Organ chip platform integrated with optical tweezers and three-dimensional cell assembling method

Abstract

The invention relates to the technical field of organ chips and tissue engineering, in particular to an organ chip platform integrated with optical tweezers and a three-dimensional cell assembling method, the platform comprises an organ chip with optical transparency and an optical tweezers system integrated with the organ chip; the organ chip comprises at least two induced differentiation culture pools and at least one assembly culture pool; the induced differentiation culture pool is used for respectively culturing the stem cells and inducing and differentiating the stem cells into at least two different types of cells; the optical tweezers system is used for capturing cells and moving the cells from the induced differentiation culture pool to the assembly culture pool; assembling the at least two different types of cells into an initial three-dimensional cell tissue according to the proportion, sequence and pattern structure in the target organ tissue in the assembly culture pool; the assembly culture pool is used for culturing the initial three-dimensional cell tissue to obtain the three-dimensional cell tissue with the target pattern structure. The technical threshold of the organ chip can be reduced, and the biological functionality of the organ chip is improved.

Description

Technical Field

[0001] This invention relates to the field of organ-on-a-chip and tissue engineering technology, and in particular to an organ-on-a-chip platform with integrated optical tweezers and a three-dimensional cell assembly method. Background Technology

[0002] Organ-on-a-chip (OIC) is a novel research model based on a microfluidic chip platform. Through intelligent control, it enables the biomimetic construction of a dynamic physiological microenvironment in vitro, controlling the spatial positioning and differentiation direction of stem cells. This guides stem cells to self-organize into more ordered and reproducible structures similar to real organs, thereby generating corresponding organ functions. This provides a low-cost model that more closely approximates real physiological and pathological conditions for disease research and drug screening. Compared to conventional three-dimensional cell culture, OIC offers significant advantages in spatial organization, cell differentiation regulation, tissue function, reproducibility, and scalability.

[0003] Leveraging the self-organization and self-patterning capabilities of stem cells, organ-on-a-chip technology can be used to simulate advanced stages of organ development through physical or chemical induction methods. Existing methods for preparing organ-on-a-chip mainly fall into the following five categories:

[0004] (i) By utilizing microfluidics and microstructure design, the position and arrangement of cells in three-dimensional space can be controlled to form a more ordered and reproducible tissue structure.

[0005] (ii) By regulating the physicochemical properties of the microenvironment (such as stiffness, biochemical signals, etc.), the differentiation process of cells can be affected, and cells can be guided to self-organize to form the tissue structure required.

[0006] (iii) Using micro-patterning techniques, such as microprinting and photo-oxidation, micron-scale protein patterns are created on the matrix surface to provide guidance for the spatial positioning of cells.

[0007] (iv) Using micro-molding technology, such as creating micropores and microchannels, cells can be spatially confined and guided in a three-dimensional environment to promote orderly cell arrangement and tissue formation.

[0008] (v) Adding biodegradable microfibers and other structures to cell culture provides a scaffold for directional cell growth, guiding cells to self-organize into more ordered tissue structures.

[0009] All five methods described above are based on the regulation of the microenvironment (mechanics), indirectly affecting stem cells. Their effectiveness depends on the stem cells' responsiveness to changes in the microenvironment and their self-organizing ability, which stems from extremely complex molecular mechanisms (biological signaling pathways). On the one hand, due to precision limitations, regulating smaller-scale microenvironments (microfabrication) remains a challenge, and the cost of using advanced technologies is also very high, making organ-on-a-chip fabrication a technically demanding process for application. On the other hand, the aforementioned indirect methods involve more variables, are not universally applicable across different types of organs, and have different developmental patterns (molecular mechanisms), requiring the development of corresponding complex microenvironment regulation methods, which remains challenging with current technologies. Summary of the Invention

[0010] (a) Technical problems to be solved

[0011] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an organ-on-a-chip platform with integrated optical tweezers and a three-dimensional cell assembly method, which solves the technical problems of high technical threshold, insufficient functionality and limited spatiotemporal control capability of the prior art in preparing organ-on-a-chip.

[0012] (II) Technical Solution

[0013] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0014] In a first aspect, embodiments of the present invention provide an organ-on-a-chip platform with integrated optical tweezers, comprising an organ-on-a-chip with optical transparency and an optical tweezers system integrated with the organ-on-a-chip;

[0015] The organ-on-a-chip includes at least two induction differentiation culture pools and at least one assembly culture pool; the induction differentiation culture pools are connected to the assembly culture pools; the at least two induction differentiation culture pools are used to culture and induce stem cells to differentiate into at least two different types of cells respectively;

[0016] The optical tweezers system is used to capture the different types of cells and move the different types of cells from the induction differentiation culture tank to the assembly culture tank; and, in the assembly culture tank, to assemble at least two different types of cells into an initial three-dimensional cell tissue according to the proportion, order and pattern structure in the target organ tissue.

[0017] The assembly culture tank is used to culture the initial three-dimensional cell tissue to obtain a three-dimensional cell tissue with the target pattern structure.

[0018] Optionally, the induction differentiation culture tank and the assembly culture tank are connected by a connecting channel, and a control valve is provided on the connecting channel.

[0019] Optionally, a clearance cavity is provided on the connecting channel;

[0020] The control valve includes an electromagnet and a magnetic block for blocking the connection channel, wherein the magnetic block is matched with the shape of the relief cavity;

[0021] When the electromagnet is energized, the magnet block moves from the connecting channel to the clearance cavity under the action of the electromagnet's magnetic field, so that the connecting channel is opened.

[0022] When the electromagnet is de-energized, the magnet block moves from the clearance cavity to the connection channel under the action of gravity to block the connection channel.

[0023] Optionally, each of the induced differentiation culture tanks or assembly culture tanks further includes:

[0024] The liquid inlet channel is used to introduce new culture medium liquid or drugs from the outside into the induction differentiation culture tank and the assembly culture tank;

[0025] The liquid outlet channel is used to drain the old culture medium liquid from the induction differentiation culture tank and the assembly culture tank to the outside.

[0026] Optionally, the optical tweezers system includes:

[0027] Laser source, used to generate laser beams and control laser power;

[0028] An optical path assembly is used to transmit and adjust the laser beam so that the laser beam illuminates the organ-on-a-chip and forms one or more optical traps for capturing and moving cells; the optical path assembly includes optical fibers, optical paths, and optical lens assemblies.

[0029] A spatial light modulator is used to encode the light field distribution of the laser beam to modulate the light trap to capture one or more cells; and to encode the light field distribution of the laser beam to form a preset light field pattern, thereby controlling the position and movement path of the light trap.

[0030] A detection component is used to monitor the position and movement of cells in the organ-on-a-chip;

[0031] The control system is communicatively connected to the laser source, spatial light modulator, optical path assembly, and detection assembly, and is used to control the laser source, spatial light modulator, optical path assembly, and detection assembly.

[0032] Optionally, the laser beam generated by the laser source has a wavelength range of 1064nm to 1500nm and a power of 30mW to 3W.

[0033] In a second aspect, embodiments of the present invention provide a three-dimensional cell assembly method, the method being implemented based on any of the organ-on-a-chip platforms described in the first aspect, the method comprising:

[0034] S1. Inject stem cells into at least two induction differentiation culture pools, and culture and induce differentiation to obtain at least two different types of cells;

[0035] S2. Use an optical tweezers system to capture the different types of cells and move them to the assembly culture tank;

[0036] S3. Using an optical tweezers system, different types of cells in the assembly culture tank are assembled according to the proportion, order, and pattern structure of the target organ tissue to obtain the initial three-dimensional cell tissue.

[0037] S4. The initial three-dimensional cell tissue is cultured in the assembly culture tank to obtain a three-dimensional cell tissue with the target pattern structure.

[0038] Optionally, the target organ tissue is a multi-chambered cardiac organoid, and S1 includes:

[0039] S101. Human pluripotent stem cells were injected into three induction differentiation culture pools respectively.

[0040] S102. Human pluripotent stem cells in three induction differentiation culture pools were cultured using culture medium, and induced to differentiate into left ventricular cell spheres, right ventricular cell spheres, and cardiac cell spheres with atrial characteristics, respectively, during culture.

[0041] Optionally, the induction differentiation culture tank includes a first induction differentiation culture tank, a second induction differentiation culture tank, and a third induction differentiation culture tank, and S102 includes:

[0042] For human pluripotent stem cells in the first induction differentiation culture pool, the cell density was first controlled to differentiate human pluripotent stem cells into first cardiac field precursor cells. Then, by regulating the Activin / Nodal, WNT, BMP and RA signaling pathways, the differentiation of first cardiac field precursor cells was induced to obtain left ventricular cell spheres.

[0043] For human pluripotent stem cells in the second induction differentiation culture pool, the WNT and Nodal / Activin signaling pathways were first inhibited to obtain second cardiac field precursor cells. Formic acid was then added during the culture of the second cardiac field precursor cells to induce their differentiation and obtain right ventricular cell spheres.

[0044] For human pluripotent stem cells in the third induction differentiation culture pool, retinoic acid, FGF2, LDN-193189 and LY-411575 were injected into the culture medium on day 7. On day 10, dexamethasone, indomethacin, T3 hormone and lipid concentrate were injected into the culture medium. Culture continued until day 21 to obtain cardiac cell spheres with atrial characteristics.

[0045] Optionally, S4 includes:

[0046] The three-dimensional cell tissue was cultured in a culture tank using a culture medium to obtain the target organ tissue.

[0047] The components of the culture medium include at least one of dextran, mannan, chitosan, pectin, and gelatin.

[0048] (III) Beneficial Effects

[0049] The organ-on-a-chip platform proposed in this invention integrates an optical tweezers system and an organ-on-a-chip. It first culturees stem cells into 3D spherical cells with specific functions in an induction differentiation culture chamber, then transfers them to an assembly culture chamber to directly assemble them into three-dimensional cell tissues according to the proportions and order of the target organ tissue. Compared to existing technologies, it eliminates the need for microfabrication techniques. The optical tweezers system enables more precise control of cell tissue structure in both time and space dimensions in a rapid, easy-to-operate, and low-cost manner, effectively reducing costs and technical barriers while enhancing functionality. Furthermore, the independent culture setup using induction differentiation and assembly culture chambers at different growth stages of cells or tissues effectively reduces the difficulty of microenvironment control, further lowering the technical threshold for organ-on-a-chip technology and holding significant importance for disease modeling and regenerative medicine research. In addition, the manipulation of cell sphericals using the optical tweezers system is non-contact, minimizing cell damage and preventing contamination. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the architecture of an organ-on-a-chip platform with integrated optical tweezers provided in the embodiment;

[0051] Figure 2 This is a schematic diagram of the organ-on-a-chip structure in the embodiment;

[0052] Figure 3 This is a schematic diagram of the organ-on-a-chip structure in the embodiment;

[0053] Figure 4 This is a schematic diagram of the optical tweezers system in the embodiment;

[0054] Figure 5 This is a flowchart illustrating a three-dimensional cell assembly method provided in the embodiment;

[0055] Figure 6 This is a schematic diagram of an assembly method for a multi-chambered cardiac organoid provided as an embodiment;

[0056] Figure 7 A schematic diagram of an assembly method for a biomimetic liver lobule provided for an embodiment;

[0057] Figure 8 A schematic diagram of an assembly method for a biomimetic liver lobule provided for an embodiment;

[0058] Figure 9 A schematic diagram illustrating a method for assembling vascularized liver organoid lobules as provided in an embodiment;

[0059] Figure 10 This is a schematic diagram of a method for assembling vascularized liver organoid lobules, provided as an example.

[0060] [Explanation of Labels in the Attached Image]

[0061] 10. Organ-on-a-chip; 101. Induction differentiation culture tank; 102. Assembly culture tank; 103. Connecting channel; 104. Upper plate; 105. Middle plate; 106. Lower plate; 107. Displacement cavity; 108. Electromagnet; 109. Magnet block; 110. Inlet channel; 111. Outlet channel;

[0062] 20. Optical tweezers system; 201. Laser source; 202. Spatial light modulator; 203. Dichroic mirror; 204. Objective lens; 205. Illumination source; 206. Collimating lens; 207. CCD camera;

[0063] 30. Three-dimensional cellular tissue. Detailed Implementation

[0064] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0065] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0066] Example 1

[0067] like Figure 1 and 2 As shown, this embodiment provides an organ-on-a-chip platform with integrated optical tweezers, including an organ-on-a-chip 10 with optical transparency and an optical tweezers system 20 integrated with the organ-on-a-chip.

[0068] The organ-on-a-chip 10 includes at least two induction differentiation culture pools 101 and at least one assembly culture pool 102; the induction differentiation culture pools 101 and the assembly culture pools 102 are connected; the at least two induction differentiation culture pools 101 are used to culture and differentiate stem cells into at least two different types of cells. It should be noted that the cells here can be single cells or 3D cell spheres with a three-dimensional shape.

[0069] The optical tweezers system 10 is used to capture the different types of cells and move the different types of cells from the induction differentiation culture pool 101 to the assembly culture pool 102; and, in the assembly culture pool 102, to assemble the at least two different types of cells into an initial three-dimensional cell tissue 30 according to the proportion, order and pattern structure in the target organ tissue.

[0070] The assembly culture tank 102 is used to culture the initial three-dimensional cell tissue 30 to obtain a three-dimensional cell tissue with the target pattern structure.

[0071] Generally, the organ-on-a-chip 10 is horizontally disposed on the stage of the organ-on-a-chip platform, and the optical tweezers system 20 is integrated on the platform of the organ-on-a-chip. The objective lens of the optical tweezers system 20 is vertically oriented toward the organ-on-a-chip 10, specifically located directly above or below the organ-on-a-chip 10.

[0072] The organ-on-a-chip platform proposed in this embodiment integrates an optical tweezers system and an organ-on-a-chip. It first culturees stem cells into 3D spherical cells with specific functions in an induction differentiation culture chamber, then transfers them to an assembly culture chamber to directly assemble them into three-dimensional cell tissues according to the proportions and order found in the target organ tissue. Compared to existing technologies, it eliminates the need for microfabrication techniques. The optical tweezers system enables more precise control of cell and tissue structures in a rapid, easy-to-operate, and low-cost manner, effectively reducing costs and technical barriers. Furthermore, the independent culture setup using induction differentiation and assembly culture chambers at different growth stages of cells or tissues effectively reduces the difficulty of microenvironment control, further lowering the technical threshold for organ-on-a-chip technology. This is of great significance for disease modeling and regenerative medicine research.

[0073] In one specific embodiment of this invention, the organ-on-a-chip 10 is made of highly optically transparent glass or a high-molecular polymer, specifically one of polystyrene (PS), polycarbonate (PC), or polydimethylsiloxane (PDMS), to ensure that the organ-on-a-chip 10 is sterile, pyrogen-free / endotoxin-free, and cytotoxic. The induction differentiation culture tank 101 and the assembly culture tank 102 are connected by a connecting channel 103, on which a control valve is provided. The dimensions of the connecting channel are 2mm × 2mm.

[0074] Specifically, such as Figure 2and 3 As shown, the organ-on-a-chip includes an upper plate 104, a middle plate 105, and a lower plate 106 stacked sequentially. The differentiation induction culture tank 101, the assembly culture tank 102, and the connecting channel 103 are grooves or windows carved into the top or bottom surface of the middle plate 105, and are sandwiched between the upper plate 104 and the lower plate 106 to form corresponding cavities for the differentiation induction culture tank 101, the assembly culture tank 102, and the connecting channel 103. A clearance cavity 107 is provided at the position of the upper plate 104 or the lower plate 106 corresponding to the connecting channel. The control valve includes an electromagnet 108 and a magnet 109 for sealing the connecting channel, and the shape of the magnet 109 matches that of the clearance cavity 107.

[0075] When the electromagnet 108 is energized, the magnet block 109 moves from the connecting channel to the clearance cavity 107 under the influence of the magnetic field of the electromagnet 108, so that the connecting channel 103 is open. When the electromagnet 108 is de-energized, the magnet block 109 moves from the clearance cavity 107 to the connecting channel under the influence of gravity, so as to block the connecting channel 103.

[0076] In addition, such as Figure 2 As shown, each of the induced differentiation culture tank 101 or assembly culture tank 102 further includes:

[0077] The liquid inlet channel 110 is used to introduce new culture medium liquid or drugs into the induction differentiation culture tank 101 and the assembly culture tank 102 from the outside.

[0078] The liquid outlet channel 111 is used to discharge the old culture medium liquid from the induction differentiation culture tank 101 and the assembly culture tank 102 to the outside.

[0079] Each culture tank has a separate inlet and outlet channel, which can be used to inject different drugs to induce differentiation into different types of cells.

[0080] The aforementioned inlet and outlet channels allow the induction differentiation culture pool and assembly culture pool to independently control the input substances during the cell or tissue culture stage. Initially, stem cells are injected into different induction differentiation culture pools with corresponding differentiation-inducing agents to induce different types of cells, which are then assembled together later. Existing technologies can only achieve patterning through organ-on-a-chip patterning or special configurations, relying on the self-organizing properties of stem cells; this process is usually uncontrollable. This embodiment, however, uses the aforementioned independent induction differentiation culture pools and assembly culture pools, combined with an optical tweezers system, to provide a two-step cell and tissue culture stage, thereby controlling the structure of cells and tissues, thus offering higher control precision compared to existing technologies. Furthermore, the induction differentiation culture pool and assembly culture pool are connected by a communication channel, eliminating the need for external contact during the overall manipulation of cells and tissues, resulting in advantages such as low damage, aseptic operation, rapid and easy execution, and high controllability.

[0081] Optical tweezers are tools that use the force generated by a laser to manipulate microparticles, enabling high-precision, low-damage, and non-contact capture and manipulation of biological cells. The basic principle of optical tweezers is that a focused laser forms a three-dimensional optical trap, exerting a force on an object—a light radiation pressure—which controls the object within the trap. The movement of the laser beam allows manipulation of objects ranging in size from micrometers to nanometers. This embodiment creatively integrates an optical tweezers system into an organ-on-a-chip platform, using optical tweezers to manipulate cells or cell tissues in induced differentiation and assembly culture chambers. On one hand, this non-contact cell manipulation method reduces the possibility of cell contamination, damage, or injury during manipulation; on the other hand, it lowers the technical difficulty of organ-on-a-chip fabrication, enabling the organ-on-a-chip platform to assemble nanoscale three-dimensional cells, achieving more precise control in a rapid, easy-to-operate, and low-cost manner.

[0082] In one specific implementation of this embodiment, such as Figure 4 As shown, the 20 optical tweezers system is a holographic optical tweezers system, comprising: a laser source 201, a spatial light modulator 202, an optical path assembly, a detection assembly, and a control system, as detailed below:

[0083] The laser source 201 is used to generate a laser beam. The wavelength range of the laser beam generated by the laser source is 1064 nm to 1500 nm, and the power is 30 mW to 3 W. More preferably, the wavelength of the laser beam is 1064 nm to reduce damage to cells or cell spheroids. For cell spheroids of 5 to 500 micrometers, the larger the size, the greater the beam energy required, and the greater the power of the laser source. Existing optical tweezers commonly use laser sources with a wavelength of 540 nm, which can damage cell spheroids at high power. This embodiment selects a 1064 nm light source, which neither damages cell spheroids nor reduces the power, thereby increasing the beam energy and enabling manipulation of micrometer-sized cell spheroids.

[0084] The optical path assembly is used to transmit and adjust the laser beam, so that the laser beam illuminates the organ-on-a-chip and forms one or more optical traps for capturing and moving cells; the optical path assembly includes optical fibers, an optical path, and an optical lens assembly. The optical lens assembly specifically includes optical devices such as a dichroic mirror 203, a reflecting mirror, and an objective lens 204. The objective lens 204 is positioned directly above the organ-on-a-chip 10. The optical path is arranged between the optical lens assemblies.

[0085] The spatial light modulator 202 (SLM) is used to encode the light field distribution of the laser beam to regulate the capture of one or more cells by the light trap; and to encode the light field distribution of the laser beam to form a preset light field pattern, controlling the position and movement path of the light trap. Specifically, the spatial light modulator can simultaneously move cells from multiple induction differentiation culture tanks 101 to assembly culture tank 102 based on the preset light field pattern, and form a special pattern in the assembly culture tank 102 according to the proportion, order, and pattern structure in the target organ tissue. The light field pattern is determined according to the requirements for cell movement.

[0086] The detection component is used to monitor the position and movement of 3D spheroid cells in the organ-on-a-chip. Specifically, the detection component includes an illumination source 205, a collimating lens 206, and a CCD camera 207. The illumination source 205 is positioned directly below the organ-on-a-chip 10. The collimating lens 206 is positioned between the organ-on-a-chip 10 and the illumination source 205 to collimate the illumination beam emitted by the illumination source 205. The CCD camera 207 is positioned above the organ-on-a-chip 10 to receive the illumination beam transmitted through the organ-on-a-chip 10, thereby acquiring images containing information such as the morphology and position of cells, spheroids, or other cellular tissues in the organ-on-a-chip 10.

[0087] The control system is communicatively connected to the laser source 201, spatial light modulator 202, optical path assembly, and detection assembly. It is used to control the laser source 201, spatial light modulator 202, optical path assembly, and detection assembly. Specifically, it includes adjusting the laser source 201, programming holographic optical elements such as the spatial light modulator 202, adjusting the optical path assembly, and real-time monitoring and feedback control of the movement of cells or cell spheres, so as to help users manipulate cells or cell spheres.

[0088] Based on the aforementioned optical tweezers system, when cell assembly is required, the system is manipulated to illuminate the cells or cell spheres with a laser beam. The refraction, scattering, and absorption effects of the beam generate an optical field gradient around the cells or cell spheres, forming an optical potential well that confines the cells or cell spheres to the optical field. The cells or cell spheres displace as the beam moves or the stage moves, thereby achieving the capture, movement, or assembly of the cells or cell spheres.

[0089] Example 2

[0090] like Figure 5 As shown, based on the organ-on-a-chip platform provided in Embodiment 1, this embodiment provides a three-dimensional cell assembly method, specifically including steps S1 to S4:

[0091] S1. Inject stem cells into at least two induction differentiation culture pools, and culture and induce differentiation to obtain at least two types of cells.

[0092] S2. Use an optical tweezers system to capture the cells and move them to the assembly culture tank. Specifically, there are usually multiple cells in an induction differentiation culture tank. Before capture, the morphology of these cells can be observed using the detection component of the optical tweezers system, and healthy cell spheres with good shape and condition can be selected as target cell spheres for capture.

[0093] S3. Using an optical tweezers system, assemble the cells in the assembly culture tank according to the proportions and order of the target organ tissue to obtain a three-dimensional cell tissue.

[0094] S4. The three-dimensional cell tissue is cultured in the assembly culture tank to obtain the target organ tissue.

[0095] Specifically, the three-dimensional cell tissue can be cultured in a culture medium in an assembly culture tank. In addition to the nutrients required to maintain cell viability, the culture medium also requires the addition of 1%-20% stabilizers. These stabilizers include one or more of the following: polysaccharides such as dextran, mannan, and chitosan, and pectin and gelatin. The pH value is adjusted as needed. This eliminates the repulsion between cell spheres and generates an electrostatic adsorption effect, allowing the assembled three-dimensional cell tissue to maintain its three-dimensional morphology.

[0096] Furthermore, the culture and differentiation induction steps in S1 described above differ slightly depending on the target organ tissue. In one specific embodiment of this example, when the target organ tissue is a multi-chambered cardiac organoid, such as... Figure 6 As shown, step S1 includes steps S101 and S102, as detailed below:

[0097] S101. Human pluripotent stem cells (PSCs) were injected into three induction differentiation culture pools respectively.

[0098] S102. Human pluripotent stem cells in three induction differentiation culture pools were cultured in liquid culture medium and induced to differentiate into left ventricular cell spheres, right ventricular cell spheres, and cardiac cell spheres with atrial characteristics, respectively.

[0099] Preferably, the concentration of the stabilizer bulk in the culture medium liquid, and the ratio (mass ratio) of each component in the stabilizer bulk, can be any one of the component proportions in Table 1:

[0100] Table 1. Concentration of stabilizer bulk in culture medium liquid and ratio of each component in stabilizer bulk.

[0101]

[0102] It should be noted that all the culture media mentioned above are existing commercial culture media.

[0103] Specifically, the induction differentiation culture pool includes a first induction differentiation culture pool, a second induction differentiation culture pool, and a third induction differentiation culture pool, and S102 includes:

[0104] S102-A: For human pluripotent stem cells in the first induction differentiation culture pool, the cell density is first controlled to differentiate human pluripotent stem cells into first heart field (FHF) precursor cells. Then, by regulating the Activin / Nodal, WNT, BMP and RA signaling pathways, the differentiation of first heart field precursor cells is induced to obtain left ventricular cell spheres.

[0105] S102-B: For human pluripotent stem cells in the second induction differentiation culture pool, the WNT and Nodal / Activin signaling pathways are first inhibited to obtain anterior second heart field (aSHF) precursor cells. Formic acid is then added during the culture of the anterior second heart field precursor cells to induce their differentiation and obtain right ventricular cell spheres.

[0106] For human pluripotent stem cells in the third induction differentiation culture pool, on day 7 of culture, retinoic acid, FGF2 (basic fibroblast growth factor), LDN-193189 (BMP signaling pathway inhibitor) and LY-411575 (γ-secretase inhibitor) were injected into the culture medium. On day 10 of culture, dexamethasone, indomethacin, T3 hormone and lipid concentrate were injected into the culture medium. Culture continued until day 21 to obtain cardiac cell spheres with atrial characteristics.

[0107] Corresponding to the three types of cell spheres mentioned above, in step S3, the three types of cell spheres can be assembled according to the structure of the heart into a three-dimensional structure with the left ventricular cell sphere in the lower left, the right ventricular cell sphere in the lower right, and the cardiac cell sphere with atrial characteristics on top of the two, so as to ensure that the structure of the target organ tissue cultured in S4 is closer to the actual human heart.

[0108] In another specific embodiment of this example, when the target organoid tissue is a biomimetic liver lobule, it can be induced to differentiate, assemble, and cultured according to steps S1 to S4 provided in this example. The difference is as follows:

[0109] Two differentiation induction culture tanks were used to induce differentiation into hepatocytes and endothelial cells, respectively. Hepatocytes were first transferred from the connecting channel into the assembly culture tank, and then arranged in sections using optical tweezers. Figure 7 The pattern shown has six regions. Endothelial cells are then filled into the spaces and centers of these six regions, forming a hexagonal pattern of hepatocytes and endothelial cells, as shown. Figure 8 As shown. The induction differentiation culture tank contains the corresponding hepatocyte culture medium or endothelial cell culture medium, and the assembly culture tank contains liver organoid culture medium with added stabilizers (the stabilizers prepared in Table 1 can be used).

[0110] In another specific embodiment of this example, when the target organ tissue is a vascularized liver organoid lobule, induction differentiation, assembly, and culture can be performed according to steps S1 to S4 provided in this example. The difference is as follows:

[0111] Two induction differentiation culture tanks were used to induce differentiation into liver organoids and vascular organoid fragments (tiny tubes), respectively. The liver organoids were first transferred into the assembly culture tank and arranged into a planar pattern using an optical tweezers system. Figure 9 The four blue sections on the left), followed by stacking vascular organoid fragments on a planar pattern. Figure 9 The pink section on the right side forms a double-layered structure. (For example...) Figure 10 As shown, vascular organoid fragments are then arranged on the sides of the double-layered structure, corresponding to the sides of the liver organoids. New liver organoids are then stacked on top of the newly arranged vascular organoids, forming a structure as shown. Figure 10 The right side shows a three-dimensional cellular structure with overlapping vascular organoid fragments and liver organoids.

[0112] This embodiment of the three-dimensional cell assembly method, based on the organ-on-a-chip platform with an integrated optical tweezers system provided in Embodiment 1, offers a method for first culturing and inducing differentiation of stem cells into different cell types in an induced differentiation culture pool, and then transferring the cells to an assembly culture pool for assembly and culture according to specific structures in the target organ tissue. Compared to existing technologies that rely on the self-organizing properties of stem cells to achieve patterning, this method offers higher precision in controlling cell tissues, and the resulting target organ tissue better meets user needs.

[0113] Those skilled in the art will understand that embodiments of the present invention can be provided as methods, systems, or computer program products. Therefore, the present invention can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present invention can take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0114] This invention is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, as well as combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions.

[0115] It should be noted that any reference numerals placed between parentheses in the claims should not be construed as limiting the claims. The word "comprising" does not exclude the presence of components or steps not listed in the claims. The word "a" or "an" preceding a component does not exclude the presence of a plurality of such components. The invention can be implemented by means of hardware comprising several different components and by means of a suitably programmed computer. In claims that enumerate several means, several of these means may be embodied by the same hardware. The use of the terms first, second, third, etc., is merely for convenience of expression and does not indicate any order. These terms can be understood as part of the component names.

[0116] Furthermore, it should be noted that in the description of this specification, the terms "one embodiment," "some embodiments," "embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0117] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the claims should be interpreted to include both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0118] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, then this invention should also include these modifications and variations.

Claims

1. An organ chip platform with integrated optical tweezers, characterized in that: Includes organ chips with optical transparency and optical tweezers systems integrated with organ chips; The organ chip comprises at least two induced differentiation culture pools and at least one assembly culture pool; the induced differentiation culture pool is connected to the assembly culture pool; the at least two induced differentiation culture pools are used to culture stem cells separately and induce differentiation into at least two different types of cells; The optical tweezers system is used to capture the different types of cells and move the different types of cells from the induced differentiation culture tank to the assembly culture tank; and assemble at least two different types of cells into an initial three-dimensional cell tissue in the assembly culture tank according to the proportion, sequence and pattern structure in the target organ tissue; The assembly culture tank is used to culture the initial three-dimensional cell tissue to obtain a three-dimensional cell tissue with a target pattern structure.

2. The organ chip platform according to claim 1, characterized in that: The induction differentiation culture tank is connected with the assembly culture tank through a connecting channel, and a control valve is arranged on the connecting channel.

3. The organ chip platform according to claim 2, characterized in that: A clearance cavity is provided on the connecting channel; The control valve comprises: an electromagnet and a magnet block for blocking the connecting channel, and the magnet block matches the shape of the give-way cavity; When the electromagnet is energized, the magnet block moves from the connecting channel to the giving way cavity under the action of the magnetic field of the electromagnet, so that the connecting channel is conductive; When the electromagnet is powered off, the magnet block moves from the clearance cavity to the connecting channel under the action of gravity to block the connecting channel.

4. The organ chip platform according to claim 2, characterized in that: Each of the differentiation induction culture tanks or assembly culture tanks further comprises: A liquid inlet channel, used for inputting new culture medium liquid or medicine from the outside into the induction differentiation culture tank and the assembly culture tank; The liquid outlet channel is used to discharge old culture medium liquid from the induced differentiation culture tank and the assembled culture tank to the outside.

5. The organ chip platform according to claim 1, characterized in that: The optical tweezers system comprises: Laser light source, used to generate laser beam and control laser power; An optical path component, used for transmitting and adjusting the laser beam, so that the laser beam is irradiated on the organ chip and forms one or more light traps for capturing and moving cells; the optical path component includes an optical fiber, an optical path and an optical lens component; A spatial light modulator, used to encode the light field distribution of the laser beam to regulate the light trap to capture one or more cells; and to encode the light field distribution of the laser beam to form a preset light field pattern to control the position and movement path of the light trap; A detection component for monitoring the position and movement state of cells in the organ chip; The control system is communicatively connected with the laser light source, the spatial light modulator, the optical path component and the detection component, and is used to control the laser light source, the spatial light modulator, the optical path component and the detection component.

6. The organ chip platform according to claim 5, characterized in that: The laser light source generates a laser beam with a wavelength ranging from 1064nm to 1500nm and a power ranging from 30mW to 3W.

7. A three-dimensional cell assembly method, characterized in that: The method is implemented based on any one of the organ chip platforms described in claims 1 to 6, and the method comprises: S1. Injecting stem cells into at least two differentiation induction culture pools, and culturing and inducing differentiation to obtain at least two different types of cells; S2, using an optical tweezers system to capture the different types of cells and move them to an assembly culture tank; S3, using an optical tweezers system to assemble different types of cells in the assembly culture pool according to the proportion, sequence and pattern structure of the target organ tissue to obtain an initial three-dimensional cell tissue; S4. Cultivating the initial three-dimensional cell tissue in the assembly culture tank to obtain a three-dimensional cell tissue with a target pattern structure.

8. The method according to claim 7, characterized in that The target organ tissue is a multi-chambered cardiac organoid, and S1 comprises: S101, injecting human pluripotent stem cells into three induced differentiation culture pools respectively; S102. Using culture medium to culture human pluripotent stem cells in three induced differentiation culture pools, and inducing them to differentiate into left ventricular cell spheres, right ventricular cell spheres, and cardiac cell spheres with atrial characteristics, respectively.

9. The method according to claim 8, characterized in that The induced differentiation culture pool includes a first induced differentiation culture pool, a second induced differentiation culture pool and a third induced differentiation culture pool, and S102 includes: For the human pluripotent stem cells in the first induced differentiation culture pool, the cell density is first controlled to differentiate the human pluripotent stem cells into the first heart field progenitor cells, and then the first heart field progenitor cells are induced to differentiate by regulating the Activin / Nodal, WNT, BMP and RA signaling pathways to obtain left ventricular cell spheres; For the human pluripotent stem cells in the second induced differentiation culture pool, the WNT and Nodal / Activin signaling pathways are firstly inhibited to obtain the second heart field progenitor cells, and then formic acid is added during the culture process of the second heart field progenitor cells to induce their differentiation to obtain right ventricular cell spheres; For the human pluripotent stem cells in the third induced differentiation culture pool, retinoic acid, FGF2, LDN-193189 and LY-411575 were injected into the culture medium on the 7th day of culture, and dexamethasone, indomethacin, T3 hormone and lipid concentrate were injected into the culture medium on the 10th day of culture. The culture was continued to the 21st day to obtain cardiac cell spheres with atrial characteristics.

10. The method according to claim 7, characterized in that The S4 includes: Cultivating the three-dimensional cell tissue in an assembled culture tank using a culture medium to obtain a target organ tissue; The components of the culture medium include at least one of glucan, mannan, chitosan, pectin and gelatin.

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