A 3D cell cloning culture screening method and screening system
Patent Information
- Application Number
- CN202510277155.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-07-04
AI Technical Summary
The traditional 3D cell culture screening methods have problems such as non-standardization of operations, cloning loss or amplification stagnation, separation accuracy and low success rate, and the existing screening systems are unable to accurately detect the height position of 3D cells, which is slow and has a risk of contamination.
The inverted microscope is used to obtain images of 3D cells, and the three-axis moving mechanism is driven to automatically select cells, and the height position of cells is calibrated by the inverted microscope focusing mechanism to achieve precise selection.
It improves the efficiency and yield of cell screening, realizes precise selection of cloned cells, reduces the risk of contamination, and meets the needs of batch experiments.
Smart Images
Figure CN120098887A8_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a 3D cell clone culture screening method and screening system. Background Art
[0002] 3D cell culture, also known as three-dimensional cell culture, refers to the co-culturing of animal cells with scaffold materials with three-dimensional structures or the culturing of cells in materials such as matrix gel to form three-dimensional cell clone clusters, which enables cells to grow, proliferate and migrate in three-dimensional space, forming three-dimensional cell-cell or cell-carrier complexes, thereby better simulating the growth environment of cells in the body.
[0003] At present, there are at least the following technical problems in the culture and screening of 3D cells: 1) Traditional manual selection or limiting dilution methods have non-standardized operation problems, which can easily lead to clone loss or amplification stagnation, affecting separation accuracy and success rate. At the same time, the manual screening process is cumbersome and time-consuming, and it is difficult to meet the needs of batch experiments.
[0004] 2) Since the cultured 3D cells are located inside the matrix gel, and the existing screening system mainly targets the culture on the upper layer of the culture plate, it is unable to detect the height position of the 3D cells, and thus cannot accurately select the 3D cells. In addition, the existing screening system uses steel needles for selection, which has the problems of slow speed and high risk of contamination.
[0005] Based on the above technical problems, the present application proposes a 3D cell clone culture screening method and screening system. Summary of the invention
[0006] The purpose of the present invention is to provide a 3D cell clone culture screening method and screening system to solve the technical problems mentioned in the background technology. The purpose of the present invention is achieved through the following technical solutions: A 3D cell clone culture screening method comprises the following steps: Step S1, obtaining an image of the cloned cells to be selected through an inverted microscope, and calculating the X-direction position information x and the Y-direction position information y of the cloned cells to be selected according to the conversion formula between pixels and millimeters; Step S2: Obtain the Z-axis position Z of the focusing mechanism of the inverted microscope when the cloned cells to be selected are clearest in the inverted microscope. 1 , and the Z-axis position Z of the focusing mechanism of the inverted microscope when calibrating the bottom of the culture well 2 , calculate the position information z of the cloned cells to be selected from the bottom of the culture well, Where a is the conversion factor of the inverted microscope; Step S3: Based on the position information x, position information y and position information z obtained in steps S1 and S2, the three-axis moving mechanism is used to drive the pipette to absorb the cloned cells to be selected and transfer them to the clone receiving plate.
[0007] Furthermore, before step S1, the following steps are also included: Step S01, loading a TIP head on a pipette; Step S02, moving the TIP head to a calibration position through a three-axis moving mechanism; Step S03, acquiring an image of the TIP head, and obtaining an offset between the TIP head and the center of the image; Step S04, compensating the offset obtained in step S03.
[0008] Furthermore, after step S3, the following steps are further included: Step S4: remove the used TIP head and reload a new TIP head.
[0009] A 3D cell clone culture screening system comprises a base, a screening platform is installed on the base, a clone receiving plate is installed on the screening platform, and a plurality of receiving holes are provided on the clone receiving plate; a hollow window is provided on the screening platform, an electric stage is installed at the hollow window, a clone culture plate is installed on the electric stage, a plurality of culture holes are provided on the clone culture plate, and the electric stage can drive the clone culture plate to move along the X direction and the Y direction; an inverted microscope and an image processing unit are installed on the base, the inverted microscope is located below the screening platform, an image acquisition unit is installed on the inverted microscope, and the image acquisition unit is electrically connected to the image processing unit; the inverted microscope has a focusing mechanism, and the focusing mechanism can drive the inverted microscope to move along the Z direction; a three-axis moving mechanism is installed above the screening platform, a pipette is installed on the three-axis moving mechanism, a TIP head is detachably installed on the pipette, and the three-axis moving mechanism can drive the pipette to move along the X direction, the Y direction and the Z direction.
[0010] Furthermore, an observation window is provided on the electric stage, a positioning mechanism is provided on the periphery of the observation window, and the cloning culture plate is installed at the observation window through the positioning mechanism so that the culture wells are located in the observation window.
[0011] Furthermore, a TIP head box and a waste box are installed on the screening platform. Clean TIP heads are placed in an array in the TIP head box. The three-axis moving mechanism can move the pipette to the TIP head box to load the TIP head. A material return mechanism is installed on the pipette, which can separate the TIP head from the pipette and drop it into the waste box.
[0012] Furthermore, any culture well of the cloning culture plate is in an empty state.
[0013] Furthermore, it also includes an isolation cover, a side of the isolation cover is provided with an isolation door, and a precision filter is installed on the top of the isolation cover.
[0014] The technical solution provided in the embodiments of the present application has at least the following technical effects or advantages: 1. Use an inverted microscope to mark the cloned cells to be selected, and use a three-axis moving mechanism to drive the pipette to automatically select the marked cloned cells, which improves the efficiency and yield of cell screening and has strong versatility, extensiveness and use value; 2. By calibrating the height position of cloned cells by adjusting the focusing distance of an inverted microscope, the height information of cloned cells to be selected can be accurately obtained, thereby achieving accurate selection of cloned cells and ensuring the accuracy of cell screening; 3. Through the three-axis moving mechanism and the material return mechanism, the automatic replacement of the TIP head is realized, which reduces the risk of contamination during the clone cell selection process and ensures the yield of clone cell screening. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0016] Figure 1 This is a schematic diagram of the appearance of an embodiment of the present application; Figure 2 This is a schematic diagram of the structure of an embodiment of the present application; Figure 3 This is a schematic diagram of the screening platform structure of an embodiment of the present application.
[0017] Figure numerals: 1. base; 2. screening platform; 21. hollow window; 22. TIP head box; 23. waste box; 3. electric stage; 31. observation window; 4. cloning culture plate; 41. culture well; 5. cloning receiving plate; 51. receiving well; 6. inverted microscope; 61. objective lens; 62. light source; 7. image processing unit; 8. three-axis moving mechanism; 81. pipette; 82. TIP head; 9. isolation cover; 91. isolation door; 92. precision filter. DETAILED DESCRIPTION
[0018] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.
[0019] Example 1 like Figure 2 , Figure 3 A 3D cell clone culture screening device shown includes a base 1, and a screening platform 2 is installed on the upper end surface of the base 1 through a pillar. The screening platform 2 is a rectangular plate. A hollow window 21 is opened on the left side of the screening platform 2, and an electric stage 3 is installed at the hollow window 21. An observation window 31 is opened on the electric stage 3, and a positioning mechanism (not shown) is fixed on the periphery of the observation window 31. The positioning mechanism clamps and fixes the clone culture plate 4 at the observation window 31 so that the culture hole 41 of the clone culture plate 4 is located in the observation window 31. The electric stage 3 can drive the clone culture plate 4 to move along the X direction and the Y direction to adjust the position of the culture hole 41. The structure and principle of the electric stage 3 and the positioning mechanism are both existing technologies and will not be repeated here.
[0020] like Figure 2 , Figure 3 As shown, the right side of the screening platform 2 is sequentially placed with a clone receiving plate 5, a TIP head box 22 and a waste box 23 from front to back. The clone receiving plate 5 has 96 receiving holes 51 arranged in an 8*12 array, which can select 96 cloned cells at a time. Several clean TIP heads are arranged in an array in the TIP head box 22 for easy replacement; the waste box 23 is used to receive used TIP heads.
[0021] like Figure 2 As shown, an inverted microscope 6 and an image processing unit 7 are installed on the base 1. The inverted microscope 6 is located between the base 1 and the screening platform 2. The objective lens 61 of the inverted microscope 6 is located at the hollow window 21 of the screening platform 2. The electric stage 3 drives the cloning culture plate 4 to move along the X direction and the Y direction so that different culture wells 41 and different positions of the culture wells 41 are located in the field of view of the inverted microscope 6. The light source 62 of the inverted microscope 6 is installed above the screening platform 2 to illuminate the inverted microscope 6. An image acquisition unit (not shown) is installed on the inverted microscope 6. The image acquisition unit is an industrial camera. The industrial camera is electrically connected to the image processing unit 7 and is used to collect and process the fiber image of the inverted microscope 6.
[0022] The inverted microscope 6 also has a focusing mechanism (not shown), which can drive the inverted microscope 6 as a whole or the objective lens 61 of the inverted microscope 6 to move along the Z direction, so that the objective lens 61 of the inverted microscope 6 is close to or away from the electric stage 3, thereby achieving focusing of the inverted microscope 6.
[0023] like Figure 2As shown, a three-axis moving mechanism 8 is installed above the screening platform 2. The three-axis moving mechanism 8 is an XYZ three-axis moving module. A pipette 81 is installed on the three-axis moving mechanism 8. A TIP head 82 is detachably installed at the lower end of the pipette 81. The three-axis moving mechanism 8 can drive the pipette to move along the X direction, the Y direction and the Z direction to achieve the change of the position of the TIP head. The culture well 41 at the upper left corner of the cloning culture plate 4 is in an empty state, and the empty culture well 41 is used as a calibration position of the TIP head.
[0024] The pipette 81 is provided with a material return mechanism (not shown), which is installed at the nozzle of the pipette 81. The material return mechanism can be reciprocated and extended along the axis direction of the nozzle of the pipette 81. When the material return mechanism is extended, the TIP head 82 can be pushed out of the pipette 82. The material return mechanism can also adopt other structures, such as using a material return fixture to grab and return the TIP head 82. The structure and principle of the material return mechanism are all existing technologies and will not be described here.
[0025] like Figure 1 As shown, the screening device further includes an isolation cover 9, which covers the screening platform 2, the inverted microscope 6 and the three-axis moving mechanism 8. The side of the isolation cover 9 located at the screening platform 2 is provided with an isolation door 91 that can be pushed up and down to facilitate the placement of the clone culture plate 4, the clone receiving plate 5, the TIP head box 22 and the waste box 23. A precision filter 92 is installed on the top of the isolation cover 9 to ensure the cleanliness of the inside of the screening device.
[0026] When using: First, the operator places the clone culture plate 4 on the electric stage 3 above the inverted microscope 6, and places the clone receiving plate 5, the TIP head box 22, and the waste box 23 at designated positions.
[0027] Then, an image of cloned cells in the clone receiving plate 5 is obtained by an inverted microscope 6, and the operator selects and marks the clones that he wants to select by using a mouse; Finally, the device is started, and the three-axis moving mechanism 8 drives the pipette 81 to the TIP head box 22 to load the clean TIP head 82, and moves the TIP head 82 to the calibration position for calibration. After the calibration is completed, the TIP head 82 is moved to the clone culture plate 4 to absorb the cloned cells and discharge them into the receiving hole 51 of the clone receiving plate 5, and then the pipette 81 is moved to the waste box 23 to discharge the used TIP head 82. Repeat the above actions to complete the selection of the remaining cloned cells.
[0028] Example 2 A 3D cell clone culture screening method comprises the following steps: Step S01, loading a clean TIP head on a pipette; Step S02, moving the TIP head to a calibration position through a three-axis moving mechanism; Step S03, acquiring an image of the TIP head, and obtaining an offset between the TIP head and the center of the image; By imaging with an inverted microscope, the center pixel position of the TIP head on the image is obtained, and then the X and Y pixel offsets between the center of the TIP head and the center of the image are obtained based on the center pixel of the image. The actual offsets of the TIP head in the X and Y directions are calculated using the conversion formula between pixels and millimeters.
[0029] The specific conversion formula is: number of pixels / DPI (dots per inch) = number of inches, and the number of inches multiplied by 25.4 is the number of millimeters.
[0030] Step S04: Compensate the offset obtained in step S03. By compensating the actual offset obtained by calibration, cloned cells can be selected accurately.
[0031] Step S1, obtaining an image of the cloned cells to be selected through an inverted microscope, and calculating the X-direction position information x and the Y-direction position information y of the cloned cells to be selected according to the conversion formula between pixels and millimeters; Step S2: Obtain the Z-axis position Z of the focusing mechanism of the inverted microscope when the cloned cells to be selected are clearest in the inverted microscope. 1 , and the Z-axis position Z of the focusing mechanism of the inverted microscope when calibrating the bottom of the culture well 2 , calculate the position information z of the cloned cells to be selected from the bottom of the culture well, Where a is the conversion coefficient of the inverted microscope, which can be obtained by setting calibration objects of different heights in the culture well and conducting multiple tests to correlate the moving distance of the focusing mechanism of the inverted microscope with the height change in the culture well.
[0032] Step S3: Based on the position information x, position information y and position information z of the cloned cells to be selected obtained in steps S1 and S2, the three-axis moving mechanism is used to drive the pipette to absorb the cloned cells to be selected and transfer them to the clone receiving plate.
[0033] Step S4: remove the used TIP head and reload a new TIP head.
[0034] The technical solution provided in the embodiments of the present application has at least the following technical effects or advantages: 1. Use an inverted microscope to mark the cloned cells to be selected, and use a three-axis moving mechanism to drive the pipette to automatically select the marked cloned cells, which improves the efficiency and yield of cell screening and has strong versatility, extensiveness and use value; 2. The height position of cloned cells can be calibrated by adjusting the focusing distance of the inverted microscope, so that cloned cells can be selected accurately, ensuring the accuracy of cell screening; 3. Through the three-axis moving mechanism and the material return mechanism, the automatic replacement of the TIP head is realized, which reduces the risk of contamination during the clone cell selection process and ensures the yield of clone cell screening.
[0035] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A 3D cell clone culture screening method, characterized in that: The following steps are involved: Step S1, obtaining an image of the cloned cells to be selected through an inverted microscope, and calculating the X-direction position information x and the Y-direction position information y of the cloned cells to be selected according to the conversion formula between pixels and millimeters; Step S2, obtaining the Z-axis position Z1 of the focusing mechanism of the inverted microscope when the clone cells to be selected are clearest in the inverted microscope, and the Z-axis position Z2 of the focusing mechanism of the inverted microscope when the bottom of the culture well is calibrated, and calculating the position information z of the clone cells to be selected from the bottom of the culture well, Where a is the conversion factor of the inverted microscope; Step S3: Based on the position information x, position information y and position information z obtained in steps S1 and S2, the three-axis moving mechanism is used to drive the pipette to absorb the cloned cells to be selected and transfer them to the clone receiving plate.
2. A 3D cell clone culture screening method according to claim 1, characterized in that: Before step S1, the method further includes: Step S01, loading a TIP head on a pipette; Step S02, moving the TIP head to a calibration position through a three-axis moving mechanism; Step S03, acquiring an image of the TIP head, and obtaining an offset between the TIP head and the center of the image; Step S04, compensating the offset obtained in step S03.
3. A 3D cell clone culture screening method according to claim 1, characterized in that: After step S3, the following steps are also included: Step S4: remove the used TIP head and reload a new TIP head.
4. A 3D cell clone culture screening system, characterized in that: The invention comprises a base, on which a screening platform is installed, on which a clone receiving plate is installed, and on which a plurality of receiving holes are provided; a hollow window is provided on the screening platform, on which an electric stage is installed, on which a clone culture plate is installed, and on which a plurality of culture holes are provided, and the electric stage can drive the clone culture plate to move along the X direction and the Y direction; an inverted microscope and an image processing unit are installed on the base, the inverted microscope is located below the screening platform, on which an image acquisition unit is installed, and the image acquisition unit is electrically connected to the image processing unit; the inverted microscope has a focusing mechanism, and the focusing mechanism can drive the inverted microscope to move along the Z direction; a three-axis moving mechanism is installed above the screening platform, on which a pipette is installed, on which a TIP head is detachably installed, and the three-axis moving mechanism can drive the pipette to move along the X direction, the Y direction and the Z direction.
5. A 3D cell clone culture screening system according to claim 4, characterized in that: An observation window is provided on the electric stage, a positioning mechanism is provided on the periphery of the observation window, and the cloning culture plate is installed at the observation window through the positioning mechanism so that the culture well is located in the observation window.
6. A 3D cell clone culture screening system according to claim 4, characterized in that: A TIP head box and a waste box are installed on the screening platform. Clean TIP heads are placed in an array in the TIP head box. The three-axis moving mechanism can move the pipette to the TIP head box to load the TIP head. A material return mechanism is installed on the pipette, and the material return mechanism can separate the TIP head from the pipette and drop it into the waste box.
7. A 3D cell clone culture screening system according to claim 4, characterized in that: Any of the culture wells of the cloning culture plate is in an empty state.
8. The 3D cell clone culture screening system according to claim 4, characterized in that: It also includes an isolation cover, a side of which is provided with an isolation door, and a precision filter is installed on the top of the isolation cover.