Monoclonal cell culture method capable of realizing accurate positioning and long-term tracking

Abstract

The invention belongs to the technical field of biological cell culture, and particularly relates to a monoclonal cell culture method capable of realizing accurate positioning and long-term tracking. According to the monoclonal cell culture method capable of realizing accurate positioning and long-term tracking, the positioning adhesive film capable of being attached to the bottom of the culture plate is utilized, and the hard film prepared from an adsorption soft film or a special material is used at the bottom of the culture plate, so that the monoclonal cell culture method can be applied to culture of various types of cells; the invention aims to solve the problems of accuracy and efficiency of positioning and tracking cells in the prior art, and provides a more efficient, reliable and universal tool for researchers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological cell culture, and particularly relates to a monoclonal cell culture method capable of achieving precise positioning and long-term tracking. Background Art

[0002] With the rapid development of cell culture technology, various types of cells have been widely used in the fields of biomedical research, disease modeling, drug screening, and regenerative medicine. For example, in cancer research, by selecting and tracking monoclonal tumor cells, their heterogeneity and treatment responses can be explored in depth; in the field of immunology, the functional positioning and tracking of individual immune cells can reveal their mechanisms of action; and in stem cell research, monoclonal long-term tracking and selection are important steps in establishing disease models and standardizing cell resource banks.

[0003] Currently, whether in the culture of stem cells, tumor cells, or other types of cells, the positioning, tracking, and precise selection of monoclonal cells all face similar technical bottlenecks. Most of the existing cell tracking and selection methods require manual marking on the bottom of the culture plate using a marker pen, but the marking itself is prone to obscuring the target clone, affecting subsequent observation and image acquisition. Especially for closely located clone cells, it is very difficult to effectively distinguish them using the marker pen marking method. It can be seen that the marker pen manual marking method is not conducive to clearly marking multiple densely growing clones one by one. Moreover, another drawback of this method is that it takes a long time, resulting in the cells being out of the incubator for too long, which may reduce the cell survival rate, especially for cell types sensitive to the environment (such as immune cells or primary cultured cells). In addition, for cells cultured for a long time, such as cells carrying fluorescent labels, it is often necessary to track their fluorescent signals for a long time to assist in judgment, and the traditional method of marking with a marker pen is also not suitable. For example, if the signal may be weak in the early stage, causing the operator to misjudge it as the target clone and mark it with a marker pen, and later it is found that the clone is negative and needs to modify the mark, it is more complicated; or, the traditional method of marking with a marker pen is difficult to distinguish the time node when the clone is identified as a candidate clone, resulting in the time information being difficult to record.

[0004] Currently, in the face of the problems of positioning and tracking in the process of single-cell cloning culture, for example, Chinese Patent CN104232486A discloses a culture plate for single-cell cloning culture, which includes a culture plate cover and a culture plate bottom. A cell culture part is arranged on the culture plate bottom. The cell culture part has a plurality of cell culture single chambers arranged in an array. Its peripheral wall and bottom wall enclose a culture hole for accommodating cells. The plurality of cell culture single chambers arranged in an array are numbered in the horizontal and vertical directions for cell positioning. However, this culture plate is a product with a fixed size and needs to be designed and prepared in advance or purchased in advance. The preparation operation is complex and it cannot be reused. Therefore, there is an expectation in the field to develop a monoclonal cell culture method with more flexible application that can achieve precise positioning and long-term tracking. Summary of the Invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a monoclonal cell culture method that can achieve precise positioning and long-term tracking. The method uses a paste film that can be attached to the bottom of the culture plate for cloning cell positioning and can be applied to the positioning culture of various types of cloned cells.

[0006] To solve the above technical problems, a monoclonal cell culture method that can achieve precise positioning and long-term tracking according to the present invention includes the following steps:

[0007] (1) Select a positioning paste film provided with a positioning grid and paste it on the bottom of the cell culture plate for cell culture;

[0008] (2) Fix the cell culture plate and observe it under a microscope. Select candidate cloned cells, and at the corresponding position of the positioning paste film, perform positioning marking on the cloned cells and continue cell culture;

[0009] (3) At the selected culture time point, confirm the cloned cells to be picked according to the positioning marking of the positioning paste film, and perform picking and peeling;

[0010] (4) Aspirate the cloned cells for cloning culture.

[0011] Specifically, for the monoclonal cell culture method that can achieve precise positioning and long-term tracking, the positioning paste film includes a polypropylene film (PP film), polyurethane (PU), or polydimethylsiloxane (PDMS) film.

[0012] Specifically, for the monoclonal cell culture method that can achieve precise positioning and long-term tracking, the grid size of the positioning paste film includes various sizes. Preferably, the grid size of the positioning paste film is a 2-5 mm grid.

[0013] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, grid or number markings are embedded on the membrane surface of the positioning adhesive membrane for precise positioning of cell colonies.

[0014] Preferably, grid or number markings with micron-level resolution are embedded on the membrane surface of the positioning adhesive membrane.

[0015] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (2), there is also a step of pre-changing the culture medium for adherent-dependent cells.

[0016] Preferably, the adherent-dependent cells include pluripotent stem cells, tumor cells, immune cells or primary cultured cells.

[0017] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, the method further includes: a step of cell fluorescence labeling for the cloned cells in step (2); and a step of fluorescence signal detection for the cloned cells in step (3).

[0018] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, the fluorescent proteins carried in the fluorescence labeling step include GFP, RFP, EGFP or Tdtomato.

[0019] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (3), there is also a step of preparing a glass micropipette puller.

[0020] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (3):

[0021] The picking step includes: transferring the cell culture plate under a microscope, finding the clone to be picked according to the positioning mark, and picking the clone with a glass micropipette puller.

[0022] The peeling step includes: moving the glass micropipette puller to the vicinity of the clone, pushing open the clone edge from the four sides of the clone, and then gently pushing the clone to separate the whole clone from the bottom of the cell culture plate and peel the clone from the cell culture plate.

[0023] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (4), the clone culture step includes: culturing at a constant temperature of 35 - 40 °C and 5% carbon dioxide.

[0024] Preferably, in step (4), the clone culture step further includes a step of coating and incubating the cell culture plate.

[0025] The coating step includes coating with Matrigel; and / or,

[0026] The temperature of the incubation and culture step is 35 - 40 °C; and / or,

[0027] The time of the incubation and culture step is 20 - 40 min.

[0028] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (4), it further includes the step of aspirating the coating solution and adding fresh cell culture medium.

[0029] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, in step (4), it further includes the step of regularly recording the growth condition of the monoclonal cells and analyzing the growth trend of the monoclonal cells;

[0030] Preferably, an automated image acquisition system is used to record the growth condition of the monoclonal cells;

[0031] Preferably, the growth trajectory includes analyzing the growth dynamics, division pattern or migration trajectory of the cells.

[0032] Specifically, in the monoclonal cell culture method capable of achieving precise positioning and long-term tracking, the method further includes the step of tracking and positioning the cloned cells;

[0033] Preferably, the tracking and positioning step includes:

[0034] In step (2), the initial distribution of the cells is precisely positioned through the positioning sticker film, and the corresponding relationship with the grid coordinates on the positioning sticker film is recorded; and,

[0035] In step (3), cell images of the same field of view are obtained at different time points, and the growth and migration conditions of the cloned cells are analyzed through image comparison.

[0036] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention utilizes a positioning sticker film that can adhere to the bottom of the culture plate. By using an adsorption soft film or a hard film prepared from a special material at the bottom of the culture plate, it can be applied to various types of cell culture, including but not limited to pluripotent stem cells, tumor cells, immune cells, and other adherent-dependent cells. The present invention aims to solve the problems of precision and efficiency in positioning and tracking cells in the prior art, and provides a more efficient, reliable and general tool for researchers. This innovation will provide new ideas for single-cell research and large-scale cell screening, and is expected to play a wide role in the fields of biology and medicine.

[0037] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention. By virtue of the labeling function of the sticking film, through the grids or numbers on the sticking film and combined with microscope imaging, a clear and intuitive monoclonal positioning tool is provided, which can directly achieve precise positioning of the target monoclonal at the bottom of the plate, avoid blocking of cell colonies, has the advantage of precise positioning, simplifies the operation process, and overcomes the problem of blocking the target by traditional manual labeling.

[0038] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention. In combination with cell fluorescence labeling or image processing technology, and combined with gene-edited cells labeled with fluorescence such as GFP, it can track the cell growth, differentiation status and the effect of gene editing in real time, can perform long-term dynamic tracking on single cells or clones, realize more efficient and intuitive data collection and analysis, and provide a reliable basis for screening the most suitable monoclonal cell line.

[0039] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention can achieve precise positioning, tracking and selection in a single operation. By reducing the operation steps and shortening the residence time outside the culture plate, the efficiency is improved and the survival of environment-sensitive cells is ensured.

[0040] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention is adapted to multiple cell types and can be applied to the culture and screening requirements of various types of cells, including the rapid positioning and screening of special cells such as human pluripotent stem cells. It can not only improve the efficiency of cell selection in basic scientific research, but also provide technical support for fields such as clinical treatment and drug screening, and has broad application prospects.

[0041] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention does not require frequent manual labeling or complex operations, shortens the selection cycle and improves cell survival rate, simplifies the operation and improves the efficiency.

[0042] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention has developed an adsorption film that can be pasted on the bottom of the culture plate (the material can be polypropylene film (PP film), polyurethane (PU) or polydimethylsiloxane (PDMS) film), which has labeling and positioning functions, can precisely position adherent cell monoclonals, avoid the blocking problem of traditional manual labeling with a marker pen, and combine fluorescence labeling or image processing technology to achieve long-term dynamic tracking of cells.

[0043] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention effectively overcomes the problems of unclear marking and difficulty in long-term marking when using a marker pen to pick up pluripotent stem cell clones compared with the traditional method. The method provided by the present invention provides a simpler and more precise positioning method through a paste film attached to the bottom of the culture plate. This method does not require picking up the culture plate, nor does it require using a marker pen to circle or mark above or beside the clone, thus avoiding the problem that the marker pen may block the cell clone. Through the grid coordinates on the paste film, the position of adherent cell monoclonals can be directly determined; in addition, the problem that the marker pen marking is difficult to maintain for a long time (such as the marking may fade over time or the growth of cell clusters exceeds the marking range) is also overcome. The paste film marking technology is transparent, light and durable, not only overcomes these defects, but also can support the long-term dynamic cell tracking requirements.

[0044] A monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to the present invention, compared with the traditional culture plate for single-cell clone culture, the method of the present invention uses a paste film attached to the bottom of the culture plate, which does not require complex process preparation, has higher flexibility, and is easy to replace or reuse. The paste film is easy to operate, without complex design or preparation steps, and can directly complete the marking at the bottom of the culture plate, thus simplifying the operation process and saving a large amount of time and cost. At the same time, this method significantly improves the convenience of experimental operation, can accurately mark the cell position, and realizes the dynamic tracking of monoclonals. Brief Description of the Drawings

[0045] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in conjunction with the drawings, where

[0046] Figure 1 is a schematic structural diagram of the positioning paste film of the present invention, where the red grid is the film pasted at the bottom, and A, B, and C in the figure are divided areas;

[0047] Figure 2 is a flowchart of the method of the present invention. Detailed Embodiments

[0048] In the following embodiments of the present invention, a paste film with positioning grids is used for the positioning, memory and tracking of monoclonal cells.

[0049] In the following embodiments of the present invention, the material of the positioning paste film is preferably a material with high permeability and ultra-thin to meet the requirements of precise positioning and long-term tracking of monoclonal adherent cells. Specifically:

[0050] High permeability: Ensure that the material does not interfere with microscopic observation, while reducing light scattering and improving the acquisition efficiency of fluorescence signals;

[0051] Thinner thickness: Select materials with a thickness in the range of 10 - 50 microns to reduce interference in the optical path, ensure the clarity of fluorescence and morphological signals, and avoid affecting the culture environment (such as temperature and pressure).

[0052] Example 1

[0053] As Figure 1 shown in the structure of the positioning sticker film, as an exemplary embodiment, the positioning sticker film can be selected as an optimized polypropylene film (PP film) or polyurethane (PU) film. These materials have high permeability, flexibility, and chemical stability, and can not only achieve good adhesion to the culture plate but also meet the requirements of long-term tracking.

[0054] As an exemplary embodiment, the traditional PET material is excluded from the positioning sticker film. The PET film performs okay in terms of permeability, but it is rigid and difficult to adhere to the bottom of the culture plate, affecting the positioning accuracy and operation convenience. At the same time, it is not recommended to choose the silicone rubber film, which has high flexibility, but its thickness is usually greater than 100 microns, and the surface is not suitable for printing high-resolution grid marks, unable to meet the requirements of precise positioning and clear observation.

[0055] As Figure 1 shown in the structure of the positioning sticker film, in this embodiment, in terms of the processing method and the size division of the positioning grid of the positioning sticker film, an adjustable grid size with a unit of 2 - 5 mm is designed respectively to adapt to the needs of different experiments (for example, smaller grids are used when monoclonal adherent cells are small, and larger grids are used for observation after the cell clumps increase).

[0056] As Figure 1 shown in the structure of the positioning sticker film, the width of its grid lines is controlled within the range of 10 - 20 microns to ensure clear marking without interfering with cell observation.

[0057] As Figure 1 shown in the structure of the positioning sticker film, the processing of its positioning lines adopts high-resolution printing technology: using laser or high-resolution UV printing technology, the marks are directly printed on the surface of the sticker film to ensure that the grid lines are fine and the resolution is higher than 1200 dpi, thereby reducing the blurring and irregular edge problems that may be caused by traditional screen printing. Lightfast inks or coating materials can also be used to avoid the grid marks from fading or becoming blurred due to long-term culture or microscope irradiation. The grid line and interval design can optimize the contrast between the grid line interval and the line color by printing different colors for the grid interval lines, ensuring easy recognition under different microscopes without interfering with the acquisition of fluorescence signals.

[0058] Example 2

[0059] As Figure 2 shown in the flowchart, the monoclonal cell culture method described in this example includes the following steps:

[0060] (1) For the culture of target cells, the human embryonic stem cells (hESC H9) are pre-treated with a medium change: before picking clones, the medium in the cell culture plate is replaced with fresh cell medium to maintain the state of H9 cells when observing, photographing and picking clones under the microscope; the culture plate is fixed, and a positioning adhesive film with appropriate material and size (marking sites through the grid or number on the PP film) is selected for fastening and pasting;

[0061] (2) Place the cell culture plate under the microscope for observation. Find the clones with good morphology that need to be picked under the microscope. After fixing the culture plate, at the corresponding position of the positioning adhesive film, select a positioning adhesive film with appropriate material and size to record the positions of the clones. The grid information of the positioning adhesive film can be used for information recording;

[0062] (3) Prepare a glass pulling needle: light an alcohol lamp, place the glass Pasteur pipette on the outer flame of the alcohol lamp and burn about 3 / 4 of its part, then stretch it until it breaks into a filamentous shape and stop burning to prepare a glass pulling needle; transfer the cell culture plate to under the microscope, find the clone to be picked under the microscope according to the previous positioning marks, and pick the clone with the glass pulling needle; slowly move the glass pulling needle to the vicinity of the clone, gently push open the clone edge from around the clone, and then gently push the clone to separate the whole clone from the bottom of the cell culture plate and peel the clone out of the cell culture plate;

[0063] (4) Place the cell culture plate coated with Matrigel in an incubator at 37 °C for 30 min, aspirate the coating solution, and add fresh stem cell medium for the subsequent culture of the clones; insert a pipette tip into a 200 μL pipette, place it under a stereomicroscope, observe under the stereomicroscope to find the pipette tip in the stereomicroscope, slowly move the pipette tip to the vicinity of the peeled clone, then aspirate the clone and transfer the aspirated clone to the cell culture plate coated with Matrigel, and culture it in a constant temperature incubator at 37 °C and 5% carbon dioxide.

[0064] Example 3

[0065] As Figure 2 shown in the flowchart, this example is used to detect the differentiation state of the H7 embryonic stem cell line during daily culture, and the specific operation steps are the same as those in Example 1.

[0066] Example 4

[0067] As Figure 2The flow chart shown is used in this example to detect the differentiation status of the H1 embryonic stem cell line during daily culture. The specific operation steps are the same as those in Example 1.

[0068] Example 5

[0069] As Figure 2 shown in the flow chart, in this example, a paste film made of an adsorption-type soft film (based on polydimethylsiloxane PDMS material) is used for cell positioning.

[0070] Prepare an adsorption film with regular grid (5mm×5mm) fluorescence labeling on the surface, cut it to the size of the bottom of the cell culture plate, and directly attach the film to the bottom of the culture plate using the adhesiveness of PDMS.

[0071] Perform the positioning and culture of monoclonal cells according to the method in Example 2 above.

[0072] Example 6

[0073] As Figure 2 shown in the flow chart, in this example, a paste film made of PVC hard film is used for cell positioning.

[0074] Cut the PVC hard film (with a thickness of about 200μm) to the size of the bottom of the culture plate, and use laser etching technology to engrave regular grids (each grid size is 5mm×5mm) on the film surface. Each unit in the grid is marked with a unique micro number for subsequent precise labeling. Fix the processed film piece to the bottom of the culture plate using biosecurity glue to prevent the film from falling off in the liquid.

[0075] Perform the positioning and culture of monoclonal cells according to the method in Example 2 above.

[0076] Example 7

[0077] In this example, referring to the method in Example 2, the monoclonal observation and precise positioning of human pluripotent stem cells are carried out.

[0078] Before picking the clones, replace the medium in the cell culture plate with fresh human pluripotent stem cell medium to maintain the cell state during microscopic observation, photographing, and picking of the clones.

[0079] In this example, the selected PVC hard film (each grid size is 5mm×5mm) is pasted on the bottom of the cell culture plate and fixed.

[0080] Under the microscope, observe the clones with large area, compactness, and clear edges: the cells in the clones are plump in morphology, with a high nuclear-cytoplasmic ratio and no obvious differentiation; try to select clones that are far away from other clones for easy picking operation to prevent cross "contamination" between different clones.

[0081] Take pictures of the selected clones and record their positions using the grid markings on the membrane surface, such as in the C3 area.

[0082] Example 8

[0083] In this example, the method in Example 2 is referred to for the picking and passage of human pluripotent stem cells.

[0084] Transfer the cell culture plate to an ordinary microscope. Locate the clone to be picked under the microscope according to the marked grid marking information, and pick the clone with a 200 μL pipette with a pipette tip inserted.

[0085] Slowly move the pipette near the clone, gently push aside the clone edge from all around the clone, and then gently push the clone to separate the whole clone from the bottom of the cell culture plate. Peel the clone out of the cell culture plate, then aspirate the clone and transfer the aspirated clone to a cell culture plate coated with Matrigel, and culture it in a 37 °C, 5% carbon dioxide constant temperature incubator.

[0086] Example 9

[0087] Taking the culture method in Example 2 as an example, this example uses cell fluorescence labeling to track the growth dynamics of cells and verify the labeling accuracy and long-term tracking effect of the membrane.

[0088] Use a dual-fluorescent reporter cell line carrying dual fluorescent proteins (EGFP and Tdtomato) - H9-NKX2.5GFP / TBX5Td-T2A-18, (abbreviated as T2A), which is based on the H9 human embryonic stem cell line. The observation of EGFP and Tdtomato fluorescence respectively marks the expression of NKX2.5 and TBX5, proving that T2A cells are successfully differentiated into cardiomyocyte lineages under specific induction conditions.

[0089] Cultivate under appropriate culture medium and environment (such as 37 °C, 5% CO), maintain the cells in good condition, and do not cause a decrease in fluorescence expression due to environmental stress or variation in culture conditions. Conduct preliminary observation through a fluorescence microscope to quickly judge whether fluorescence is expressed. For example, the excitation wavelength of GFP is about 488 nm and the emission wavelength is 507 nm; the excitation wavelength of Tdtomato is about 555 nm and the emission wavelength is 581 nm. When observing under the microscope, monitor the fluorescence expression of GFP and Tdtomato respectively by switching filter plates of different wavelengths.

[0090] After observing the dual-fluorescent expression of EGFP and Tdtomato, which mark NKX2.5 and TBX5 respectively, under the microscope, switch to the bright field mode, and accurately correspond the grid coordinates with the fluorescence signal of the cells through the marking membrane pasted on the bottom of the culture plate. For example, it is determined that the T2A cell line in the C3 area expresses NKX2.5 and TBX5.

[0091] In this embodiment, an automated image acquisition system is further used to regularly record the growth status of monoclonal cells, and a machine learning algorithm is used to automatically analyze the cell growth dynamics, division patterns, and migration trajectories, thereby reducing the errors that may be brought about by manual operations.

[0092] Example 10

[0093] Taking the culture method in Example 2 as an example, in this embodiment, the migration of adherent cells within the marked grid is observed by time-lapse imaging to track the cell growth dynamics.

[0094] First, an adherent cell line (such as stem cells, fibroblasts, or tumor cells, etc.) is inoculated onto a culture plate, and the culture environment is ensured to be suitable (such as 37 °C, 5% CO₂) to maintain the normal growth state of the cells. In the experiment, a cell line carrying a fluorescent protein (such as GFP, RFP) can be selected for fluorescence labeling, or bright-field imaging can be used to record the dynamic changes of non-fluorescent cells.

[0095] After the cells adhere and meet the experimental requirements, the initial distribution of the cells is accurately positioned through the labeling film pasted on the bottom of the culture plate, and the corresponding relationship with the grid coordinates on the pasted film is recorded.

[0096] Subsequently, using a microscope combined with an automated image acquisition system, cell images of the same field of view are obtained at different time points (such as 0 h, 6 h, 12 h), and whether the cells deviate from the original grid area is analyzed through image comparison to judge the migration of the cells. This method can monitor the growth status of various adherent cells in a long-term and stable manner, providing reliable technical support for single-cell positioning and function research.

[0097] Comparative Example 1

[0098] In this comparative example, referring to the culture method in Example 2, a PET film is used for pasting and labeling.

[0099] However, due to its relatively high rigidity and lack of good flexibility and conformability, it may cause the pasting film to not fit tightly to the bottom of the culture plate, thereby affecting the accurate alignment of the grid and long-term stability.

[0100] Therefore, the present invention preferably uses a polypropylene film (PP film) or a polyurethane (PU) film with moderate flexibility and stable adhesion to ensure the accuracy of positioning and long-term tracking effect.

[0101] Comparative Example 2

[0102] This comparative example refers to the culture method in Example 2, and the only difference is that the grid size designed for the positioning pasting film is less than 2 mm or greater than 5 mm.

[0103] However, it was found in the experiment that when the grid size is less than 2 mm, the available space within a single grid is limited, and it is easy for cell colonies to span multiple grids after amplification, resulting in complex tracking analysis and affecting the accurate recording of cell growth status. When the grid size is greater than 5 mm, it is difficult to accurately mark the initial positions of monoclonal cells, reducing the accuracy of long-term tracking.

[0104] Therefore, the present invention preferably selects a grid size within the range of 2 - 5 mm to achieve a balance between precise positioning and data operability.

[0105] Comparative Example 3

[0106] This comparative example follows the culture method in Example 2, with the only difference being that the grid markings on the surface of the adhesive film only have millimeter-level accuracy, which is difficult to meet the precise positioning requirements at the single-cell level and may cause positioning errors, especially when observed under a high-power microscope.

[0107] Therefore, this solution uses a grid or number marking with a resolution higher than 1200 dpi to ensure the precise positioning of cell colonies and support long-term tracking and quantitative analysis.

[0108] In summary, the method described in the present invention utilizes a positioning adhesive film that can be attached to the bottom of the culture plate. By using an adsorption soft film or a hard film made of special materials at the bottom of the culture plate, it can be applied to various types of cell cultures, including but not limited to pluripotent stem cells, tumor cells, immune cells, and other adherent-dependent cells, and has high accuracy in positioning and tracking cells.

[0109] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A monoclonal cell culture method capable of achieving precise positioning and long-term tracking, characterized in that: The steps include: (1) Select a positioning adhesive film provided with a positioning grid and stick it to the bottom of a cell culture plate for cell culture; (2) fixing the cell culture plate and observing under a microscope, selecting candidate clone cells, and positioning and marking the clone cells at corresponding positions of the positioning adhesive film, and continuing cell culture; (3) At the selected culture time point, the cloned cells to be picked are identified according to the positioning marks on the positioning adhesive film, and the cloned cells are picked and peeled off; (4) aspirating the cloned cells for clonal culture.

2. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to claim 1, characterized in that: The positioning adhesive film includes a polypropylene film (PP film), a polyurethane (PU) or a polydimethylsiloxane (PDMS) film.

3. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to claim 2, characterized in that: The mesh size of the positioning adhesive film is a mesh of 2-5 mm.

4. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 3, characterized in that: The surface of the positioning adhesive membrane is embedded with a grid or number mark for accurately positioning the cell colony; Preferably, a grid or number mark with a micron-level resolution is embedded in the film surface of the positioning adhesive film.

5. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 4, characterized in that: The step (2) further includes the step of preliminarily exchanging the medium for the anchorage-dependent cells; Preferably, the anchorage-dependent cells include pluripotent stem cells, tumor cells, immune cells or primary culture cells.

6. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 5, characterized in that: The method further comprises: a step of performing cell fluorescence labeling on the cloned cells in step (2); and a step of performing fluorescence signal detection on the cloned cells in step (3).

7. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to claim 6, characterized in that: The fluorescent protein carried in the fluorescent labeling step includes GFP, RFP, EGFP or Tdtomato.

8. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 7, characterized in that: In step (4), the cloning culture step includes: constant temperature culture at 35-40°C and 5% carbon dioxide; Preferably, in step (4), the cloning culture step further comprises the steps of coating and incubating the cell culture plate; The coating step comprises coating with Matrigel; and / or, The temperature of the incubation step is 35-40°C; and / or, The incubation time is 20-40 minutes.

9. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 8, characterized in that: The step (4) further includes the steps of regularly recording the growth of the monoclonal cells and analyzing the growth trend of the monoclonal cells; Preferably, an automated image acquisition system is used to record the growth of the monoclonal cells; Preferably, the growth trajectory includes analyzing the growth dynamics, division pattern or migration trajectory of cells.

10. The monoclonal cell culture method capable of achieving precise positioning and long-term tracking according to any one of claims 1 to 9, characterized in that: The method further comprises the step of tracking and locating the cloned cells; Preferably, the tracking and positioning step includes: In the step (2), the initial distribution of cells is accurately located by the positioning adhesive film, and the corresponding relationship between the initial distribution of cells and the grid coordinates on the positioning adhesive film is recorded; and, In the step (3), cell images of the same visual field are acquired at different time points, and the growth and migration of the cloned cells are analyzed by image comparison.

Images