Manufacturing method of pathological tissue microarray chip of 3D cell model

Abstract

The embodiment of the invention discloses a manufacturing method of a pathological tissue microarray chip of a 3D cell model, a culture chip for preparing the 3D cell model is adopted for manufacturing, the culture chip is dried after being subjected to high-temperature and high-pressure sterilization, and low-adhesion treatment is performed for standby application; selecting a cell sample with a good growth state, and culturing a 3D cell model by utilizing the treated culture chip and the cell sample; directly performing embedding pretreatment on the 3D cell model in the culture chip to obtain a pre-embedded block comprising the 3D cell model; carrying out sample embedding treatment on the pre-embedded block by adopting OCT (Optical Coherence Tomography) glue to obtain a sample embedded block; and carrying out frozen section operation on the sample embedding block by adopting a freezing slicer to obtain the pathological tissue microarray chip. The steps of sample collection, transfer, centrifugation and the like can be omitted, the problems of sample loss, damage and the like are effectively avoided, the slicing operation is simplified, and the slicing efficiency is improved; besides, by utilizing array positioning of the culture micropores, the conditions of sample aggregation, deformation, extrusion damage, overlapping shielding and the like are avoided, and the requirements of visualization, data acquisition and statistics, spatial positioning and the like of sample monomers are met.

Description

Technical Field

[0001] This application relates to the field of bioculture technology, and in particular to a method for fabricating a 3D cell model pathological tissue microarray chip. Background Technology

[0002] With the surge in demand for precision medicine and personalized drug screening, 3D (three-dimensional) cell models, due to their high degree of simulation of the in vivo tissue microenvironment, such as cell-cell interactions, oxygen gradient distribution, and drug permeation kinetics, have become a key carrier for tumor drug sensitivity testing, toxicity assessment, and disease mechanism research. This helps reduce reliance on animal experiments, thereby avoiding interspecies differences and limitations. 3D cell models are mainly prepared through two channels: cell lines and organoids. 3D cell models are either 3D organoids or 3D cell spheres. However, current technologies only focus on pathological sections of organoids, lacking integration with cell lines, especially tumor cells, and cultured 3D cell models. This presents several technical bottlenecks in achieving large-scale, standardized pathological analysis of 3D cell models. Specific existing solutions can be found in the organoid tissue frozen section embedding kit and embedding method (application number CN202110611852.4) and the histopathological embedding method suitable for 3D culture models and cytology samples (application number CN202311680969.3).

[0003] Regarding cell lines, they are mostly cultured in a 2D adherent state. Under normal conditions, they cannot spontaneously form 3D cell spheroids. Scaffold-free culture (such as the hanging drop method and ultra-low adsorption culture plates) and scaffold-assisted culture (such as hydrogel microspheres and matrix gel) are needed to force cell clusters to grow into spheroids. However, the spheroidization ability of different cell lines varies greatly and is highly dependent on the number and function of tumor stem cells. Their tolerance and response to different culture methods also differ. The prepared cell spheroids are small in size, usually only tens to hundreds of micrometers, and of poor quality, often exhibiting loose spheroids and central necrosis, with significant batch-to-batch variability. Currently, most cell lines are prepared into 3D cell spheroids by embedding them in scaffold materials (such as matrix gel) in plate culture containers (such as well plates and culture dishes). The appearance, size, morphology, biological characteristics, and spatial location of these spheroids are largely heterogeneous, leading to differences in sample biological characteristics, data quality, and statistical analysis, resulting in decreased throughput and poor visualization. When cell suspensions are mixed with scaffold materials for seeding, the resulting cell spheres exhibit random morphology and size due to cell proliferation or cell cluster fusion. The varying initial cell numbers and fluctuating culture conditions lead to significant differences in self-organization behavior, internal structure, and biological function development. Furthermore, the random dispersion of seeded cells within the gel material results in random cell sphere positioning, hindering observation, evaluation, and collection. These methods have low compatibility with traditional embedding and sectioning techniques, lacking suitable embedding molds and presenting considerable difficulties in sample processing. Embedding samples in gel scaffolds requires recovery and removal of the gel material; due to random growth and positioning, pretreatment cannot guarantee sample aggregation on a single plane, resulting in only a few or no samples being sectioned. Moreover, centrifugation to a single plane before histopathological sectioning can lead to excessive sample aggregation, overlap, and even breakage. Repeated centrifugation and manipulation can also damage and lose samples, ultimately leading to experimental failure.

[0004] Meanwhile, existing technologies lack integration with cell lines, particularly 3D cell spheroids from tumor cell culture, and fail to consider the organic unity of sample preparation and post-processing. Sample collection may require the use of digestive enzymes and cell recovery solutions to dissolve the matrix gel, followed by multiple centrifugations and washes, a time-consuming process that can lead to sample loss and structural damage. Furthermore, cell culture plates are unsuitable for direct embedding; existing embedding molds and methods require multiple transfers or component manipulations, making the process complex and potentially causing sample damage and loss, ultimately leading to experimental failure. Since samples rely on centrifugation to be positioned on the same horizontal plane, their random locations are prone to overlap and obstruction, hindering pathological visualization, data acquisition, and statistical analysis, and failing to ensure that sections meet the diverse experimental requirements of immunofluorescence protein detection, in situ gene detection, and special staining. The random spatial arrangement of cell spheroid monomers results in inconsistent section sizes and morphologies, reduced effective throughput, decreased effective data, and unfriendly visualization, impacting data acquisition. When facing specialized analyses such as drug sensitivity testing and toxicity assessment, the acquired data is insufficient for comparative analysis between sample monomers and groups, further hindering in situ information tracing and spatial proteomics analysis.

[0005] In the case of organoids, organoids utilize tissue-derived cells, guided by in vitro cell differentiation induction (e.g., pluripotent stem cells) or culture condition induction (e.g., growth factors, matrix gel) to induce self-organization and form 3D cell clusters, which more closely resemble the physiological state. However, the culture cycle is long and the technical threshold is high. Currently, most organoids (especially tumor organoids) are prepared by embedding 3D organoid spheres in slab culture containers using scaffold materials. Their appearance, size, morphology, biological characteristics, and spatial location are mostly heterogeneous, leading to differences in sample biological characteristics, data quality, and statistical analysis. When cell suspensions are mixed with scaffold materials for seeding, cell proliferation or cell cluster fusion results in random morphology and size of the formed cell spheres. Due to different initial cell quantities and fluctuations in culture conditions, the formed cell spheres vary significantly in self-organization behavior, internal structure, and biological functional development. Furthermore, the random dispersion of seeded cells in the gel material results in random positioning of the prepared cell spheres, leading to inconsistent cross-sectional size, uneven morphology, reduced effective throughput, poor visualization, and impact on data acquisition. Organoid culture is characterized by long cycles, high costs, small scale, and small cell model size, limiting the number of samples available for histological analysis and increasing the difficulty of histopathological embedding. After fixation, organoid cell spheres are mostly transparent, and their small number and size make it difficult to visually determine whether they are lost during collection, transfer, and centrifugation. Proper sample handling during pathological embedding and sectioning also increases the difficulty of creating pathological tissue arrays. In organoid drug sensitivity testing and other analyses, organoids are often cultured in 96-well or even 384-well plates. The sample size per well is usually small, and sometimes it is necessary to compare differences between wells, groups, and even individual 3D cell models. Due to the numerous and difficult steps involved, and the low data usability, it is not conducive to evaluating drug efficacy using histological microscopic analysis. While centrifugation can be used to bring samples to a single plane before histopathological sectioning, centrifugation easily leads to sample aggregation, overlap, and even damage. The data obtained is also insufficient to meet the needs of comparative analysis between sample monomers and groups in drug sensitivity, toxicity and other testing and evaluation, and is not conducive to in-situ information tracing and spatial proteomics analysis.

[0006] Furthermore, existing technical solutions focus on pathological sections of organoids, concentrating solely on pathological operations after the preparation of 3D organoid spheres. They fail to organically integrate sample preparation and post-processing. Sample collection requires dissolving the matrix gel with digestive enzymes and cell recovery fluid, followed by multiple centrifugations and washes, a time-consuming process that may result in sample loss and structural damage. Cell culture plates are unsuitable for direct embedding, and the volume and number of organoids cultured on different plates vary significantly. While similar solutions disclose different embedding molds and methods, improving the integrity of organoid structures to some extent, they still require multiple transfers, centrifugations, sedimentation, or component manipulations, making them relatively complex and prone to sample aggregation, deformation, crushing, and overlapping. Since organoid samples rely on centrifugation to be positioned on the same horizontal plane, their random locations are prone to overlap and occlusion, hindering pathological visualization, data acquisition, and statistical analysis. This also fails to ensure that sections meet the diverse experimental requirements for immunofluorescence protein detection, in situ gene detection, and special staining. The random spatial arrangement of organoids leads to inconsistent section sizes and morphologies, reducing effective throughput and data volume, resulting in poor visualization and impacting data acquisition. When faced with professional analyses such as drug sensitivity testing and toxicity assessment, the data obtained is insufficient to meet the needs of comparative analysis between individual samples and groups, and is even less conducive to in-situ information tracing and spatial proteomics analysis. Summary of the Invention

[0007] To address the aforementioned issues, this application proposes a method for fabricating a 3D cell model pathological tissue microarray chip.

[0008] In a first aspect, a method for fabricating a pathological tissue microarray chip for a 3D cell model, comprising: fabrication using a culture chip for preparing a 3D cell model, including:

[0009] The cultured chips were sterilized under high temperature and high pressure, dried, and then treated with low adhesion before being put into use.

[0010] Select cell samples in good growth condition, and use the processed culture chip and cell samples to culture a 3D cell model;

[0011] The 3D cell model was pre-embedded in the culture chip to obtain a pre-embedded block containing the 3D cell model.

[0012] The pre-embedded blocks were embedded using OCT adhesive to obtain the embedded sample blocks;

[0013] The embedded blocks of the samples were frozen and sectioned using a cryostat to obtain a pathological tissue microarray chip.

[0014] In an optional implementation, the culture chip undergoes a low-adhesion treatment, including:

[0015] Fill the culture wells, reservoir wells, and connection channels of the culture chip with anti-adhesion washing solution and immerse them;

[0016] After aspirating the anti-adhesion washing solution, rinse the culture chip with PBS solution at least once before use.

[0017] In an optional implementation, culturing a 3D cell model includes:

[0018] Select the appropriate digestion solution according to the cell sample type, count and adjust the cell quantity, centrifuge and collect the cell suspension.

[0019] Add cell suspension to the culture wells of the processed culture chip and place the culture chip in an incubator until the cells in the cell suspension settle and are evenly distributed into the culture microwells. Remove excess liquid and cells that have not entered the culture microwells, except for the liquid in the culture microwells.

[0020] Add culture medium to the culture wells, place them in an incubator, and change the medium regularly until the cells in the culture wells aggregate and grow into a 3D cell model.

[0021] In an optional implementation, when the 3D cell model is an organoid, the process further includes the following step before "adding culture medium to the culture wells":

[0022] Add the required concentration of matrix gel to the culture wells and spread it evenly over the culture microwells at the bottom of the wells until it solidifies in the incubator.

[0023] In an optional implementation, an embedding pretreatment is performed, including:

[0024] Discard the culture medium from the culture wells of the culture chip, rinse the culture wells at least once with PBS solution, then add eosin staining solution to the culture wells to pre-stain the 3D cell model, and then discard the eosin staining solution.

[0025] Cool the culture chip to equilibrate with the environment inside the cryosection chamber, and slowly add OCT glue from the side of the culture well until the culture well is filled.

[0026] After the OCT adhesive has completely solidified, a pre-embedded block is formed, which is then peeled off from the culture chip.

[0027] In an optional implementation, after rinsing the culture wells with PBS solution at least once, the following is also included:

[0028] Tissue fixative was added to the culture wells to fix the 3D cell model, and then the tissue fixative was aspirated.

[0029] In an optional implementation, sample embedding is performed, including:

[0030] Flip the pre-embedded block and fix it onto the sample holder;

[0031] The space around the pre-embedded block is filled with OCT glue, and then a second embedding is performed using OCT glue to obtain the sample embedding block.

[0032] In an optional implementation, "performing cryosections on the embedded sample blocks using a cryostat" includes:

[0033] Place the sample embedding block into the sample head of the cryostat, adjust the sample head to trim and level the sample embedding block until a 3D cell model stained with eosin appears.

[0034] The embedded blocks of the exposed stained 3D cell model were frozen sectioned to obtain a pathological tissue microarray chip.

[0035] In an optional implementation, the culture chip includes a chip body, culture wells, liquid storage wells, and connection channels;

[0036] The chip body is provided with culture wells, connection channels and liquid storage wells. Each culture well is connected to an adjacent liquid storage well through a connection channel. The number of connection channels corresponds to the number of culture wells. The depth of the culture well is greater than or equal to the depth of the liquid storage well, and the depth of the liquid storage well is greater than the depth of the connection channel.

[0037] Both the culture well and the liquid storage well have openings at the top. The surface of the culture well opening and the surface of the liquid storage well opening are located on the same side of the chip body. The bottom of the culture well has culture micropores arranged in an equally spaced array. The culture micropores are concave spherical surfaces.

[0038] In an optional embodiment, the number of culture microwells provided in each culture well is any one or more of 12 wells, 16 wells, 24 wells, and 40 wells.

[0039] The embodiments of this application have the following beneficial effects:

[0040] This application discloses a method for preparing pathological tissue microarray chips using 3D cell models. The method involves using a culture chip for preparing the 3D cell model. The culture chip is sterilized under high temperature and pressure, dried, and then subjected to low-adhesion treatment before use. Cell samples in good growth condition are selected, and the 3D cell model is cultured using the treated culture chip and the cell samples. The 3D cell model is directly embedded in the culture chip to obtain a pre-embedded block containing the 3D cell model. The pre-embedded block is then embedded using OCT gel to obtain a sample embedding block. The sample embedding block is then frozen and sectioned using a cryostat to obtain the pathological tissue microarray chip. For cell lines, this application utilizes the culture micropore structure to promote cell aggregation and growth, preparing relatively uniform cell spheres with a relatively dense structure and rich self-organizing characteristics under scaffold-free culture conditions. For organoids, the culture micropore structure is used to prepare relatively uniform organoid spheres in morphology and function, and the cells are covered but not completely embedded with matrix gel for subsequent processing. Furthermore, by unifying the procedures for cell culture and pathological embedding, the culture chip can be directly used as an embedding mold. After preparing a 3D cell model in the culture chip, sample pretreatment and embedding of frozen sections can be completed directly, eliminating steps such as sample collection, transfer, and centrifugation. This effectively prevents sample loss and damage. The physical structure of the culture chip ensures that the sliced ​​samples are always on the same horizontal plane, simplifying the slicing operation and improving slicing efficiency. In terms of results, by utilizing the array positioning of the culture microwells, micron-sized 3D cell models can be made into pathological tissue microarray chips, avoiding sample aggregation, deformation, compression damage, and overlapping occlusion. This facilitates the visualization of individual samples, data acquisition and statistics, spatial positioning, and comparison between individual samples. Attached Figure Description

[0041] To more clearly illustrate the technical solution of this application, the accompanying drawings required in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and therefore should not be regarded as a limitation on the scope of protection of this application. In the various drawings, similar components are numbered similarly.

[0042] Figure 1 Schematic diagrams of culture chip structure one and culture chip structure two in this application are shown;

[0043] Figure 2 This is a schematic diagram of the configuration of the culture micropores used in this application;

[0044] Figure 3 This is a schematic diagram of the operation flow of Embodiment 1 of this application;

[0045] Figure 4 This is a schematic diagram of the operation flow of Embodiment 2 of this application;

[0046] Figure 5 This is a schematic diagram of the key steps in the operation of slicing human cervical cancer organoids and human lung cancer organoids in the operation process of Embodiment 1 of this application;

[0047] Figure 6 These are microscopic images of human cervical cancer organoids cultured in Example 1 of this application, along with their pathological tissue microarray chip images and pathological tissue microarray chip images of cultured human lung cancer organoids.

[0048] Figure 7 These are continuous micrographs of the tumor organoid culture process in the culture chip, taking human colon cancer tissue sample organoids as an example, in the operation flow of Embodiment 1 of this application;

[0049] Figure 8 This is a microarray image of 3D human liver cancer cells Huh7 cultured in Example 2 of this application and their pathological tissue.

[0050] Figure 9 This is a pathological tissue microarray chip image of the human colon cancer organoid spheres prepared using the 16-well specification in Embodiment 3 of this application;

[0051] Figure 10 This is an immunofluorescence staining image of a pathological tissue microarray chip made from human cervical cancer organoid spheres prepared with a 12-well configuration, as described in Example 4 of this application.

[0052] Figure 11 This is a schematic diagram of the operation process of Comparative Example 1 of this application;

[0053] Figure 12 This is a schematic diagram of the operation process of Comparative Example 2 of this application;

[0054] Figure 13 This is a schematic diagram of the operation process of Comparative Example 3 of this application;

[0055] Figure 14 These are microarray images of pathological tissues made from organoids cultured in Comparative Examples 1, 2 and 3 of this application.

[0056] Figure 15 These are microscopic images of cell spheres and microarray results of pathological tissues from Comparative Example 4 of this application;

[0057] Reference numerals: 1. Chip body; 2. Culture well; 3. Culture microwell; 4. Liquid storage well; 5. Connecting channel. Detailed Implementation

[0058] 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. In order to more clearly demonstrate the implementation steps and advantages of the invention, specific implementation methods will be described below with reference to the drawings.

[0059] To address the systemic bottlenecks in current 3D cell models for pathological research, this application proposes a method for fabricating a microarray chip of pathological tissue based on 3D cell models. This microarray chip comprises micron-sized pathological tissue slices, belonging to the interdisciplinary field of pathological analysis and biomedical engineering. It mainly involves the preparation of 3D cell model culture, the fabrication of pathological slices from small amounts of tissue, and the fabrication technology of micron-sized tissue culture chips. The 3D cell model is a 3D organoid or a 3D cell sphere. By using an appropriate culture chip, relatively uniform morphological and structurally arranged, arrayed 3D cell sample monomers are prepared. Pre-treatment such as sample staining, pre-cooling, and OCT (Optimal Cutting Temperature Compound) embedding are directly performed within the chip without recovery or centrifugation, enabling the fabrication of frozen sections and creating a micron-sized pathological tissue chip with an array of samples. This chip can be used for subsequent analyses such as routine pathology and spatial proteomics. The method for fabricating the microarray chip of pathological tissue based on 3D cell models in this application utilizes a culture chip for 3D cell model fabrication and specifically includes the following steps:

[0060] S100: The culture chip is sterilized by high temperature and high pressure, dried, and then treated with low adhesion before use.

[0061] First, the culture chip will be prepared. The appropriate microwell size will be selected based on requirements. The culture chip will be fabricated using PDMS (Polydimethylsiloxane Molding), sterilized by high temperature and autoclaving, and then dried for later use. To prevent cells from adhering to the microwells of the culture chip, a low-adhesion treatment will be performed before use. In other words, the culture wells, reservoir wells, and connecting channels of the culture chip will be filled with anti-adhesion washing solution. This involves soaking the microwells in the anti-adhesion washing solution for at least 12 hours to reduce cell adhesion and promote cell aggregation into spheres. The soaking process also eliminates any air bubbles that may be present in the microwells. If air bubbles remain in the microwells, they can be manually removed using a pipette tip to ensure sufficient contact between the culture medium and the microstructure of the culture chip. After treatment, the anti-adhesion washing solution will be discarded, and the microwells will be rinsed at least once with PBS (phosphate buffered saline) solution, or 2-3 times if necessary, to keep the microwells moist.

[0062] The culture chip used above for preparing 3D cell models can be used as a cell culture chip for 3D cell models, or as a pre-embedding mold for frozen tissue sections. The culture chip includes a chip body, culture wells, reservoir wells, and connecting channels. The number of connecting channels corresponds to the number of culture wells, the depth of the culture wells is greater than or equal to the depth of the reservoir wells, and the depth of the reservoir wells is greater than the depth of the connecting channels. The chip body has at least one culture well and at least one reservoir well, with a reservoir well next to each culture well for storing culture medium. Each culture well is connected to an adjacent reservoir well via a connecting channel. The shape, size, and volume of the culture wells and reservoir wells can be adjusted as needed, as can their number and arrangement; there are no restrictions on this, such as the culture wells and reservoir wells being cylindrical, cuboid, or similar shapes. Both the culture wells and the reservoir wells have openings at the top, and the surfaces of the culture well openings and reservoir well openings are located on the same side of the chip body. The bottom of the culture wells features an equally spaced array of micropores, called culture micropores, which are concave spherical in shape. Optionally, the number, diameter, depth, density, and other parameters of the culture micropores can be adjusted as needed to prepare cell culture chips of appropriate specifications for use. Figure 2 As shown, when the culture wells are cylindrical, there are four commonly used specifications. Each culture well contains one or more of the following: 12 wells with a diameter of 1000 μm; 16 wells with a diameter of 800 μm; 24 wells with a diameter of 600 μm; or 40 wells with a diameter of 400 μm. The wells are evenly spaced at 400 μm intervals and arranged in an array, with a depth of 400 μm for each well.

[0063] Specifically, the culture chip used is also the carrier of the pre-embedded mold, and there are two commonly used structures. The first structure is as follows: Figure 1 As shown in the diagram, the chip includes a chip body 1, culture wells 2, culture micropores 3, liquid storage holes 4, and connecting channels 5. The chip body has multiple culture wells, with adjacent wells grouped into a group. Any well in each group serves as a liquid storage hole, and the two wells in each group are connected by the connecting channels. The chip body 1 is a cuboid with horizontal top and bottom surfaces. It has multiple culture wells 2, for example, eight wells 2 in a row. The position of the culture wells 2 can be set according to requirements, such as at the center line of any side of the chip body 1. The size of the chip body 1 can be adjusted according to the number and position of the culture wells 2. The culture wells 2 can be top-opening cylinders with an inner diameter of 6.5 mm and a depth of 6 mm. Multiple culture micropores 3 are located on their inner bottom surface. The culture micropores 3 are arrayed concave spherical surfaces, and can be constructed using methods such as... Figure 2The four commonly used sizes shown are: 12 wells (1000 μm diameter), 16 wells (800 μm diameter), 24 wells (600 μm diameter), and 40 wells (400 μm diameter). The depth of the culture wells 3 in each size is 400 μm. The culture wells 3 can also use any one or more combinations of the four commonly used sizes. Connecting channels 5 connect two adjacent culture wells 2, and the depth of the connecting channels 5 is less than the depth of the culture well 2. If only one of the two connected culture wells 2 is used, the other culture well 2 can be used as a substitute for the reservoir well 4. Adjacent culture wells can be used as reservoir wells when no cells are seeded. The eight culture wells 2 are divided into four groups, with two culture wells 2 in each group interconnected. A cross-sectional view of any group of reservoir wells, connecting channels 5, and culture wells 2 is shown below. Figure 1 As shown in AA of structure 1, the bottom surface of the four sets of culture wells 2 can be provided with four commonly used culture microwells 3, and the specifications and number of culture microwells 3 can also be adjusted as needed.

[0064] The second structure of the chip is as follows Figure 1 As shown in Structure 2, it includes a chip body 1, culture wells 2, culture microwells 3, liquid storage wells 4, and connecting channels 5. Compared to the first structure, the second structure can use only one type of culture microwell 3, such as 16-well culture microwells with a diameter of 800μm. A row of five groups of culture wells 2, connecting channels 5, and liquid storage wells 4 is located at the center of the chip body 1. The spacing between each group of culture wells 2, connecting channels 5, and liquid storage wells 4 can be adjusted according to requirements, and the size of the chip body 1 can also be adjusted according to the number and position of the culture wells 2 and liquid storage wells 4. A cross-sectional view of any group of culture wells 2, connecting channels 5, and liquid storage wells is shown below. Figure 1 As shown in BB of structure two, the liquid storage hole 4 is located beside the culture hole 2, with an open top surface, and can be cylindrical. The inner diameter of the cylindrical liquid storage hole 4 can be 5mm, the depth can be 3mm, the distance between it and the culture hole 2 can be 2.5mm, and it is connected to the culture hole 2. A connecting channel 5 connects the culture hole 2 and the liquid storage hole 4, and the depth of the connecting channel 5 is less than that of the culture hole 2.

[0065] S200: Select cell samples in good growth condition and use the processed culture chip and cell samples to culture a 3D cell model.

[0066] Select well-grown cell samples cultured under conventional methods, i.e., organoid or cell spheroid samples in good condition. Choose an appropriate digestion solution according to the type of cell sample, following the passage procedure for different sample types. If matrix gel is present, remove it. Count and adjust the cell volume, then centrifuge to collect the cell pellet. Resuspend the cells in culture medium to a high-density cell suspension. Add the cell suspension to the wells of the processed culture chip, ensuring the cell suspension covers the entire bottom of the well. Place the culture chip in an incubator and allow it to stand for an adjustable time. Once the cells have settled and evenly distributed into the culture wells, use a pipette tip to aspirate excess liquid and any cells that have not entered the wells.

[0067] In addition, during organoid culture, a suitable concentration of matrix gel or other gel material required for organoid culture is added to the culture wells, evenly covering the bottom of the culture microwells, and then allowed to solidify in an incubator. The gel material is well-known to those skilled in the art and will not be described further here. The culture microwells are independent of each other, and the solidification time can be adjusted as needed. Cell samples aggregate into spheres in the culture microwells, gradually growing into relatively uniform 3D organoid spheres, evenly distributed within the arrayed culture microwells. When culturing cell spheres with cell lines, it is not necessary to add matrix gel to the culture microwells; the above-mentioned matrix gel addition step is skipped. The culture microwells are independent of each other, and the cell lines will autonomously aggregate into 3D cell spheres within the cell microwells, gradually growing into relatively uniform spheres, evenly distributed within the arrayed culture microwells. Add culture medium to the culture microwells of the above-mentioned cultured organoids or cell lines, place them in an incubator for normal culture, and change the medium regularly, such as once a day, until the cells in the culture microwells aggregate and grow into a 3D cell model that meets the requirements, thus obtaining 3D organoid spheres or 3D cell spheres.

[0068] The above method allows for the simple and rapid preparation of corresponding 3D cell models from organoid and cell line-derived cell samples. The physical structure of the culture micropores promotes cell aggregation into spheres, resulting in relatively uniform morphology and richer self-organizing characteristics. After being used for drug sensitivity testing, toxicity assessment, and other experiments, the prepared cell models can be used as microarray samples for pathological tissues.

[0069] Specifically, the 3D cell model culture methods in this application include Method 1 (organ culture) and Method 2 (cell line culture).

[0070] Method 1: Organ culture steps are as follows Figure 3Steps ② and ③ are shown: (1) After rinsing the culture chip for later use, select organoids that are growing well in the culture plate, and digest them with a suitable digestion solution according to the sample requirements. If there is matrix gel, remove the matrix gel and collect the cells by centrifugation to obtain a cell suspension. Resuspend the cell suspension in the culture medium to obtain a high-density suspension. (2) Add the above cell suspension to culture well 2, which can be 50 μl / well, ensuring that the liquid surface covers the bottom of the entire well. Let it stand in the incubator for 15 minutes until the organoids settle and are evenly distributed into the culture microwells 3. Use the pipette tip to aspirate excess liquid and organoids that have not entered the culture microwells. (3) Mix the matrix gel and culture medium at a volume ratio of 1:2 and add it to culture well 2, spreading it evenly to cover the bottom and all culture microwells 3. Let it solidify in the incubator for 15 minutes. (4) Add culture medium to the storage well 4 until the culture well 2 is filled with culture medium. Place the culture chip in the incubator for normal culture. Change the medium 1-2 times a day until the organoids in the culture microwell 3 aggregate and grow into a 3D cell model that meets the requirements. If it can be cultured for 7 days, 3D organoid spheres can be obtained in the culture microwell.

[0071] In this application, the primary culture steps for organoids can be as follows: 1. Obtain tumor tissue samples that meet ethical and preservation standards from a hospital tumor biobank, and culture organoids from the freshly collected tumor tissue samples. Rinse the sample tissue thoroughly with physiological saline, trim away necrotic and contaminated edges around the specimen with tissue scissors, and then rinse thoroughly with physiological saline again. Subsequently, cut the tumor sample into fragments of 2 mm in size using tissue scissors. 3 1. Transfer tissue fragments to primary tissue digestion solution and digest at 37°C for 30-40 minutes until cell clusters or single cells are reached. Add basal culture medium, stop digestion, and then filter. 2. Centrifuge the filtered cell suspension to remove the supernatant, add 1 ml of culture medium, count and adjust the cell number (e.g., 10,000-30,000 cells / well for a 24-well plate). 3. Take an appropriate volume of cell suspension and centrifuge again to remove the supernatant. 4. Add a mixture of matrix gel and culture medium at a volume ratio of 1:2, seed 25 μL per well in a 24-well plate, and incubate for 15 minutes. 5. After curing, add organoid culture medium, change the medium every 3-5 days, and closely observe the growth of organoids daily until the experimental requirements are met.

[0072] Method 2: Cell line culture of cell spheroids - steps as follows Figure 4 As shown in steps ② and ③, specifically, step (1) in method one is replaced with: selecting adherent cells that are growing well on the culture plate, digesting them, centrifuging to collect them, and resuspending them in the culture medium to 1~2×10⁻⁶. 6Cells / mL, cell spheroid culture does not require the addition of matrix gel, and step (3) in Method 1 is not performed. Except for the above-mentioned distinguishing steps, the steps in Method 2 are the same as those in Method 1. When culturing cell lines, the medium can be changed once a day for 3 days until 3D cell spheroids are obtained in the culture microwells.

[0073] Step S300: The 3D cell model is pre-embedded in the culture chip to obtain a pre-embedded block containing the 3D cell model. The specific steps of the pre-embedding process are as follows:

[0074] like Figure 3 or Figure 4 As shown in steps ④ and ⑤, gently aspirate the culture medium from the culture wells. Since the matrix gel is not mixed with the organoids or cell spheres in the culture wells, if the organoids in the culture wells are covered with matrix gel, gently aspirate the matrix gel covering the culture wells. If no matrix gel is added when culturing 3D cell spheres in cell lines, this step can be skipped. Rinse the culture wells at least once with PBS solution to keep the 3D cell model in the culture wells undisturbed and without displacement. In addition, tissue fixative can be added to the culture wells to fix the 3D cell model. The tissue fixative can be 4% paraformaldehyde. Fixation before operation can ensure the stability of the cell model's morphology and structure. Then aspirate the fixative. The fixation time can be adjusted according to the actual situation, such as fixing for 10 minutes and then aspirating the fixative. Gently handle to keep the 3D cell model in the culture wells from shifting. Add eosin staining solution to the culture wells to pre-stain the organoid spheres or cell spheres. The amount of eosin staining solution can be 100 μl / well. The pre-staining time can be adjusted according to the actual situation, such as 10 minutes. Handle gently to keep the 3D cell model in the culture wells undisturbed and without displacement, and then aspirate the staining solution.

[0075] In addition, the culture chip containing the 3D cell model is placed in a cryostat to allow it to cool to equilibrium with the cryostat chamber environment. The placement time can be adjusted as needed; for example, the culture chip can be pre-cooled at 4 degrees Celsius for 10 minutes, or placed in the cryostat for 15 minutes to reach equilibrium. OCT glue is slowly added from the side of the culture wells of the culture chip until it is full, and then placed in the cryostat to allow it to solidify slowly. After the OCT glue has completely solidified, a pre-embedded block containing the 3D cell model (i.e., 3D organoid spheres or 3D cell spheres) is formed. The complete pre-embedded block is then peeled off from the culture chip, thus completing the cell sample extraction while maintaining the arrayed positioning of the culture microwells.

[0076] While ensuring the efficiency, quality, and uniformity of 3D cell model culture, the culture of 3D organoids or 3D cell spheres is unified with the corresponding pathological embedding operation process. The cell culture chip is directly used as a pathological embedding tool, that is, as a sample pretreatment and pre-embedding mold. This not only omits steps such as sample recovery, centrifugation, and transfer, but also reduces sample loss and damage risk, lowers the complexity of operation, and facilitates the visualization of individual samples through staining operations.

[0077] S400: The pre-embedded block is embedded using OCT adhesive to obtain the embedded sample block. The specific steps for sample embedding are as follows:

[0078] like Figure 3 or Figure 4 As shown in step ⑥, the detached pre-embedded block is flipped over so that the 3D cell model, i.e., the 3D organoid or cell spherical surface, is placed horizontally with its surface facing upwards and fixed on the frozen section sample holder. OCT glue is used to fill the space around the pre-embedded block to reinforce it, and then allowed to solidify. The pre-embedded block is then covered again with OCT glue to create a sample embedding block, thus completing the sample embedding, i.e., the secondary embedding. It is permissible to cover the pre-embedded block with OCT glue at least once.

[0079] S500: The sample embedding block is frozen and sectioned using a cryostat to obtain a pathological tissue microarray chip.

[0080] like Figure 3 or Figure 4 As shown in steps ⑦ and ⑧, following standard frozen sectioning procedures, the embedded block of the stained 3D cell model is placed in the sample head of the cryostat. The sample head is adjusted to trim and level the entire embedded block until eosin-stained arrayed sample dots are visible near the 3D cell model. In other words, since all samples are now on the same level, the sample head of the cryostat can be slightly adjusted to ensure that all stained samples are exposed simultaneously and can be excised. Frozen sectioning is then performed on the embedded block exposing the stained 3D cell model to obtain frozen sections, thus creating a micron-scale tissue array microarray chip for pathological tissue. The frozen sectioning procedure can be a standard procedure, well-known to those skilled in the art, and will not be described further here.

[0081] Furthermore, the aforementioned pathological tissue microarray chip can be stained with hematoxylin-eosin (HE) and the corresponding images can be acquired using a microscope. This pathological tissue microarray chip is suitable for histopathological analysis such as HE staining, immunohistochemistry, and immunofluorescence staining. The organoid or cell spheroid tissue microarray chip prepared in this application, i.e., the pathological tissue microarray chip, can provide more visualized 3D cell model slices for subsequent analysis, facilitate pathological detection and analysis, enable data acquisition and statistical comparison, and open up new possibilities for spatial proteomics research and clinical applications.

[0082] This application combines pathological frozen sectioning technology with 3D cell culture chips, utilizing the microstructure of the culture chip to ensure that cell samples are always on the same horizontal plane. This solves the problem that most existing technologies require centrifugation or other methods to position samples on the same horizontal plane. This not only simplifies the slicing process and effectively avoids potential sample loss and damage, but also reduces the probability of sample aggregation or overlap, thereby improving slicing efficiency while preserving sample integrity and more original data. Furthermore, this application utilizes the arrayed arrangement of microwells in the culture chip to spatially position organoids or cell spheroids growing within it. Through direct embedding and slicing operations, micron-scale pathological tissue microarray chips are fabricated, greatly improving slicing quality and efficiency.

[0083] The HE staining procedure in this application is as follows: The sample is fixed during pretreatment. Frozen sections are washed at least once with PBS solution, each time for the appropriate washing time (e.g., three times, 3 minutes each time). The sections are then immersed in hematoxylin staining solution for 5–10 minutes, followed by differentiation with 0.5% hydrochloric acid-ethanol solution for 1–5 seconds. Finally, the sections are treated with tap water for 5–10 minutes for inversion blueing. The sections are then immersed in eosin staining solution for 5–10 seconds. The stained sections are then sequentially placed in ethanol solutions of different concentrations for different times for dehydration treatment. For example, they can be placed in 95% ethanol solution for 3 seconds; 95% ethanol solution for 10 seconds; 100% ethanol solution for 5 minutes; and 100% ethanol solution for another 5 minutes. The dehydrated sections are then placed in xylene for immediate clearing treatment, which can be 5 minutes initially. After clearing, the sections were mounted with neutral resin, observed and collected under a microscope, and the acquired images were analyzed. The washing time, staining time, differentiation time, inverted blue treatment time, and initial staining time can all be adjusted according to requirements.

[0084] The steps for immunofluorescence staining are as follows:

[0085] Immerse the obtained frozen sections in PBS solution and wash on a shaker at least once, for a second time each time, for example, wash 3 times, 5 minutes each time, to remove embedding gel and other impurities from the sample. Immerse the washed sections in EDTA antigen retrieval buffer, perform antigen retrieval by water bath at the appropriate temperature for a third time, then allow to cool naturally and wash with PBS solution, for example, perform antigen retrieval by water bath at 90°C for 30 minutes, allow to cool naturally, and wash 3 times with PBS solution. Then, add Triton working solution to the sections for permeabilization, and wash with PBS solution at least once, for example, permeabilize for 10 minutes, and wash 3 times with PBS solution. Add BSA (bovine serum albumin) to the sections for the appropriate blocking time, such as 30 minutes. After discarding the blocking solution, add primary antibody working solution to cover the sample and incubate overnight in a humidified chamber at the preset temperature, such as 4°C, and then wash with PBS solution at least once, for example, 3 times. Add secondary antibody working solution to the sections and incubate at room temperature in a humidified chamber for 60 minutes, and then wash with PBS solution at least once, for example, 3 times. In addition, DAPI was added to the slides for counterstaining, followed by washing with PBS solution. For example, counterstaining for 10 minutes and washing with PBS solution three times was performed. The cleared slides were then mounted with neutral resin, and observed and collected under a microscope. The collected images were then analyzed.

[0086] Example 1

[0087] like Figure 3 As shown, this embodiment describes the fabrication method of a micron-scale pathological tissue microarray chip for 3D cell model using the method applied for. The method involves culturing, embedding, sectioning, and HE staining of human cervical cancer organoids and human lung cancer organoids.

[0088] This embodiment uses the first structure of the culture chip of this application. The culture chip of the first structure has four sizes of culture microwells, that is, there are four sets of culture microwells, reservoirs and connecting channels. In each set, only one well is seeded with cells, and the other well is used as a reservoir. In order to verify the universality of the culture chip, human cervical cancer organoids and human lung cancer organoids can be cultured simultaneously in 12-well, 16-well and 24-well sizes and in 40-well size on the same culture chip. After 7 days of culture, 3D organoid spherical models are obtained. The culture medium in the culture wells is aspirated, the matrix gel covering the culture microwells is aspirated, the samples in the culture wells are rinsed and fixed with paraformaldehyde, then stained with eosin, pre-cooled in a cryostat, and then pre-embedded with OCT gel. After solidification, the pre-embedded block with cell samples is carefully peeled off, then flipped and fixed on the sample holder, and the pre-embedded block is covered and embedded again with OCT gel. After the second embedding, the sample head is adjusted and the embedding block is leveled to completely cut out the sample points. Then, sectioning, HE staining and other operations can be performed.

[0089] like Figure 5 As shown, Example 1 uses the culture chip of this application to culture human cervical cancer organoids in three sizes (12-well, 16-well, and 24-well) of culture microwells and human lung cancer organoids in one size (40-well), simultaneously preparing two 3D organoid spherical models. During the culture process, the culture microwells are covered with matrix gel, which facilitates the removal of excess matrix gel during sample pretreatment, simplifying the washing steps. The organoids gradually grow into 3D cell models in the culture microwells, exhibiting relatively uniform morphology and size, and remaining on the same plane. The culture wells of the culture chip serve as pre-embedding molds, and both sample pretreatment and pre-embedding processes are completed within the culture wells of the culture chip, eliminating sample collection and transfer steps and maximizing sample slicing efficiency. Furthermore, as... Figure 5 As shown in steps ④, ⑥, and ⑦, after staining with eosin, the positioning of the 3D organoid model in the culture microwell, the stained 3D organoid sphere model after inversion, and the sample points removed after trimming the gel block following secondary embedding can all be manipulated with the naked eye. The sample position can be directly observed in key steps such as sample fixation, pre-staining, embedding, and sectioning, ensuring operational accuracy and greatly improving the operational experience.

[0090] like Figure 6 The illustration shows the cultivation of micron-scale 3D tumor organoids in Example 1, and the presentation of various sizes of pathological tissue microarrays using the slicing method of this application to display the 3D tumor organoids as tissue array chips. The organoids grow in the culture microwells into relatively uniform morphology and size 3D tumor organoids. Using individual spheres as the unit of analysis ensures both the cell population effect and facilitates the statistical, reproducible, and comparable data under relatively standardized conditions. The slicing method of this application greatly improves sample visibility, allows for statistical analysis at the individual 3D tumor organoid sphere level, facilitates inter-monolithic comparisons, and provides the possibility of acquiring spatial proteomics information.

[0091] Furthermore, in one implementation, such as Figure 7 As shown, continuous microscopic images of human colon cancer tissue samples as organoids were taken in a culture chip on day 1 (day 1), day 3 (day 3), day 5 (day 5), and day 7 (day 7), and finally, 3D organoid spherical models were obtained in the culture microwells.

[0092] Example 2

[0093] like Figure 4As shown, the method for fabricating a micron-scale pathological tissue microarray chip based on the 3D cell model of this application was used to culture, embed, section, and stain human liver cancer cell line (Huh7) with 3D cell spheres.

[0094] Using the second culture chip structure of this application, the human hepatocellular carcinoma cell line Huh7 was seeded into 16-well culture microwells with a diameter of 800 μm. After 3 days of culture, 3D cell spheroids were obtained in the culture microwells. The culture medium in the culture wells was aspirated, and the 3D cell spheroid samples were rinsed and fixed with 4% paraformaldehyde, then stained with eosin. After pre-cooling in a cryostat, OCT gel was added for pre-embedding. After solidification, the pre-embedded block with the sample was carefully peeled off, then flipped and fixed on a sample holder. The pre-embedded block was then covered and embedded again with OCT gel. Sectioning and HE staining were performed. The specific process is not described in this embodiment.

[0095] like Figure 8 As shown, this is a 3D human liver cancer cell line, Huh7 (3D tumor cell spheres), cultured in Example 2, and the 3D tumor cell spheres are presented in the form of a tissue microarray chip using the pathological tissue microarray chip fabrication method of this application. It can be seen that the cell line grows into 3D cell spheres with relatively uniform morphology and size in the culture microwells. Using a single sphere as the unit of analysis ensures both the population effect of cells and facilitates the statistical, reproducible, and comparable data under relatively standardized conditions. Using the slicing method of this application greatly improves sample visibility, allows for statistical analysis at the unit of individual 3D cell spheres, facilitates comparisons between individual cells, and provides the possibility of collecting spatial proteomics information.

[0096] Example 3

[0097] like Figure 9 The image shows the HE staining results of tissue array sections prepared from 3D organoid culture of human colon cancer organoids using the second culture chip structure of this application. This embodiment uses 16-well culture microwells with a diameter of 800 μm. This embodiment utilizes the pathological tissue microarray chip fabrication method of this application to perform 3D organoid culture, embedding, sectioning, and HE staining of human colon cancer organoids. The sectioning method of this application greatly improves sample visibility, allows for statistical analysis on a per-unit basis for individual 3D organoid spheres, facilitates inter-monolithic comparisons, and provides the possibility of acquiring spatial proteomics information.

[0098] Example 4

[0099] like Figure 10As shown, the pathological tissue microarray chip fabrication method of the 3D cell model of this application was used to select a 12-well specification to prepare a human cervical cancer organoid spherical tissue microarray chip. The results of immunofluorescence staining were performed (blue is DAPI, red is panCK, and Merge is the superimposed image of the two). Figure 10 The top-middle image is a composite image, and the bottom image is a magnified view of a part. It can be seen that the staining is uniform and the internal protein and other structures are stable. This shows that the 3D spherical model cultured and the pathological tissue microarray chip fabricated in this application are suitable for routine pathological analysis.

[0100] Comparative Example 1

[0101] like Figure 11 As shown in Comparative Example 1, human cervical cancer organoids cultured in well plates were collected in 1.5 ml EP tubes, washed, digested, and excess matrix gel was removed. After centrifugation and discarding the supernatant, 4% paraformaldehyde was added for fixation for 10 minutes, and the fixative was discarded. Eosin staining solution was then added to stain the sample for 10 minutes, and the staining solution was discarded. OCT embedding gel was added, and the sample was cooled and solidified in a cryostat. The bottom and top of the EP tube were cut off, and the solidified embedding gel block was removed from the bottom opening with forceps. The sample was turned over (sample facing up) and fixed on the sample holder with OCT gel. The sample was then embedded and covered again. After solidification, the sample was frozen sectioned and stained with HE.

[0102] Comparative Example 2

[0103] like Figure 12 As shown in Comparative Example 2, human cervical cancer organoids cultured in well plates were selected. Weighing paper was folded and rolled into a tube and placed in a 1.5 ml EP tube beforehand. After collecting, cleaning, and centrifuging the organoid samples, they were transferred to the weighing paper tube in the EP tube. The samples were fixed, dehydrated (stained with eosin), and cleared according to routine histopathological procedures. The weighing paper containing the samples was removed and the excess part at the top was cut off. The bottom containing the samples was embedded in an embedding cassette for paraffin embedding, sectioning, and HE staining.

[0104] Comparative Example 3

[0105] like Figure 13 As shown in Comparative Example 3, the second culture chip structure used in this application was selected. Human colon cancer organoids were cultured in culture microwells with a specification of 40 wells (400 μm in diameter). After obtaining the 3D organoid model, the corresponding pathological tissue microarray chip was prepared using the same operating procedure as Comparative Example 2, and HE staining was performed.

[0106] Comparative Example 4

[0107] Following the standard procedure for tumor cell spheroidization assays, human liver cancer cell line (Huh7) cultured on conventional plates was selected. After normal digestion, the cells were collected by centrifugation. The resuspended cell suspension was mixed with matrix gel at a volume ratio of 1:1 and seeded into well plates. After solidification, culture medium was added and the cells were cultured normally to prepare 3D cell spheroids. After cell spheroids formed on day 7, the cells were observed and photographed under a microscope. Subsequent sample collection and frozen sectioning procedures were the same as in Comparative Example 1, and HE staining was performed.

[0108] In this application, as Figure 6 , Figure 7 and Figure 9 As shown, HE staining of sections from Examples 1 and 3 reveals that the organoid spheroids from cervical cancer, lung cancer, and colon cancer exhibit complex self-organizing structures, indicating that they possess certain histological functions. Figure 8 The tumor cell spheres in Example 2 also exhibit a certain degree of complex self-organized structure, demonstrating the advantages of their preparation method; however, their structure is noticeably looser compared to organoids. Furthermore, as... Figure 14 As shown, organoids cultured using conventional well plates in Comparative Examples 1 and 2 exhibit randomness in morphology, size, and other characteristics; Comparative Example 4 uses a common tumor cell spheroidization assay protocol, such as... Figure 15 As shown in the diagram, based on the tumor cell spheroidization and detailed images of tumor cell spheroidization in Comparative Example 4, it can be seen that a large number of scattered cells are densely distributed in the matrix gel. Not all cells can form spheroids, the spheroidization rate is low, and the diameter of the spheroids varies from tens to hundreds of micrometers. They are different in shape and size and have significant differences in characteristics. After sectioning and HE staining and local magnification, it can be seen that the interior is mostly solid and does not show complex self-organized structure.

[0109] Example 1 uses the culture chip structure one of this application to culture tumor organoids in four different sizes of culture microwells, and tissue array sections can be produced in all of them, demonstrating the usability of each size. The appropriate size can be freely selected based on actual conditions and experimental needs, such as cell type, spheroidization ability, size and number of spheroid models. Example 2 uses the culture chip structure two of this application to culture human liver cancer cells (Huh7) in 16-well (800 μm diameter) culture microwells, and tissue array sections are also produced. It can be seen that liver cancer cells can also grow into 3D cell spheroid structures and maintain a relatively uniform cross-sectional morphology. Comparative Example 4, such as... Figure 15As shown, in conventional plate cell spheroidization experiments, although liver cancer cells exhibit spheroidal growth, their morphological structures vary considerably. Furthermore, the repeated collection, transfer, and centrifugation of the cultured spheroids significantly impacts subsequent pathological sectioning results. HE staining results show substantial loss of cell spheroids, low sectioning efficiency, uneven sample distribution in the field of view, numerous fragmented spheroids with severely damaged morphology, or excessive concentration, close proximity, or overlap, along with numerous impurities. Additionally, a large number of small spheroids accumulate and fill the gaps between larger spheroids, making it difficult to identify and effectively distinguish individual spheroids. Moreover, this application utilizes single-size culture microwells, which improves embedding efficiency and sample throughput, facilitating control experiments on a well-by-well basis.

[0110] Furthermore, in Examples 1 and 2, the tissue array culture chips maintained all 3D cell model samples at the same horizontal plane during the growth and pre-embedding of the 3D cell models, eliminating the need for sample collection and transfer. Samples were arranged in an array on the slice section using 3D cell spheres as individual units; the number of prepared 3D cell spheres matched the number of individual sample units in the slice, achieving a theoretical slice rate of 100% with no sample loss; the individual sample sections were clear, retaining their internal structures, preserving spatial information, and exhibiting neat arrangement and high visualization, facilitating data acquisition and comparative statistics. In contrast, Comparative Examples 1 and 4 concentrated the samples at the bottom of the embedding mold (1.5ml EP tube) by centrifugation. However, the bottom of the mold was approximately hemispherical, causing sample deflection during centrifugation. This made it difficult to adjust the sample plane to a horizontal plane before fixing it to the sample holder during OCT gel embedding, and also made it difficult to ensure the slice section covered all samples, ultimately resulting in low slice efficiency. Comparative Examples 2 and 3 use a weighing paper wrapping method to concentrate the samples at the bottom of the paper roll. When the samples are transferred from the EP tube to the paraffin embedding box, the possibility of the concentrated samples diffusing again during the paraffin impregnation and embedding process is reduced. Even so, the position of the samples in the paper roll is still random, making it difficult to concentrate the samples on the slicing plane, ultimately resulting in low slicing efficiency.

[0111] Depend on Figure 14 and Figure 15As can be seen, in the slice images of Comparative Examples 1-4, the number, arrangement, and cross-section of samples are random. Some are too concentrated or overlap, resulting in unclear edge contours between monomers, while others are too scattered, leading to insufficient sample quantity in the same field of view. Due to the collection, transfer, and repeated centrifugation processes, samples are lost or damaged, resulting in obvious sample fragmentation and impurities in the slices. Furthermore, Comparative Examples 1, 2, and 4 used well plate culture, which exhibited significant randomness in growth, resulting in varying monomer morphology and size. Comparative Example 3 used the culture chip described in this application; although the sample morphology and size were relatively uniform, the number of samples decreased significantly after transfer, centrifugation, and slicing, while the number of fragments and impurities increased markedly. The sample distribution was also uneven or scattered, resulting in insufficient sample quantity in the field of view, poor visualization, and difficulty in analysis and statistics.

[0112] In summary, this application introduces a cell culture method utilizing culture chips to prepare relatively homogeneous 3D cell models. For cell lines (especially tumor cell lines), the culture microporous structure promotes cell aggregation and growth, preparing relatively homogeneous cell spheres with a relatively dense structure and rich self-organization features under scaffold-free culture conditions. For organoids (especially tumor organoids), the culture microporous structure is used to prepare organoid spheres with relatively homogeneous morphology and function, and the cells are covered but not completely embedded in matrix gel for subsequent processing. This application utilizes the array-based positioning of culture microporous pores to fabricate micron-sized 3D cell models (organoids / cell spheres) into pathological tissue microarray chips, avoiding sample aggregation, deformation, compression damage, overlapping and occlusion, etc., which helps meet the needs of individual sample visualization, data acquisition and statistics, spatial positioning, and comparison between individual samples. Furthermore, by unifying the cell culture and pathological embedding procedures, the culture chip can be directly used as an embedding mold. After preparing a 3D cell model in the culture chip, the sample pretreatment and embedding operations of frozen sections can be completed directly, eliminating steps such as sample collection, transfer, and centrifugation, effectively preventing sample loss and damage. The physical structure of the culture chip ensures that the sliced ​​samples are always on the same horizontal plane, simplifying the slicing operation and improving slicing efficiency.

[0113] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application. Any changes, modifications, substitutions and variations made by those skilled in the art to various elements (including but not limited to chips, configurations, steps, methods, etc.) in the above embodiments are within the protection scope of the present application.

[0114] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for fabricating a 3D cell model pathological tissue microarray chip, characterized in that, Fabricated using a culture chip for preparing 3D cell models, including: The culture chip was sterilized by high temperature and high pressure, dried, and then subjected to low adhesion treatment before use. Select cell samples in good growth condition, and use the processed culture chip and the cell samples to culture a 3D cell model; The 3D cell model is pre-embedded in the culture chip to obtain a pre-embedded block including the 3D cell model; The pre-embedded block was embedded with OCT adhesive to obtain the sample embedding block; The sample embedding block was frozen sectioned using a cryostat to obtain a pathological tissue microarray chip.

2. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, The culture chip is subjected to a low-adhesion treatment, including: p Fill the culture wells, reservoir wells, and connection channels of the culture chip with anti-adhesion washing solution and immerse them; After aspirating the anti-adhesion washing solution, the culture chip is rinsed at least once with PBS solution before use.

3. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, Cultured 3D cell models, including: According to the cell sample type, select the appropriate digestion solution for digestion treatment, count and adjust the cell quantity, centrifuge and collect the cell suspension; Add the cell suspension to the culture wells of the culture chip after processing, and place the culture chip in an incubator until the cells in the cell suspension settle and are evenly distributed into the culture microwells of the culture chip. Remove excess liquid and cells that have not entered the culture microwells, except for the liquid in the culture microwells. Culture medium is added to the culture microwells, and the cells are placed in an incubator for culture. The medium is changed regularly until the cells in the culture microwells aggregate and grow into a 3D cell model.

4. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 3, characterized in that, When the 3D cell model is an organoid, the procedure before "adding culture medium to the culture microwells" further includes: The required concentration of matrix gel for culturing the organoids is added to the culture wells and evenly spread over the culture micropores at the bottom of the culture wells until it solidifies in the incubator.

5. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, Pre-embedding treatment includes: Discard the culture medium in the culture wells of the culture chip, rinse the culture wells at least once with PBS solution, then add eosin staining solution to the culture wells to pre-stain the 3D cell model, and then discard the eosin staining solution. Cool the culture chip to equilibrate with the environment inside the cryostat chamber, and slowly add OCT gel from the side of the culture well until the culture well is filled; After the OCT adhesive has completely solidified, a pre-embedded block is formed, which is then peeled off from the culture chip.

6. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 5, characterized in that, Following the statement "rinse the culture wells at least once with PBS solution", the following is also included: Tissue fixation solution was added to the culture wells to fix the 3D cell model, and then the tissue fixation solution was aspirated.

7. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, Sample embedding processing includes: Flip the pre-embedded block and fix it onto the sample holder; The space around the pre-embedded block is filled with OCT glue, and then the OCT glue is used again for secondary embedding to obtain the sample embedding block.

8. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, The phrase "performing cryosectioning of the embedded sample block using a cryostat" includes: Place the sample embedding block into the sample head of the cryostat, and adjust the sample head to trim and level the sample embedding block until a 3D cell model stained with eosin appears. The embedded blocks of the exposed stained 3D cell model were frozen sectioned to obtain a pathological tissue microarray chip.

9. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 1, characterized in that, The culture chip includes a chip body, culture wells, liquid storage wells, and connection channels; The chip body is provided with the culture well, the connection channel and the liquid storage well. Each culture well is connected to the adjacent liquid storage well through the connection channel. The number of connection channels corresponds to the number of culture wells. The depth of the culture well is greater than or equal to the depth of the liquid storage well. The depth of the liquid storage well is greater than the depth of the connection channel. Both the culture well and the liquid storage well have openings at the top. The surface of the opening of the culture well and the surface of the opening of the liquid storage well are located on the same side of the chip body. The bottom of the culture well has culture micropores arranged in an equally spaced array. The culture micropores are concave spherical surfaces.

10. The method for fabricating a pathological tissue microarray chip for a 3D cell model according to claim 9, characterized in that, The number of culture micropores in each culture well is any one or more of 12, 16, 24, and 40 wells.

Images