurrence and development of tumors. Cell migration is affected by a variety of external environmental factors and internal factors. Research on cell migration can improve the treatment exploration of diseases closely related to cell migration, such as tumors.
[0003] The purpose of cell migration ability testing is to study the factors affecting cell migration, verify the regulatory mechanism and explore the molecular mechanism. The detection of cell migration ability has broad application prospects in the fields of drug development, cancer treatment and tissue engineering. Therefore, the effect of its detection has an important impact on subsequent research.
[0004] Currently, commonly used methods for detecting cell migration ability include scratch assays and Transwell assays. The scratch assay method is to artificially create a blank area, namely a "scratch", on a fused monolayer of cells. Cells at the edge of the scratch will gradually enter the blank area to allow the "scratch" to heal. By capturing images at the beginning and regular intervals of the cell migration process, measuring the scratch spacing at different time points and calculating the difference, the cell migration ability can be easily obtained. Although the above method can conveniently calculate the cell migration ability, it can only detect the invasion and metastasis ability of tumor cells growing on the wall, and has high requirements for the scratching force. At the same time, during the cell migration process, its migration direction is random, which makes the calculation of its migration ability have certain Due to the deviation of the cell migration assay, the commonly used method for detecting cell migration is the Transwell assay, which uses a microporous filter membrane (pore size 8.0 μm) in the cell chamber to separate the upper and lower chambers, and uses chemokines (such as FBS) to drive cell migration. Cells in a low-nutrient medium are inoculated in the upper chamber, and high-nutrient medium or stimulating factors are added to the lower chamber. If the filter membrane is covered with Matrigel matrix (simulating basement membrane), the cell invasion ability can be detected (the matrix gel needs to be enzymatically hydrolyzed before passing through the membrane). In the absence of Matrigel, the migration ability is detected, and the cells in the lower chamber are counted by staining to quantify the migration rate. However, the accuracy of membrane experiments in different brands of chambers varies, and it is difficult to obtain the number of cells after migration is completed. Therefore, it is necessary to optimize the detection method of cell migration ability. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a cell migration ability testing and evaluation method based on cell chambers that can compare the migration effects of chambers of different brands, has high detection accuracy, and can efficiently and reliably detect cell migration effects.
[0006] To achieve the above-mentioned object, the present invention provides the following technical solution: a cell chamber-based cell migration ability test and evaluation method, comprising the following steps: S1, pre-treating the cells to be evaluated: resuscitating the cells to be evaluated, passage the cells to a density of 85%-90%, and resuspending them in serum-free DMEM medium after trypsin digestion to obtain a serum-free DMEM medium cell suspension;
[0007] S2. Cell inoculation: Add the serum-free DMEM cell suspension obtained in step S1 into the upper chamber of the cell chamber, add the culture medium containing FBS into the lower chamber of the cell chamber, and culture for 24 hours;
[0008] S3. Migration assay: After the culture is completed, the culture medium in the upper and lower chambers is removed, the cells in the upper chamber are wiped off, and the cells on the back of the cell chamber membrane are stained to calculate the migration percentage.
[0009] The present invention is further configured as follows: the subculture method in step S1 comprises the following steps: S11, adding pre-warmed PBS to the culture medium for rinsing to remove residual serum in the culture medium;
[0010] S12, adding trypsin to the culture medium and covering the cell layer in the culture medium, and incubating at 37°C for T time;
[0011] S13, after the T time period, observing the cell status in the culture medium. If the intercellular gap increases, the cultivation is determined to be complete, and the process jumps to S14 to terminate. Otherwise, the cultivation is determined to be incomplete, and the cultivation is continued.
[0012] S14. Add culture medium containing serum to neutralize the activity of trypsin, and repeatedly pipette until the cells are completely detached;
[0013] S15. Centrifuge the cultured cells for a time period of T. After the T time period, add the cells at the bottom into fresh culture medium for resuspending.
[0014] The present invention is further configured as follows: the culture environment in step S2 is 37° C. and 5% CO 2 .
[0015] The present invention is further configured as follows: Step S3 further includes a method for calculating cell migration rate, comprising the following steps: S31, placing the stained chamber on a glass slide, covering the chamber with neutral gum and a cover glass, and photographing the cell image on the back of the cell chamber membrane using an optical microscope;
[0016] S32, converting the captured image into a grayscale image, and adjusting the image threshold to distinguish cells from the background;
[0017] S33, counting the number of migrating cells, recording the cell data in the current field of view, and calculating the average value of cell migration in the current field of view;
[0018] S34. Calculate the cell migration rate based on the average value of cell migration.
[0019] Preferably, the step S33 further includes deviation value detection, including the following steps: S331, recording the number of cells within each field of view, and recording the cell number value within each field of view respectively, and arranging the cell number values in descending order as X1, X2, X3, X4, and X5;
[0020] S332. Compare the cell number values. If X1>2X2, it is determined that the cells in the field of view corresponding to X1 are accumulated. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to X1 is discarded and a new microscopic image is captured. Conversely, if X5<0.5X4, it is determined that the complete chamber membrane is not captured in the field of view corresponding to X5 or the cells in the field of view corresponding to X5 fall off during the staining process. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to S5 is discarded and a new microscopic image is captured.
[0021] Preferably, in step S33, the calculation formula for the average value of cell migration is as follows:
[0022]
[0023] Where A is the average value of cell migration, X is the number of cells in each field of view, and Z is the number of recorded fields of view.
[0024] Preferably, in step S34, the calculation formula of mobility is as follows:
[0025]
[0026] Among them, Q is the migration rate of the current cells, B is the number of migrated cells, and J is the initial number of cells.
[0027] The present invention is further configured as follows: in step S2, the amount of cell suspension in the upper chamber of the cell chamber is 150 μL, and the volume of the cell suspension is 7.5×10 4 / well, and the volume of culture medium in the lower chamber of the cell chamber was 800 μL.
[0028] By adopting the above technical solution, the beneficial effects are as follows: 1. By comparing multiple groups of cell chambers, the present application can conveniently and quickly evaluate the effects of cell chambers of different brands on cell migration experiments, thereby improving the accuracy of experimental results. At the same time, the present application establishes a complete standardized process system, so that a full-process closed-loop control system for cell pretreatment, acquisition of migration parameters, and calculation of migration amount is formed. Specifically, by combining serum-free culture medium with cells of fixed density, the interference of serum on migration movement can be clearly determined during cell migration, thereby ensuring the consistency of the basal state of cell chambers of different brands. Moreover, during the culture process, different volumes of culture medium are respectively provided in the upper / lower chambers, and the precise volume ratio of the culture medium in the upper and lower chambers can be used to stabilize the chemical chemotactic gradient during cell migration, and the cell seeding density (5×10 5 The system improves the repeatability of the experiment by adjusting various parameters such as FBS chemoattractant concentration (10%), staining time (10-15 minutes), and centrifugation parameters (1500 rpm / 3 minutes). Furthermore, after cell migration is completed, the cells are stained to facilitate the acquisition of cell number. By intercepting multiple fields of view to calculate the average value of migrating cells, errors caused by uneven migrating cells in some areas are avoided. After the interception is completed, the precise cell number is obtained through intelligent image processing, avoiding the limitations and errors of manual measurement.
[0029] 2. Furthermore, during the cell migration experiment, the digestion time in step S1 can be dynamically adjusted by real-time monitoring of the gaps between cells, effectively preventing the digestion time from being too long and causing cell damage. Furthermore, during the preparation of the cell suspension, by adding trypsin to the culture medium and covering the cell layer in the culture medium, the above operation can ensure that the obtained cell suspension has high activity and monodispersity, laying the foundation for subsequent cell inoculation.
[0030] 3. At the same time, the present application forms a precise parameter model for cell migration experiments by quantifying culture parameters. For example, in step S2, the culture environment of the cells is set to a 37°C environment with 5% CO2, which reduces the impact of environmental factors on the culture medium. Specifically, the 5% CO2 environment and the DMEM culture medium containing sodium bicarbonate constitute a carbonate buffer system, which can stabilize the pH of the culture medium within the physiological range of 7.2-7.4 through dynamic equilibrium. In addition, in step S1, the cells are resuspended in serum-free DMEM culture medium, which lacks the additional buffering capacity of serum proteins. The control of 5% CO2 can avoid pH fluctuations, ensure the stability of cell membrane potential and the normal operation of ion channels, and the constant temperature control of 37°C can avoid secondary damage to cell membrane proteins caused by temperature fluctuations or residual trypsin, and can maintain the overall migration movement of the cells. The above parameter settings not only ensure the repeatability of the experiment, but also ensure the high precision of the cell migration experiment.
[0031] 4. Moreover, after the cell migration is completed, the amount of cell migration is analyzed by machine vision technology. Specifically, by covering the chamber with neutral gum and coverslip, the chamber membrane can be corrected to eliminate the imaging distortion caused by the curvature of the chamber membrane. At the same time, the dynamic threshold segmentation algorithm is implemented by adjusting the image threshold. By converting the intercepted image into a grayscale image, the migrating cells can be easily obtained. The problem of uneven cell distribution at some chamber membranes is avoided by the multi-data sampling method, which reduces the detection error and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is a flow chart of a cell migration method according to an embodiment of a cell chamber-based cell migration ability testing and evaluation method of the present invention;
[0033] Figure 2 This is a flow chart of a passage method according to an embodiment of a cell chamber-based cell migration ability test and evaluation method of the present invention;
[0034] Figure 3 This is a flow chart of a method for calculating migration rate according to an embodiment of a cell chamber-based method for testing and evaluating cell migration ability of the present invention;
[0035] Figure 4 This is a flow chart of cell number deviation value detection according to an embodiment of a cell migration ability test and evaluation method based on a cell chamber of the present invention;
[0036] Figure 5 This is an experimental rendering of the migration effects of multiple groups of cell chambers according to an embodiment of a cell chamber-based cell migration ability testing and evaluation method of the present invention; DETAILED DESCRIPTION
[0037] Reference Figures 1 to 5The present invention further illustrates an embodiment of a cell migration ability testing and evaluation method based on a cell chamber.
[0038] For ease of explanation, spatial relative terms such as "upper", "lower", "left", and "right" are used in the embodiments to illustrate the relationship between one element or feature shown in the figures and another element or feature. It should be understood that, in addition to the orientation shown in the figures, spatial terms are intended to include different orientations of the device in use or operation. For example, if the device in the figure is inverted, the element described as being "under" other elements or features will be positioned "above" other elements or features. Therefore, the exemplary term "under" can include both upper and lower orientations. The device can be positioned in other ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used here can be interpreted accordingly.
[0039] Moreover, relational terms such as “first” and “second” are merely used to distinguish one component from another having the same name, but do not necessarily require or imply any actual relationship or order between these components.
[0040] A cell migration ability test and evaluation method based on a cell chamber comprises the following steps:
[0041] S1. Pretreatment of cells to be evaluated: resuscitate the cells to be evaluated, passage the cells to a density of 85%-90%, and resuspend them in serum-free DMEM medium after trypsin digestion to obtain a serum-free DMEM medium cell suspension;
[0042] S2. Cell inoculation: Add the serum-free DMEM cell suspension obtained in step S1 into the upper chamber of the cell chamber, add the culture medium containing FBS into the lower chamber of the cell chamber, and culture for 24 hours;
[0043] S3. Migration assay: After the culture is completed, the culture medium in the upper and lower chambers is removed, the cells in the upper chamber are wiped off, and the cells on the back of the cell chamber membrane are stained to calculate the migration percentage.
[0044] The passaging method in step S1 comprises the following steps: S11, adding pre-warmed PBS to the culture medium for rinsing to remove residual serum in the culture medium;
[0045] S12, adding trypsin to the culture medium and covering the cell layer in the culture medium, and incubating at 37°C for T time;
[0046] S13, after the T time period, observing the cell status in the culture medium. If the intercellular gap increases, the cultivation is determined to be complete, and the process jumps to S14 to terminate. Otherwise, the cultivation is determined to be incomplete, and the cultivation is continued.
[0047] S14. Add culture medium containing serum to neutralize the activity of trypsin, and repeatedly pipette until the cells are completely detached;
[0048] S15. Centrifuge the cultured cells for a time period of T. After the T time period, add the cells at the bottom into fresh culture medium for resuspending.
[0049] The culture environment in step S2 is 37° C. and 5% CO 2 .
[0050] The present invention is further configured as follows: Step S3 further includes a method for calculating cell migration rate, comprising the following steps: S31, placing the stained chamber on a glass slide, covering the chamber with neutral gum and a cover glass, and photographing the cell image on the back of the cell chamber membrane using an optical microscope;
[0051] S32, converting the captured image into a grayscale image, and adjusting the image threshold to distinguish cells from the background;
[0052] S33, counting the number of migrating cells, recording the cell data in the current field of view, and calculating the average value of cell migration in the current field of view;
[0053] S34. Calculate the cell migration rate based on the average value of cell migration.
[0054] Preferably, the step S33 further includes deviation value detection, including the following steps: S331, recording the number of cells within each field of view, and recording the cell number value within each field of view respectively, and arranging the cell number values in descending order as X1, X2, X3, X4, and X5;
[0055] S332. Compare the cell number values. If X1>2X2, it is determined that the cells in the field of view corresponding to X1 are accumulated. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to X1 is discarded and a new microscopic image is captured. Conversely, if X5<0.5X4, it is determined that the complete chamber membrane is not captured in the field of view corresponding to X5 or the cells in the field of view corresponding to X5 fall off during the staining process. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to S5 is discarded and a new microscopic image is captured.
[0056] Preferably, in step S33, the calculation formula for the average value of cell migration is as follows:
[0057]
[0058] Where A is the average value of cell migration, X is the number of cells in each field of view, and Z is the number of recorded fields of view.
[0059] Preferably, in step S34, the calculation formula of mobility is as follows:
[0060]
[0061] Among them, Q is the migration rate of the current cells, B is the number of migrated cells, and J is the initial number of cells.
[0062] The present invention is further configured as follows: in step S2, the amount of cell suspension in the upper chamber of the cell chamber is 150 μL, and the volume of the cell suspension is 7.5×10 4 / well, and the volume of culture medium in the lower chamber of the cell chamber was 800 μL.
[0063] By comparing multiple groups of cell chambers, the present application can quickly and easily evaluate the effects of different brands of cell chambers on cell migration experiments, thereby improving the accuracy of experimental results. At the same time, the present application establishes a complete standardized process system to form a full-process closed-loop control system for cell pretreatment, acquisition of migration parameters, and calculation of migration amount. Specifically, by combining serum-free culture medium with cells of a fixed density, the interference of serum on migration movement can be clearly determined during cell migration, ensuring the consistency of the basal state of cell chambers of different brands. Moreover, during the culture process, by setting different volumes of culture medium in the upper / lower chambers respectively, the chemical chemotactic gradient during cell migration can be stabilized by accurately matching the volume ratio of the culture medium in the upper and lower chambers, and the cell seeding density (5×10 5 The system improves the repeatability of the experiment by adjusting various parameters such as FBS chemoattractant concentration (10%), staining time (10-15 minutes), and centrifugation parameters (1500 rpm / 3 minutes). Furthermore, after cell migration is completed, the cells are stained to facilitate the acquisition of cell number. By intercepting multiple fields of view to calculate the average value of migrating cells, errors caused by uneven migrating cells in some areas are avoided. After the interception is completed, the precise cell number is obtained through intelligent image processing, avoiding the limitations and errors of manual measurement.
[0064] Furthermore, during the cell migration experiment, the digestion time in step S1 can be dynamically adjusted by real-time monitoring of the gaps between cells, effectively preventing the digestion time from being too long and causing cell damage. Furthermore, during the preparation of the cell suspension, by adding trypsin to the culture medium and covering the cell layer in the culture medium, the above operation can ensure that the obtained cell suspension has high activity and monodispersity, laying the foundation for subsequent cell inoculation.
[0065] At the same time, the present application forms a precise cell migration experiment parameter model by quantifying culture parameters. For example, in step S2, the cell culture environment is set to a 37°C environment with 5% CO2, which reduces the impact of environmental factors on the culture medium. Specifically, the 5% CO2 environment and the DMEM culture medium containing sodium bicarbonate constitute a carbonate buffer system, which can stabilize the pH of the culture medium within the physiological range of 7.2-7.4 through dynamic balance. In addition, in step S1, the cells are resuspended in serum-free DMEM culture medium, which lacks the additional buffering capacity of serum proteins. The control of 5% CO2 can avoid pH fluctuations, ensure the stability of cell membrane potential and the normal operation of ion channels, and the constant temperature control of 37°C can avoid secondary damage to cell membrane proteins caused by temperature fluctuations or residual trypsin, and can maintain the overall migration movement of cells. The above parameter settings not only ensure the repeatability of the experiment, but also ensure the high precision of the cell migration experiment.
[0066] Moreover, after the cell migration is completed, the amount of cell migration is analyzed by machine vision technology. Specifically, by covering the chamber with neutral gum and coverslip, the chamber membrane can be corrected to eliminate the imaging distortion caused by the curvature of the chamber membrane. At the same time, the dynamic threshold segmentation algorithm is realized by adjusting the image threshold. By converting the intercepted image into a grayscale image, the migrating cells can be easily obtained. The problem of uneven cell distribution at some chamber membranes is avoided by the multi-data sampling method, which reduces the detection error and improves the detection efficiency. Figure 5 As shown, in the experiment, the migration effects of multiple groups of cells in the cell chamber were extracted for experiment and the sample images were intercepted. After processing the images, the distribution of cells can be clearly obtained. For example, the darker part in the image is the cell, which can be clearly distinguished from the light background after grayscale adjustment. In addition, the cells in the above-collected pattern are evenly distributed without obvious accumulation, which can facilitate the subsequent evaluation and calculation of migration ability and has high detection accuracy.
[0067] It is worth noting that when the above three cell types were tested, the parameters during the migration process were kept consistent with those mentioned in the specification. Figure 5 In the figure, the image on the right is the blank control group, which is mainly used for comparison with the embodiment to obtain cell migration data. At the same time, the staff can select 3 groups of different cell chambers and test the same cells while ensuring that the environment and experimental parameters remain unchanged. The control group is selected as a cell chamber of a different brand from the cell chamber to be tested. The above scheme can not only detect the migration rate of different cell chambers for the same cells, but also detect the migration rate of different cells in the same cell chamber, so that the staff can dynamically select cell chambers according to the migration rate data.
[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Common changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included in the protection scope of the present invention.
Claims
1. A cell migration ability test and evaluation method based on a cell chamber, characterized in that: The following steps are involved: S1. Pretreatment of cells to be evaluated: resuscitate the cells to be evaluated, passage the cells to a density of 85%-90%, and resuspend them in serum-free DMEM medium after trypsin digestion to obtain a serum-free DMEM medium cell suspension; S2. Cell inoculation: Add the serum-free DMEM cell suspension obtained in step S1 into the upper chamber of the cell chamber, add the culture medium containing FBS into the lower chamber of the cell chamber, and culture for 24 hours; S3. Migration assay: After the culture is completed, the culture medium in the upper and lower chambers is removed, the cells in the upper chamber are wiped off, and the cells on the back of the cell chamber membrane are stained to calculate the migration percentage; The step S3 further includes a method for calculating cell migration rate, comprising the following steps: S31, placing the stained chamber on a glass slide, covering the chamber with neutral gum and a cover glass, and photographing the cell image on the back of the cell chamber membrane using an optical microscope; S32, converting the captured image into a grayscale image, and adjusting the image threshold to distinguish cells from the background; S33, counting the number of migrating cells, recording the cell data in the current field of view, and calculating the average value of cell migration in the current field of view; S34. Calculate the cell migration rate based on the average value of cell migration.
2. The cell migration ability test and evaluation method based on a cell chamber according to claim 1, characterized in that: The step S33 further includes deviation value detection, including the following steps: S331, recording the number of cells within each field of view, and recording the cell number value within each field of view respectively, and arranging each cell number value in descending order as X1, X2, X3, X4, and X5; S332. Compare the cell number values. If X1>2X2, it is determined that the cells in the field of view corresponding to X1 are accumulated. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to X1 is discarded and a new microscopic image is captured. Conversely, if X5<0.5X4, it is determined that the complete chamber membrane is not captured in the field of view corresponding to X5 or the cells in the field of view corresponding to X5 fall off during the staining process. The cell migration data will affect the calculation of the migration rate. The field of view image corresponding to S5 is discarded and a new microscopic image is captured.
3. The cell migration ability test and evaluation method based on a cell chamber according to claim 1, characterized in that: In step S34, the calculation formula of mobility is as follows: Among them, Q is the migration rate of the current cells, B is the number of migrated cells, and J is the initial number of cells.
4. The cell migration ability test and evaluation method based on a cell chamber according to claim 1, characterized in that: The passaging method in step S1 comprises the following steps: S11, adding pre-warmed PBS to the culture medium for rinsing to remove residual serum in the culture medium; S12, adding trypsin to the culture medium and covering the cell layer in the culture medium, and incubating at 37°C for T time; S13, after the T time period, observing the cell status in the culture medium. If the intercellular gap increases, the cultivation is determined to be complete, and the process jumps to S14 to terminate. Otherwise, the cultivation is determined to be incomplete, and the cultivation is continued. S14. Add culture medium containing serum to neutralize the activity of trypsin, and repeatedly pipette until the cells are completely detached; S15. Centrifuge the cultured cells for a time period of T. After the T time period, add the cells at the bottom into fresh culture medium for resuspending.
5. The cell migration ability test and evaluation method based on a cell chamber according to claim 1, characterized in that: The culture environment in step S2 is 37° C. and 5% CO 2 .
6. The cell migration ability test and evaluation method based on a cell chamber according to claim 1, characterized in that: In step S2, the amount of cell suspension in the upper chamber of the cell chamber was 150 μL, and the volume of the cell suspension was 7.5×10 4 / well, and the volume of culture medium in the lower chamber of the cell chamber was 800 μL.