An experimental method for three-dimensional migration of cells in hydrogels

Abstract

The application relates to an experimental method for three-dimensional migration of cells in a hydrogel, comprising the following steps: preparing a confocal culture dish, and processing a fluorescent positioning line on the bottom of the confocal culture dish, which is used for interface reference positioning; installing a demarcation blocking mechanism in the confocal culture dish, forming a hydrogel in a demarcation area in the confocal culture dish through the demarcation blocking mechanism, and the hydrogel comprises a cell-containing hydrogel corresponding to the demarcation area and a cell-free hydrogel; adding a culture medium to the upper space of the hydrogel, carrying out three-dimensional culture of cells in the hydrogel, and completing migration of the cells during the culture process; placing the confocal culture dish on a confocal microscope platform, determining the interface position through the fluorescent positioning line, collecting the cell distribution position in a time sequence, and measuring the migration track and distance in the subsequent process.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, and more specifically, to an experimental method for three-dimensional migration of cells in a hydrogel. Background Technology

[0002] With the development of cell biology and tissue engineering, cell culture methods are transitioning from traditional two-dimensional planar culture to three-dimensional culture.

[0003] Compared to two-dimensional conditions, three-dimensional culture can more realistically simulate the in vivo microenvironment, making cells more closely resemble their in-situ physiological state in terms of spatial structure, intercellular interactions, extracellular matrix deposition, and signal transduction. Therefore, three-dimensional hydrogel matrices are widely used in stem cell research, tissue regeneration, tumor invasion and metastasis mechanism studies, and drug sensitivity evaluation.

[0004] 1) Three-dimensional hydrogel culture: The core of three-dimensional hydrogel culture lies in allowing cells to grow, migrate, and differentiate in a three-dimensional space that more closely resembles the in vivo microenvironment, unlike two-dimensional planar culture which is limited to growth in a two-dimensional plane. Hydrogels possess a high water content, permeability, and extracellular matrix-like three-dimensional network structure. Cells are directly encapsulated within this structure, allowing them to extend and adhere three-dimensionally and interact mechanically with the matrix through traction forces. This activates integrins and mechanotransmission pathways that are more consistent with in vivo conditions. This system also allows nutrients, metabolites, and drugs to diffuse freely within the gel, sustaining long-term cell growth. The advantage of three-dimensional hydrogel culture lies not only in its "more three-dimensional" imaging but also in its "more realistic" biological aspects. Compared to two-dimensional culture, which only allows horizontal migration and signals deviating from the in vivo environment, the three-dimensional environment can reconstruct cell morphology, migration behavior, cell-cell and cell-matrix interactions, mechanotransmission, and more reliable gene and protein expression patterns.

[0005] However, one of the drawbacks of three-dimensional culture is the difficulty in imaging. Three-dimensional culture media such as hydrogels have high deep scattering and reduced resolution, which may prevent ordinary optical microscopes from imaging, requiring confocal microscopy for imaging.

[0006] 2) Due to the need for confocal microscopy imaging, ordinary petri dishes have excessively thick bottoms and unstable refractive indices. Furthermore, the uneven thickness and refractive index of ordinary plastic dishes at the bottom, deviating from glass, cause light scattering and focus drift, resulting in blurred images. Additionally, the excessive thickness of the bottom of ordinary dishes prevents the objective lens from getting close enough, leading to either no image formation or extremely poor image quality.

[0007] 3) Three-dimensional migration experiments: In three-dimensional culture systems, cell migration characteristics are important indicators for studying cell behavior, and experimental scenarios for evaluating cell migration ability are increasingly common. Cell migration under three-dimensional culture conditions requires cells to migrate from one side of a cell-containing culture substrate (such as hydrogel) to another side of a cell-free culture substrate. Existing studies typically require constructing an interface between a cell-containing hydrogel and a cell-free hydrogel to observe the behavior of cells migrating across the interface to the cell-free region. However, existing experimental methods for constructing the cell-containing hydrogel-cell-free hydrogel matrix interface often require manual, sterile operation, which is cumbersome and yields poor interface construction results. The following problems are commonly encountered: ① Interface construction is difficult and has poor repeatability; dual-region hydrogel molding is difficult and has poor repeatability. Currently, it is common to rely on manual application of adhesives in a sterile environment or pre-made molds. This type of operation is cumbersome, depends on the operator's experience, and makes it difficult to control the interface position and shape, resulting in poor usability, weak experimental repeatability, and a high failure rate.

[0008] Relying on manual division or external molds to form cellular / cell-free regions, the interface position, thickness, and shape are easily affected by the operator's experience and operating speed, making it difficult to guarantee the reproducibility of experimental results.

[0009] ②The operation process is cumbersome, time-consuming, and carries a high risk of contamination. Before the hydrogel initially sets, multiple steps such as zoning, area positioning, cutting, or mold removal are performed. The time window is extremely short, which requires a sterile environment and high skill levels from the experimental personnel. This increases the risk of experimental errors and contamination, resulting in a high failure rate and making it unsuitable for high-throughput experiments or long-term continuous experiments.

[0010] ③ Interface imaging is limited in three-dimensional migration experiments. Unlike two-dimensional migration experiments, three-dimensional migration experiments cannot be imaged under a bright field microscope due to the thickness and light transmittance of the hydrogel. Therefore, fluorescence imaging is required under a laser confocal microscope. Since there is no fluorescence signal at the cell migration interface, it does not develop during fluorescence imaging. Existing imaging methods can only see the cells and not the interface, which leads to migration distance measurement relying on subjective judgment or the superposition of bright field / fluorescence images, resulting in significant errors and making it difficult to perform objective quantitative or automated analysis.

[0011] In subsequent quantitative or semi-quantitative analysis, it is difficult to determine the interface location. In the past, the interface location could only be inferred from the cell distribution location, or the images could be superimposed after switching to bright field, which introduced subjectivity and uncertainty into the calculation of experimental results.

[0012] ④ There is still a lack of integrated experimental consumables for three-dimensional transfer experiments. Currently, there are no confocal culture dishes on the market with structural designs specifically for the needs of three-dimensional migration experiments. Researchers still need to assemble various accessories or make temporary modifications, which is inefficient and costly.

[0013] In summary, current technologies still lack a dedicated culture dish device and an effective and accurate experimental method for rapidly, stably, and reproducibly constructing dual-region hydrogel systems in cell migration experiments in three-dimensional hydrogels, while also enabling subsequent confocal imaging and interface visualization analysis. Summary of the Invention

[0014] The purpose of this application is to provide an experimental method for three-dimensional cell migration in hydrogels, which can conveniently and quickly form bilateral cell-containing / cell-free hydrogel matrices while constructing a good separating interface. Furthermore, for the purpose of subsequent cell migration observation, fluorescent lines are placed on the bottom slide of the well plate to facilitate visualization of the cell migration interface and assist in subsequent quantitative and semi-quantitative analysis.

[0015] To achieve the above objectives, the present invention provides an experimental method for three-dimensional cell migration in hydrogels, comprising the following steps: A confocal culture dish was prepared, and a fluorescent positioning line was formed on the bottom of the confocal culture dish. The fluorescent positioning line was used for interface reference positioning. A boundary barrier mechanism is installed in a confocal culture dish, and a hydrogel is formed in the partitioned area of ​​the confocal culture dish through the boundary barrier mechanism. The hydrogel includes cell-containing hydrogels and cell-free hydrogels corresponding to the partitioned areas. Culture medium is added to the upper space of the hydrogel to carry out three-dimensional cell culture in the hydrogel, and cell migration is completed during the culture process. The confocal culture dish was placed on the confocal microscope platform, and the interface position was determined by the fluorescence positioning line. The cell distribution position was collected according to the time series for subsequent migration trajectory and distance measurement.

[0016] In an optional embodiment, the confocal culture dish includes a dish body, a glass slide base plate, and a dish lid. The dish body is constructed as an annular dish body with annular steps inside. The sink space at the bottom of the annular steps constitutes a hydrogel space, and the remaining space of the annular dish body constitutes a culture medium space. The fluorescent positioning line is formed on the glass slide base plate of the hydrogel space. The bottom of the outer wall of the dish has a mating step, and the dish lid is attached to the mating step.

[0017] In an optional embodiment, the dividing barrier mechanism includes a barrier sheet, and the annular dish body is provided with a positioning and limiting part. The barrier sheet is detachably installed in the positioning and limiting part and the barrier sheet abuts and adheres to the glass slide base plate. The edge of one side of the barrier corresponds vertically to the fluorescent positioning line.

[0018] In an optional embodiment, the forming of the fluorescent positioning line includes: cleaning the glass slide substrate, constructing a surface pattern of the fluorescent positioning area, fixing fluorescent molecules / dyes, rinsing and drying; The cleaning of the glass slide base plate is used to remove organic contaminants and expose the glass to silanol groups; A surface pattern is constructed for the fluorescent localization region to expose the slit region corresponding to the fluorescence. Immobilized fluorescent molecules / dyes are used to bind fluorescent molecules or fluorescent dyes to a glass slide substrate.

[0019] In optional implementations, the fluorescent positioning area is patterned and fixed by photolithography / metal masking, microcontact printing, inkjet printing, laser patterning, screen printing, plasma patterning, imprinting, or photopolymer exposure and development. The binding of fluorescent molecules to the surface of the glass slide substrate is achieved through silanization-NHS coupling, glutaraldehyde crosslinking, acrylamide grafting, polymer brush fixation, or charge adsorption to enhance covalent end-capping. Fluorescent molecules / dyes, including organic fluorescent dyes, quantum dots, self-luminescent nanomaterials, fluorescent polymers, or embedded fluorescent microstructures, are used as imaging calibration signals.

[0020] In an optional embodiment, after the silanol groups of the slide substrate are exposed, a metal mask with slits is applied to the surface of the slide substrate to expose the slit area corresponding to the fluorescent positioning line. Add silane coupling agent ethanol solution dropwise and react to form an amino functional group layer in the exposed area of ​​the slit; Remove the metal mask and clean with anhydrous ethanol; An organic fluorescent dye solution was prepared, dropped onto the surface of a glass slide substrate, and incubated at room temperature to achieve amide bond coupling and fixation of the fluorescent dye with amino groups; Fluorescent positioning lines of constant width were obtained by rinsing with phosphate buffer solution and air drying.

[0021] In an optional implementation, the process of forming the hydrogel includes: Install the barrier sheet on the positioning and limiting part of the dish body, and make the bottom surface of the barrier sheet fit tightly against the glass slide base plate; The cell-containing hydrogel precursor solution is injected into the pre-defined septum of the barrier sheet, and gelled after being filled evenly. Remove the barrier sheet after the hydrogel has initially solidified; Inject cell-free hydrogel precursor solution into the blank partition region and complete gelation.

[0022] In an optional embodiment, the positioning and limiting part includes a slot, a groove, or a magnetic component; The barrier plate is fixedly connected to the dish body via a slot or groove; The barrier sheet is magnetically attached to the dish body. The barrier sheet is elastically clamped or adhered to the dish body.

[0023] In an optional embodiment, the barrier sheet is made of glass, a polymer, a metal, or a surface hydrophobic coating material.

[0024] In optional embodiments, the fluorescent positioning lines include a single dividing line, multiple parallel lines, intersecting lines, ring marks, serrated marks, cross positioning marks, or dot matrix marks.

[0025] The experimental method for three-dimensional cell migration in hydrogels in this invention can conveniently and quickly complete the molding of bilateral cell-containing / cell-free hydrogel matrices by adding an auxiliary boundary barrier mechanism, while constructing a good separation interface.

[0026] In addition, to facilitate the observation of cell migration in the later stages, fluorescent positioning lines are fabricated at the bottom of the confocal culture dish to make the cell migration interface more visible and to assist in subsequent quantitative and semi-quantitative analysis.

[0027] Other features and advantages of this application will be described in detail in the following detailed description section. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a diagram showing the results of a three-dimensional cell migration experiment in the existing technology; Figure 2 This is a schematic diagram of the structure of the CCP-focused petri dish in this application; Figure 3 This is a schematic diagram of the fluorescent positioning line in this application; Figure 4 This is a schematic diagram of the hydrogel formation and three-dimensional cell migration process in this application; Figure 5 This is a flowchart illustrating the experimental method used in this application.

[0030] icon: 1-Dish body; 11-Annular step; 12-Matching step; 13-Positioning and limiting part; 2-Slide base plate; 3-Dish lid; 4-Fluorescent positioning line; 5-Cell-containing hydrogel; 6-Cell-free hydrogel; 7-Barrier sheet. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0032] In the description of this application, it should be noted that the terms "inner" and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product is in use. They are used only for the convenience of describing this application and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0033] In the description of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "setup" and "connection" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0034] The experimental method for three-dimensional cell migration in hydrogels in this application is mainly used to improve the experimental conditions for three-dimensional cell migration in hydrogels. It can solve the technical problems in existing three-dimensional migration experiments, such as difficulty in interface construction and poor reproducibility, cumbersome operation procedures, time consumption and high risk of contamination, limited interface imaging in three-dimensional migration experiments, and lack of integrated experimental consumables for three-dimensional migration experiments.

[0035] See Figure 2 and combined Figures 3-5 The experimental method for three-dimensional cell migration in hydrogels in this application includes the following steps: A confocal culture dish was prepared, and a fluorescent positioning line 4 was fabricated on the bottom of the confocal culture dish. The fluorescent positioning line 4 was used to locate the interface between the cell-containing hydrogel 5 and the cell-free hydrogel 6.

[0036] The confocal culture dish in this application can meet the imaging conditions of laser confocal microscopy. Existing ordinary culture dishes have too thick a bottom and unstable refractive index, while confocal microscopy requires the laser to be precisely focused on a certain layer of the cell.

[0037] Furthermore, the uneven thickness of the bottom of ordinary plastic dishes and their refractive index deviating from that of glass can cause light scattering and focus drift, resulting in blurred images. Confocal culture dishes include a glass slide base plate 2, which is specifically a finely ground flat surface, allowing for a stable and consistent focal plane during laser scanning.

[0038] Preferably, the slide base plate 2 is a glass base plate of standard thickness. By using a glass base plate of standard thickness (170±5 μm), it can be matched with the optical design of high numerical aperture microscope objectives. Therefore, the propagation of laser on the slide base plate 2 has almost no refraction deviation, the focus is stable, and the imaging resolution is high.

[0039] Meanwhile, high numerical aperture microscope objectives need to be close to the sample, and confocal microscopes commonly use oil immersion or water immersion (NA 1.2–1.4), resulting in extremely short working distances. Ordinary dishes have very thick bottoms, making it difficult for the objective to get close enough, leading to either no image formation or extremely poor image quality.

[0040] Live-cell imaging requires an undisturbed environment. In the past, live-cell imaging could only be performed by temporarily transferring cells to a glass slide, which significantly altered the cell state. Confocal culture dishes allow cells to be cultured, stimulated, and imaged directly in the experimental environment, maintaining a more stable physiological state.

[0041] The fabrication and shaping of the fluorescent positioning line 4 enables real-time observation of cell migration distance and direction, as well as subsequent quantitative analysis, providing a precise and reliable interface reference for the cell-containing hydrogel 5 and the cell-free hydrogel 6. In existing observations of three-dimensional cell migration, cell migration imaging generally relies on confocal microscopy to observe fluorescently labeled cells, roughly determining the migration interface through cell distribution and bright-field images, followed by quantitative or semi-quantitative calculations. Figure 1 The image shown is a result of a three-dimensional cell migration experiment using existing methods. It can be seen that the cell migration interface is not visualized, only fluorescently labeled cells are visible. Therefore, the migration interface can only be roughly defined by cell distribution (shown by the dotted line in the middle), followed by calculations. This makes it difficult to determine the interface location in quantitative or semi-quantitative analysis; previously, the interface location could only be inferred from the cell distribution (e.g., ...). Figure 1 (As shown by the dashed line in the middle), or by switching to bright field and then overlaying the image, the calculation of experimental results is mixed with subjectivity and uncertainty.

[0042] Current observations of three-dimensional cell migration mainly focus on fluorescent labeling of cells and observing cell migration behavior from hydrogel matrix to cell-free areas. They do not perform interface datum localization on the initial interface on slide plate 2, nor do they consider the angle of visual observation of fluorescent labeling at the interface datum localization.

[0043] This application, by processing and shaping the fluorescent positioning line 4 at the initial stage of three-dimensional cell culture before hydrogel formation, can provide an interface benchmark for hydrogel processing. It also facilitates the boundary formation of cell-containing hydrogel 5 and cell-free hydrogel 6. In subsequent cell migration observation, the fluorescent positioning line 4 and the fluorescently labeled cells can be placed in the same imaging field of a confocal microscope, thereby enabling more objective and accurate quantitative or semi-quantitative analysis.

[0044] Most conventional fluorescent labeling techniques focus on cells whose migration activity can be observed. However, fluorescent localization on glass slides goes beyond the conventional technique of fluorescent labeling only cells. By extending the fluorescent labeling to the glass slide substrate, it can facilitate the boundary formation of different types of hydrogels, providing a basis for hydrogel formation. On the other hand, it can observe cell migration in the same confocal microscopy field of view.

[0045] After the fluorescent positioning line 4 is formed, a boundary barrier mechanism is installed in the confocal culture dish. The hydrogel is formed in the partition area of ​​the confocal culture dish through the boundary barrier mechanism. The hydrogel includes a cell-containing hydrogel 5 and a cell-free hydrogel 6 corresponding to the partition area, and the fluorescent positioning line 4 is positioned at the boundary between the cell-containing hydrogel 5 and the cell-free hydrogel 6.

[0046] Current research on the three-dimensional migration behavior of cells in hydrogel matrices typically requires constructing two hydrogel regions, one containing cells and the other not, within the same culture system to observe cross-boundary migration of cells from the initial region to the adjacent region. Existing techniques mainly employ a manual process of removing and re-adding the hydrogel. On a standard glass-bottomed culture dish, a cell-containing hydrogel (5) is added. After initial solidification, it is manually cut, removing half of the hydrogel while maintaining a clean cut surface. Then, a cell-free hydrogel (6) is added. This method relies on manual cutting, which often results in uneven cut surfaces and poor fusion at the interface between the two hydrogel parts, potentially affecting subsequent cell migration. Furthermore, it requires a high degree of aseptic technique, and the introduction of microorganisms greatly increases the probability of contamination during the three-dimensional culture process.

[0047] By installing a boundary barrier mechanism in a confocal culture dish, the hydrogel forming process can be effectively improved, allowing both cell-containing hydrogel 5 and cell-free hydrogel 6 to be formed in a fixed space in one step, with a smooth interface and accurate positioning, thus improving experimental repeatability and success rate.

[0048] Specifically, the demarcation barrier mechanism is detachably installed in the confocal culture dish and can form a separation area corresponding to the fluorescence positioning line 4, thereby enabling the boundary between the cell-containing hydrogel 5 and the cell-free hydrogel 6 to be aligned with the position of the fluorescence positioning line 4 after the hydrogel is formed.

[0049] Specifically, after the fluorescent positioning line 4 is processed and formed, the boundary barrier mechanism is installed through the necessary positioning and limiting part 13, so that the installation position of the boundary barrier mechanism is opposite to the position of the fluorescent positioning line 4. Furthermore, one part of the boundary barrier mechanism is preferably the edge opposite to the position of the fluorescent positioning line 4, so that the boundary of the hydrogel on different sides corresponds to the position of the fluorescent positioning line 4.

[0050] During the hydrogel forming process, cell-containing hydrogel is first added to one side of the fluorescent positioning line 4 or the preset area of ​​the fluorescent positioning line 4. After solidification, the boundary barrier mechanism is removed, and cell-free hydrogel is added to the other side or the preset area of ​​the fluorescent positioning line 4. After processing and forming different types of hydrogels, a three-dimensional culture space for cells in a confocal culture dish can be formed, allowing cells to move in the hydrogel matrix.

[0051] Simultaneously, after the hydrogel is formed, culture medium is added to the upper space of the hydrogel to carry out three-dimensional cell culture in the hydrogel. During the culture process, the cells migrate from the cell-containing hydrogel 5 to the blank hydrogel space.

[0052] During the observation process, the confocal culture dish was placed on the confocal microscope platform, and the position of the predetermined hydrogel interface was determined by the fluorescence positioning line 4. The cell distribution position was collected according to the time sequence for subsequent migration trajectory and distance measurement.

[0053] From the perspective of the composition of the confocal culture dish, the confocal culture dish includes a dish body 1, a glass slide base plate 2, and a dish lid 3. The dish body 1 is constructed as an annular dish body 1 with an annular step 11 inside. The sink space at the bottom of the annular step 11 constitutes the bottom space for forming hydrogel. The remaining space of the annular dish body 1 constitutes the space for injecting culture medium. The fluorescent positioning line 4 is formed on the glass slide base plate 2 in the hydrogel space.

[0054] Meanwhile, in order to facilitate the closing of the lid 3, the bottom of the outer wall of the dish body 1 is constructed with a mating step 12, and the lid 3 is closed and overlapped on the mating step 12.

[0055] The boundary barrier mechanism in this application includes a barrier plate 7 and a positioning and limiting part 13 provided on the annular dish body 1. The barrier plate 7 can be detachably positioned and installed in the annular dish body 1 by the positioning and limiting part 13.

[0056] Taking the form of a dividing barrier mechanism that equally divides the hydrogel space on both sides as an example, in order to form an effective molding space, the bottom of the barrier 7 is in contact with the wall of the glass slide base plate 2. At the same time, in order to make the dividing part correspond to the fluorescence positioning line 4, the edge of one side of the barrier 7 corresponds vertically to the fluorescence positioning line 4. After the cell-containing hydrogel 5 is formed, the barrier 7 is removed and the cell-free hydrogel 6 is injected, a hydrogel with the same volume on both sides is formed, thus forming the corresponding dividing line between the fluorescence positioning line 4 and the hydrogel on both sides.

[0057] The process of forming the fluorescent positioning line 4 includes: cleaning the glass slide substrate 2, constructing the surface pattern of the fluorescent positioning area, fixing the fluorescent molecules / dye, rinsing and drying.

[0058] The cleaning of the glass slide base plate 2 is carried out by using Piranha solution. The surface of the glass slide base plate 2 is cleaned with Piranha solution to remove organic contaminants and expose the silanol groups of the glass. Construct a surface pattern for the fluorescent positioning region to expose the slit region corresponding to the fluorescent positioning line 4; Immobilize fluorescent molecules / dyes to stably bind fluorescent molecules or fluorescent dyes to the glass slide substrate 2.

[0059] Specifically, in the processing and molding process, the fluorescent positioning area is patterned and fixed through photolithography / metal masking, micro-contact printing, inkjet printing, laser patterning, screen printing, plasma patterning, imprinting, or photosensitive polymer exposure and development.

[0060] Meanwhile, fluorescent molecules are bound to the surface of the glass slide substrate 2 through silanization-NHS coupling, glutaraldehyde crosslinking, acrylamide grafting, polymer brush fixation, or charge adsorption to enhance covalent end-capping.

[0061] Fluorescent molecules / dyes include organic fluorescent dyes such as FITC, Cy3, and Alexa Fluor (fluorescein isothiocyanate, cyanine dye, AlexaFluor dye), quantum dots, self-luminescent nanomaterials, fluorescent polymers, or embedded fluorescent microstructures, which are used as imaging calibration signals.

[0062] In one specific embodiment, a polystyrene dish body 1 is used, and the bottom of the dish is a high optical transmittance glass slide base plate 2 with a thickness of 170±5μm. The glass slide base plate 2 is bonded to the dish body 1 using medical-grade shadowless adhesive to ensure sealing and optical flatness. Positioning and limiting grooves are provided on both sides of the dish body 1 for installing auxiliary barrier glass slides as a separation and blocking mechanism. The auxiliary barrier glass slides are 0.5 mm thick, and their length matches the inner diameter of the dish. Their lower end face is polished to ensure flat attachment with the glass slide base plate 2.

[0063] A fluorescence positioning line 4 is prepared at the center of the inner surface of the slide base plate 2, directly opposite the position of the barrier slide, including the following preparation steps: The surface of the slide substrate 2 was cleaned with Piranha solution to remove organic contaminants and expose the silanol groups in the glass. After the silanol groups in the slide substrate 2 were exposed, a metal mask with a 100 μm wide slit was covered on the surface of the slide substrate 2 to expose the slit area corresponding to the fluorescence positioning line 4. Add 2% (v / v) silane coupling agent ethanol solution and react for 40 min to form an amino functional group layer in the exposed area of ​​the slit; Remove the metal mask and clean with anhydrous ethanol; Prepare a 100 μg / mL NHS-Alexa Fluor 555 dye solution, drop it onto the surface of glass slide base plate 2 and incubate at room temperature for 30 min to achieve amide bond coupling and fixation of fluorescent dye with amino groups; The fluorescent positioning line 4 with constant width was obtained by rinsing with phosphate buffer solution and air drying.

[0064] Dual-region hydrogel molding: After the confocal culture dish is prepared, the following steps are taken for the shaping of the cell three-dimensional migration experiment: Install the auxiliary barrier glass slide into the positioning and limiting groove of the main body of the dish 1, so that the lower end face of the barrier glass slide is tightly attached to the glass slide base plate 2; Inject the cell-containing hydrogel 5 precursor solution into one side of the barrier slide, fill it evenly, and then place it under appropriate conditions to gel. Remove the auxiliary barrier glass slide after the hydrogel has initially solidified; An equal volume of cell-free hydrogel precursor solution was injected into the blank partition area on the other side and gelation was completed. After the formation was completed, a dual-region hydrogel structure with different components on both sides and a smooth interface was formed in the dish. The interface between the cell-containing region and the cell-free region was on the same straight line as the fluorescence positioning line 4.

[0065] Add complete culture medium for three-dimensional cell culture until cell migration is complete.

[0066] Three-dimensional migration experiments and imaging: The process of observing cell migration is as follows: Place the confocal culture dish on a confocal microscope platform equipped with an environmental control device; Images of fluorescent positioning line 4 were acquired using the corresponding fluorescence channel, followed by the acquisition of the imaging channel used for cell fluorescence labeling; Cell signals and fluorescence localization line 4 signals were recorded in the same field of view, and the interface position was determined by fluorescence localization line 4. Cell distribution locations were collected based on time series data for subsequent migration trajectory and distance measurements.

[0067] The fluorescent positioning line 4 maintains a stable spatial position throughout the entire culture cycle and imaging phase, and does not shift with changes in hydrogel, cell, or culture medium conditions. It can serve as a fixed coordinate reference for cell migration direction and distance.

[0068] This application does not limit the specific form of the positioning and limiting part 13, or the connection form between the barrier 7 and the dish body 1. The positioning and limiting part 13 may be in the form of a slot, a groove, or a magnetic component.

[0069] The barrier sheet 7 can be fixedly connected to the dish body 1 through a slot or groove, or it can be magnetically attached to the dish body 1. It can also be elastically clamped or adhered to the dish body 1. Any alternative solution is that it can maintain stable adhesion during the glue injection stage and can be detached after the first part of the gel is formed.

[0070] The barrier sheet 7 can be made of glass, polycarbonate or other high molecular polymers, metals or surface hydrophobic coating materials. Any material can be used as long as the surface can prevent hydrogel from adhering and the interface can remain flat.

[0071] In addition to the single fluorescent positioning line 4 mentioned above, the fluorescent positioning line 4 also includes multiple parallel lines, intersecting lines, ring marks, serrated marks, cross positioning marks, or dot matrix marks. As long as they can be used for interface reference positioning, they are all within the scope of protection of this invention.

[0072] The fluorescent positioning structure is not limited to completely coinciding with the interface; it can also be arranged on both sides of the interface to form a reference interval positioning mark, which will not be elaborated here.

[0073] The experimental method for three-dimensional cell migration in hydrogels described in this application, through structural improvements to the culture dish and surface chemical modification of the slide substrate 2, achieves the following objectives: A dual-region hydrogel system can be quickly constructed without manual cutting. By using a detachable auxiliary barrier slide, the cell-containing / cell-free hydrogel 6 can be formed in a fixed space in one step, with a smooth interface and accurate positioning, which improves the repeatability and success rate of experiments.

[0074] Meanwhile, the removable barrier 7 forms a confined space at the bottom of the culture dish, allowing the hydrogels containing cells and those without cells to form separately, preventing the mixing of gels of different components, and achieving stable and controllable interface positions.

[0075] To reduce cross-contamination and operation time in three-dimensional experiments, dual-region hydrogel molding can be completed under normal aseptic conditions, eliminating the need for repeated disassembly and assembly of devices during the initial hydrogel setting period, thus significantly reducing the risk of contamination and improving experimental efficiency.

[0076] The boundary barrier mechanism can be fixed to the main body of the dish 1 by means of a slot, groove, magnetic limit or other means and can be detached to ensure stable adhesion during the glue injection stage. It can be removed after the first part of the gel has initially cured without disturbing the gel.

[0077] Optically stable fluorescent positioning lines 4 are set on the bottom slide 2 of the dish to visualize the migration interface. Simultaneously, the fluorescent positioning lines 4 are used to calibrate the spatial position of the hydrogel interface and serve as a reference for measuring cell migration direction and distance. Through photolithography / metal mask patterning, silanization surface modification, and covalent coupling of NHS-activated fluorescent dyes, fluorescent molecules or dyes are covalently fixed to the slide surface, forming fluorescently labeled lines with constant width and accurate position. These lines are used for real-time observation of cell migration distance and direction and subsequent quantitative analysis, ensuring that the fluorescent positioning lines 4 have stable width, fixed position, and do not detach or diffuse.

[0078] Dual-region hydrogel molding is more reliable, uniform, and reproducible. Existing technologies rely on manual cutting and other operations, which can easily lead to deviations in interface position and geometry. This application employs a detachable auxiliary barrier 7 that fits into the culture dish body, creating a fixed space for region separation before gelation. This ensures consistent interface position, linearity, and interface width in each molding process, significantly improving experimental repeatability.

[0079] To eliminate the procedural risks during the initial setting stage of hydrogels, existing technologies require rapid operation during this phase, otherwise gel tearing, mixing, or contamination are likely to occur. This application achieves separation of the two sides in one step using a fixed barrier structure, eliminating the need for complex operations during the initial setting period and fundamentally reducing experimental failure rates and contamination risks.

[0080] This application aims to visualize and directly image the 3D migration interface. In existing technologies, the cell migration interface is not visible in confocal imaging and can only be inferred from cell distribution. This application sets fluorescent positioning lines 4 on the surface of the slide substrate 2, ensuring stable position through covalent chemical fixation. This allows the hydrogel interface to be directly imaged during imaging, providing a spatial reference and achieving absolute interface positioning. In existing technologies, the invisible interface leads to inconsistent analytical standards among different experimenters, resulting in significant deviations in results. In this application, the fluorescent positioning lines 4 are imaged simultaneously with the cell signal, allowing direct measurement of the cell's position and distance relative to the interface, eliminating subjective human judgment and enabling data quantification and automated analysis.

[0081] The integrated setup for three-dimensional culture, interface calibration, and confocal imaging currently requires multiple independent devices or manual labeling to complete the culture and imaging processes. This application integrates a hydrogel separator and a fluorescent localization labeling structure within the confocal culture dish, allowing all experimental steps to be performed within the same container, avoiding physiological disturbances and mechanical damage caused by sample transfer. The covalently immobilized layer, formed using photolithography / silanization / NHS-fluorescent molecule coupling, remains stable over long periods under culture medium, PBS, hydrogel, and laser irradiation conditions, without affecting the focal length and numerical aperture matching of the confocal microscope.

[0082] It should be noted that, where there is no conflict, the features in the embodiments of this application can be combined with each other.

[0083] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An experimental method for three-dimensional cell migration in hydrogels, characterized in that, Includes the following steps: A confocal culture dish was prepared, and a fluorescent positioning line was formed on the bottom of the confocal culture dish. The fluorescent positioning line was used for interface reference positioning. A boundary barrier mechanism is installed in a confocal culture dish, and a hydrogel is formed in the partitioned area of ​​the confocal culture dish through the boundary barrier mechanism. The hydrogel includes cell-containing hydrogels and cell-free hydrogels corresponding to the partitioned areas. Culture medium is added to the upper space of the hydrogel to carry out three-dimensional cell culture in the hydrogel, and cell migration is completed during the culture process. The confocal culture dish was placed on the confocal microscope platform, and the interface position was determined by the fluorescence positioning line. The cell distribution position was collected according to the time series for subsequent migration trajectory and distance measurement.

2. The experimental method according to claim 1, characterized in that, The confocal culture dish includes a dish body, a glass slide base plate, and a dish lid. The dish body is constructed as an annular dish body with annular steps inside. The sink space at the bottom of the annular steps constitutes a hydrogel space, and the remaining space of the annular dish body constitutes a culture medium space. The fluorescent positioning line is formed on the glass slide base plate of the hydrogel space. The bottom of the outer wall of the dish has a mating step, and the dish lid is attached to the mating step.

3. The experimental method according to claim 2, characterized in that, The dividing barrier mechanism includes a barrier sheet, and a positioning and limiting part is provided on the annular dish body. The barrier sheet is detachably installed in the positioning and limiting part and the barrier sheet abuts and adheres to the glass slide base plate. The edge of one side of the barrier corresponds vertically to the fluorescent positioning line.

4. The experimental method according to claim 2, characterized in that, The formation of the fluorescent positioning line includes: cleaning the glass slide substrate, constructing the surface pattern of the fluorescent positioning area, fixing the fluorescent molecules / dye, rinsing and drying; The cleaning of the glass slide base plate is used to remove organic contaminants and expose the glass to silanol groups; A surface pattern is constructed for the fluorescent localization region to expose the slit region corresponding to the fluorescence. Immobilized fluorescent molecules / dyes are used to bind fluorescent molecules or fluorescent dyes to a glass slide substrate.

5. The experimental method according to claim 4, characterized in that, The fluorescent positioning area can be patterned and fixed through photolithography / metal masking, micro-contact printing, inkjet printing, laser patterning, screen printing, plasma patterning, imprinting, or photosensitive polymer exposure and development. The binding of fluorescent molecules to the surface of the glass slide substrate is achieved through silanization-NHS coupling, glutaraldehyde crosslinking, acrylamide grafting, polymer brush fixation, or charge adsorption to enhance covalent end-capping. Fluorescent molecules / dyes, including organic fluorescent dyes, quantum dots, self-luminescent nanomaterials, fluorescent polymers, or embedded fluorescent microstructures, are used as imaging calibration signals.

6. The experimental method according to claim 5, characterized in that, After the silanol groups on the slide substrate are exposed, a metal mask with slits is placed over the surface of the slide substrate to expose the slit area corresponding to the fluorescent positioning line. Add silane coupling agent ethanol solution dropwise and react to form an amino functional group layer in the exposed area of ​​the slit; Remove the metal mask and clean with anhydrous ethanol; An organic fluorescent dye solution was prepared, dropped onto the surface of a glass slide substrate, and incubated at room temperature to achieve amide bond coupling and fixation of the fluorescent dye with amino groups; Fluorescent positioning lines of constant width were obtained by rinsing with phosphate buffer solution and air drying.

7. The experimental method according to claim 5, characterized in that, The process of forming a hydrogel includes: Install the barrier sheet on the positioning and limiting part of the dish body, and make the bottom surface of the barrier sheet fit tightly against the glass slide base plate; The cell-containing hydrogel precursor solution is injected into the pre-defined septum of the barrier sheet, and gelled after being filled evenly. Remove the barrier sheet after the hydrogel has initially solidified; Inject cell-free hydrogel precursor solution into the blank partition region and complete gelation.

8. The experimental method according to claim 3, characterized in that, The positioning and limiting part includes a card slot, a slot, or a magnetic component; The barrier plate is fixedly connected to the dish body via a slot or groove; The barrier sheet is magnetically attached to the dish body. The barrier sheet is elastically clamped or adhered to the dish body.

9. The experimental method according to claim 3, characterized in that, The barrier sheet is made of glass, polymer, metal, or a hydrophobic coating material.

10. The experimental method according to claim 3, characterized in that, The fluorescent positioning lines include single dividing lines, multiple parallel lines, intersecting lines, ring marks, serrated marks, cross positioning marks, or dot matrix marks.

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