Dual-channel organ-like dynamic co-culture micro-fluidic chip and application thereof

Abstract

The invention provides a dual-channel organ-like dynamic co-culture micro-fluidic chip and application thereof. The chip comprises at least one group of parallel micro-fluidic channels, namely a micro-fluidic channel I and a micro-fluidic channel II; the distance between the parallel micro-fluidic channels is 50 to 2000 [mu] m; at least one semispherical concave structure is arranged at the bottom of the microfluidic channel, and the semispherical concave structure is used for directly fixing the organoid or embedding the organoid by hydrogel; a gap structure is arranged between the hemispherical concave structures on the microfluidic channel I and the microfluidic channel II, and allows organoid in the microfluidic channel I and the microfluidic channel II to be in direct contact and secreted cell factors to be diffused. The microfluidic chip solves the problems of controllable contact, independent nutrition supply and dynamic microenvironment simulation of heterologous organs in a three-dimensional space, and provides a precise technical platform for drug interaction research, tumor-matrix interaction analysis, organ development model construction and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological microfluidic chips, specifically relating to a dual-channel organoid dynamic co-culture microfluidic chip and its applications. Background Technology

[0002] Organoids are three-dimensional cell cultures formed in vitro using adult stem cells, pluripotent stem cells, or tissue progenitor cells, which can mimic the structural and functional characteristics of real organs. They induce cell self-organization and differentiation through the extracellular matrix and specific growth factors, forming spatial structures containing organ-specific cell types. Their main applications are in developmental biology, disease modeling, drug screening, and regenerative medicine research.

[0003] CN202311328131.8 discloses a microfluidic chip for co-culturing multiple organoids, comprising an organoid microfluidic chip monomer. The monomer consists of two bonded layers: the upper layer sequentially comprises organoid culture medium perfusion channels, a first channel barrier, an organoid culture chamber, a second channel barrier, and a vascular endothelial culture channel; the lower layer comprises a microperfusion network channel. This microfluidic chip ensures specific nutrition and microenvironment for various organs, helping to maintain their specific phenotypes. Simultaneously, it can simulate blood flow in vivo, enabling signal communication and interaction between organoids, fully simulating the in vivo environment and interactions of multi-organ diseases.

[0004] CN202320408917.X discloses a microfluidic-based vascularized organoid-peripheral vascular interaction model chip, comprising a vascularized placental organoid, a peripheral vascular organoid, and a microfluidic chip. The microfluidic chip has a first culture region I and a second culture region II. The vascularized placental organoid is in the first culture region I, and the peripheral vascular organoid is in the second culture region II. A channel connects the first culture region I and the second culture region II, allowing for controllable fluid interaction between them using microfluidic technology. Based on the established vascularized placental organoid, the chip further combines the vascularized placental organoid with peripheral target organoids to evaluate the influence of the placenta on peripheral blood vessels in the pathogenesis of placental-derived diseases.

[0005] The existing organoid co-culture technology has the following technical problems: (1) Traditional co-culture devices (such as Transwell) are difficult to achieve physical contact and dynamic microenvironment control of three-dimensional organoids, which limits the study of cell-cell interactions. (2) Existing microfluidic chips mostly adopt the mixed culture medium perfusion mode, which cannot provide an independent nutrient supply environment for heterologous organoids. (3) There is a lack of standardized platforms that can simultaneously accommodate hydrogel-embedded organoids and direct organoid grafts, which affects the reproducibility of experiments.

[0006] Therefore, there is an urgent need to solve the problems of controllable contact, independent nutrient supply and dynamic microenvironment simulation of xenogeneic organoids in three-dimensional space, and to develop new organoid culture devices. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a dual-channel organoid dynamic co-culture microfluidic chip and its applications. This invention solves the problems of controllable contact, independent nutrient supply, and dynamic microenvironment simulation of heterologous organoids in three-dimensional space by constructing a dual-channel independent perfusion system and an organoid fixation structure, providing a precise technical platform for drug interaction research, tumor-matrix interaction analysis, and organ development model construction.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a dual-channel organoid dynamic co-culture microfluidic chip, wherein the chip includes at least one set of parallel microfluidic channels, denoted as microfluidic channel one and microfluidic channel two; the spacing between the parallel microfluidic channels is 50-2000 μm, for example, it can be 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, 1500 μm or 2000 μm, etc.

[0010] The bottom of the microfluidic channel is provided with at least one hemispherical recess structure, which is used to directly fix organoids or embed organoids in hydrogel.

[0011] A gap structure is provided between the hemispherical recesses on the microfluidic channel one and the microfluidic channel two, which allows the organoids in the microfluidic channel one and the microfluidic channel two to come into direct contact and for the secreted cytokines to diffuse.

[0012] The microfluidic chip design of this invention is based on the physiological structure of spatially adjacent yet functionally independent organs in vivo, aiming to reconstruct a three-dimensional co-culture environment with independent nutrient supply and controllable signal interaction in vitro. The chip employs a dual-channel parallel layout structure to achieve liquid isolation and controllable diffusion of secretory factors. Each channel has multiple hemispherical depressions at its bottom to fix organoids or hydrogel-embedded organoids, preventing displacement under fluid conditions. The two channels are connected to a programmable perfusion system, allowing for independent nutrient supply and shear force control through flow rate adjustment, thereby simulating metabolic differences between different organs. The gap structure between the two channels creates a bidirectional diffusion channel for signal molecules while ensuring liquid isolation, reconstructing the in vivo paracrine factor exchange environment.

[0013] This invention achieves a dynamic process of heterologous organoids from long-distance signal interaction to contact fusion through a dual-mode "independent perfusion-controlled contact" approach. It is compatible with various hydrogel matrices such as Matrigel, collagen, and sodium alginate, and supports co-culture of multiple organoid systems, including kidney, liver, intestine, brain, and tumor organoids. This chip can be integrated with automated culture and imaging systems, significantly improving the standardization and scalability of organoid models, and providing a precise and reliable platform for multi-organ interaction, drug interaction research, and disease modeling.

[0014] In this invention, cytokines are a class of small molecule proteins or polypeptides synthesized by cells and released outside the cells. Their core function is to transmit signals between cells and regulate the growth, differentiation, migration or functional state of other cells. Cytokines can move in the interstitial structure and achieve bidirectional diffusion.

[0015] Preferably, the two ends of the microfluidic channel one and the microfluidic channel two are respectively connected to a programmable perfusion system; the programmable perfusion system is used to perfuse organoid-specific culture medium.

[0016] In this invention, a programmable perfusion system is connected to both ends of the channel, supporting independent flow rate control for both channels; the flow rate of the culture medium in different channels can be adjusted according to actual needs.

[0017] Preferably, the width of the first microfluidic channel is 200-1500 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm, 1000 μm, 1200 μm, 1400 μm or 1500 μm; the depth is 200-1500 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm, 1000 μm, 1200 μm, 1400 μm or 1500 μm.

[0018] Preferably, the width of the second microfluidic channel is 200-1500 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm, 1000 μm, 1200 μm, 1400 μm or 1500 μm; the depth is 200-1500 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm, 1000 μm, 1200 μm, 1400 μm or 1500 μm.

[0019] In this invention, the chip body contains at least two parallel microfluidic channels, and the channels are separated by physical structures to achieve material exchange between different culture media and between organoids.

[0020] Preferably, the width of the void structure is 50-500 μm, for example, it can be 50 μm, 100 μm, 200 μm, 300 μm, 400 μm or 500 μm.

[0021] In this invention, the gap structure design between the hemispherical concave structures allows direct contact between organoids and diffusion of cell-secreted factors, but restricts organoid displacement.

[0022] Preferably, in the first microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 500-2000 μm; for example, it can be 500 μm, 1000 μm, 1500 μm or 2000 μm, etc.

[0023] The depth of the hemispherical recessed structure is 200-1000 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm or 1000 μm.

[0024] Preferably, in the second microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 500-2000 μm; for example, it can be 500 μm, 1000 μm, 1500 μm or 2000 μm, etc.

[0025] The depth of the hemispherical recessed structure is 200-1000 μm, for example, it can be 200 μm, 400 μm, 600 μm, 800 μm or 1000 μm.

[0026] In this invention, the bottom of the two channels is provided with a connected hemispherical recess structure for directly fixing organoids or embedding organoids in hydrogel.

[0027] In this invention, the working process of the dual-channel organoid dynamic co-culture microfluidic chip is as follows: organoids or hydrogel-organoid mixtures are precisely seeded into the connected hemispherical depressions in the two channels; channel 1 is perfused with a special culture medium for the first organoid, and channel 2 is perfused with a culture medium for the second organoid; the exchange intensity of paracrine factors is controlled by adjusting the flow rate ratio of the two channels for culture.

[0028] In a second aspect, the present invention provides an organoid co-culture method based on the dual-channel organoid dynamic co-culture microfluidic chip described in the first aspect, the method comprising:

[0029] (1) Different organoid inoculants were inoculated into the hemispherical concave structures in microfluidic channel one and microfluidic channel two, respectively;

[0030] (2) The corresponding organoid culture medium was injected into microfluidic channel one and microfluidic channel two respectively for culture.

[0031] Preferably, in step (1), the organoid inoculum comprises: organoid cells or hydrogel-organoid mixture.

[0032] Preferably, the organoid inoculum is a hydrogel-organoid mixture.

[0033] In this invention, when the inoculum is a hydrogel-organoid mixture, a curing step is also included.

[0034] In this invention, the curing step serves to establish a stable three-dimensional fixed structure between the organoid and its surrounding hydrogel matrix within a hemispherical depression. This prevents displacement or detachment due to fluid shear forces during subsequent perfusion, while maintaining its original morphology and spatial positioning. By performing temperature- or ion-induced curing (e.g., Matrigel or collagen curing at 37°C) after inoculation, localized gel microregions can be formed at the bottom of the chip, ensuring physical support and uniform nutrient distribution for the organoid under dynamic culture conditions. This step not only improves the reproducibility and stability of organoid culture but also provides a reliable structural basis for long-term live-cell imaging and subsequent immunostaining.

[0035] Preferably, the volume fraction of organoid cell suspension in the hydrogel-organoid mixture is 10%-50%, more preferably 20%-40%; the remainder is hydrogel matrix; and the concentration of organoid cell suspension is 500-5000 cells / μL.

[0036] In this invention, the hydrogel-organoid mixture is preferably obtained by mixing organoid cell suspension and hydrogel matrix in a certain volume fraction ratio. The hydrogel matrix can be a gelling material such as Matrigel, collagen, or sodium alginate. To simultaneously ensure the spatial fixation and subsequent growth capacity of organoids within the microfluidic channel, the volume fraction of the hydrogel matrix in the hydrogel-organoid mixture is preferably 50%-90%; the volume fraction of the organoid cell suspension (containing organoid fragments, organoid clumps, or recombinant structures of single cells / clumps) is preferably 10%-50%, more preferably 10%-20%. The above ratio can form a local gelling support structure within the hemispherical depression, ensuring rapid solidification after sample loading and confining the organoids to a predetermined position, preventing them from being washed away by shear force during the continuous perfusion stage; at the same time, the hydrogel concentration is not so high as to completely inhibit organoid expansion, branching growth, or migration and fusion between organoids in the two channels, thereby meeting the needs of the subsequent dynamic perfusion culture stage and static fusion stage.

[0037] Preferably, the hydrogel is selected from Matrigel, collagen, or sodium alginate.

[0038] The hydrogel described in this invention can be selected from natural or synthetic hydrogel materials such as Matrigel, collagen, sodium alginate, GelMA, fibrin, or polyethylene glycol (PEG). Its concentration, degree of cross-linking, and mixing ratio can be adjusted according to different organoid types and culture requirements to achieve a suitable mechanical support and signal transduction environment.

[0039] Preferably, in step (2), the organoid culture medium is perfused into the corresponding microfluidic channels through a programmable perfusion system.

[0040] Preferably, in step (2), the culture steps include: a continuous perfusion culture stage and a static culture stage; during the continuous perfusion culture stage, the organoids gradually grow; during the static culture stage, perfusion is stopped to achieve contact co-culture and induce organoid migration and fusion.

[0041] Thirdly, the present invention provides the application of the dual-channel organoid dynamic co-culture microfluidic chip described in the first aspect in organoid culture.

[0042] The dual-channel organoid dynamic co-culture microfluidic chip described in this invention has broad application prospects. By realizing independent perfusion and controllable substance exchange between organoids in a microscale space, this chip can accurately simulate the physiological interactions between multiple organs in vitro, making it suitable for various biomedical research and drug development scenarios.

[0043] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) This invention pioneers a dual-mode "independent perfusion-controllable contact" approach, which achieves independent nutrient supply and precise control of fluid shear force for different organoids through a parallel dual-channel structure and a programmable perfusion system. Compared with traditional Transwell co-culture or single-channel microfluidic systems, this invention can simultaneously achieve independent nutrient supply, controllable signal exchange, and dynamic contact fusion for organoids, significantly improving the accuracy and reproducibility of three-dimensional co-culture.

[0046] (2) The present invention has high material compatibility and structural adaptability. The hemispherical depression in the chip makes it compatible with a variety of natural and synthetic hydrogels, including Matrigel, collagen, sodium alginate, GelMA, fibrin and polyethylene glycol (PEG), etc., which can be flexibly selected according to the source and culture requirements of different organoids, significantly improving the versatility and scalability of the system.

[0047] (3) The gap structure between the two channels designed in this invention realizes the controllable diffusion of signal molecules and the physical isolation of organoids, which can significantly improve the repeatability of experiments and the consistency of data.

[0048] (4) The chip structure of the present invention can be modularly integrated into an automated culture and imaging system, which facilitates batch preparation and standardized operation, and can be used for multi-organ interaction research, disease modeling and drug screening, etc. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the dual-channel planar structure of a microfluidic chip.

[0050] Figure 2 This is a schematic diagram of the dual-channel three-dimensional structure of a microfluidic chip.

[0051] Figure 3 This is an enlarged planar schematic diagram of the hemispherical recessed structures on microfluidic channel one and microfluidic channel two.

[0052] Figure 4 This is a magnified three-dimensional schematic diagram of the hemispherical recessed structures on microfluidic channel one and microfluidic channel two.

[0053] Figure 5 Bright-field image of renal organoids and ureteral buds after 7 days of co-culture on a microarray.

[0054] Figure 6 Immunofluorescence image of GFP-labeled ureteral bud fused with renal unit organoids. Detailed Implementation

[0055] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0057] Example 1

[0058] This embodiment provides a dual-channel organoid dynamic co-culture microfluidic chip. The chip includes at least one set of parallel microfluidic channels, denoted as microfluidic channel one and microfluidic channel two; the spacing between the parallel microfluidic channels is 1000 μm.

[0059] The bottom of the microfluidic channel is provided with at least one hemispherical recess structure, which is used to directly fix organoids or embed organoids in hydrogel.

[0060] A gap structure is provided between the hemispherical recesses on the microfluidic channel one and the microfluidic channel two, which allows the organoids in the microfluidic channel one and the microfluidic channel two to come into direct contact and for the secreted cytokines to diffuse.

[0061] The first microfluidic channel has a width of 550 μm and a depth of 1000 μm; the second microfluidic channel has a width of 500 μm and a depth of 1000 μm; the pore structure has a width of 370 μm; in the first microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 1000 μm; the depth of the hemispherical recessed structure is 500 μm; in the second microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 500 μm; the depth of the hemispherical recessed structure is 300 μm.

[0062] A schematic diagram of the dual-channel planar structure of the microfluidic chip is shown below. Figure 1 As shown; Figure 1 In the diagram, 1-1 and 1-5 are the inlets of the microfluidic channel one connecting to the programmable irrigation system; 1-2, 1-3 and 1-4 are hemispherical recessed structures on the microfluidic channel one; 1-6 and 1-10 are the inlets of the microfluidic channel two connecting to the programmable irrigation system; and 1-7, 1-8 and 1-9 are hemispherical recessed structures on the microfluidic channel two. Among these, there are gap structures between 1-2 and 1-7, between 1-3 and 1-8, and between 1-4 and 1-9.

[0063] A schematic diagram of the dual-channel three-dimensional structure of the microfluidic chip is shown below. Figure 2 As shown. Figure 3 This is an enlarged planar schematic diagram of the hemispherical recessed structures on microfluidic channel one and microfluidic channel two.

[0064] Figure 4 This is a magnified three-dimensional schematic diagram of the hemispherical recessed structures on microfluidic channel one and microfluidic channel two.

[0065] Example 2

[0066] This embodiment provides a dual-channel organoid dynamic co-culture microfluidic chip. The chip includes at least one set of parallel microfluidic channels, denoted as microfluidic channel one and microfluidic channel two; the spacing between the parallel microfluidic channels is 500 μm.

[0067] The bottom of the microfluidic channel is provided with at least one hemispherical recess structure, which is used to directly fix organoids or embed organoids in hydrogel.

[0068] A gap structure is provided between the hemispherical recesses on the microfluidic channel one and the microfluidic channel two, which allows the organoids in the microfluidic channel one and the microfluidic channel two to come into direct contact and for the secreted cytokines to diffuse.

[0069] The first microfluidic channel has a width of 1000 μm and a depth of 1000 μm; the second microfluidic channel has a width of 900 μm and a depth of 1000 μm; the pore structure has a width of 500 μm; in the first microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 1500 μm; the depth of the hemispherical recessed structure is 500 μm; in the second microfluidic channel, the diameter of the maximum cross-section of the hemispherical recessed structure is 1500 μm; the depth of the hemispherical recessed structure is 400 μm.

[0070] Example 3

[0071] This embodiment uses a dual-channel organoid dynamic co-culture microfluidic chip as described in Example 1 to co-culture renal unit organoids (NO) and ureteral bud organoids (UBO).

[0072] 1. Experimental procedures.

[0073] (1) Prepare two differentiation culture media.

[0074] The stage II renal unit differentiation medium consists of DMEM, KOSR, NEAA, Glutamax, HEPES, and PVA. The concentrations are as follows: DMEM: 1×; KOSR: 15%; NEAA: 1×; Glutamax: 1×; HEPES: 1×; PVA: 2.5 mg / mL.

[0075] The sources of each component in the above-mentioned renal unit differentiation culture medium stage II are as follows: DMEM: Gibco, 12440053; KOSR: Gibco, A3181502; NEAA: Gibco, 11140050; Glutamax: Gibco, 35050061; HEPES: Gibco, 15630080; PVA: Sigma, P8136.

[0076] The ureteral bud differentiation medium consists of: F12, Glutamax, NEAA, β-mercaptoethanol, B-27 supplement minus vitamin A, ITS, LDN-193189, TTNPB, CHIR99021, JAK inhibitor, GDNF, A83-01, R-Spondin 1, FGF7, SB202190, and EGF. Among them, the dosage is F12: 1×; Glutamax: 1×; NEAA: 1×; β-Mercaptoethanol: 1 mM; B-27 supplement minus vitaminA: 1×; ITS: 1×; ng / mL; A83-01: 0.2 μM; R-Spondin 1: 100 ng / mL; FGF7: 50 ng / mL; SB202190: 5 μM; EGF: 50 ng / mL.

[0077] The sources of each component in the above-mentioned ureteral bud differentiation culture medium are as follows:

[0078] F12: Gibco, 12440053; NEAA: Gibco, 11140050; β-Mercaptoethanol: Gibco, 21985023; B-27 supplement minus vitaminA: Invitrogen, 12587-010; ITS: Sigma, I3146; LDN-193189: Reagents Direct, 36-F52; TTNPB: TOCRIS, 0761; CHIR99021: Stemcell Technologies, 72054; JAK inhibitor: Stemcell Technologies, 74022; GDNF: PeproTech, 450-10-50; A83-01: Stemcell, 04-0014; R-Spondin 1: R&D Systems, 4645-RS-100; FGF7: PeproTech, 100-19; SB202190: Sigma, S7067; EGF: R&D Systems, 236-EG-200.

[0079] (2) In vitro fusion of renal unit organoids and ureteral bud organoids: Early day 8 renal unit organoids obtained from hPSC differentiation were selected under a stereomicroscope. The organoids were 300-500 μm in diameter and well differentiated. A small notch was carefully torn open with sterile surgical forceps. A small piece of ureteral bud organoid was placed into the notch with sterile forceps. After incubation overnight at 37°C, the organoids were transferred to a microarray.

[0080] (3) Transfer to chip co-culture: Transfer the fused organoids after overnight culture to the chip using a Pasteur pipette. Under a microscope, use sterile surgical forceps to adjust the renal unit organoids in the channel one recessed structure and the ureteral bud organoids in the channel two recessed structure.

[0081] (4) Set different flow rates for the culture medium: the flow rate of the renal unit organoid culture medium is 100 μL / h, the flow rate of the ureter bud culture medium is 50 μL / h, and the dynamic culture is carried out for 5-10 days.

[0082] 2. Experimental results.

[0083] Structural fusion: On day 6, microscopy revealed that the two organoids formed a connecting tubular structure. The dual-channel organoid dynamic co-culture microfluidic chip can be used for the co-culture of renal unit organoids (NO) and ureteral bud organoids (UBO), realizing the exchange of secretory factors and three-dimensional fusion of the two organoids.

[0084] Figure 5 Bright-field image of renal organoids and ureteral buds after 7 days of co-culture on a microarray. Figure 5 The data shows the fusion morphology of the renal unit organoid (NO) and the ureteral bud (UB) obtained from H1-GFP differentiation after 7 days of co-culture on a dual-channel microfluidic chip. The GFP-UB is located on one side of the UB culture medium channel on the chip, and the renal unit tissue morphology is clear and its positioning is stable.

[0085] Figure 6 Immunofluorescence image of GFP-labeled ureteral bud fusion with renal unit organoids, from Figure 6 As can be seen from the microarray, GFP-labeled UB (green) and kidney unit organoid sections, after immunostaining, showed PODXL (blue, podocyte marker), CD31 (red, vascular endothelial marker), and CDH1 (red, distal duct marker). The results indicate that the two organoids achieved fusion growth and tissue connection within the microarray, forming a composite kidney development model with structural continuity.

[0086] The above results demonstrate that organoids with different culture conditions, namely renal units and ureteral buds, can be cultured and fused together in this chip.

[0087] Example 4

[0088] This embodiment uses a dual-channel organoid dynamic co-culture microfluidic chip from Embodiment 2 to co-culture vascular organoids and liver organoids.

[0089] The dual-channel microfluidic chip of this invention is selected: Channel 1 and Channel 2 are arranged in parallel; the two channels are separated by a trapezoidal gap structure (width 200 μm); Channel 1 has a width of 600 μm, a depth of 1000 μm, and a bottom hemispherical recess diameter of 500 μm; Channel 2 has a width of 500 μm, a depth of 1000 μm, and a bottom hemispherical recess diameter of 400 μm; the chip material is PDMS-glass bonding, and both ends of the channels are connected to a programmable perfusion system.

[0090] 1. Experimental procedures.

[0091] (1) Organoid preparation.

[0092] Vascular organoids (VO): Vascular organoids derived from ips were combined with fibrin to prepare a hydrogel suspension, wherein the volume fraction of fibrin in the mixture was 70%.

[0093] Liver organoid (HepAO) inoculum: liver organoids derived from ips are suspended in Matrigel; wherein Matrigel constitutes 60% of the mixture by volume.

[0094] Chip pretreatment and inoculation: The inner surface of the chip was pre-washed with organoid culture medium for 10 min. VO inoculum (0.5 μL per pit) was dropwise injected into each hemispherical depression of channel one (wide channel) for in-situ curing. HepAO inoculum (0.4 μL per pit) was dropwise injected into each hemispherical depression of channel two (narrow channel). The chips were incubated at 37°C for 20 min to complete Matrigel / collagen curing. After curing, the respective culture medium was slowly added to cover the channels.

[0095] Perfusion culture strategy:

[0096] Independent perfusion phase (Day 0-Day 2): Channel 1 was perfused with endothelial / vascular culture medium at a flow rate of 30 μL / h; Channel 2 was perfused with liver organoid culture medium at a flow rate of 10 μL / h; a constant flow direction was maintained to remove metabolic waste and stabilize the early morphology of the two organoids. The above independent culture media were kept unmixed, maintaining a flow rate ratio of 3:1; the interstitial structure was used to achieve bidirectional diffusion of secretory factors.

[0097] Signal interaction phase (Day 3-Day 5): Keep the above-mentioned independent culture media separate and maintain a flow rate ratio of 3:1; utilize the interstitial structure to achieve bidirectional diffusion of secreted factors. An angiogenesis-promoting formula (such as VEGF) can be added to the Channel 2 culture medium to promote endothelial budding.

[0098] Contact fusion phase (Day 6-Day 7): A cycle of "12 h resting / 12 h perfusion" was adopted; during resting, the pump was turned off to reduce shear and promote tissue contact and cell migration in the interstitial zone; during perfusion, the flow rate ratio was restored to the aforementioned ratio to maintain overall homeostasis.

[0099] (4) Observation and detection: After co-culturing for 5-10 days, morphological and functional tests were performed on the various organoids. The results showed that the vascular organoids and liver organoids, which were cultured under different conditions, could be cultured and fused together in this chip.

[0100] In summary, this invention presents a dual-channel organoid dynamic co-culture microfluidic chip, pioneering a dual-mode "independent perfusion-controllable contact." Through a parallel dual-channel structure and a programmable perfusion system, it achieves independent nutrient supply and precise control of fluid shear force for different organoids. This dual-channel organoid dynamic co-culture microfluidic chip has broad application prospects in biomedical research and drug development.

[0101] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A dual-channel organoid dynamic co-culture microfluidic chip, characterized in that, The chip comprises at least one set of parallel microfluidic channels, denoted as microfluidic channel one and microfluidic channel two; the spacing between the parallel microfluidic channels is 50-2000 μm; The bottom of the microfluidic channel is provided with at least one semispherical recess structure, which is used for directly fixing the organoids or hydrogel-embedded organoids; The positions between the semispherical recess structures on the microfluidic channel one and the microfluidic channel two are provided with gap structures, which allow the organoids in the microfluidic channel one and the microfluidic channel two to directly contact and the secreted cytokines to diffuse.

2. The dual-channel organoid dynamic co-culture microfluidic chip of claim 1, wherein, The two ends of the microfluidic channel one and the microfluidic channel two are respectively connected to a programmable perfusion system; the programmable perfusion system is used for perfusing organoid-specific culture medium.

3. The dual-channel organoid dynamic co-culture microfluidic chip according to claim 1 or 2, characterized in that, The width of the microfluidic channel one is 200-1500 μm; the depth is 200-1500 μm.

4. The dual-channel organoid dynamic co-culture microfluidic chip according to any one of claims 1-3, wherein, The width of the microfluidic channel two is 200-1500 μm; the depth is 200-1500 μm.

5. The dual-channel organoid dynamic co-culture microfluidic chip according to any one of claims 1-4, wherein, The width of the gap structure is 50-500 μm.

6. The dual-channel organoid dynamic co-culture microfluidic chip according to any one of claims 1-5, wherein, In the microfluidic channel one, the diameter of the maximum cross section of the semispherical recess structure is 500-2000 μm; the depth of the semispherical recess structure is 200-1000 μm.

7. The dual-channel organoid dynamic co-culture microfluidic chip according to any one of claims 1-5, wherein, In the microfluidic channel two, the diameter of the maximum cross section of the semispherical recess structure is 500-2000 μm; the depth of the semispherical recess structure is 200-1000 μm.

8. An organoid co-culture method based on the double-channel organoid dynamic co-culture microfluidic chip according to any one of claims 1-7, characterized in that, The method comprises: (1) inoculating different organoid inocula into the semispherical recess structures in the microfluidic channel one and the microfluidic channel two, respectively; (2) perfusing the corresponding organoid-specific culture medium into the microfluidic channel one and the microfluidic channel two, respectively, for culture.

9. The organoid co-culture method according to claim 8, wherein In step (1), the organoid inocula comprise organoids or hydrogel-organoid mixtures; Preferably, the organoid inocula are hydrogel-organoid mixtures; Preferably, the hydrogel-organoid mixtures are obtained by mixing an organoid cell suspension with a hydrogel matrix at a certain volume fraction ratio; Preferably, the hydrogel is selected from Matrigel, collagen or sodium alginate; Preferably, in step (2), the organoid-specific culture medium is perfused into the corresponding microfluidic channel through the programmable perfusion system; Preferably, in step (2), the culture step comprises a continuous perfusion culture stage and a static culture stage; the organoids gradually grow in the continuous perfusion culture stage; the perfusion is stopped in the static culture stage to realize contact co-culture and induce organoid migration and fusion.

10. Use of the double-channel organoid dynamic co-culture microfluidic chip of any one of claims 1-7 in organoid culture.

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