Three-dimensional cell microtissue culture method, microplate and culture apparatus

Abstract

The application discloses a three-dimensional cell microtissue culture method, a microplate and a culture device, and relates to the technical field of chemistry, and comprises the steps of injecting cells, incubating the cells and injecting a culture solution; by adopting a target microplate with a first layer of hole positions, a second layer of hole positions and a tapered portion connecting the two, and injecting a target volume of a mixed solution of target cell suspension and matrix glue into the first layer of hole positions, the mixed solution can be laid in the first layer of hole positions under the limiting and / or guiding action of the tapered portion. Then, after in-situ incubation of the mixed solution, a planar gel layer is formed, and then a culture solution is added to the second layer of hole positions, so that the culture solution is located above the planar gel layer, and nutrients in the culture solution can penetrate into the planar gel layer through diffusion, so as to promote the growth of target cells in the target microplate to form organoids. The application can meet the organoid culture demand, and improve the consistency and detection convenience in the organoid culture process.

Description

Technical Field

[0001] This application relates to the field of chemical technology, and more specifically, to a three-dimensional cell microtissue culture method, microplate, and culture equipment. Background Technology

[0002] Three-dimensional cellular microtissues, such as organoids, are three-dimensional culture models that can simulate the partial structure and function of source tissues in vitro. They have been widely used in disease research, drug screening, and personalized medicine evaluation. Microplate-based organoid culture has become a common technique for high-throughput organoid applications due to its suitability for multi-well parallel experiments.

[0003] Currently, organoid culture typically employs the matrix gel droplet method. This involves mixing target cells or organoid clumps with matrix gel, then seeding the mixture as droplets into the wells of a microplate. After the matrix gel solidifies, culture medium or drug-containing medium is added for culturing and analysis. This method provides a three-dimensional growth environment for organoids. However, the droplets usually have a certain thickness, which may affect the distribution of organoids within the droplets, the diffusion of nutrients and drugs, and subsequent imaging and quantification processes. Furthermore, in batch operations using multi-well plates, droplet seeding requires high precision in positioning and consistency of operation; differences between wells may affect the stability of experimental results.

[0004] Therefore, how to improve the consistency and ease of detection in the organoid culture process while meeting the needs of organoid culture has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a three-dimensional cell microtissue culture method to improve the consistency and detection convenience in the organoid culture process while meeting the requirements of organoid culture.

[0006] Another objective of this application is to provide a microplate for three-dimensional cell microtissue culture that is suitable for the above-mentioned three-dimensional cell microtissue culture method.

[0007] Another objective of this application is to provide a three-dimensional cell microtissue culture device suitable for the above-mentioned three-dimensional cell microtissue culture method.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] A three-dimensional cell microtissue culture method is provided, wherein the three-dimensional cell microtissue culture method utilizes a target microplate, the target microplate comprising a plate body, the plate body having at least one target well, the target well comprising a first layer of wells and a second layer of wells extending upward from the first layer of wells, the first layer of wells and the second layer of wells being connected and transitioned by a tapered portion; the three-dimensional cell microtissue culture method includes the following steps:

[0010] Injecting cells, a mixture of target cell suspension and matrix gel of target volume is injected into the first layer of pores of the target pores, wherein the target volume matches the volume of the first layer of pores, so that the mixture injected into the first layer of pores is spread evenly in the first layer of pores under the limiting and / or guiding action of the conical portion;

[0011] Cells are incubated in situ with the mixture located in the pores of the first layer to form a planar gel layer;

[0012] Inject culture medium into the second layer of wells, so that the culture medium is above the planar gel layer, and the nutrients in the culture medium diffuse into the planar gel layer to support the growth of the target cells in the target microplate to form microtissues.

[0013] Optionally, in the above-described three-dimensional cell microtissue culture method, the height of the first layer of pores is 0.75 mm to 1.25 mm; and / or,

[0014] The first layer of holes are circular holes, and the diameter of the first layer of holes is 2.66mm to 4.44mm.

[0015] Optionally, in the above-described three-dimensional cell microtissue culture method, the sidewall of the target well is a light-shielding material; or, the sidewall of the target well is provided with a light-shielding layer.

[0016] Optionally, in the above-described three-dimensional cell microtissue culture method, the taper of the conical portion is 33.75° to 56.25°; and / or,

[0017] The height of the tapered portion is 0.375mm to 0.625mm.

[0018] A microplate for three-dimensional cell microtissue culture, suitable for the three-dimensional cell microtissue culture method as described in any of the preceding claims, comprising the target microplate, wherein:

[0019] The target microplate includes a plate body, on which at least one target hole is provided. The target hole includes a first layer of holes and a second layer of holes extending upward from the first layer of holes. The first layer of holes and the second layer of holes are connected and transitioned by a tapered portion.

[0020] Optionally, in the microplate for three-dimensional cell microtissue culture described above, the volume of the first layer of wells can match the target volume of the mixture, and the first layer of wells is configured such that after receiving the target volume of the mixture, the mixture spreads evenly within the first layer of wells and forms a planar gel layer.

[0021] The target cell suspension includes an organoid suspension, and the second layer pores are configured to contain culture medium after the planar gel layer has solidified, such that the culture medium diffuses into the planar gel layer to support the growth of organoids in the planar gel layer.

[0022] Optionally, in the microplate described above for three-dimensional cell microtissue culture, the diameter of the planar gel layer is greater than the thickness of the planar gel layer.

[0023] Optionally, in the microplate for three-dimensional cell microtissue culture described above, the height of the first layer of wells is 0.75 mm to 1.25 mm; and / or,

[0024] The first layer of holes are circular holes, and the diameter of the first layer of holes is 2.66mm to 4.44mm.

[0025] Optionally, in the microplate for three-dimensional cell microtissue culture described above, the sidewall of the target well is made of a light-shielding material; or, the sidewall of the target well is provided with a light-shielding layer.

[0026] Optionally, in the microplate described above for three-dimensional cell microtissue culture, the taper of the conical portion is 33.75° to 56.25°; and / or,

[0027] The height of the tapered portion is 0.375mm to 0.625mm.

[0028] A three-dimensional cell microtissue culture device, suitable for the three-dimensional cell microtissue culture method as described in any of the preceding claims, includes a liquid handling module, a transport module, and a worktable, wherein:

[0029] The liquid processing module is used to process and transfer the target liquid, which includes at least one of target cell suspension, matrix gel, and culture medium;

[0030] The transport module is used to transport the target object to a designated location, the target object including a container holding the target liquid and / or the target microporous plate;

[0031] The workbench is used to support the liquid handling module, the transport module, the target object, and / or the target liquid;

[0032] The liquid processing module is configured to transfer a mixture of the target cell suspension and the matrix gel into the first layer of wells of the target microplate, and to transfer the culture medium into the second layer of wells of the target microplate.

[0033] Optionally, in the above-mentioned three-dimensional cell microtissue culture device, a first temperature control zone and a second temperature control zone are provided on the worktable. The first temperature control zone is used to maintain the fluid state of the matrix gel, and the second temperature control zone is used for organoid culture.

[0034] Optionally, in the above-mentioned three-dimensional cell microtissue culture device, the worktable is further provided with a loading area and a centrifugation device. The loading area is used to store the target liquid and / or a container holding the target liquid, and the centrifugation device is used to centrifuge the biological sample to obtain the target cell suspension.

[0035] Optionally, in the above-described three-dimensional cell microtissue culture device, the liquid handling module includes a pipetting component and / or a mixing component, wherein the pipetting component is used to aspirate and dispense the target liquid, and the mixing component is used to mix the target cell suspension and the matrix gel; and / or,

[0036] The handling module includes a robotic arm, which is used to handle the target object between the feeding area, the first temperature control area, the second temperature control area, the centrifuge, and the liquid handling module.

[0037] The three-dimensional cell microtissue culture method provided in this application employs a target microplate with a first layer of wells, a second layer of wells, and a conical portion connecting the two. A mixture of a target volume of target cell suspension and matrix gel is injected into the first layer of wells, allowing the mixture to spread evenly within the wells under the constraint and / or guidance of the conical portion. The mixture within the first layer of wells is then incubated in situ to form a planar gel layer. Finally, culture medium is added to the second layer of wells, placing the culture medium above the planar gel layer. Nutrients in the culture medium can diffuse into the planar gel layer, promoting the growth of target cells and the formation of microtissues within the target microplate. As can be seen from the above example, the three-dimensional cell microtissue culture method provided in this application enables target cells to be distributed more evenly and uniformly within the gel layer, which helps reduce the differences in the culture environment of target cells in different regions. Furthermore, because the target volume matches the volume of the first layer of wells, the injected mixture is less likely to form irregular accumulations within the wells. Instead, it is constrained by the first layer of wells and the conical portion to form a stable planar gel layer. This improves the consistency of the plate morphology, reduces inter-well differences, and consequently enhances the operational stability and reliability of experimental results during batch culture in multi-well plates. Simultaneously, the thickness and spatial distribution of the planar gel layer are easier to control, resulting in shorter and more balanced diffusion paths for nutrients or subsequent drug components to organoids and other micro-tissues, thus improving the consistency of nutrient or drug supply during culture. In addition, the planar gel layer allows the formed organoids and other micro-tissues to be distributed in a relatively monolayer or near-monolayer, reducing overlap and occlusion in the height direction. This facilitates subsequent imaging observation, quantity statistics, size measurement, and growth status analysis of organoids and other micro-tissues, improving detection convenience and quantitative analysis accuracy.

[0038] The technical features mentioned above, those to be mentioned below, and those shown individually in the accompanying drawings can be combined arbitrarily, provided that the combined technical features are not contradictory. All feasible combinations of features are those explicitly described herein. Any one of the multiple sub-features contained in the same statement can be applied independently, without necessarily being applied together with other sub-features. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0040] Figure 1A flowchart of a three-dimensional cell microtissue culture method provided in the embodiments of this application;

[0041] Figure 2 Axonometric view of a microplate for three-dimensional cell microtissue culture provided in an embodiment of this application;

[0042] Figure 3 A top view of a microplate for three-dimensional cell microtissue culture provided in an embodiment of this application;

[0043] Figure 4 A cross-sectional view of a microplate for three-dimensional cell microtissue culture provided in an embodiment of this application;

[0044] The annotations in the attached figures are explained as follows:

[0045] Among them, 10 is the orifice plate body, 11 is the target hole position, 111 is the first layer hole position, 112 is the second layer hole position, and 113 is the conical part. Detailed Implementation

[0046] The core of this application is to provide a three-dimensional cell microtissue culture method, which can improve the consistency and detection convenience in the organoid culture process while meeting the needs of organoid culture.

[0047] Another key aspect of this application is to provide a microplate for three-dimensional cell microtissue culture that is suitable for the aforementioned three-dimensional cell microtissue culture method.

[0048] Another core aspect of this application is to provide a three-dimensional cell microtissue culture device suitable for the above-mentioned three-dimensional cell microtissue culture method.

[0049] 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. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0050] In traditional three-dimensional cell microtissue culture processes, such as organoid culture, cell suspensions are typically mixed with matrix gel and seeded into ordinary culture wells in droplet, clumping, or irregular gel form. While this method can achieve organoid culture, the viscosity of matrix gel and the influence of factors such as temperature, operating speed, sample placement, and well bottom shape during gelation can easily lead to differences in gel morphology, thickness, and cell distribution between different well locations. Furthermore, organoids may overlap along their height within gel clumps, resulting in inconsistent focal planes, severe occlusion, difficulty in counting, and significant dimensional statistical errors during microscopic imaging. These differences can reduce the consistency and reliability of experimental results, especially in high-throughput drug screening, toxicity evaluation, or personalized drug testing scenarios.

[0051] Therefore, such as Figure 1 As shown in the embodiments of this application, a three-dimensional cell microtissue culture method is disclosed. This method employs a target microplate having a first layer of wells 111, a second layer of wells 112, and a conical portion 113 connecting the two. A target volume of a target cell suspension and a matrix gel mixture is injected into the first layer of wells 111, allowing the mixture to spread evenly within the first layer of wells 111 under the limiting and / or guiding effect of the first layer of wells 111 and the conical portion 113. Then, after in-situ incubation of the mixture to form a planar gel layer, culture medium is added to the second layer of wells 112, placing the culture medium above the planar gel layer. Nutrients in the culture medium diffuse into the planar gel layer, supporting the growth of target cells into microtissues within the target microplate. It should be noted that, in this application, three-dimensional cell microtissue culture refers to culturing cells under three-dimensional conditions to form cell aggregates with a three-dimensional structure, the specific forms of which include, but are not limited to, organoids, spheroids, and multicellular cultured microtissues.

[0052] Through the aforementioned three-dimensional cell microtissue culture method, target cells can be distributed more uniformly and stably in the planar gel layer, reducing the differences in the culture environment of target cells in different regions. Furthermore, because the volume of the target cell suspension and matrix gel mixture matches the volume of the first layer pores 111, the mixture is less likely to form irregular accumulations within the pores. Instead, it is constrained by the first layer pores 111 and the conical portion 113 to form a planar gel layer with controllable thickness and stable boundaries, thereby improving the consistency of the plating morphology and reducing inter-pore differences. In addition, the planar gel layer allows organoids to be distributed in a relatively monolayer or near-monolayer manner, reducing overlap and occlusion of organoids in the height direction, thus facilitating subsequent imaging observation, quantity statistics, size measurement, and growth status analysis.

[0053] Among them, such as Figures 2 to 4As shown, the target microplate, used for three-dimensional cell microtissue culture, may include a plate body 10, which may have at least one target well 11. The target well 11 may include a first layer of wells 111, a second layer of wells 112, and a conical portion 113 connecting the first layer of wells 111 and the second layer of wells 112. The first layer of wells 111 may be located at the bottom region of the target well 11 to contain a mixture of target cell suspension and matrix gel, and to form a planar gel layer. The second layer of wells 112 may be located above the first layer of wells 111 to contain culture medium. The conical portion 113 is disposed between the first layer of wells 111 and the second layer of wells 112 to limit and / or guide the mixture when it is injected into the first layer of wells 111. Specifically, the plate body 10 may be made of a transparent material to facilitate subsequent microscopic observation, bright-field imaging, fluorescence imaging, confocal imaging, or high-content imaging. The transparent material can be polystyrene, cyclic olefin polymers, cyclic olefin copolymers, polycarbonate, polymethyl methacrylate, glass, or other materials suitable for cell culture and optical detection. To improve cell culture compatibility, the surface of the well plate body 10 can be treated with hydrophilicity, low adsorption, biocompatible coating, or sterilization. Simultaneously, the target microplate can be a single-well plate or a multi-well plate; for example, the well plate body 10 can be a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, or 1536-well plate. To adapt to high-throughput organoid culture and drug screening, the well plate body 10 is preferably a 96-well plate, 384-well plate, or 1536-well plate. Of course, the number, arrangement, and spacing of the target wells 11 can be adjusted according to the actual culture system, imaging equipment, or automated equipment, and this application does not impose any limitations on these aspects.

[0054] The following will combine Figures 1 to 4 The three-dimensional cell microtissue culture method disclosed in the embodiments of this application will be explained and described in detail.

[0055] Step S100: Inject cells;

[0056] A mixture of target cell suspension and matrix gel of target volume is injected into the first layer of wells 111 of target well 11, wherein the target volume matches the volume of the first layer of wells 111, so that the mixture injected into the first layer of wells 111 is spread evenly in the first layer of wells 111 under the limiting and / or guiding action of the conical portion 113.

[0057] In some embodiments, the target cell suspension is a liquid suspension medium containing three-dimensional cellular micro-tissues, which may include, but is not limited to, organoid suspensions, spherical suspensions, or multicellular aggregate suspensions. The target cells may be derived from primary cells, tumor cells, stem cells, induced pluripotent stem cells, tissue-derived cells, passaged organoid cells, or gene-edited engineered cells from humans or animals. These target cells can be used to form intestinal organoids, gastric organoids, liver organoids, pancreatic organoids, lung organoids, brain organoids, kidney organoids, breast organoids, prostate organoids, ovarian organoids, bladder organoids, or tumor organoids. The matrix gel provides a three-dimensional or quasi-three-dimensional growth microenvironment for the target cells. The matrix gel may be collagen, gelatin methacrylamide hydrogel, alginate hydrogel, hyaluronic acid hydrogel, fibrin gel, laminin gel, basement membrane extract, or a combination of the above materials. The target cell suspension and matrix gel can be mixed in a preset ratio, and the mixture can remain flowable at low temperatures (e.g., 4°C) and gel at a preset incubation temperature (e.g., 37°C).

[0058] In some embodiments, the target volume of the mixture of target cell suspension and matrix gel can be matched with the volume of the first layer of pores 111, i.e., the target volume is not greater than the volume of the first layer of pores 111, or slightly smaller than the volume of the first layer of pores 111, or it can be equal to the volume of the first layer of pores 111 within an allowable error range, so that the mixture can substantially fill the first layer of pores 111 and form a planar distribution under the limiting and / or guiding effect of the conical portion 113. Specifically, when the target volume of mixture is injected into the first layer of pores 111, the mixture can spread under the constraint of the bottom and sidewalls of the first layer of pores 111. The conical portion 113 can form a gradually contracting transition region between the first layer of pores 111 and the second layer of pores 112, thereby limiting the edge of the mixture and preventing the mixture from forming obvious protrusions at the pore walls. At the same time, the conical portion 113 can also provide a buffer transition structure for the subsequent addition of culture medium, reducing the risk of the culture medium directly impacting the planar gel layer.

[0059] In some embodiments, the sampling needle or pipette tip can extend into the second layer of wells 112 and apply the sample close to the central region of the first layer of wells 111; alternatively, the sampling needle or pipette tip can slowly release the mixture along the sidewall of the target well 11, allowing the mixture to enter the first layer of wells 111 under the constraints of gravity, surface tension, and the well structure. Of course, the sample application process can be performed manually using a pipette or using an automated liquid handling module.

[0060] Step S200: Incubate cells;

[0061] The mixture located within the wells 111 of the first layer is incubated in situ to allow the matrix gel to solidify and form a planar gel layer. In situ incubation refers to the process where the mixture remains in its current position within the wells 111 and undergoes gelation. During this process, the target microplate can be placed in the second temperature-controlled zone or incubator of the culture device to allow the matrix gel to solidify and form a gel layer. The incubation temperature can be set according to the type of matrix gel, and can be between 35℃ and 39℃, for example, a specific value among 35℃, 37℃, and 39℃, or a value between these specific values. The incubation time can be between 10 min and 30 min, for example, a specific value among 10 min, 20 min, and 30 min, or a value between these specific values.

[0062] In some embodiments, the target microplate can be kept horizontal during in-situ incubation to prevent the mixture from flowing out of its proper place before gelation, thus affecting the uniformity of the planar gel layer thickness. Alternatively, the target microplate can be allowed to stand for a short period before in-situ incubation to allow the mixture to spread and stabilize further within the first layer of pores 111.

[0063] In some embodiments, after the mixture is injected into the first layer of wells 111, the target microplate can be centrifuged to promote the uniform distribution of target cells in the planar gel layer. The centrifugation can be performed before the mixture is fully gelled or during the partial gelation stage to ensure a more uniform distribution of target cells while avoiding excessive cell sedimentation that could lead to localized accumulation.

[0064] Step S300: Inject culture medium;

[0065] Culture medium is added to the second layer well 112, positioning it above the planar gel layer. Nutrients in the culture medium diffuse into the planar gel layer, promoting the growth of target cells and the formation of organoids and other microtissues. After the mixture forms a planar gel layer, more culture medium is added to the second layer well 112, covering the planar gel layer. The culture medium may include basal culture medium, serum, serum-free additives, growth factors, small molecule compounds, antibiotics, buffer systems, nutritional factors, and other components required for organoid and other microtissue culture. Depending on the target cell type, the culture medium can be intestinal organoid culture medium, tumor organoid culture medium, liver organoid culture medium, lung organoid culture medium, or other specialized culture media. Specifically, after the culture medium is added to the second layer well 112, nutrients in the culture medium diffuse into the planar gel layer, promoting the growth, proliferation, differentiation, and formation of organoids and other microtissues within the gel layer. Because the planar gel layer is relatively thin, the diffusion paths of nutrients, oxygen, and subsequently added drug molecules into the gel layer are shorter and more uniform, which is beneficial for improving the consistency of nutrient supply and drug action during the culture of organoids and other micro-tissues. After adding the culture medium, the target microplate can be placed in the second temperature-controlled zone of the culture device or in a cell culture incubator. The culture temperature can be between 35℃ and 39℃, such as specific values ​​within 35℃, 37℃, and 39℃, or values ​​between specific values. During the culture process, the culture medium can be changed or culture factors supplemented periodically according to the growth status of the organoids.

[0066] In some embodiments, the test drug, fluorescent dye, live / dead dye, immunostaining reagent, fixative, or lysis buffer may be added to the second layer well 112 during the culture process for drug screening, toxicity evaluation, imaging analysis, molecular detection, or downstream sequencing. Because organoids and other micro-tissues are distributed in a relatively monolayer or near-monolayer, focal plane differences and occlusion can be reduced during detection, improving the accuracy of image recognition and quantitative analysis.

[0067] In some embodiments, the cultured organoids and other microtissues can be observed using bright-field microscopy, fluorescence microscopy, high-content imaging systems, confocal microscopy, or automated imaging equipment. Detectable parameters include, but are not limited to, the number, diameter, area, roundness, estimated volume, number of branches, fluorescence intensity, activity signal, death signal, proliferation signal, apoptosis signal, and drug response curve of organoids and other microtissues.

[0068] As can be seen from the above embodiments, the three-dimensional cell microtissue culture method provided in this application, by injecting a mixture of target cell suspension and matrix gel of the target volume into the first layer of wells 111, and using the first layer of wells 111 and the conical portion 113 to form a planar gel layer, allows the target cells to be distributed more uniformly in the gel layer, reducing the differences between wells caused by irregular gel clumps. Simultaneously, by setting a second layer of wells 112 above the gel layer and adding culture medium, stable contact between the culture medium and the gel layer can be ensured, allowing nutrients and drug components to diffuse into the planar gel layer along a relatively even path, thereby improving culture consistency and detection convenience.

[0069] like Figure 2 As shown in the embodiments, this application also discloses a microplate for three-dimensional cell microtissue culture, which is applicable to the three-dimensional cell microtissue culture method described in the above embodiments. Therefore, it has all the technical effects of the above-mentioned three-dimensional cell microtissue culture method, which will not be repeated here. The microplate for three-dimensional cell microtissue culture may include a target microplate, and the target microplate may include a plate body 10. By setting target wells 11 with a first layer of wells 111 and a second layer of wells 112 on the plate body 10, and by using a tapered portion 113 to achieve the connection and transition between the first layer of wells 111 and the second layer of wells 112, the mixture can have a relatively clear containing area in the target wells 11, and the culture medium can be located above the area where the mixture is located. This reduces the culture differences caused by the instability of the position or shape of the droplets during the traditional droplet inoculation process, and improves the consistency and ease of operation of organoid and other microtissue culture. Meanwhile, the conical part 113 can guide and limit the mixture during the sample addition process, making it easier for the mixture to enter the first layer well 111 and remain in the first layer well 111. This is conducive to the formation of a relatively stable culture state of organoids and other micro-tissues in the target well 11, which facilitates subsequent drug processing, image acquisition and quantitative analysis.

[0070] The following is combined Figures 2 to 4 The microplates for three-dimensional cell microtissue culture disclosed in the embodiments of this application will be explained and described in detail.

[0071] like Figures 2 to 4 As shown, at least one target well 11 can be provided on the well plate body 10. That is, one, two, three or more target wells 11 can be used so that the target cell suspension (such as organoid suspension) and matrix gel mixture can be seeded, gel solidified, culture medium added, and subsequent drug treatment, image acquisition and detection analysis can be performed through the target well 11.

[0072] The target well 11 may include a first layer of wells 111 and a second layer of wells 112 extending upward from the first layer of wells 111. The first layer of wells 111 can hold a mixture of target cell suspension and matrix gel, while the second layer of wells 112 can hold culture medium. The first layer of wells 111 and the second layer of wells 112 are connected and transitioned by a tapered portion 113. By setting the target wells 11, including the first layer of wells 111 and the second layer of wells 112, on the well plate body 10, the mixture of target cell suspension and matrix gel can be contained in the first layer of wells 111, while the culture medium can be contained in the second layer of wells 112. This allows for the formation of a relatively well-defined layered culture structure within the target wells 11, reducing the problem of the droplet position, shape, and thickness being easily affected by manual dispensing in traditional gel droplet methods, and improving the consistency of organoid culture status among different target wells 11. Meanwhile, the first layer of wells 111 and the second layer of wells 112 are connected and transitioned by a conical part 113. This conical part 113 can guide and limit the mixture of target cell suspension and matrix gel during sample addition, making it easier for the mixture to enter the first layer of wells 111 and remain within it. This facilitates stable spreading of the mixture in the central area of ​​the well, reducing displacement, adhesion to the wall, or uneven distribution. Consequently, it facilitates subsequent drug processing, microscopic imaging, fluorescence detection, or quantitative analysis of cell viability.

[0073] In some embodiments, such as Figure 2 and Figure 3 As shown, the plate body 10 can adopt a multi-well plate structure, such as a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, or other multi-well plate structures suitable for cell culture, organoid culture, and drug screening. In this embodiment, the plate body 10 can adopt a 96-well plate structure to be used in conjunction with existing automated sample loading equipment, culture equipment, ELISA readers, fluorescence detection equipment, or high-content imaging equipment. Of course, the number of wells in the plate body 10 is not limited to this. Those skilled in the art can adjust the number and arrangement of the target wells 11 according to the scale of microtissue culture, detection throughput, and equipment adaptation requirements.

[0074] In some embodiments, such as Figure 2 and Figure 3 As shown, the target wells 11 can be arranged in an array along the length and width of the plate body 10, so that multiple experimental groups or multiple drug concentration groups can be cultured and detected simultaneously. At the same time, the arrangement of the target wells 11 can be consistent with the well arrangement of a standard microplate, thus facilitating compatibility with conventional laboratory instruments.

[0075] In some embodiments, such as Figure 4As shown, the first layer of pores 111 can be located in the lower region of the target pores 11, the second layer of pores 112 is located in the upper region of the first layer of pores 111, and the conical portion 113 can be located between the first layer of pores 111 and the second layer of pores 112. In use, the target cell suspension, such as organoid suspension, can be mixed with matrix gel in a preset ratio to form a mixture. This mixture is then added to the target pores 11, and guided by the conical portion 113, it enters the first layer of pores 111 and spreads within them. After the matrix gel solidifies to form a gel layer, culture medium is added to the second layer of pores 112, thus providing a nutrient environment for the culture of organoids and other micro-tissues. The culture medium diffuses and permeates into the planar gel layer, supporting the uniform growth of organoids and other micro-tissues within the planar gel layer.

[0076] In some embodiments, such as Figure 4 As shown, the volume of the first layer of pores 111 can match the target volume of the mixture. That is, when the target volume of the organoid suspension and matrix gel mixture is added into the target pores 11, the mixture can basically fill the first layer of pores 111, or form a gel layer of predetermined thickness and area in the first layer of pores 111. This can reduce the difference in droplet volume, thickness or spreading area caused by manual dispensing and improve the consistency of micro-tissue inoculation such as organoids. Specifically, the first layer of pores 111 is configured to allow the mixture to spread evenly within the first layer of pores 111 after receiving the target volume of the mixture, forming a planar gel layer. Simultaneously, the second layer of pores 112 is configured to contain the culture medium after the planar gel layer has solidified, so that the culture medium is located above the planar gel layer. This allows the gel layer formed by the mixture to no longer exist primarily in the form of traditional hemispherical or protruding droplets, but to form a relatively flat planar gel layer with more controllable thickness within the first layer of pores 111. This enables organoids and other micro-tissues to have a more stable and consistent spatial distribution within the target pores 11, reducing the longitudinal dispersion of organoids and other micro-tissues in thick droplets, which is beneficial for subsequent microscope focusing, image acquisition, and quantitative analysis.

[0077] In some embodiments, the planar gel layer may be a pancake-shaped gel layer, that is, the gel layer formed after the mixture is spread in the first layer pores 111 is flat in shape, and its diameter or lateral dimension may be greater than its thickness, thereby shortening the distance that the culture medium, drug or detection reagent diffuses into the gel, which is beneficial to improving the consistency of drug processing and detection results.

[0078] In some embodiments, such as Figure 4As shown, the height of the first layer of wells 111 can be between 0.75 mm and 1.25 mm. For example, the height of the first layer of wells 111 can be a specific value among 0.75 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, and 1.25 mm, or a value between these specific values. By controlling the height of the first layer of wells 111 within the above range, a thin and stable gel layer can be formed within the first layer of wells 111. This is beneficial for maintaining the three-dimensional matrix environment required for the culture of organoids and other micro-tissues, and also for subsequent imaging and detection. Preferably, the height of the first layer of wells 111 can be 1 mm. When the height of the first layer of wells 111 is 1 mm, the mixture of target cell suspensions such as organoid suspensions and matrix gel can form a thin, cake-like gel layer with a thickness of approximately 1 mm, which is easier to control and helps reduce the difference in gel thickness between different wells.

[0079] In some embodiments, such as Figure 4 As shown, the first layer of pores 111 can be circular, and the diameter of the first layer of pores 111 can be 2.66 mm to 4.44 mm. For example, the diameter of the first layer of pores 111 can be a specific value among 2.66 mm, 3.0 mm, 3.2 mm, 3.55 mm, 3.8 mm, 4.0 mm, 4.2 mm, and 4.44 mm, or a value between these specific values. By setting the first layer of pores 111 as circular, the mixture can be spread more uniformly in the circumferential direction within the first layer of pores 111, which is beneficial for forming a stable circular or near-circular planar gel layer. Preferably, the diameter of the first layer pore 111 can be 3.55 mm, and the height of the first layer pore 111 can be 1 mm. In this case, the first layer pore 111 can match the volume of the mixed liquid of about 10 μL. That is, when about 10 μL of the mixture of target cell suspension such as organoid suspension and matrix gel is added into the first layer pore 111, the mixture can spread in the first layer pore 111 to form a thin cake-shaped gel layer with a diameter of about 3.55 mm and a thickness of about 1 mm.

[0080] In some embodiments, the target volume of the mixture can be 5 μL to 10 μL, meaning that the first layer well 111 can accommodate 5 μL to 10 μL of target cell suspensions such as organoid suspensions and matrix gel mixtures. Depending on different experimental requirements, organoid types, matrix gel concentrations, and culture cycles, those skilled in the art can select specific values ​​from 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL, or values ​​between these specific values, as the target volume of the mixture. For example, 10 μL of the mixture can be added to the target well 11 to form a pancake-like gel layer with a diameter of approximately 3.55 mm and a thickness of approximately 1 mm within the first layer well 111; alternatively, 5 μL of the mixture can be added to the target well 11 to form a gel layer with a thickness of less than 1 mm or a spreading area smaller than the complete bottom area of ​​the first layer well 111. It should be noted that the different volumes of the mixture can all achieve relatively stable positioning and spreading using the first layer well 111 and the conical portion 113.

[0081] In some embodiments, such as Figure 4 As shown, the tapered portion 113 can be disposed between the first layer of holes 111 and the second layer of holes 112, forming a transition structure from large to small between the first layer of holes 111 and the second layer of holes 112. By disposing of the tapered portion 113, abrupt steps can be avoided between the first layer of holes 111 and the second layer of holes 112, thereby reducing the wall adhesion or retention of the mixed liquid during the sample addition process and improving the smoothness of the mixed liquid entering the first layer of holes 111.

[0082] In some embodiments, such as Figure 4 As shown, the taper of the conical portion 113 can be between 33.75° and 56.25°. For example, the taper of the conical portion 113 can be a specific value among 33.75°, 35°, 40°, 45°, 50°, 55°, and 56.25°, or a value between these specific values. By controlling the taper of the conical portion 113 within the above range, both the guiding effect and the limiting effect of the mixture can be taken into account, making it easier for the mixture to converge towards the first layer well 111 during sample addition, while reducing the possibility of the mixture entering the sidewall region of the second layer well 112 due to over-spreading. Preferably, the taper of the conical portion 113 can be 45°. When the taper of the conical portion 113 is 45°, the conical portion 113 can form a structure similar to an inclined chamfer, allowing the mixture to transition along the conical portion 113 towards the first layer well 111 during sample addition, thereby making it easier for the target cell matrix gel mixture to be spread in the central region of the target well 11.

[0083] In some embodiments, such as Figure 4As shown, the height of the tapered portion 113 can be between 0.375mm and 0.625mm. For example, the height of the tapered portion 113 can be a specific value among 0.375mm, 0.4mm, 0.45mm, 0.5mm, 0.55mm, 0.6mm, and 0.625mm, or a value between these specific values. By setting the tapered portion 113 within this height range, a sufficient guiding transition area can be formed between the first layer pore position 111 and the second layer pore position 112. Preferably, the height of the tapered portion 113 can be 0.5mm, which means that a chamfered structure with a height of 0.5mm and a taper of 45° can be set between the first layer pore position 111 and the second layer pore position 112, thereby guiding 5μL to 10μL of the target cell matrix gel mixture into the first layer pore position 111 and helping to ensure that the target cell matrix gel mixture is laid in the center of the target pore position 11.

[0084] In some embodiments, such as Figure 4 As shown, the second layer of wells 112 can hold the culture medium, and the volume of the second layer of wells 112 can be designed according to the amount of culture medium added. For example, in a 96-well plate structure, the second layer of wells 112 can be used to hold the volume of culture medium required for conventional organoid culture, so that the culture medium can cover the solidified planar gel layer in the first layer of wells 111. The culture medium can be organoid culture medium, drug-treated culture medium, culture medium containing detection substrate, or other liquid systems suitable for organoid culture and detection.

[0085] In some embodiments, such as Figure 4 As shown, the second layer pore 112 can have a large-diameter end and a small-diameter end, with the small-diameter end located close to the first layer pore 111. The diameter gradually decreases from the large-diameter end to the small-diameter end, meaning the second layer pore 112 can adopt a structure that is larger at the top and smaller at the bottom. This allows the upper part of the second layer pore 112 to have a larger opening, facilitating the addition of culture medium by the pipette tip or automatic sample dispensing device. Simultaneously, the lower part of the second layer pore 112, near the first layer pore 111, has a smaller dimension to cooperate with the conical portion 113 and the first layer pore 111, reducing excessive impact on the planar gel layer after the culture medium is added. Of course, the second layer pore 112 can be, but is not limited to, a conical pore; it can also be a cylindrical pore or a stepped pore. As long as the second layer pore 112 can accommodate the culture medium and forms a transitional connection with the first layer pore 111 through the conical portion 113, it should fall within the scope of protection of this application.

[0086] In some embodiments, the sidewalls of the target wells 11 may be made of a light-shielding material, or a light-shielding layer may be provided on the sidewalls of the target wells 11. The light-shielding material or light-shielding layer can reduce optical crosstalk between adjacent target wells 11, making it suitable for experimental scenarios such as fluorescence detection, chemiluminescence detection, absorbance detection, or high-content imaging. Specifically, the well plate body 10 may be made of a transparent material, while the sidewalls of the target wells 11 may be partially or entirely provided with a light-shielding layer; alternatively, at least a portion of the well plate body 10 may be made of black or other opaque materials. For example, the sidewalls of the target wells 11 may be made of a black polymer material, or a black coating, light-shielding film layer, or light-shielding filling layer may be formed on the surface of the sidewalls of the target wells 11, so as to reduce the interference of detection signals between adjacent wells and improve detection accuracy when performing organoid drug sensitivity experiments or cell viability detection.

[0087] In some embodiments, the bottom of the target well 11 may be made transparent to facilitate microscopic observation, image acquisition, or bottom-reading optical detection. That is, the sidewalls of the target well 11 may be made light-shielding, while the bottom may be made transparent. This can reduce crosstalk between wells while still allowing observation of the planar gel layer and organoids and other micro-tissues within the first layer of wells 111 from the bottom of the target well 11.

[0088] In some embodiments, the well plate body 10 may be made of a material suitable for cell culture, such as polystyrene, polycarbonate, cyclic olefin copolymer, polypropylene or other biocompatible materials, and the surface of the well plate body 10 may be subjected to cell culture treatment, low adsorption treatment, hydrophilic treatment or hydrophobic treatment to meet the needs of different organoid and other micro-tissue culture systems.

[0089] In some embodiments, the bottom surface of the first layer of pores 111 can be a planar structure, so as to facilitate the formation of a planar gel layer with a relatively uniform thickness in the first layer of pores 111. Of course, the bottom surface of the first layer of pores 111 can also be a micro-concave surface or other structures that can help the mixture to be centered and positioned, as long as the mixture can form a relatively stable gel layer.

[0090] The following describes the microplate for organoid culture provided in this application embodiment with reference to a specific usage process.

[0091] First, the organoid suspension is mixed with matrix gel to obtain a mixture of organoid suspension and matrix gel. The volume of the mixture can be 5 μL to 10 μL. Then, the mixture is added into the target pore site 11. Since the target pore site 11 includes a first layer of pore sites 111, a second layer of pore sites 112, and a conical portion 113 connecting the two, the mixture can be guided into the first layer of pore sites 111 along the conical portion 113 during the addition process.

[0092] After the mixture enters the first layer of pores 111, it can spread out within the pores and form a pancake-like structure. For example, when the diameter of the first layer of pores 111 is 3.55 mm and the height is 1 mm, approximately 10 μL of the mixture can form a pancake-like gel layer with a diameter of approximately 3.55 mm and a thickness of approximately 1 mm. Subsequently, the microplate is placed in a suitable temperature environment, such as 37°C, to allow the matrix adhesive to cure and form a planar gel layer.

[0093] After the planar gel layer solidifies, culture medium is added to the second layer pores 112. The culture medium is located above the planar gel layer and can cover it. At the same time, the culture medium diffuses into the planar gel layer to support the uniform growth of organoids in the planar gel layer to obtain organoids.

[0094] Finally, drug addition, culture, medium change, microscopic imaging, fluorescence detection, cell viability detection, or other analytical operations can be performed according to experimental needs.

[0095] As can be seen from the above usage process, the microplate provided in this application embodiment enables the mixture of organoid suspension and matrix gel to form a relatively stable planar gel layer in the first layer well 111, reducing the problems of droplet displacement, uneven thickness, or inconsistent shape in the traditional droplet method. Since the culture medium is located in the second layer well 112 and above the planar gel layer, the culture medium, drugs, and detection reagents can act relatively uniformly on the organoids in the gel layer, which helps to improve the reproducibility of drug processing and detection results.

[0096] This application also discloses a three-dimensional cell microtissue culture device, which is applicable to the three-dimensional cell microtissue culture method disclosed above. Therefore, it has all the technical effects of the above-described three-dimensional cell microtissue culture method, and will not be repeated here. The three-dimensional cell microtissue culture device may include at least some of the following structures: a feeding area, a first temperature control area, a second temperature control area, a liquid handling module, a centrifuge device, a transport module, and a workbench.

[0097] The loading area can store at least one target liquid from the target cell suspension, matrix gel, and culture medium, or place a container containing the aforementioned target liquid. The loading area may be equipped with an identification component to identify the location, type, specifications, or barcode information of the container containing the target liquid and / or the target microplate or other target objects. A first temperature control area is located on the worktable and can perform low-temperature control on the target cell suspension, matrix gel, mixture, pipette tips, and / or target microplate. The first temperature control area can maintain a temperature between 0°C and 10°C, such as a specific value within the range of 0°C, 2°C, 3°C, 4°C, and 10°C, or a value between these specific values, depending on the type of matrix gel, to maintain the fluid state of the matrix gel and prevent premature gelation before mixing or sample addition. The first temperature control area may include a cooling plate, a semiconductor cooling component, a circulating cooling component, or an ice box adapter structure to achieve low-temperature control. The second temperature control zone, located on the worktable, incubates and cultivates the target microplate, allowing the matrix gel to gel. This zone maintains a suitable temperature for organoid culture, specifically between 35°C and 39°C, or within a range of these values. The second temperature control zone can be an incubator, temperature-controlled chamber, constant-temperature platform, or closed culture module to facilitate organoid culture. The liquid processing module processes and transfers the target liquid, which may include at least one of the following: target cell suspension, matrix gel, and culture medium. Specifically, the target liquid can be one or a combination of these components. The liquid processing module transfers a mixture of target cell suspension and matrix gel to the first well 111 of the target microplate and transfers the culture medium to the second well 112. Furthermore, a centrifuge device centrifuges the biological sample to obtain the target cell suspension. The transport module can move the container holding the target liquid and / or the target microplate or other target objects to a designated location. Specifically, the transport module can move the target objects between the loading area, the first temperature control area, the second temperature control area, the centrifuge, and the liquid handling module. The liquid handling module, the transport module, the target objects, and / or the target liquid can all be located on the workbench, thus providing a reliable operating platform for the culture of organoids and other micro-tissues, ensuring the reliability and safety of organoid and other micro-tissue cultures.

[0098] In some embodiments, the liquid handling module may include a pipetting assembly and / or a mixing assembly. The pipetting assembly can aspirate and dispense the target liquid, while the mixing assembly can mix the target cell suspension and matrix gel. Specifically, the pipetting assembly may include a single-channel pipette tip, a multi-channel pipette tip, a needle pipette tip, a syringe pump, a plunger pump, a peristaltic pump, or a pressure-driven dispensing structure. The pipetting assembly can inject the mixture into the first layer wells 111 according to a preset volume, and can add culture medium to the second layer wells 112 after the gel layer has formed. The mixing assembly can mix the target cell suspension and matrix gel, which can be achieved through repeated aspiration, rotational stirring, oscillation mixing, static mixing tubes, or microfluidic mixing structures.

[0099] In some embodiments, a centrifuge can centrifuge biological samples to obtain a target cell suspension. Specifically, the biological sample can be a tissue digestion solution, a cell mixture, an organoid passage digestion solution, a body fluid sample, or other samples containing target cells. The centrifuge can cause the target cells to settle or accumulate through centrifugation, thereby facilitating the removal of supernatant, digestive enzymes, tissue debris, cell metabolites, impurities, and / or liquid components unsuitable for subsequent culture. Subsequently, a predetermined volume of culture medium, buffer, or other resuspension solution can be added to the centrifuged cell pellet to resuspend the target cells into a target cell suspension with a predetermined cell concentration. This allows the centrifuge to complete the separation, washing, and / or concentration of target cells before mixing them with the matrix gel, making the obtained target cell suspension more suitable for subsequent mixing with the matrix gel and injection into the first layer wells 111 of the target microplate for organoid culture.

[0100] In some embodiments, the centrifugation device may include a rotor or support frame for carrying centrifuge tubes, centrifuge plates, sample tubes, or multiwell plates, and the centrifugation speed, centrifugation force, and centrifugation time can be set according to the processing requirements of different biological samples. The centrifugation device can be used in conjunction with a transport module to enable the transport module to transfer containers containing biological samples between the loading area, the centrifugation device, and the liquid handling module. By incorporating a centrifugation device, the three-dimensional cell microtissue culture equipment can complete sample centrifugation, target cell suspension preparation, target cell suspension mixing with matrix gel, and sample loading of the mixture on the same workbench, thereby improving the automation level and operational consistency of the organoid culture process.

[0101] In some embodiments, the handling module can move target objects between the loading area, the first temperature control area, the second temperature control area, the centrifuge, and the liquid handling module. The handling module may include a robotic arm. The robotic arm can be a Cartesian coordinate robotic arm, a six-axis robotic arm, or other automated handling structures, and can grasp and transfer target microplates, reagent containers, etc., using grippers, suction cups, electromagnetic clamps, or tray structures.

[0102] In some embodiments, the three-dimensional cell microtissue culture device may further include a control module, which may be communicatively connected to a liquid handling module, a transport module, a first temperature control zone, a second temperature control zone, and a centrifuge device to control the organoid culture process. The control module may preset or receive culture programs, which may include, but are not limited to, pre-cooling time, mixing ratio, sample volume, sample loading speed, incubation temperature, incubation time, culture medium addition volume, centrifugation parameters, medium replacement cycle, and drug addition scheme.

[0103] In some embodiments, the three-dimensional cell microtissue culture device may further include an imaging module. The imaging module can image the planar gel layer and organoids within the target well 11, and may include a bright-field imaging unit, a fluorescence imaging unit, a phase-contrast imaging unit, or a high-content imaging unit. Since organoids are mainly distributed within the planar gel layer, the imaging module can obtain more complete organoid images with fewer focal planes, improving the efficiency of automatic identification and statistical analysis.

[0104] The following section provides further explanation of this application in the context of specific application scenarios.

[0105] In some embodiments, in the gastric cancer organoid drug sensitivity screening experiment, digested gastric cancer organoid agglomerates can be prepared into organoid agglomerates with a particle size of less than 70 μm, and mixed with matrix gel at a density of approximately 100 agglomerates / 10 μL to obtain an organoid matrix gel suspension. Subsequently, 5 μL to 10 μL of the organoid matrix gel suspension is added to the first layer pores 111 of the target microplate, allowing it to spread evenly within the first layer pores 111 to form a planar gel layer. The diameter of the first layer pores 111 can be 3.55 mm and the height can be 1 mm, so that the organoid matrix gel suspension forms an approximately "pancake" shaped gel layer. A conical portion 113 is provided between the first layer pores 111 and the second layer pores 112. The height of the conical portion 113 can be 0.5 mm and the taper can be 45°, so as to limit and / or guide the organoid matrix gel suspension through the inclined chamfer structure, so that it is stably spread in the center of the pore. After the matrix gel solidified in situ at 37°C, culture medium or drug-containing culture medium was added to well 112 of the second layer, and six concentration gradients of the test drug were set for drug treatment. Five days after drug treatment, organoid cell viability was detected using the CTG method, and the IC50 was calculated. 50 The half-maximal inhibitory concentration (WMC) and area under the curve (AUC) were used to evaluate the sensitivity of gastric cancer organoids to the test drug. Because the resulting planar gel layer is relatively thin and uniformly distributed, the diffusion pathway of drugs and nutrients to the organoids is shorter, reducing the likelihood of central necrosis due to local hypoxia or malnutrition. Furthermore, it facilitates monolayer or near-monolayer imaging and quantitative analysis, thereby improving the consistency and accuracy of drug sensitivity testing results.

[0106] The terminology used in the above embodiments is for the purpose of describing specific embodiments only and is not intended to be limiting of this application. As used in the specification and appended claims of this application, the singular expressions "a," "an," "the," "the," "the," and "this" are intended to also include expressions such as "one or more," unless the context clearly indicates otherwise. It should also be understood that in the embodiments of this application, "one or more" refers to one, two, or more; "and / or" describes the relationship between related objects, indicating that three relationships may exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship.

[0107] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0108] The "multiple" mentioned in the embodiments of this application refers to two or more. It should be noted that in the description of the embodiments of this application, terms such as "first" and "second" are used only for the purpose of distinguishing descriptions and should not be construed as indicating or implying relative importance, nor should they be construed as indicating or implying order.

[0109] The terms "parallel" and "perpendicular" used in this application refer to "basically parallel" and "basically perpendicular" in practical operation. "Basically parallel" can be understood as parallelism with a certain degree of error, and similarly, "basically perpendicular" can be understood as perpendicularity with a certain degree of error.

[0110] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A three-dimensional cell microtissue culture method, characterized in that, The three-dimensional cell microtissue culture method utilizes a target microplate, which includes a plate body (10) and at least one target well (11) on the plate body (10). The target well (11) includes a first layer of wells (111) and a second layer of wells (112) extending upward from the first layer of wells (111). The first layer of wells (111) and the second layer of wells (112) are connected and transitioned by a tapered portion (113). The three-dimensional cell microtissue culture method includes the following steps: Injecting cells, a mixture of target cell suspension and matrix gel of target volume is injected into the first layer of pores (111) of the target pores (11), wherein the target volume matches the volume of the first layer of pores (111) so that the mixture injected into the first layer of pores (111) is spread flat in the first layer of pores (111) under the limiting and / or guiding action of the cone (113); Incubate cells in situ with the mixture located in the first layer pores (111) to form a planar gel layer; Inject culture medium into the second layer well (112) so that the culture medium is above the planar gel layer, and allow the nutrients in the culture medium to diffuse into the planar gel layer to support the growth of the target cells in the target microplate to form micro-tissues.

2. The three-dimensional cell microtissue culture method according to claim 1, characterized in that, The height of the first layer of holes (111) is 0.75mm to 1.25mm; and / or, The first layer hole (111) is a circular hole, and the diameter of the first layer hole (111) is 2.66mm to 4.44mm.

3. The three-dimensional cell microtissue culture method according to claim 1, characterized in that, The sidewall of the target hole (11) is made of light-shielding material; or, the sidewall of the target hole (11) is provided with a light-shielding layer.

4. The three-dimensional cell microtissue culture method according to claim 1, characterized in that, The taper of the tapered portion (113) is 33.75° to 56.25°; and / or, The height of the tapered portion (113) is 0.375 mm to 0.625 mm.

5. A microplate for three-dimensional cell microtissue culture, characterized in that, The method for three-dimensional cell microtissue culture according to any one of claims 1 to 4, comprising the target microplate, wherein: The target microplate includes a plate body (10), on which at least one target hole (11) is provided. The target hole (11) includes a first layer hole (111) and a second layer hole (112) extending upward from the first layer hole (111). The first layer hole (111) and the second layer hole (112) are connected and transitioned by a tapered portion (113).

6. The microplate for three-dimensional cell microtissue culture according to claim 5, characterized in that, The volume of the first layer pores (111) can match the target volume of the mixture, and the first layer pores (111) are configured such that after receiving the target volume of the mixture, the mixture is spread out in the first layer pores (111) and forms a planar gel layer. The target cell suspension includes an organoid suspension, and the second layer pores (112) are configured to contain culture medium after the planar gel layer has solidified, such that the culture medium diffuses into the planar gel layer to support the growth of organoids in the planar gel layer.

7. The microplate for three-dimensional cell microtissue culture according to claim 6, characterized in that, The diameter of the planar gel layer is greater than the thickness of the planar gel layer.

8. The microplate for three-dimensional cell microtissue culture according to claim 5, characterized in that, The height of the first layer of holes (111) is 0.75mm to 1.25mm; and / or, The first layer hole (111) is a circular hole, and the diameter of the first layer hole (111) is 2.66mm to 4.44mm.

9. The microplate for three-dimensional cell microtissue culture according to claim 5, characterized in that, The sidewall of the target hole (11) is made of light-shielding material; or, the sidewall of the target hole (11) is provided with a light-shielding layer.

10. The microplate for three-dimensional cell microtissue culture according to claim 5, characterized in that, The taper of the tapered portion (113) is 33.75° to 56.25°; and / or, The height of the tapered portion (113) is 0.375 mm to 0.625 mm.

11. A three-dimensional cell microtissue culture device, characterized in that, The method for three-dimensional cell microtissue culture as described in any one of claims 1 to 4 includes a liquid handling module, a transport module, and a workbench, wherein: The liquid processing module is used to process and transfer the target liquid, which includes at least one of target cell suspension, matrix gel, and culture medium; The transport module is used to transport the target object to a designated location, the target object including a container holding the target liquid and / or the target microporous plate; The workbench is used to support the liquid handling module, the transport module, the target object, and / or the target liquid; The liquid processing module is configured to transfer a mixture of the target cell suspension and the matrix gel into the first layer well (111) of the target microplate and to transfer the culture medium into the second layer well (112) of the target microplate.

12. The three-dimensional cell microtissue culture device according to claim 11, characterized in that, The workbench is provided with a first temperature control zone and a second temperature control zone. The first temperature control zone is used to maintain the fluid state of the matrix gel, and the second temperature control zone is used for micro-tissue culture.

13. The three-dimensional cell microtissue culture device according to claim 12, characterized in that, The workbench is also equipped with a loading area and a centrifuge. The loading area is used to store the target liquid and / or a container holding the target liquid. The centrifuge is used to centrifuge the biological sample to obtain the target cell suspension.

14. The three-dimensional cell microtissue culture device according to claim 13, characterized in that, The liquid processing module includes a pipetting assembly and / or a mixing assembly, wherein the pipetting assembly is used to aspirate and dispense the target liquid, and the mixing assembly is used to mix the target cell suspension and the matrix gel; And / or, The handling module includes a robotic arm, which is used to handle the target object between the feeding area, the first temperature control area, the second temperature control area, the centrifuge, and the liquid handling module.

Images