Preparation method and device of high-throughput suspension cell sphere assembly

Abstract

The invention relates to a preparation method and device of a high-throughput suspension cell sphere assembly, and belongs to the technical field of biological cell three-dimensional culture. In order to solve the problems that an existing three-dimensional cell sphere assembly technology is complex in operation, low in flux, depends on external field guidance and has large interference on cell activity, the invention provides a method for realizing non-contact capture, transfer and space collection of cell spheres by utilizing the physical characteristics that the density of suspension cell spheres is lower than that of a culture solution and the suspension cell spheres stably float to the top ends of liquid drops. And finally, fusing to form the three-dimensional cell assembly. The device is a horizontal assembling mechanism or a vertical assembling mechanism, the horizontal assembling mechanism achieves pairing of spheres in the horizontal direction through liquid drop sandwich transfer formed by a first substrate, a second substrate and a spacer, and the vertical assembling mechanism achieves stacking of the spheres in the vertical direction through a capillary tube and a positioner under the capillary action. The three-dimensional co-culture system is used for constructing a high-throughput drug screening model, tumor invasion and microenvironment interaction research and tissue engineering related three-dimensional co-culture system.

Description

Technical Field

[0001] This invention belongs to the field of three-dimensional culture technology of biological cells, specifically relating to a method and apparatus for preparing high-throughput suspended cell sphere assemblies. Background Technology

[0002] Cell spheroids are three-dimensional aggregates formed by the self-aggregation of cells under non-adherent conditions. They can simulate cell interactions and metabolic environments in in vivo tissues to a certain extent, and are therefore often used as in vitro models for drug screening and disease research. To achieve more complex tissue simulations, multiple cell spheroids need to be assembled and fused according to specific spatial relationships. Existing assembly methods, such as those relying on manual splicing, 3D bioprinting positioning, or external field-guided assembly, typically suffer from cumbersome operation steps and limited parallel processing capabilities. These methods often require sophisticated instruments or the application of external physical fields such as magnetic fields and acoustic fields, which not only increases the complexity and cost of the operation, but also may introduce external interventions that unnecessarily interfere with cell activity and function. How to achieve high-throughput, low-damage, and spatially controllable assembly of cell spheroids without relying on complex external fields and delicate mechanical manipulation is a challenge that needs to be solved in current 3D cell culture technology. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a method for preparing high-throughput suspended cell sphere assemblies. This method enables non-contact capture, transfer, and controllable aggregation of suspended cell spheres through simple interfacial contact operations without the need for external magnetic, acoustic, or electric field guidance, and ultimately fuses them to form a three-dimensional cell assembly. This method is characterized by its ease of operation, high throughput, and minimal interference with cell activity.

[0005] To achieve these objectives and other advantages of the present invention, a method for preparing a high-throughput suspended cell sphere assembly is provided, comprising the following steps: Multiple droplets are provided, each droplet carrying a suspended cell sphere; wherein the suspended cell sphere is stably suspended in the top region of the droplet due to its lower density than the culture medium; Based on the spatial position of each of the suspended cell spheres in the droplet tip region, an interface contact operation is performed so that at least two suspended cell spheres are introduced into the internal cavity of the same capillary or the same droplet without the need for an external magnetic field, acoustic field or electric field. At least two suspended cell spheres that have been introduced into the internal cavity of the same capillary or the same droplet are placed in a culture environment and cultured for 1-3 days to allow the at least two suspended cell spheres to come into contact and fuse, thereby forming a three-dimensional cell assembly.

[0006] Preferably, when the interface contact operation is a droplet sandwich transfer operation, it includes the following steps: A first substrate and a second substrate are provided, each having an array of micropores for carrying droplets; A first droplet carrying a first suspended cell sphere is placed in a micropore array of a first substrate, and a second droplet carrying a second suspended cell sphere is placed in a micropore array of a second substrate. The first substrate is configured with the droplet facing upwards, and spacers are disposed on it; The second substrate is flipped so that its droplets face downwards, and the second substrate is stacked with the first substrate through a spacer so that the micropore arrays of the first substrate and the second substrate are aligned, and the first droplet is brought into contact with the two droplets to form a liquid bridge. Maintain the first and second substrates in an overlapping state for 1-120 seconds to allow cell spheroids to transfer or pair through the liquid bridge; The second substrate is separated from the first substrate so that the transferred or paired cell spheres are located within the same droplet of the first substrate.

[0007] Preferably, when the interface contact operation is a capillary sequential aspiration operation, it includes the following steps: A culture medium substrate is provided, the culture medium substrate having an array of micropores for holding droplets; Fix the capillary tube in the positioner and tilt the open end of the capillary tube to align with the tip of the target droplet. The angle between the capillary axis and the horizontal plane is 75-85°. The open end of the capillary is brought into contact with the tip of the target droplet, and the cell spheroid is drawn into the capillary through capillary action. Repeat the above contact aspiration steps in a preset order to sequentially aspirate multiple cell spheres into the same capillary, forming a linear stack within the capillary. Seal both ends of the capillary.

[0008] Preferably, the diameter of the cell spheres is 200-550 micrometers, and the number of cell spheres used for assembly is 2-10. Cells in a spheroid include tumor cells, tumor microenvironment-related cells, tissue regeneration-related cells, and organoid construction-related cells.

[0009] Preferably, the tumor cells include colorectal cancer cells, cervical cancer cells, lung cancer cells, breast cancer cells, and liver cancer cells; Tumor microenvironment-related cells include fibroblasts, endothelial cells, immune cells, and mesenchymal stem cells; Cells involved in tissue regeneration include mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, neural stem cells, hematopoietic stem cells, endothelial progenitor cells, and their differentiated cells; Cells involved in organoid construction include epithelial cells and their stem-progenitor cells, organ-specific parenchymal cells and their stem-progenitor cells, and supporting cells.

[0010] A device for forming high-throughput suspended cell sphere assemblies, the device being a horizontal assembly mechanism or a vertical assembly mechanism; The horizontal assembly mechanism includes a first substrate, a second substrate, and a spacer. Both the first substrate and the second substrate are provided with micropore arrays, and the spacer is used to limit the distance between the first substrate and the second substrate. In the horizontal assembly process, a first droplet containing cell spheres is added to the micropore arrays of the first and second substrates, respectively. The first substrate is placed with the droplet facing upwards and a spacer is installed. The second substrate is flipped so that the droplet faces downwards and is aligned with the pores of the first substrate, so that the upper and lower droplets come into contact to form a liquid bridge. After holding for 1-120 seconds, the two substrates are separated, and paired cell spheres are obtained on the first substrate, thus realizing the horizontal transfer and pairing assembly of cell spheres. The vertical assembly mechanism includes a capillary tube and a positioner. The positioner is equipped with a groove array and is used to fix the capillary tube and realize the arrayed arrangement of the capillary tube. In the vertical assembly process, suspended cell spheres are cultured in a micro-pore array of droplets at the bottom of the culture medium, allowing the cell spheres to float to the top of the droplets. A capillary tube is then inserted into the groove of the locator and kept tilted so that the open end of the capillary tube contacts the top of the droplet. Multiple cell spheres are then sequentially drawn into the capillary tube through capillary action to form a stack, thereby achieving the stacking and fusion assembly of cell spheres in the vertical direction.

[0011] Preferably, both the first substrate and the second substrate are composite substrates, and the composite substrate is formed by bonding a polydimethylsiloxane layer with a microporous array to a glass plate after plasma surface activation treatment; The spacer material is one or more of polylactic acid, acrylonitrile-butadiene-styrene, modified polyethylene terephthalate, polypropylene, polyethylene, polycarbonate, polyamide, polyoxymethylene, and thermoplastic polyurethane.

[0012] Preferably, the composite substrate is prepared by the following method: The micro-pore array mold is made of one of the following materials: copper, brass, aluminum alloy, stainless steel, nickel, or electroformed nickel. The micro-pore array mold is formed by CNC machining to obtain a forming surface with a micro-pore array structure. Mix polydimethylsiloxane (PDMS) based adhesive and curing agent at a mass ratio of 5-10:1, stir and degas to obtain PDMS prepolymer; PDMS prepolymer was poured onto a molding surface with a microporous array structure, cured at 80 °C for 2 hours, and then peeled off to obtain a PDMS substrate with a microporous array. The PDMS substrate with micropore array is cut along the preset outer peripheral positioning boundary of the micropore array mold to obtain a PDMS chip that meets the size requirements of the droplet micropore array. A glass plate is provided, and plasma activation treatment is performed on the surface of the PDMS chip and the surface of the glass plate. The activated PDMS chip is bonded to a glass plate to obtain the composite substrate.

[0013] Preferably, the diameter of each culture well in the microwell array is 4-6 mm, the depth is 1.5-2.5 mm, and the center-to-center distance between adjacent culture wells is 2-5 mm; in the groove array of the locator, the center-to-center distance between adjacent grooves matches the center-to-center distance between adjacent culture wells in the microwell array.

[0014] Preferably, the inner diameter of the capillary is 1-4 mm and the length is 10-15 cm; During vertical assembly, the capillary tubes are loaded with cell spheres and then sealed, with a culture time of 1-3 days.

[0015] The present invention has at least the following beneficial effects: First, this invention utilizes the inherent physical property that suspended cell spheres, due to their lower density than the culture medium, stably float to the top of the droplet. By designing a sequential interface contact operation matched to this spatial position, it achieves precise capture and transfer of cell spheres. This simplifies the operation process and reduces equipment costs and technical barriers without relying on external magnetic fields, acoustic fields, electric fields, or complex precision instruments. The method completes the assembly steps solely through simple droplet contact or capillary action, avoiding the cumbersome mechanical positioning or external field control steps of traditional methods, making the experimental process more direct and easier to implement.

[0016] Secondly, this invention is based on a microporous array droplet system and programmable interface contact operations. This method can process multiple cell spheroids in parallel, achieving high-throughput assembly. Through horizontal droplet sandwich transfer or vertical capillary sequential aspiration, a large number of cell spheroids can be introduced into the internal cavity of the same capillary or the same droplet at one time, significantly improving assembly efficiency. This method is suitable for the needs of large-scale, batch construction of three-dimensional cell models and overcomes the bottleneck of limited throughput in existing technologies.

[0017] Third, because the entire assembly process of this invention relies entirely on the natural buoyancy of the cell spheres and interfacial physical interactions, there is no need to introduce external physical fields or chemical modifications that may affect cell viability, thus minimizing mechanical disturbance and physiological interference to the cells. This helps maintain the original phenotype and function of the cells, ensuring that the assembled cell spheres maintain a high survival rate and biological activity in fusion culture, providing a more reliable three-dimensional model basis for subsequent drug screening or mechanism research.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 In the diagram, A represents the structural parameters of the base mold for the horizontal assembly device; B represents a schematic diagram of the base fabrication process for the horizontal assembly device. Figure 2 This is a schematic diagram of a horizontal assembly process based on droplet sandwich transfer. Figure 3 The histograms show the changes in contact neck length and contact angle of horizontal cell assemblies over time; where A is a micrograph at different fusion times; B is a histogram of contact neck length over time; and C is a histogram of contact angle over time. Figure 4 These are confocal images of type I collagen, cytoskeleton (F-actin), and cell nuclei stained with fluorescent dye in horizontal cell assemblies; where A is Hodgest dye; B is type I collagen; C is fibrous actin; D is an overlay of images A, B, and C; and E is a magnified overlay image. Figure 5 Images are scanning electron microscope (SEM) images of the surface morphology and magnified local areas of horizontal cell assemblies; where A is a scanning electron microscope image of the surface morphology of horizontal cell assemblies; B and C are magnified scanning electron microscope images of local areas at different locations. Figure 6 The growth of horizontal assemblies composed of HCT116 cell spheroids prepared by the method of the present invention is shown from 1 to 4 days; wherein, A is 3 HCT116 cell spheroids; B is 4 HCT116 cell spheroids; and C is 5 HCT116 cell spheroids. Figure 7 The diagram shows the structure of the vertical assembly device and the process of cell spheroid aspiration; where A is a structural diagram of the vertical assembly device; and B is a schematic diagram of the process of aspirating cell spheroids using the vertical assembly device. Figure 8This presents a schematic diagram of vertical assemblies of HCT116 cell bodies of different sizes prepared by the method of this invention; wherein, A1 is a structure of two equally sized cell spheres vertically assembled within a capillary, which is the basic unit of quantity encoding; A2 is a ternary encoding string formed by vertically assembling three equally sized cell spheres; A3 is a quaternary encoding string formed by vertically assembling four equally sized cell spheres; A4 is a quinary encoding string formed by vertically assembling five equally sized cell spheres; B1 is an asymmetric composite encoding structure of "single large sphere – small sphere cluster" consisting of one large cell sphere at the bottom and a cluster of small cell spheres at the top. The structures are as follows: B2 is an alternating series structure of "single large sphere – small sphere cluster – single large sphere"; B3 is a two-segment alternating series structure of "single large sphere – small sphere cluster – single large sphere – small sphere cluster"; B4 is a symmetrical composite series structure of "(single large sphere – small sphere cluster) – single large sphere – (single large sphere – small sphere cluster)"; C1 is a compact series structure of 6 dense small sphere clusters; C2 is a dense series structure of "large sphere – small sphere – large sphere – small sphere – large sphere"; C3 is a segmented dense series structure of "large sphere – (small sphere cluster) – large sphere – large sphere"; C4 is an alternating ultra-dense series structure of "large sphere – large sphere – (small sphere cluster) – large sphere – large sphere". Figure 9 This is a schematic diagram illustrating the effect of drugs based on horizontal assemblies on tumor invasion behavior; where A is the control group; B is the omeprazole group; and C is the tumor necrosis factor group. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0023] A method for preparing a high-throughput suspended cell sphere assembly includes the following steps: Multiple droplets are provided, each droplet carrying a suspended cell sphere; wherein the suspended cell sphere is stably suspended in the top region of the droplet due to its lower density than the culture medium; Based on the spatial position of each of the suspended cell spheres in the droplet tip region, an interface contact operation is performed so that at least two suspended cell spheres are introduced into the internal cavity of the same capillary or the same droplet without the need for an external magnetic field, acoustic field or electric field. At least two suspended cell spheres that have been introduced into the internal cavity of the same capillary or the same droplet are placed in a culture environment and cultured for 1-3 days to allow the at least two suspended cell spheres to come into contact and fuse, thereby forming a three-dimensional cell assembly.

[0024] Existing three-dimensional cell spheroid assembly techniques typically rely on sophisticated instruments or external fields such as magnetic fields and acoustic fields for guidance. The process is complex, making high-throughput processing difficult, and external field interventions or mechanical manipulations may adversely affect cell viability. To address this, this invention leverages the physical phenomenon of suspended cell spheroids naturally rising to the top of the droplet due to their lower density than the culture medium. By designing a matching sequential interface contact operation, it achieves non-contact capture, transfer, and spatial aggregation of cell spheroids without the need for complex external fields or sophisticated mechanics. This approach not only simplifies the operation and increases assembly throughput but also minimizes mechanical and physical interference with cells, helping to maintain their original physiological state and function. It provides a simple, efficient, and gentle technical foundation for subsequently constructing three-dimensional cell models that more closely resemble the in vivo environment.

[0025] In practice, 96-well or 384-well plates can be used as the microwell array to hold the droplets. The volume of each droplet can be 10-50 μL. Suspended cell spheroids can be seeded onto the substrate by mixing cells with polylysine-modified microbubbles at a density of 5000-20000 cells per well, and cultured in an incubator at 37 °C and 5% CO2 concentration for 3-5 days. The culture medium can be Duchenne modified Eagle (DMEM) medium containing 10% fetal bovine serum. The resulting cell spheroids typically have a diameter between 200-550 μm. Because their average density is lower than that of the culture medium, they will float stably and adhere to the upper surface of the droplets after standing.

[0026] There are two main methods for interface contact operation. The first is horizontal droplet sandwich transfer: Two identical PDMS-glass composite substrates with a regular array of micropores on their surfaces are prepared. Using a pipette, droplets of culture medium containing different cell spheroids are added to corresponding micropores on the two substrates. One substrate is placed horizontally (droplets facing upwards), and polylactic acid spacers with a height of 1 mm are placed at its four corners. The other substrate is flipped over (droplets facing downwards), precisely aligned with the wells, and stacked on the spacers, allowing the corresponding droplets to contact and form liquid bridges. This state is maintained for 10 seconds, allowing cell spheroids to transfer or pair via the liquid bridges. The upper and lower substrates are then separated, and the paired cell spheroids are located in the same droplet on the lower substrate. The second method is vertical capillary sequential aspiration: A glass capillary with an inner diameter of 1.5 mm is fixed in a specially designed polylactic acid locator groove, with the long axis of the capillary tilted at an 80° angle to the horizontal plane. The substrate containing the prepared cell spheroid droplets was placed under a microscope, and the positioner was manually moved so that the open end of the capillary gently touched the tip of the target droplet in sequence. With the help of capillary force, the cell spheroids were sequentially drawn into the same capillary, forming a linear stack. The two ends of the capillary were then sealed with a sealing film.

[0027] After completing the above introduction steps, the droplet plate containing multiple cell spheroids or the sealed capillary tube is placed back into an incubator at 37 °C, 5% CO2, and humidity greater than 90% for incubation. The incubation time can be 24-72 hours. During this period, the cell spheroids in contact with each other gradually fuse to form a structurally continuous three-dimensional cell assembly. The fusion process can be observed daily using a microscope, and the fusion efficiency can be evaluated by measuring the change in the length of the fusion neck using image analysis software. This method utilizes the natural physical properties of cell spheroids and simple interface operations, avoiding complex external field devices and precise robotic arm operations, thus reducing technical difficulty and equipment costs. Simultaneously, the microplate or capillary array format allows for the parallel processing of dozens to hundreds of assembly units, significantly improving throughput. The entire process causes minimal mechanical disturbance to the cell spheroids, which helps maintain high cell viability and provides a more reliable in vitro three-dimensional model for subsequent experiments.

[0028] In another technical solution, when the interface contact operation is a droplet sandwich transfer operation, it includes the following steps: A first substrate and a second substrate are provided, each having an array of micropores for carrying droplets; A first droplet carrying a first suspended cell sphere is placed in a micropore array of a first substrate, and a second droplet carrying a second suspended cell sphere is placed in a micropore array of a second substrate. The first substrate is configured with the droplet facing upwards, and spacers are disposed on it; The second substrate is flipped so that its droplets face downwards, and the second substrate is stacked with the first substrate through a spacer so that the micropore arrays of the first substrate and the second substrate are aligned, and the first droplet is brought into contact with the two droplets to form a liquid bridge. Maintain the first and second substrates in an overlapping state for 1-120 seconds to allow cell spheroids to transfer or pair through the liquid bridge; The second substrate is separated from the first substrate so that the transferred or paired cell spheres are located within the same droplet of the first substrate.

[0029] In the horizontal assembly of cell spheroids, existing methods often rely on precise robotic arms or complex microfluidic systems to achieve spheroid transfer and pairing. This is not only cumbersome and has limited throughput, but also involves high equipment costs. The droplet sandwich transfer operation proposed in this invention aligns and abuts two microporous array substrates carrying different cell spheroids, allowing droplets in corresponding pores to contact and form stable liquid bridges. This process cleverly utilizes the interfacial tension and capillary action of the liquid bridges, enabling cell spheroids to spontaneously and rapidly migrate or pair between the upper and lower droplets. This method requires only simple alignment and abutment steps, without relying on external power or precision drive devices, and can complete the parallel transfer and precise pairing of a large number of cell spheroids within seconds, significantly improving operational efficiency and throughput. Simultaneously, it greatly reduces mechanical compression and physical disturbance to the cells, which helps maintain the integrity and viability of the cell spheroids.

[0030] In another technical solution, when the interface contact operation is a capillary sequential aspiration operation, it includes the following steps: A culture medium substrate is provided, the culture medium substrate having an array of micropores for holding droplets; Fix the capillary tube in the positioner and tilt the open end of the capillary tube to align with the tip of the target droplet. The angle between the capillary axis and the horizontal plane is 75-85°. The open end of the capillary is brought into contact with the tip of the target droplet, and the cell spheroid is drawn into the capillary through capillary action. Repeat the above contact aspiration steps in a preset order to sequentially aspirate multiple cell spheres into the same capillary, forming a linear stack within the capillary. Seal both ends of the capillary.

[0031] Existing methods for the controllable stacking and assembly of cell spheres in the vertical direction typically face challenges such as complex operation, difficulty in controlling stacking precision, and difficulty in achieving programmable sequential arrangement of multiple spheres. This invention employs a capillary sequential aspiration operation. By aligning a capillary at a specific tilt angle with the tip of a droplet, capillary action is used to draw the cell spheres located on top of the droplet one by one into the capillary. This process leverages the spatial characteristic of the cell spheres stably suspended at the droplet tip and the pressure difference generated when the capillary opening contacts the droplet's gas-liquid interface, enabling the gentle capture and transfer of cell spheres without applying external active suction. By contacting different droplets in a preset sequence, multiple cell spheres can form a stable linear stacked structure within the same capillary. This method simplifies the vertical assembly operation, reduces reliance on expensive or complex instruments, and allows for controllable programming of the number, type, and stacking order of cell spheres, providing an efficient and gentle technical means for constructing three-dimensional cell assemblies with specific layered or linear topological structures.

[0032] In another technical solution, the diameter of the cell spheres is 200-550 micrometers, and the number of cell spheres used for assembly is 2-10. Cells in a spheroid include tumor cells, tumor microenvironment-related cells, tissue regeneration-related cells, and organoid construction-related cells.

[0033] When assembling cell spheroids from different sources or culture conditions, differences in size and quantity can lead to poor spatial adaptation within the droplet or low contact fusion efficiency, thus affecting the reproducibility and success rate of assembly. By limiting the diameter of the cell spheroids to the range of 200-550 micrometers and controlling the number of spheroids used in each assembly to 2-10, it is possible to ensure that the spheroids are stably suspended within the droplet and have suitable physical dimensions, enabling them to maintain consistent fluid behavior and space occupancy during subsequent interfacial contact operations. Furthermore, the method clearly defines the cell types that can be used for assembly, including tumor cells, tumor microenvironment-related cells, tissue regeneration-related cells, and organoid construction-related cells, thus defining a clear and broad scope for its application. This dual definition of the spheroid's physical parameters and biological origin not only enhances the operational compatibility and fusion controllability between different batches and different cell spheroids but also provides a clear material basis and adaptation standard for constructing diverse, physiologically relevant three-dimensional co-culture models or tissue-like assemblies.

[0034] In another technical solution, tumor cells include colorectal cancer cells, cervical cancer cells, lung cancer cells, breast cancer cells, and liver cancer cells; Tumor microenvironment-related cells include fibroblasts, endothelial cells, immune cells, and mesenchymal stem cells; Cells involved in tissue regeneration include mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, neural stem cells, hematopoietic stem cells, endothelial progenitor cells, and their differentiated cells; Cells involved in organoid construction include epithelial cells and their stem-progenitor cells, organ-specific parenchymal cells and their stem-progenitor cells, and supporting cells.

[0035] In constructing complex three-dimensional cell models for drug screening or disease simulation, simply limiting cell types may lead to unclear model construction direction, poor experimental reproducibility, or the inability of the constructed assemblies to accurately simulate the physiological and pathological environment of specific tissues due to the lack of clarity regarding specific cell types. Therefore, this method further explicitly lists specific cell examples available for use, such as common tumor cell lines like colorectal cancer and cervical cancer, as well as tumor microenvironment components like fibroblasts and endothelial cells. It also includes various stem cells, progenitor cells, and their differentiated cells for tissue regeneration, and various epithelial cells and organ-specific parenchymal cells for organoid construction. By providing such a specific, rather than exhaustive, list of cell types, it offers operators a clear and reliable basis for cell selection and combination. This not only ensures that the constructed assemblies possess high biological relevance and reproducibility in cellular composition, enabling more accurate simulation of the target tissue or disease, but also significantly broadens the application boundaries of this assembly method from a practical perspective, allowing it to stably serve multiple specific and important cutting-edge fields, from basic tumor biology research to tissue engineering and regenerative medicine.

[0036] A device for forming high-throughput suspended cell sphere assemblies, the device being a horizontal assembly mechanism or a vertical assembly mechanism; The horizontal assembly mechanism includes a first substrate, a second substrate, and a spacer. Both the first substrate and the second substrate are provided with micropore arrays, and the spacer is used to limit the distance between the first substrate and the second substrate. In the horizontal assembly process, a first droplet containing cell spheres is added to the micropore arrays of the first and second substrates, respectively. The first substrate is placed with the droplet facing upwards and a spacer is installed. The second substrate is flipped so that the droplet faces downwards and is aligned with the pores of the first substrate, so that the upper and lower droplets come into contact to form a liquid bridge. After holding for 1-120 seconds, the two substrates are separated, and paired cell spheres are obtained on the first substrate, thus realizing the horizontal transfer and pairing assembly of cell spheres. The vertical assembly mechanism includes a capillary tube and a positioner. The positioner is equipped with a groove array and is used to fix the capillary tube and realize the arrayed arrangement of the capillary tube. In the vertical assembly process, suspended cell spheres are cultured in a micro-pore array of droplets at the bottom of the culture medium, allowing the cell spheres to float to the top of the droplets. A capillary tube is then inserted into the groove of the locator and kept tilted so that the open end of the capillary tube contacts the top of the droplet. Multiple cell spheres are then sequentially drawn into the capillary tube through capillary action to form a stack, thereby achieving the stacking and fusion assembly of cell spheres in the vertical direction.

[0037] In practical implementation, the first and second substrates of the horizontal assembly mechanism can be composite substrates formed by bonding a polydimethylsiloxane layer to a glass plate. The pore size of each culture well in the microwell array can be selected as 4 mm, 5 mm, or 6 mm, the pore depth as 1.5 mm, 2.0 mm, or 2.5 mm, and the center-to-center spacing between adjacent culture wells as 2 mm, 3 mm, or 5 mm. Spacers can be installed at the edges or corners of the first substrate, and their material can be polylactic acid, acrylonitrile-butadiene-styrene, or polycarbonate, with a thickness of 0.5 mm, 1.0 mm, or 1.5 mm to precisely control the substrate spacing. During horizontal assembly, firstly, using a pipette, 10 μL, 20 μL, or 30 μL of culture medium containing first cell spheres is added to the microwells of the first substrate to form droplets. Then, an equal volume of culture medium containing second cell spheres is added to the corresponding microwells of the second substrate. The first substrate is placed horizontally with the droplets facing upwards, and the spacers are installed. The second substrate is then flipped over so that its droplet faces downwards. By observing the well markings or using an alignment clamp, its microwell array is precisely aligned with the microwell array of the first substrate. It is then slowly lowered until the corresponding droplets contact each other and form a stable liquid bridge, defined by the spacer. The two substrates are held in this overlapping state for 10, 30, or 60 seconds. During this time, cell spheroids can transfer or pair via the liquid bridge. Finally, the second substrate is separated from the first substrate. The paired or transferred cell spheroids are now located within the same droplet on the first substrate, and the substrate can then be transferred to an incubator for subsequent fusion culture.

[0038] For vertical assembly, glass capillaries with inner diameters of 1 mm, 2 mm, or 3 mm can be used, and their lengths can be 10 cm, 12 cm, or 15 cm. The locator can be a block with a parallel groove array, made of the same polylactic acid as the spacers, and fabricated via 3D printing. The width of the grooves matches the outer diameter of the capillary, and the center-to-center spacing between adjacent grooves can be consistent with the center-to-center spacing between adjacent culture wells in the microporous array, for example, 5 mm, to facilitate rapid alignment of the capillary array with the droplet wells. During vertical assembly, the prepared suspended cell spheres are first cultured in a substrate with a microporous array, ensuring the cell spheres float and remain stable at the tip of the droplet. The capillaries are then placed one by one into the grooves of the locator and fixed, with the long axis of the capillary tilted at approximately 80° to the horizontal plane. By moving the locator or the culture substrate, the open ends of the capillaries are gently brought into contact with the tip of the target droplet. Using capillary action, cell spheres at the tips of droplets are sequentially drawn into the same capillary tube and, under the influence of linear surface tension, form a stable stack within the tube. By contacting different droplets in a predetermined order, a second, third, and even tenth cell sphere can be drawn in sequence. After all target cell spheres have been drawn in, both ends of the capillary tube are sealed with sealing film or hot melt adhesive. The entire capillary tube is then placed horizontally or at an angle in an incubator and cultured at 37 °C with 5% carbon dioxide for 1, 2, or 3 days. The stacked cell spheres within the tube will gradually come into contact and fuse, forming a vertically oriented assembly.

[0039] This technical solution, through the aforementioned specific structural design and working process, enables the controllable assembly of cell spheres in both horizontal and vertical directions. The horizontal assembly mechanism utilizes the droplet sandwich transfer principle, avoiding complex mechanical operations and enabling the parallel pairing of a large number of cell spheres within seconds to minutes, achieving high throughput. The vertical assembly mechanism utilizes simple capillary action to achieve controllable, programmable stacking of multiple cell spheres in sequence and quantity. The entire assembly process does not rely on external physical fields or sophisticated fluid control systems, making operation relatively simple. The substrate, spacers, capillaries, and other components used are all common or easily processed materials, helping to reduce the cost and technical barriers of the device. Furthermore, since the assembly action mainly relies on the interfacial tension and capillary force of the liquid, the mechanical disturbance to the cell spheres is minimal, which helps maintain cell viability and provides a foundation for obtaining high-quality three-dimensional cell fusion assemblies.

[0040] In another technical solution, both the first substrate and the second substrate are composite substrates. The composite substrate is formed by bonding a polydimethylsiloxane layer with a microporous array to a glass plate after plasma surface activation treatment. The spacer material is one or more of polylactic acid, acrylonitrile-butadiene-styrene, modified polyethylene terephthalate, polypropylene, polyethylene, polycarbonate, polyamide, polyoxymethylene, and thermoplastic polyurethane.

[0041] In traditional droplet sandwich transfer assembly operations using microporous array substrates, if the substrate material is too hydrophilic or has inappropriate surface energy, droplets can easily spread or slide within the pores, making it difficult to form a stable droplet interface and controllable liquid bridges. Simultaneously, if the spacer material has poor rigidity, thickness uniformity, or contact characteristics with the substrate, it is difficult to accurately and stably control the spacing between the upper and lower substrates, thus affecting the uniformity of droplet contact and the reproducibility of cell spheroid transfer. To address this, this solution specifies that the first and second substrates are composite substrates formed by bonding a polydimethylsiloxane layer with a microporous array to a glass plate through plasma surface activation treatment. This structure combines the excellent hydrophobicity and elasticity of PDMS with the flatness and rigidity of glass, which is beneficial for droplets to form stable menisci within the micropores and maintain the spheroid's floating position. The spacers are specified to be made of a series of thermoplastic materials with appropriate hardness, dimensional stability, and processability, such as polylactic acid, acrylonitrile-butadiene-styrene, and modified polyethylene terephthalate. By selecting the aforementioned specific material combination, on the one hand, the hydrophobic properties of PDMS ensure stable positioning and support of droplets within the micropores, avoiding non-specific spreading and laying the foundation for a consistent droplet gas-liquid interface. On the other hand, the thermoplastic spacers provide precise and consistent physical thickness, reliably defining the spacing between the upper and lower substrates, ensuring that droplets in all corresponding pores can bridge with essentially the same contact area and force. This material selection enhances the controllability and repeatability of the entire horizontal assembly mechanism, ensuring consistency in cell spheroid transfer or pairing behavior between different pores during high-throughput parallel operations and reducing experimental failure rates caused by material compatibility issues.

[0042] In another technical solution, the composite substrate is prepared by the following method: The micro-pore array mold is made of one of the following materials: copper, brass, aluminum alloy, stainless steel, nickel, or electroformed nickel. The micro-pore array mold is formed by CNC machining to obtain a forming surface with a micro-pore array structure. Mix PDMS base adhesive and curing agent at a mass ratio of 5-10:1, stir and degas to obtain PDMS prepolymer; PDMS prepolymer was poured onto a molding surface with a microporous array structure, cured at 80 °C for 2 hours, and then peeled off to obtain a PDMS substrate with a microporous array. The PDMS substrate with micropore array is cut along the preset outer peripheral positioning boundary of the micropore array mold to obtain a PDMS chip that meets the size requirements of the droplet micropore array. A glass plate is provided, and plasma activation treatment is performed on the surface of the PDMS chip and the surface of the glass plate. The activated PDMS chip is bonded to a glass plate to obtain the composite substrate.

[0043] This solution provides a specific method for preparing a composite substrate. First, using metals such as copper and brass with sufficient hardness and machining precision, a precisely sized microporous array mold is fabricated via CNC machining. This provides a reliable master template for replicating high-fidelity microporous structures. Second, the method specifies a PDMS base adhesive and curing agent mixed at a mass ratio of 5:1 to 10:1 and cured at 80 °C for 2 hours. This ratio and condition facilitate full cross-linking of the PDMS, resulting in a molded substrate with suitable elasticity and stable hydrophobicity. Subsequently, the substrate is cut according to the pre-set outer peripheral positioning boundary of the mold, ensuring high consistency and repeatability of the contour dimensions of each PDMS chip and the position of the microporous array relative to the edge, facilitating precise positioning in porous boards or assembly fixtures. Finally, by plasma-activating and bonding the PDMS surface and the glass plate surface, a strong chemical bond is formed at their interface, resulting in a composite material that combines the elasticity of the PDMS microporous structure with the rigidity of the glass support. This process effectively improves the controllability and batch-to-batch consistency of composite substrate manufacturing by standardizing key materials, clarifying process parameters, and introducing positioning and cutting. The resulting substrate not only has regular micropore size and stable surface properties, which can reliably carry droplets and maintain the stable suspension position of cell spheres, but also has a robust overall structure that can withstand a series of operations such as flipping, alignment, contact, and separation during horizontal assembly. This provides a stable and reliable basic consumable for high-throughput and reproducible cell sphere assembly experiments.

[0044] In another technical solution, the diameter of each culture well in the microwell array is 4-6 mm, the depth is 1.5-2.5 mm, and the center-to-center distance between adjacent culture wells is 2-5 mm; in the groove array of the locator, the center-to-center distance between adjacent grooves matches the center-to-center distance between adjacent culture wells in the microwell array.

[0045] This scheme specifies that the pore diameter of each culture well in the microwell array is 4 to 6 mm, and the pore depth is 1.5 to 2.5 mm. This size range can accommodate an appropriate amount of culture medium to form stable droplets and provide sufficient space for cell spheroids to suspend and move. At the same time, the center-to-center distance between adjacent culture wells is limited to 2 to 5 mm. This ensures sufficient spacing between wells to prevent droplet cross-contamination and also allows for easy access to the operating instruments. Crucially, the center-to-center distance between adjacent grooves in the groove array of the locator must match the pore spacing of the microwell array. This correspondence means that when the capillary on the locator is fixed according to the grooves, the position of its open end can automatically align with each well position in the microwell array in the horizontal direction. By pre-standardizing and matching these two sets of key spatial geometric parameters, rapid, one-time overall alignment of the capillary array and the droplet well position array can be achieved during the experimental preparation stage. Therefore, in subsequent sequential aspiration operations, there is no need for individual, time-consuming micro-position calibration of each well position. A pore size of 4 to 6 millimeters can accommodate a sufficient volume of droplets to form a stable meniscus and gas-liquid interface. This is the physical basis for ensuring that cell spheroids can float stably and remain precisely at the center of the droplet tip due to density differences. Too small a pore size would limit the droplet volume, making it difficult to form a stable floating interface and hindering subsequent liquid manipulation. This invention utilizes the fact that the density of cell spheroids is lower than that of the culture medium, allowing them to float naturally and stably position at the center of the droplet tip after static culture. This "center of the tip" becomes a predictable and repeatable physical coordinate. Whether it's the contact point between the upper and lower droplets during horizontal assembly or the aiming point of the capillary opening during vertical assembly, it's all relative to the position of the spheroid at the droplet tip, not the absolute geometric center of the micropore. Therefore, as long as the spheroid can be confined within this small area at the droplet tip, the specific pore size of the micropore, as long as it's sufficient to form a stable droplet, does not affect subsequent precise manipulation based on interface contact.

[0046] In another technical solution, the inner diameter of the capillary is 1-4 mm and the length is 10-15 cm; During vertical assembly, the capillary tubes are loaded with cell spheres and then sealed, with a culture time of 1-3 days.

[0047] This protocol limits the inner diameter of the capillary to 1-4 mm. This range ensures that the cell spheroids are gently drawn in and move within the capillary in a restricted manner to maintain stacking order, while avoiding the risk of blockage due to excessively narrow channels. Setting the capillary length to 10-15 cm provides sufficient space to sequentially accommodate multiple spheroids while maintaining the portability of the device and ease of placement in standard culture equipment. The procedure specifically specifies that both ends of the capillary must be sealed after the cell spheroids are loaded. This step effectively isolates the device from the external environment, preventing culture medium evaporation and microbial contamination, and creating a stable micro-culture environment for the spheroids. Furthermore, the culture time after vertical assembly is set at 1-3 days. This duration is based on the typical biological process of cell spheroids adhering, migrating, and eventually fusing after contact, providing the necessary time window for extracellular matrix remodeling and the establishment of intercellular connections. By comprehensively optimizing these physical parameters and culture conditions, the stability and survival rate of cell spheres in a vertically stacked state can be improved, and reliable and uniform contact and fusion between stacked spheres can be promoted, thereby ensuring that the final vertical three-dimensional assembly has the expected structural integrity and biological function.

[0048] Example 1 like Figure 1 As shown, the preparation of the composite substrate for horizontal assembly mechanisms includes the following steps: First, the mold is manufactured. Using CNC machining, a micro-hole array structure is machined onto a brass blank to form the mold required for fabrication. The mold's forming surface is designed as a 12-column × 6-row micro-hole array, with overall dimensions of 94 mm in length and 52 mm in width. Each microhole is cylindrical, with a diameter of 5 mm and a depth of 2 mm, and the spacing between the centers of adjacent microholes is 2 mm.

[0049] Next, the PDMS microporous chip is formed. Polydimethylsiloxane (PDMS) base adhesive and its corresponding curing agent are weighed at a mass ratio of 10:1, placed in a container, and stirred evenly. Vacuum degassing is then performed to remove air bubbles from the mixture, yielding a PDMS prepolymer. The degassed prepolymer is poured onto the forming surface of the brass mold, ensuring that each micropore is fully filled. The mold is then placed in an oven at 80 °C and cured for 2 hours. After curing, it is carefully peeled off from the mold to obtain a PDMS substrate with a regular array of micropores on its surface.

[0050] Next, the PDMS substrate is cut and shaped. Along the pre-set outer perimeter positioning boundary lines on the mold, the formed PDMS substrate is precisely cut and the edges are trimmed to obtain a standard-sized, fully-arrayed, independent PDMS chip. After cutting, it can be cleaned and dried as needed to remove any particles that may be attached to the surface.

[0051] Finally, plasma treatment and bonding are performed to form a composite substrate. The PDMS chip and a glass plate of matching size are cleaned with isopropanol and deionized water, respectively, and dried with nitrogen. The microporous surface of the PDMS chip and the glass plate are placed together in the plasma treatment chamber, and surface activation is performed using oxygen or air as the gas source. After treatment, the activated surface of the PDMS chip is immediately attached to the activated surface of the glass plate, and slight pressure is applied to ensure close contact, achieving permanent bonding through chemical bonds formed on the surface. To enhance the bonding strength, the bonded composite substrate can be annealed again in an 80 °C oven for 30 minutes. The final product is a composite substrate with a strong bond between the PDMS microporous array layer and the glass support plate, which is used for subsequent horizontal assembly operations.

[0052] Example 2: Preparation of horizontal cell spheroid assemblies like Figure 2 As shown, the construction of horizontal cell sphere assemblies using the PDMS microporous array-glass plate composite substrate prepared in Example 1 specifically includes the following steps: First, the suspension of cell spheroids was prepared. Cells in the logarithmic growth phase were digested, centrifuged, and resuspended to prepare a single-cell suspension. 5 × 10⁶ cells were then used to prepare the suspension. 5 Cell suspension and 10 5 After mixing the polylysine-modified microbubbles, 5000 cells were seeded into the micropores of the composite substrate prepared in Example 1 (with an appropriate amount of culture medium pre-added to the pores to form droplets). The well plates or substrate were then placed in an incubator at 37 °C, 5% CO2, and saturated humidity for 3-5 days. During this period, the cells aggregated and grew to form spherical aggregates. Due to their lower density than the culture medium, the spheres floated stably under buoyancy and suspended at the gas-liquid interface at the top of the droplets, thus obtaining suspended cell spheres with a diameter between 250-500 micrometers. These spheres could be homologous spheres formed from the same cell type or heterologous spheres obtained by culturing different cell types separately.

[0053] Next, the sandwich pairing and transfer of cell spheroids were performed. Two identical composite substrates prepared by the method in Example 1 were taken and labeled as substrate A and substrate B, respectively. Using a micropipette, 20 μL of culture medium containing cell spheroids A was added to a specific microwell of substrate A to form a first droplet; an equal volume of culture medium containing cell spheroids B was added to the corresponding microwell of substrate B to form a second droplet. Substrate A was placed horizontally (droplets facing upwards), and polylactic acid spacers with a height of 1 mm were placed at its four corners as spacers. Substrate B was flipped over so that its droplets faced downwards, and its microwell array was precisely aligned with that of substrate A. Then, substrate B was lowered smoothly until the distance defined by the spacers was reached, so that the droplets in the corresponding wells of the two substrates contacted each other and formed a stable liquid bridge. This sandwiched state was maintained for 5 seconds. During this period, the cell spheroids would migrate through the liquid bridge under the influence of interfacial tension and gravity, ultimately achieving pairing of the two spheroids in the same droplet of substrate A below.

[0054] Finally, the paired spheres were separated and fused. The upper and lower substrates were smoothly separated, with the two paired cell spheres remaining in the original droplet on substrate A. The substrate was then transferred to a cell culture incubator (37 ℃, 5% CO2, and saturated humidity for 3-5 days) for further culture. During the subsequent 1-3 days of culture, the two closely contacting cell spheres gradually formed a widening "fusion neck" at the contact interface due to cell migration and proliferation, eventually fusing into a continuous, regularly shaped three-dimensional horizontal assembly. The entire process utilized the hydrophobic properties of the PDMS surface on the substrate prepared in Example 1 to stabilize the droplet morphology, and the flatness and rigidity of its glass backing plate to ensure operational stability, thus achieving simple, efficient, and minimally damaging horizontal assembly of suspended cell spheres.

[0055] Example 3 Characterization of the two-cell level assemblies: The two-cell level assemblies prepared according to Example 2 and cultured for a predetermined time were characterized in multiple ways to evaluate their fusion process and structural integrity.

[0056] First, the dynamic parameters of the fusion process were characterized. For assemblies formed by the fusion of two cell spheroids labeled with fluorescent dyes (such as DiO and DiI) prepared in Example 2, observations were performed periodically using a fluorescence microscope during the fusion culture period (e.g., days 1, 2, and 3). Time-series images were acquired using image acquisition software and imported into image analysis software such as ImageJ. The length of the "fusion neck" in the contact area between the two spheroids and the contact angle defined by the tangents of the two spheroids' contours were measured using software tools. By statistically analyzing the changes in the fusion neck length and contact angle at different time points, the fusion kinetics of the assembly over time could be quantitatively described, thereby verifying the effectiveness of the assembly method and the stability of the fusion process in Example 2. The results are as follows: Figure 3 As shown, the two cell spheres gradually form a fusion neck and undergo stable fusion during the culture process. The length of the fusion neck increases with the increase of culture time, and the contact angle gradually tends to stabilize.

[0057] Next, fluorescent staining of the cytoskeleton and nucleus was performed. Assemblies cultured for 3 days (from Example 2) were gently washed with phosphate-buffered saline (PBSS). They were then fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, followed by washing with PBS. Next, the cells were permeabilized with a solution containing 0.1% Triton X-100 for 15 minutes to increase cell membrane permeability. After permeabilization, non-specific binding sites were blocked with 1% bovine serum albumin solution for 30 minutes. Subsequently, the cytoskeleton F-actin was stained with fluorescently labeled phalloidin, and the nuclei were stained with Hoechst 33342. The stained samples were then imaged using a confocal microscope in three dimensions. The results are as follows: Figure 4 As shown, in the fusion region of the assembly, cells from the two original spheres have established a continuous cytoskeleton network with uniformly distributed nuclei. This confirms at the subcellular level that the assembly obtained in Example 2 has achieved substantial structural fusion, rather than simple physical contact.

[0058] Finally, scanning electron microscopy (SEM) was used to observe the ultrastructure of the assembled surface. The assembled and fused components prepared according to the method in Example 2 were fixed overnight at 4 °C with 2.5% glutaraldehyde solution. After fixation, the samples were washed with phosphate buffer, followed by gradient ethanol dehydration (concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100% respectively), each treatment lasting 10 minutes. The dehydrated samples were then treated with a critical point dryer to remove residual solvent without damaging the structure. The dried assemblies were then attached to the sample stage, sputter-coated with gold to increase conductivity, and observed under a scanning electron microscope. The results are as follows: Figure 5As shown, the cells on the surface of the assembly are tightly connected with blurred boundaries, forming a continuous and dense cell layer at the fusion interface without obvious physical gaps. This further confirms at the nanoscale that the three-dimensional assembly obtained by droplet sandwich transfer and culture in Example 2 has excellent morphological integrity and structural fusion.

[0059] Example 4 The preparation of multicellular assemblies, building upon the successful construction of two-cell assemblies, can employ an iterative sandwich pairing method to sequentially introduce more cell spheroids, thereby preparing multicellular assemblies composed of three or more cell spheroids. The specific steps are as follows: First, a two-cell spheroid assembly was obtained as the starting point for assembly. Following the method of Example 2, a stable two-cell spheroid assembly was prepared and cultured. This two-cell spheroid assembly was formed by the fusion or close contact of cell spheroid A and cell spheroid B, and can be regarded as an integrated assembly unit AB.

[0060] Next, the assembly unit AB and the third cell sphere are re-paired. A new substrate carrying the droplet of the third cell sphere C is prepared (preparation method as in Examples 1 and 2). The substrate carrying assembly unit AB (droplet facing upwards) and the substrate carrying cell sphere C (droplet facing downwards) are used as a new pairing combination. Following the contact-bridging-separation droplet sandwich transfer operation described in step 2 of Example 2, the droplet containing assembly unit AB and the droplet containing cell sphere C are brought into contact and a liquid bridge is formed. After a brief contact period, the substrates are separated, at which point cell sphere C is transferred to the droplet containing assembly unit AB. All three are located in the same pore, forming a contact complex composed of three cell spheres.

[0061] Finally, fusion culture and iterative assembly are performed. The complex containing the three spheres is placed in an incubator for further culture to allow fusion, forming a more complex three-cell level assembly. If a fourth, fifth, or even more cell spheres need to be introduced, steps two and three are repeated: each time, the already formed multicellular assembly is used as an assembly unit on a receiving substrate, sandwiched and transferred to a new substrate carrying the target cell spheres, followed by fusion culture. Through this sequential iterative method, the number of cell spheres can be controllably increased stepwise to 3, 4, up to 10 or more, and complex horizontal assembly structures with specific cell arrangement patterns can be achieved by designing the type and order of cell spheres introduced each time. Figure 6 The growth of horizontal assemblies consisting of 3, 4, and 5 HCT116 cell spheres prepared by this method was shown from 1 to 4 days.

[0062] Example 5 The preparation of vertical cell spheroid assemblies depends on the sequential aspiration and stacking of cell spheroids at the tip of a droplet using a capillary tube. The specific process is as follows: First, the cell spheroids were prepared and positioned. Following the method described in Example 2, suspended cell spheroids were cultured in a droplet microwell array. The substrate used was the composite substrate prepared in Example 1; other microwell plates with hydrophobic surfaces capable of stably supporting droplets could also be selected. During culture, the cell spheroids were ensured to float stably due to their lower density than the culture medium and adhere to the central region at the tip of each droplet, providing a consistent starting position for subsequent capillary aspiration. A glass capillary with an inner diameter of 1 mm and a length of 12 cm, and a polylactic acid locator with a parallel groove array, fabricated by 3D printing, were prepared. The center-to-center spacing of adjacent grooves on the locator was designed to match the pore spacing (2 mm) of the microwell array used to achieve rapid array alignment.

[0063] Next, the capillary tubes are installed, aligned, and sequentially aspirated. The capillaries are inserted one by one into the grooves of the locator, fixing them so that the long axis of the capillary is tilted at approximately 80° to the horizontal plane. The culture medium substrate loaded with cell spheroids is placed on the microscope platform or a flat surface. The position of the locator is adjusted so that the opening of the first capillary is precisely aligned with the tip of the first target droplet in three dimensions. Using a fine-tuning device or manual, the capillary opening gently touches the center of the droplet's meniscus. Based on capillary action, the cell spheroids at the droplet tip are gently aspirated into the capillary within seconds and remain at a specific position within the tube. Maintaining the capillary posture, the locator or culture medium substrate is moved horizontally according to a pre-programmed sequence, aligning the same capillary with the tips of the second, third, and subsequent target droplets, repeating the contact aspiration operation. With each aspiration, new cell spheroids enter the capillary and arrange themselves sequentially behind or in front of the previously entered spheroids, ultimately forming a linear stacked sequence of cell spheroids within the same capillary. This process allows multiple capillaries fixed to the positioner to operate in parallel, thus enabling high-throughput preparation.

[0064] Finally, the capillary tubes are sealed and the stacked spheroids are fused for culture. After a predetermined number (e.g., two) of cell spheroids have been aspirated from a single capillary tube, the capillary is carefully removed, avoiding disturbance to the stacked structure within. Immediately, both ends of the capillary are tightly sealed using sterile sealing film or a dedicated capillary sealing adhesive to create a closed microenvironment, preventing culture medium evaporation and contamination. The sealed capillary is then placed horizontally on a culture dish or a dedicated support, and transferred to an incubator at 37°C, 5% CO2, and saturated humidity for static culture. After 1-3 days of culture, the linearly stacked cell spheroids within the tube, under conditions of close contact, will gradually migrate and fuse, ultimately forming a continuous, vertically aligned three-dimensional assembly extending along the capillary axis. Figure 7 A schematic diagram of the vertical assembly device is shown. Figure 8 Examples of vertical assemblies of HCT116 cell bodies of different sizes prepared by this method are presented.

[0065] Example 6 This embodiment demonstrates how to use the two-cell level assembly constructed by the method described in Example 2 to conduct an evaluation study on the effect of drugs on tumor invasion behavior.

[0066] First, heterologous two-cell assemblies were prepared and grouped. Following the steps in Example 2, suspension spheroids of human breast cancer cells MDA-MB-231 and normal human breast epithelial cells MCF10A were cultured separately. MDA-MB-231 and MCF10A spheroids were labeled with fluorescent cell dyes DiI and DiO, respectively, for subsequent differentiation and observation. MDA-MB-231 and MCF10A spheroids were paired one-to-one using a droplet sandwich transfer technique to construct heterologous two-cell assemblies. Successfully paired assemblies were randomly divided into three groups: a control group, a tumor necrosis factor-α (TNF-α) treatment group, and an omeprazole treatment group. Each group had at least three replicates.

[0067] Next, drug treatment and culture were performed. The control group was replaced with fresh complete culture medium (DMEM / F12 medium containing 10% fetal bovine serum). The TNF-α treatment group was replaced with complete culture medium containing 10 ng / mL TNF-α. The omeprazole treatment group was replaced with complete culture medium containing 200 μM omeprazole. During drug administration, a gentle droplet exchange method was used, carefully removing most of the old culture medium with a micropipette and slowly adding an equal volume of fresh drug-containing culture medium to minimize physical disturbance to the assemblies. Afterwards, all assemblies were returned to the incubator for further culture.

[0068] Finally, long-term dynamic monitoring and quantitative analysis of the invasion / fusion phenotype were performed. Starting from the day of drug administration (referred to as day 0), the assemblies in each group were imaged and observed using a fluorescence microscope at preset time points (e.g., days 0, 2, 4, 6, 8, and 18). Bright-field and dual-fluorescence channel images of each heterologous assembly were recorded at each imaging session. The acquired image sequences were imported into ImageJ software for quantitative analysis. Key parameters measured included: fusion neck length, i.e., the width of the contact area between the two heterologous spheres; contact angle, i.e., the angle between the tangents of the two sphere profiles at the contact point; and the equivalent radius of each assembly. By comparing the trends of these parameters over time among different treatment groups, the effect of the drug on the interaction behavior between tumor cells and normal cells was assessed. For example, if the rate of increase in fusion neck length in the TNF-α treatment group was significantly higher than that in the control group, while the increase was inhibited in the omeprazole treatment group, this indicated that TNF-α promoted and omeprazole inhibited the invasion-like phenotype based on cell sphere contact and migration, respectively. Figure 9 The diagram shows the experimental results of this study. Through this type of quantitative analysis, it can be verified that the assembly platform of the present invention is suitable for high-throughput drug screening and in vitro studies of tumor invasion mechanisms.

[0069] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a high-throughput suspended cell sphere assembly, characterized in that, Includes the following steps: Multiple droplets are provided, each droplet carrying a suspended cell sphere; wherein the suspended cell sphere is stably suspended in the top region of the droplet due to its lower density than the culture medium; Based on the spatial position of each of the suspended cell spheres in the droplet tip region, an interface contact operation is performed so that at least two suspended cell spheres are introduced into the internal cavity of the same capillary or into the same droplet. At least two suspended cell spheres that have been introduced into the internal cavity of the same capillary or the same droplet are placed in a culture environment and cultured for 1-3 days to allow the at least two suspended cell spheres to come into contact and fuse, thereby forming a three-dimensional cell assembly.

2. The method for preparing high-throughput suspended cell sphere assemblies according to claim 1, characterized in that, When the interface contact operation is a droplet sandwich transfer operation, it includes the following steps: A first substrate and a second substrate are provided, each having an array of micropores for carrying droplets; A first droplet carrying a first suspended cell sphere is placed in a micropore array of a first substrate, and a second droplet carrying a second suspended cell sphere is placed in a micropore array of a second substrate. The first substrate is configured with the droplet facing upwards, and spacers are disposed on it; The second substrate is flipped so that its droplets face downwards, and the second substrate is stacked with the first substrate through a spacer so that the micropore arrays of the first substrate and the second substrate are aligned, and the first droplet is brought into contact with the two droplets to form a liquid bridge. Maintain the first and second substrates in an overlapping state for 1-120 seconds to allow cell spheroids to transfer or pair through the liquid bridge; The second substrate is separated from the first substrate so that the transferred or paired cell spheres are located within the same droplet of the first substrate.

3. The method for preparing high-throughput suspended cell sphere assemblies according to claim 1, characterized in that, When the interface contact operation is a capillary sequential aspiration operation, it includes the following steps: A culture medium substrate is provided, the culture medium substrate having an array of micropores for holding droplets; Fix the capillary tube in the positioner and tilt the open end of the capillary tube to align with the tip of the target droplet. The angle between the capillary axis and the horizontal plane is 75-85°. The open end of the capillary is brought into contact with the tip of the target droplet, and the cell spheroid is drawn into the capillary through capillary action. Repeat the above contact aspiration steps in a preset order to sequentially aspirate multiple cell spheres into the same capillary, forming a linear stack within the capillary. Seal both ends of the capillary.

4. The method for preparing high-throughput suspended cell sphere assemblies according to claim 1, characterized in that, The diameter of the cell spheroids is 200-550 micrometers, and the number of cell spheroids used for assembly is 2-10. Cells in a spheroid include tumor cells, tumor microenvironment-related cells, tissue regeneration-related cells, and organoid construction-related cells.

5. The method for preparing high-throughput suspended cell sphere assemblies according to claim 4, characterized in that, Tumor cells include colorectal cancer cells, cervical cancer cells, lung cancer cells, breast cancer cells, and liver cancer cells; Tumor microenvironment-related cells include fibroblasts, endothelial cells, immune cells, and mesenchymal stem cells; Cells involved in tissue regeneration include mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, neural stem cells, hematopoietic stem cells, endothelial progenitor cells, and their differentiated cells; Cells involved in organoid construction include epithelial cells and their stem-progenitor cells, organ-specific parenchymal cells and their stem-progenitor cells, and supporting cells.

6. An apparatus for high-throughput formation of suspended cell sphere assemblies, characterized in that, The device can be a horizontal assembly mechanism or a vertical assembly mechanism; The horizontal assembly mechanism includes a first base, a second base, and a spacer. Both the first base and the second base are provided with micropore arrays, and the spacer is used to limit the distance between the first base and the second base. In the horizontal assembly process, a first droplet containing cell spheres is added to the micropore arrays of the first and second substrates, respectively. The first substrate is placed with the droplet facing upwards and a spacer is installed. The second substrate is flipped so that the droplet faces downwards and is aligned with the pores of the first substrate, so that the upper and lower droplets come into contact to form a liquid bridge. After holding for 1-120 seconds, the two substrates are separated, and paired cell spheres are obtained on the first substrate, thus realizing the horizontal transfer and pairing assembly of cell spheres. The vertical assembly mechanism includes a capillary tube and a positioner. The positioner is provided with a groove array and is used to fix the capillary tube and realize the arrayed arrangement of the capillary tube. In the vertical assembly process, suspended cell spheres are cultured in a micro-pore array of droplets at the bottom of the culture medium, allowing the cell spheres to float to the top of the droplets. A capillary tube is then inserted into the groove of the locator and kept tilted so that the open end of the capillary tube contacts the top of the droplet. Multiple cell spheres are then sequentially drawn into the capillary tube through capillary action to form a stack, thereby achieving the stacking and fusion assembly of cell spheres in the vertical direction.

7. The apparatus for high-throughput formation of suspended cell sphere assemblies according to claim 6, characterized in that, Both the first substrate and the second substrate are composite substrates. The composite substrate is formed by bonding a polydimethylsiloxane layer with a microporous array to a glass plate after plasma surface activation treatment. The spacer material is one or more of polylactic acid, acrylonitrile-butadiene-styrene, modified polyethylene terephthalate, polypropylene, polyethylene, polycarbonate, polyamide, polyoxymethylene, and thermoplastic polyurethane.

8. The apparatus for high-throughput formation of suspended cell sphere assemblies according to claim 7, characterized in that, The composite substrate was prepared by the following method: The micro-pore array mold is made of one of the following materials: copper, brass, aluminum alloy, stainless steel, nickel, or electroformed nickel. The micro-pore array mold is formed by CNC machining to obtain a forming surface with a micro-pore array structure. Mix PDMS base adhesive and curing agent at a mass ratio of 5-10:1, stir and degas to obtain PDMS prepolymer; PDMS prepolymer was poured onto a molding surface with a microporous array structure, cured at 80 °C for 2 hours, and then peeled off to obtain a PDMS substrate with a microporous array. The PDMS substrate with micropore array is cut along the preset outer peripheral positioning boundary of the micropore array mold to obtain a PDMS chip that meets the size requirements of the droplet micropore array. A glass plate is provided, and plasma activation treatment is performed on the surface of the PDMS chip and the surface of the glass plate. The activated PDMS chip is bonded to a glass plate to obtain the composite substrate.

9. The apparatus for high-throughput formation of suspended cell sphere assemblies according to claim 6, characterized in that, The diameter of each culture well in the microwell array is 4-6 mm, the depth is 1.5-2.5 mm, and the center-to-center distance between adjacent culture wells is 2-5 mm; in the groove array of the locator, the center-to-center distance between adjacent grooves matches the center-to-center distance between adjacent culture wells in the microwell array.

10. The apparatus for high-throughput formation of suspended cell sphere assemblies according to claim 6, characterized in that, The inner diameter of the capillary is 1-4 mm, and the length is 10-15 cm; During vertical assembly, the capillary tubes are loaded with cell spheres and then sealed, with a culture time of 1-3 days.

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