Organ-like size screening and recycling integrated chip

Abstract

The invention relates to the technical field of biomedical engineering and microfluidics, and discloses an organ-like size screening and recycling integrated chip which comprises a rigid substrate and a polymer flexible layer which is reversibly attached, and a microfluidic channel with the top surface height decreasing in a stepped mode in the fluid direction is formed between the rigid substrate and the polymer flexible layer. The method is provided with 4-12 physical interception steps with different heights, and comprises the following steps: driving an organoid suspension to flow by utilizing gravity generated by liquid level difference of a liquid inlet, and physically intercepting organoids with different sizes at the steps with corresponding heights; injecting temperature-sensitive matrigel at low temperature to wrap the organoid, and raising the temperature to cure the organoid in situ to lock the position; and the flexible layer is uncovered, and the gel strip is cut according to the step position to achieve graded recovery. According to the method, an external pumping device is not needed, the damage of fluid shear force to cells is avoided by utilizing gravity passive sorting and in-situ gel fixing technologies, and the non-destructive and rapid multi-stage sorting and soil-carrying transplanting type recovery of the organoids are realized.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and microfluidics, specifically to a chip that integrates organoid size screening and recovery. Background Technology

[0002] Organoids, as models that mimic the structure and function of human tissues and organs in vitro, have significant application value in drug screening, disease modeling, and personalized medicine. However, the size heterogeneity prevalent during organoid culture severely affects the reproducibility and accuracy of experimental results. Currently, techniques for controlling organoid uniformity mainly fall into two categories: microfluidic droplet-based techniques and culture plate optimization techniques. However, these methods still have many limitations in practical applications.

[0003] While microfluidic droplet-based methods can generate independent microreactors, they are costly and technically challenging. During droplet encapsulation, the random sedimentation and aggregation of cells make it difficult to precisely control the initial cell count within each droplet, resulting in inconsistent organoid homogeneity. Although existing techniques attempt to pretreat cells with calcium carbonate nanoparticles to form single-cell microspheres, this physically separates the cells, blocking intercellular contact and communication, and inhibiting the crucial self-organization process during organoid development. This fails to accurately reflect the interactions between cells and between organoids, as well as the heterogeneity of tumor tissue. Furthermore, existing microfluidic sorting techniques are typically based on binary decision logic, only capable of dividing samples into two components: one within a specific range and the other outside that range. This makes it difficult to achieve multi-level, refined sorting of organoids with continuous size distributions.

[0004] Methods based on culture plate optimization typically restrict the growth space of organoids through physical molds. This forced morphological constraint often ignores the inherent self-organizing characteristics of organoids, limiting their ability to simulate the in vivo microenvironment. While traditional cylindrical culture plates allow organoids to grow naturally through self-organization, they cannot effectively separate and recover organoids of different sizes growing together in the same culture system, making it difficult to meet the needs of subsequent experiments requiring samples of specific sizes. Therefore, existing technologies struggle to achieve low-cost, low-damage, and precise sorting and recovery of multi-size organoids while preserving their natural development and self-organizing characteristics. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a chip that integrates organoid size screening and recovery, solving the technical challenge of accurately sorting and completely recovering non-uniform-sized organoids at multiple levels while maintaining low cost, low damage, and preserving the self-organizing characteristics of organoids in existing organoid technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Firstly, the present invention provides a chip integrating organoid size screening and recovery, employing the following technical solution: A chip for organoid size screening and recovery includes a rigid substrate and a flexible polymer layer reversibly bonded to the surface of the rigid substrate. A microfluidic channel is formed between the flexible polymer layer and the rigid substrate. The microfluidic channel has an inlet and an outlet. The height of the top surface of the microfluidic channel decreases in a stepped manner along the direction of fluid flow from the inlet to the outlet, forming 4 to 12 steps. The height of each step in the microfluidic channel is less than the height of the step above. The height of the microfluidic channel at the inlet is 240 μm to 800 μm, and the height at the outlet is 20 μm to 200 μm.

[0007] By employing the above technical solution, passive physical screening of heterogeneous biological samples is achieved through a multi-stage stepped flow channel height design and a reversible bonding encapsulation structure. The abrupt changes in the height of the stepped steps create discrete size cutoff thresholds along the fluid path. When fluid carrying organoids flows through flow channel sections of different heights, organoids larger than the current flow channel height are physically blocked at the step of that level, while components smaller than that height move with the fluid to the next level, thus transforming a continuous particle size distribution into discrete spatial locations distributed along the longitudinal direction of the flow channel. The reversible bonding mechanism between the rigid substrate and the flexible polymer layer ensures the flow channel remains sealed during sorting, while allowing complete exposure of the flow channel interior by peeling off the flexible layer after sorting, providing an operational interface for subsequent direct physical recovery.

[0008] Preferably, the height difference between each step of the microfluidic channel and the previous step is 20μm to 150μm; the width of the microfluidic channel is 2.0mm to 5.0mm, and the total length is 30mm to 40mm.

[0009] By adopting the above technical solution, the drop setting of 20μm to 150μm provides a high-resolution size limit, which can customize the sorting accuracy according to the growth characteristics of organoids from different tissue sources; the channel width of 2.0mm to 3.0mm and the length of 30mm to 40mm ensure the throughput while ensuring the uniformity of the flow field distribution, avoiding the fluid dead zone or eddies that may be generated by the wide channel, and ensuring that the organoids can settle smoothly and remain at the edge of the step.

[0010] Preferably, the polymer flexible layer is made of polydimethylsiloxane, which is made from raw materials comprising the following parts by weight: basic prepolymer: 8 to 12 parts; curing agent: 0.8 to 1.2 parts; the preparation method of the polymer flexible layer includes the following steps: mixing the basic prepolymer and curing agent and degassing, pouring it onto the surface of a positive mold with a stepped microstructure, heating at 75°C to 85°C for 1.5 to 2.5 hours, demolding, and punching holes to obtain the final product.

[0011] By employing the above technical solution, the polydimethylsiloxane material with a specific ratio exhibits good light transmittance, gas permeability, and a suitable elastic modulus after curing. Good light transmittance facilitates real-time monitoring under an optical microscope; gas permeability maintains oxygen exchange within the flow channels; and the suitable elastic modulus allows the polymer layer to adhere tightly to the rigid substrate to prevent leakage, while also being able to be completely peeled off during recycling without easily breaking. The curing process at 75℃~85℃ ensures the consistency of the material's crosslinking degree and reduces residual stress within the polymer.

[0012] Preferably, the positive mold is prepared using a multilayer photolithography process, which includes the following steps: spin-coating photoresist on the surface of a silicon wafer, exposing and developing it to form a first basic structure; repeating the spin-coating, alignment, exposure and development steps on the first basic structure to stack 4 to 12 layers of photoresist structure, with each photoresist layer having a thickness of 20 μm to 150 μm.

[0013] By adopting the above technical solution, the multi-layer overlay process can accurately construct a stepped three-dimensional solid structure with vertical sidewalls. Compared with the slope structure formed by anisotropic wet etching, the step edge formed by photolithography is sharper and has a clear boundary, which can provide a clear physical cutoff boundary and improve the dimensional cutoff accuracy of sorting.

[0014] Preferably, the inner surface of the microfluidic channel has a bio-sealing layer, which is formed by wetting the channel surface with bovine serum albumin buffer at a concentration of 0.5% to 2% (w / v) and incubating for 20 to 40 minutes.

[0015] By adopting the above technical solution, bovine serum albumin molecules are adsorbed onto hydrophobic PDMS and glass surfaces, which masks the non-specific adsorption sites on the material surface, significantly reduces the frictional resistance and adhesion probability between organoids and the channel wall, prevents random retention of organoids in non-target areas, and improves the purity and recovery rate of the recovered samples.

[0016] Secondly, this invention provides a method for screening and recovering organoids by size using the aforementioned chip, employing the following technical solution: A method for organoid size screening and recovery using the aforementioned chip includes the following steps: S1, chip pretreatment: injecting culture medium into the chip channel and expelling air from the channel; S2, gravity-driven sorting: adding organoid suspension to the inlet to establish a liquid level difference between the inlet and outlet, and driving the suspension through the stepped channel by hydrostatic pressure, causing the organoids to remain at the corresponding step height; S3, in-situ gel fixation: injecting 80μL to 200μL of liquid thermosensitive matrix gel into the channel at a temperature of 0℃ to 4℃ to encapsulate the retained organoids, and then placing the chip in an environment of 36℃ to 38℃ for 10 to 20 minutes to allow the thermosensitive matrix gel to solidify; S4, physical recovery: peeling the polymer flexible layer off the rigid substrate, cutting the solid gel strips according to the position of the stepped steps, separating and collecting gel blocks from different regions.

[0017] By adopting the above technical solution, this method establishes a complete operational process for passive sorting, in-situ fixation, and physical extraction. Its innovative mechanism and technical advantages are mainly reflected in the synergistic effect of the following steps: First, a gravity-driven low-shear-force sorting mechanism. In step S2, the traditional mechanical pump drive is abandoned, and only the small hydrostatic pressure generated by the liquid level difference is used as the driving source. When large organoids are obstructed at the step, the flow resistance of the flow path increases instantaneously, and the fluid automatically flows around the obstructed organoids, without applying a continuous mechanical thrust to the obstructed organoids. This adaptive hydrodynamic environment prevents organoids from passing through the interception structure due to compression deformation, while also protecting the fragile organoid structure from damage by fluid shear forces to the greatest extent.

[0018] Second, the in-situ locking mechanism of the thermosensitive hydrogel. In step S3, the thermosensitive phase change properties of the matrix gel (low-temperature liquid state, high-temperature colloidal state) are utilized to introduce a liquid colloid and solidify it in situ the instant the organoids complete spatial distribution. The solidified gel acts as a three-dimensional fixation medium, freezing the dynamically distributed organoids at their sorted positions. This process prevents fluid disturbances caused by subsequent membrane peeling or chip movement, ensuring the stability of the sorting results and avoiding secondary mixing of components of different sizes.

[0019] Third, the WYSIWYG solid-phase recovery mechanism. In step S4, combining the reversible adhesion properties of the chip with gel fixation technology, the traditional liquid-phase elution recovery is transformed into solid-phase cutting recovery. The operator can directly cut the solidified gel strip according to the physical markers formed by the steps. Since the organoids are encapsulated in a matrix environment suitable for their growth, the cut gel blocks can be directly used for subsequent culture, realizing soil-in-soil transplantation, avoiding the damage to cell viability caused by repeated centrifugation and resuspension, and simplifying the operation process.

[0020] Preferably, the preparation process of the organoid suspension includes: dissociating the organoids using a dispersing enzyme or collagenase, washing and resuspending them in a basal culture medium, and adjusting the density of the organoids to 500 organs / mL to 2000 organs / mL.

[0021] By adopting the above technical solution, the suspension density is controlled within the range of 500 organs / mL to 2000 organs / mL, which not only ensures the sorting throughput, but also effectively avoids the blockage or stacking effect caused by organoid aggregation under high density, ensuring that each organoid passes through the flow channel independently as a single individual and is correctly retained.

[0022] Preferably, in step S3, the raw material components of the temperature-sensitive matrix adhesive include laminin, type IV collagen, and nestin.

[0023] By employing the above-mentioned technical solution and selecting a matrix gel rich in laminin, type IV collagen, and nestin, not only are excellent temperature-sensitive gelation properties selected, but the extracellular matrix components are also provided to closely resemble the in vivo microenvironment. This ensures that the organoids remain in a physiologically suitable microenvironment throughout the recovery process, helping to maintain their stemness characteristics and proliferative capacity.

[0024] Preferably, in step S4, the physical recycling method is selected from any of the following: Method 1: make a vertical cut along the boundary line of different height steps on the gel strip and transfer the cut gel block to the culture container; Method 2: Soak the cut gel blocks in organoid recovery solution at 2℃~6℃ for 30 minutes~50 minutes, and then collect the organoids by centrifugation.

[0025] By adopting the above technical solutions, flexible recovery pathways are provided. Method 1 is suitable for direct subsequent culture or drug screening, minimizing operational steps; Method 2 utilizes the depolymerization properties of matrix gel at low temperatures, suitable for scenarios requiring the acquisition of pure organoid particles for gene sequencing or flow cytometry analysis.

[0026] Preferably, in step S1, a pipette tip is inserted into the inlet beforehand, and an initial liquid level difference is established by injecting 100μL to 250μL of liquid; and in step S2, the fluid is driven only by the pressure generated by the liquid level difference, and the sorting process lasts for 10 to 20 minutes.

[0027] By adopting the above technical solution, a stable initial pressure head is established by using the nozzle as a reservoir, which is sufficient to maintain stable flow for 5 to 10 minutes. This time window matches the sedimentation rate and sorting throughput of general organoid suspensions, ensuring the integrity and effectiveness of the sorting process without the need for additional fluid control equipment.

[0028] This invention provides a chip for integrated organoid size screening and recovery. It has the following beneficial effects: 1. This invention reduces mechanical damage during the sorting process by combining gravity-driven passive fluid control with in-situ gel fixation technology. The weak hydrostatic pressure generated by the liquid level difference at the inlet drives the fluid, avoiding the damage to organoid structures caused by the high shear force and local high pressure generated by traditional mechanical pumps, effectively preventing cell membrane rupture or spheroid disintegration. Combined with the in-situ solidification of thermosensitive hydrogel, the organoids are directly locked at the sorting position and recovered in a solid phase by transplanting with soil, which maximizes the preservation of the three-dimensional structural integrity of the organoids, the intercellular communication network, and the heterogeneity of tumor tissue, so that the sorted organoids can quickly recover their proliferative activity in subsequent culture.

[0029] 2. This invention achieves high-throughput parallel sorting and rapid operation across multiple sizes. By setting up 4 to 12 stepped flow channels with decreasing height along the fluid flow direction, it can simultaneously separate organoid suspensions with continuous particle size distribution into multiple discrete size ranges in a single flow process. This eliminates the need for multiple sieving or complex flow path switching. The chip only needs to be used with a conventional pipette to establish an initial liquid level difference to complete sample injection and drive. It does not require complex devices such as external microfluidic pumps or pressure controllers, and is compatible with traditional matrix gel dome culture processes. It does not require changes to the existing culture system, simplifying experimental operation steps and improving sorting efficiency.

[0030] 3. This invention has excellent reusability and low cost advantages. Based on the reversible adhesion mechanism of van der Waals forces between the flexible PDMS polymer layer and the rigid substrate, after in-situ gel cutting and recycling, the PDMS chip body can be reused multiple times through simple cleaning and sterilization. Only the bottom glass slide needs to be replaced to reassemble for the next experiment. This not only reduces the consumable cost of microfluidic chips, but also achieves direct and visible recycling through physical capping, avoiding complex enzymatic elution steps. This makes the technical solution both economical and practical. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Preparation Examples 1-3: Preparation Example 1: This preparation example provides an 8-level ladder-shaped microfluidic chip for standard organoid sorting.

[0033] The chip is manufactured as follows: (1) Mold fabrication: A raised, stepped positive mold was fabricated on a clean silicon wafer surface using a multilayer photolithography process. First, SU-8 negative photoresist was spin-coated onto the silicon wafer, and then exposed and developed using a mask to form a basic flow channel structure with a height of 50 μm. Subsequently, the spin-coating, alignment, exposure, and development processes were repeated on this basis, resulting in a total of 8 layers of photoresist structure, each with a thickness controlled at 50 μm, thus forming a stepped raised structure with a total length of 40 mm and a width of 2.5 mm. The flow channel structure corresponding to this mold contains a total of 8 steps along the fluid flow direction, with the flow channel height decreasing stepwise from 400 μm at the inlet to 50 μm at the outlet, and the height difference of each step being 50 μm.

[0034] (2) PDMS casting and curing: Weigh the polydimethylsiloxane (PDMS) base prepolymer and curing agent, mix them at a mass ratio of 10:1 and put them into a vacuum mixer. Stir for 5 minutes and degas under vacuum for 20 minutes until no bubbles are present. Pour the mixture onto the silicon wafer mold prepared above, and control the PDMS layer thickness to be 4 mm. Place the mold in an 80℃ oven for heat curing for 2 hours.

[0035] (3) Demolding and punching: After the PDMS has completely cured and cooled to room temperature, carefully peel it off from the silicon wafer mold. Use a biopsy punch with a diameter of 2.0 mm to punch holes at both ends of the flow channel to form the inlet and outlet.

[0036] (4) Chip bonding: The side of the PDMS chip with the flow channel grooves is bonded to a clean borosilicate glass slide. Reversible sealing bonding is achieved by utilizing the van der Waals forces of the PDMS material itself, ensuring that the flow channel does not leak and can be manually peeled off in subsequent recycling steps, thus obtaining an 8-stage ladder-shaped microfluidic chip.

[0037] Preparation Example 2: This example provides a four-stage ladder-like microfluidic chip for coarse sorting of large-sized organoids.

[0038] The fabrication process of this chip is basically the same as that of Example 1, with the only difference being the setting of structural dimension parameters during the mold-making stage. In this example, by controlling the spin coating speed and the number of layers of photoresist, a raised structure containing four steps was fabricated on the silicon wafer. The total length of the flow channel structure corresponding to this mold is 40 mm, and the width is 3.0 mm. The height of the flow channel decreases stepwise along the fluid flow direction from 800 μm at the inlet to 200 μm at the outlet, with a total of four steps and a height difference of 150 μm between each step. The remaining PDMS casting, curing, and bonding process parameters are consistent with those of Example 1, resulting in a four-step stepped microfluidic chip suitable for organoid sorting with larger diameters and a wide size distribution.

[0039] Preparation Example 3: This preparation example provides a 12-level ladder-shaped microfluidic chip for high-precision sorting of tiny organoids.

[0040] The fabrication process of this chip is basically the same as that of Example 1, with the only difference being the setting of structural dimension parameters during the mold-making stage. In this example, a raised structure containing 12 steps was fabricated on a silicon wafer by precisely controlling a multilayer photolithography process. The total length of the flow channel structure corresponding to this mold is 30 mm, and the width is 2.0 mm. The height of the flow channel decreases stepwise along the fluid flow direction from 240 μm at the inlet to 20 μm at the outlet, with a total of 12 steps and a height difference of 20 μm between each step. The remaining PDMS casting, curing, and bonding process parameters are consistent with those of Example 1, resulting in a 12-step stepped microfluidic chip suitable for high-resolution sorting of small organoids or cell clusters sensitive to size differences.

[0041] Examples 1-4: Example 1: This embodiment provides a chip for integrated screening and recovery of organoids by size. It uses the 8-step ladder-like microfluidic chip from Preparation Example 1, and is designed for tumor organoid samples with standard size distributions. The chip includes the following steps: (1) Chip pretreatment: Take the 8-stage ladder-shaped microfluidic chip obtained in Preparation Example 1, insert a 200 μL pipette tip tightly into the chip inlet, inject 200 μL of complete culture medium into the pipette tip, let stand for 1 minute, and use gravity to expel the air in the channel so that the channel is filled with culture medium.

[0042] (2) Gravity-driven sorting: After digestion with dispersant enzyme II and washing with PBS, the mature tumor organoids were resuspended in basal culture medium to prepare a single organoid suspension with a density of 1000 organs / mL. The liquid in the original inlet pipette tip was removed and 200 μL of the above organoid suspension was added. Under the action of gravity, the suspension flowed through a stepped flow channel. Organoids of different diameters were physically trapped at the steps with a height smaller than their diameter, while cell debris with a diameter of less than 50 μm was discharged from the outlet with the fluid. The sorting process lasted for about 15 minutes.

[0043] (3) In-situ gel fixation: After sorting, the chip was placed on ice to cool for 3 minutes; the inlet pipette tip was removed and replaced with a new pipette tip containing 100 μL of soluble basement membrane matrix gel pre-cooled to 4°C. The liquid matrix gel was slowly injected into the channel by gravity to replace the original culture medium and encapsulate the retained organoids; then the chip was transferred to a 37°C constant temperature incubator and incubated for 15 minutes to allow the matrix gel to undergo thermal cross-linking and curing.

[0044] (4) Physical recovery: Remove the chip from the incubator and manually peel off the PDMS layer. At this time, the gel strip containing the organoid is attached to the glass slide. With the assistance of a stereomicroscope, use a sterile scalpel to make vertical cuts along the corresponding step junctions on the gel strip. Cut off the gel blocks of the 1-2 level (corresponding to the large size), 3-5 level (corresponding to the medium size), and 6-8 level (corresponding to the small size) areas respectively, transfer them to a new culture plate, add complete culture medium and continue culturing.

[0045] Example 2: This embodiment provides a chip for screening and recovering organoids by size. It uses the four-stage ladder-shaped microfluidic chip from Preparation Example 2, and is designed for large organoid clusters with long growth cycles. The chip includes the following steps: (1) Chip pretreatment: Take the 4-stage stepped microfluidic chip obtained in Preparation Example 2, inject culture medium through the liquid inlet to exhaust the air, and ensure that the flow channel is hydrophilic.

[0046] (2) Gravity-driven sorting: Prepare a suspension of large-sized organoids with a density of 500 organs / mL (diameter range 200-800μm); add 200μL of suspension to the inlet nozzle and use gravity to drive the fluid through the channel; due to the high height of the channel of the chip (800-200μm), the large-sized organoids are retained on 4 different stepped planes according to their size differences.

[0047] (3) In-situ gel fixation: Place the chip in a 4°C environment and cool for 5 minutes; inject 150 μL of pre-cooled liquid matrix adhesive into the flow channel; then place the chip in a 37°C environment and incubate for 20 minutes to ensure that the thick gel layer is completely cured.

[0048] (4) Dissolution and recovery: Remove the PDMS layer, immerse the slide and the gel strip on it in the cell recovery solution at 4°C for 30 minutes. After the matrix gel depolymerizes and liquefies, aspirate the liquid at the corresponding step position and centrifuge to collect the sorted organoids.

[0049] Example 3; This embodiment provides a chip for screening and recycling organoids of different sizes. It uses the 12-stage ladder-like microfluidic chip from Preparation Example 3, targeting tiny organoids or early-stage cell spheroids, and includes the following steps: (1) Chip pretreatment: Take the 12-stage ladder-shaped microfluidic chip obtained in Preparation Example 3, prefill it with culture medium and degas it.

[0050] (2) Gravity-driven sorting: Prepare a suspension of tiny organoids with a density of 2000 per mL (diameter range of 20-200 μm); add 100 μL of suspension to the inlet. Since the flow channel step drop is only 20 μm, the fluid flows slowly under gravity, and the tiny organoids are finely distributed into 12 different step regions.

[0051] (3) In-situ gel fixation: 80 μL of liquid matrix adhesive was injected into the flow channel under ice bath conditions; then cured at 37°C for 20 minutes.

[0052] (4) Physical recovery: Remove the PDMS layer and use the edge of an extremely fine coverslip as a cutting tool under a microscope to precisely cut out gel microparticles of the target size range (e.g., level 9-10, corresponding to the range of 60-100μm) for direct use in subsequent drug screening experiments.

[0053] Example 4: This embodiment provides a chip for integrated organoid size screening and recovery, using the 8-level stepped microfluidic chip from Preparation Example 1, and includes the following steps: (1) Surface sealing and pretreatment: Take the chip obtained in Preparation Example 1, inject PBS solution containing 1% bovine serum albumin (BSA) into the channel before sorting, and incubate at room temperature for 30 minutes to perform surface sealing in order to reduce non-specific adhesion between organoids and channel walls. Then rinse the channel with culture medium.

[0054] (2) Gravity-driven sorting: The operation steps are the same as in Example 1, and an organoid suspension with a density of 1000 organs / mL is introduced.

[0055] (3) In-situ gel fixation: The operation steps are the same as in Example 1, injecting the matrix adhesive and curing at 37°C.

[0056] (4) Physical recovery: The operation steps are the same as in Example 1, and the target organoids are recovered by peeling off the PDMS layer and cutting the gel. This example demonstrates that modifying the chip surface does not affect its sorting function and can improve the purity of the recovered samples.

[0057] Comparative Examples 1-3: Comparative Example 1: Compared to Example 1, the difference lies in the flow channel structure design of the microfluidic chip. This comparative example uses a ramp-shaped flow channel chip, where the channel height linearly and continuously decreases from 400 μm at the inlet to 50 μm at the outlet along the fluid flow direction. The interior of the channel has a smooth ramp structure without any step-like abrupt changes. The remaining chip fabrication materials, organoid suspension concentration, and gel fixation, cutting, and recovery procedures are the same as in Example 1.

[0058] Comparative Example 2: The difference between this comparative example and Example 1 lies in the sorting device and method. This comparative example does not use microfluidic chips or in-situ gel immobilization technology; instead, it employs a traditional nylon cell sieve for physical filtration and sorting. Specifically, the organoid suspension, identical to that in Example 1, is sequentially passed through commercially available cell sieves with pore sizes of 200 μm and 100 μm. The organoids retained on the sieve surface are the corresponding size components. Subsequently, the sieve surface is repeatedly rinsed with culture medium using a pipette to recover the organoids.

[0059] Comparative Example 3: The difference between this example and Example 1 lies in the fluid driving method. This comparative example does not employ gravity-driven methods relying on liquid level differences. Instead, the inlet of the microfluidic chip is connected to a precision injection pump via a PTFE conduit. A flow rate of 100 μL / min is set, and the organoid suspension and subsequent matrix gel are injected into the flow channel via mechanical pumping. The remaining chip structure and subsequent curing and recovery steps are the same as in Example 1.

[0060] Test Examples 1-4: Test Example 1: Verification of Sorting Accuracy and Recovered Particle Size Distribution This test case aims to verify the sorting capability of the 8-stage ladder-shaped microfluidic chip in Example 1 for organoid samples with mixed particle sizes, focusing on evaluating the size uniformity of the recovered organoids and the hit rate of the target region.

[0061] Experimental steps: Mature human pancreatic cancer organoids were selected, digested with dispersant enzyme II, and resuspended to prepare a mixed suspension with an initial particle size distribution between 40 μm and 450 μm. The initial density, as measured by a cell counter, was approximately 1500 cells / mL. The mixed suspension was divided into two groups: the control group underwent no sorting and was directly sampled for image analysis; the experimental group was operated according to the steps in Example 1, with the suspension passed through an 8-stage stepped microfluidic chip, driven by gravity. After the fluid stabilized, matrix gel was injected and in-situ solidified at 37°C.

[0062] After solidification, the PDMS layer was peeled off, and the second-level step region (designed retention range 300-350 μm) and the sixth-level step region (designed retention range 100-150 μm) in the flow channel were physically cut and recovered. The recovered gel blocks were placed in cell recovery solution at 4℃ for depolymerization, and organoids were collected. Bright-field imaging of the recovered organoids from the control and experimental groups was performed using an inverted microscope. At least 120 organoids from each group were randomly selected using ImageJ software to measure their projected area and convert it to the equivalent sphere diameter, and the particle size distribution data were statistically analyzed.

[0063] Experimental data: The particle size measurement results for each group of organoids are shown in the table below: Table 1. Statistical table of particle size distribution and coefficient of variation before and after organoid sorting. Note: Target interval hit rate refers to the proportion of particles in the recovered sample whose particle size falls within the theoretically designed rejection range.

[0064] The test data above show that the initial organoid samples without sorting have an extremely wide particle size distribution and a coefficient of variation as high as 52.95%, exhibiting heterogeneity. After processing with the chip of Example 1 of this invention, the coefficients of variation of organoid samples recovered from the second and sixth stages were reduced to 8.28% and 11.52%, respectively, and the size uniformity was improved.

[0065] This technical solution constructs a multi-level physical restraint structure along the fluid path by setting up stepped flow channels with specific height differences. When the fluid carrying organoids flows through the stepped interface, the abrupt change in channel height forms a rigid cutoff threshold. Organoids larger than this threshold cannot pass through and are retained at the current stepped plane, while organoids smaller than this threshold move with the fluid to the next level. Combined with the low flow velocity characteristics driven by gravity, this prevents organoids from being squeezed out of the restraint structure due to pressure deformation. At the same time, the in-situ solidification of the temperature-sensitive hydrogel locks the dynamically distributed organoids at the instant of sorting completion, preventing secondary mixing or displacement during the recovery process. The occasional out-of-range particle sizes observed in the experimental data (such as 288.5 μm or 364.2 μm particles in the second level) are within a reasonable error range, demonstrating that the device achieves efficient physical screening and accurate recovery of heterogeneous biological samples.

[0066] Test Example 2: Comparison Test of Sorting Efficiency and Recovery Purity This test case aims to compare the actual recovery effects of the stepped microfluidic chip of Example 1, the ramp-shaped flow channel chip of Comparative Example 1, and the traditional physical sieve of Comparative Example 2 in the organoid sorting process, focusing on the purity of the sorted samples and the sample loss during the operation.

[0067] Experimental steps: A single batch of human colorectal cancer organoid suspension was prepared, and the density was adjusted to about 1000 organs / mL. The total volume was 6mL, and it was divided into three equal parts, corresponding to the experimental group (Example 1), control group A (Comparative Example 1), and control group B (Comparative Example 2), respectively.

[0068] Experimental group (Example 1): 2 mL of suspension was passed into an 8-step stepped chip. After gravity-driven sorting, matrix gel was injected in situ and cured. The gel was cut along the front and rear physical boundaries of the 6th step (designed cutoff range 100-150 μm) under a microscope, and the organoids within this segment of gel were recovered.

[0069] Control group A (Comparative Example 1): 2 mL of suspension was introduced into the ramp-shaped chip. Since the channel height changes linearly and continuously without physical steps, the channel length range corresponding to a height of 100-150 μm (approximately 28-32 mm from the inlet) was calculated based on the channel geometry. After the matrix adhesive was injected and cured, a ruler and scalpel were used to attempt to cut and recover the material at the calculated location.

[0070] Control group B (Comparative Example 2): 2 mL of suspension was passed sequentially through nylon sieves with pore sizes of 150 μm and 100 μm. The material trapped on and above the surface of the 100 μm sieve was collected, and the sieve was repeatedly rinsed with culture medium to recover the organoids.

[0071] The three groups of recovered samples were dissociated and counted to calculate the total number of recovered particles. The diameter of each particle was measured, and the proportion (purity) of particles in the target size range (100-150 μm) and the impurity rate of non-target particles were calculated.

[0072] Experimental data: The statistical results of the recovery of each component are shown in the table below: Table 2. Statistics on Recovery Purity and Loss under Different Sorting Methods Note: Mixing rate = (Number of particles in non-target range / Total number of recovered particles) × 100%.

[0073] Conclusion Analysis: The test data revealed the significant impact of different structural designs on the sorting results. The contamination rate of the experimental group (Example 1) was only 10.16%, which was lower than that of control group A (44.39%) and control group B (22.83%).

[0074] In control group A (sloping channel), due to the continuous change in channel height, organoids of different sizes are distributed in a continuous gradient within the channel, lacking clear spatial physical separation. During gel cutting, the operator cannot visually determine the exact size boundary points, inevitably leading to the inclusion of organoids of adjacent size segments in the cutting area, thus increasing the contamination rate.

[0075] For control group B (physical sieve), although the contamination rate was better than that of the ramp group, the total number of recovered particles (92) was much lower than that of the experimental group (187), indicating serious sample loss. This is because soft and elastic organoids are easily trapped in the mesh, causing blockage, and the rinsing process cannot completely wash them away. In addition, the sieve lacks an in-situ fixation mechanism, and some small particles may adhere non-specifically.

[0076] In contrast, Embodiment 1 of this invention employs a stepped structure, transforming a continuous size distribution into a discrete spatial distribution. The abrupt changes in channel height create natural physical barriers and visual markers, strictly confining organoids of specific size ranges between two stepped sections. Combined with in-situ gel fixation technology, this structure provides precise physical guidance for subsequent cutting and recovery, ensuring high-purity sorted products without relying on precision measuring tools, while avoiding sample loss caused by mesh clogging.

[0077] Test Example 3: Evaluation of the impact of fluid-driven methods on organoid bioactivity This test case aims to compare the effects of the gravity-driven method used in Example 1 and the mechanical pump-driven method used in Comparative Example 3 on the activity and structural integrity of organoid cells, and to evaluate the degree of mechanical damage caused by different fluid dynamic environments.

[0078] Experimental steps: Human liver cancer organoids in good growth condition were selected and prepared into suspensions with an initial density of about 800 organs / mL, and divided into two groups.

[0079] Experimental group (Example 1): The suspension was added to the inlet of the stepped chip, and the fluid was driven by the hydrostatic pressure generated by the liquid level difference. The sorting process lasted for about 10 minutes.

[0080] Control group (Comparative Example 3): The suspension was loaded into a syringe and connected to the inlet of a stepped chip of the same specification through a Teflon catheter. The flow rate of the injection pump was set to 100 μL / min for constant-rate perfusion. The sorting process lasted for about 12 minutes.

[0081] After sorting, organoids within the flow channels were immediately recovered. The recovered material was divided into two parts: the first part was digested and dispersed into a single-cell suspension using trypsin, and the number of live and dead cells was counted using the trypan blue exclusion test under a hemocytometer to calculate the cell viability; the second part, maintaining the organoid morphology, was observed under an inverted microscope, and the number of organoids with intact spherical structures and the number of organoids that were broken, disintegrated, or had irregular edges within the field of view were counted to calculate the morphological integrity rate.

[0082] Experimental data: The bioactivity and morphological data of the two groups of samples are shown in the table below: Table 3. Data on organoid injury assessment under different driving modes Note: Cell viability = (number of resistant live cells / total number of cells counted) × 100%; Morphological integrity rate = (number of structurally intact granules / total number of observed organoids) × 100%.

[0083] Conclusion Analysis: Data showed that the cell survival rate and morphological integrity rate in the experimental group were significantly better than those in the control group. In the control group, the cell survival rate decreased to 81.68%, and nearly a quarter (23.44%) of the organoids showed macroscopic structural damage.

[0084] This difference stems from a fundamental difference in the fluid drive mechanism. In the control group (Comparative Example 3), the injection pump provides a constant volumetric flow rate. When a larger organoid moves to the stepped cross-section of the flow channel and is physically blocked, the mechanical thrust of the pump causes a sharp increase in the local fluid pressure at that point, applying high-intensity compressive and shear stresses to the trapped organoid, leading to cell membrane rupture or even the collapse of the entire organoid structure.

[0085] In contrast, the gravity-driven method used in Example 1 is a passive fluid control system. The driving force of the fluid depends on the liquid level at the inlet, and the resulting hydrostatic pressure is limited and constant (typically less than 500 Pa). When organoids are trapped at the step, if the flow resistance increases, the flow velocity within that path will automatically decrease or even stop. The fluid will preferentially flow through bypasses or gaps with less resistance, without continuously applying destructive mechanical thrust to the trapped organoids. This adaptive hydrodynamic characteristic effectively avoids hard compression, maximizing the preservation of the organoids' biological activity and three-dimensional structural integrity, demonstrating the superiority of this technical solution in handling fragile biological samples.

[0086] Test Example 4: Verification of Organoid Proliferative Capacity After In Situ Fixation This test case aims to verify the growth and proliferation capacity of organoids sorted and recovered in situ via microfluidic chip during subsequent culture, in order to assess whether this technical approach affects the long-term biological function of organoids.

[0087] Experimental steps: Sample preparation for the experimental group: Gel microparticles from the fourth step region (corresponding to a particle size of about 200-250 μm) that were sorted, embedded in situ matrix gel and cut and recovered in Example 1 were selected, transferred to a 24-well culture plate, and 500 μL of complete culture medium was added.

[0088] Control group sample preparation: Organoids from the same batch that were not processed by the chip were taken and resuspended in liquid matrix gel using the traditional dome culture method. They were then applied to the bottom of a 24-well plate at the same inoculation density and added with an equal amount of complete culture medium after curing.

[0089] Culture and monitoring: The two groups of samples were placed in an incubator at 37℃ and 5% CO2 saturated humidity for 5 consecutive days. During this period, the organoids within the fixed field of view were imaged every 24 hours using an inverted microscope.

[0090] Data acquisition: The projected area of ​​designated organoids was measured on day 0 (2 hours after inoculation), day 3, and day 5 using image analysis software. This area was converted into equivalent diameter, and the volume increase fold relative to day 0 was calculated to characterize the cell proliferation rate. Twenty organoids were randomly tracked and monitored in each group.

[0091] Experimental data: The growth data of the two groups of organoids during culture are shown in the table below: Table 4. Statistical table of growth trends of organoids in vitro culture after sorting and recovery. Note: Relative volume ratio = (Diameter at current time point / Diameter on day 0) 3 Data are expressed as mean ± standard deviation.

[0092] Conclusion Analysis: Table 4 shows that the experimental group organoids exhibited continuous volume growth during the subsequent 5 days of culture. Although the relative volume fold of the experimental group (2.11) was slightly lower than that of the control group (2.35) on day 3, there was no statistically significant difference between the two, and the growth fold of the experimental group reached 4.18 on day 5, indicating that it maintained a vigorous ability to divide and proliferate.

[0093] This result validates the advantages of the integrated screening and recovery design in our technical approach. Traditional sorting methods typically involve cumbersome processes such as digestion and dissociation, sieving, centrifugation, resuspension, and re-embedding. Repeated centrifugation and changes in the matrix environment can disrupt the organoid microenvironment, leading to prolonged lag phases. Our approach utilizes the thermosensitive properties of matrix gel to directly re-embed organoids within the chip channels. During recovery, the organoids are already anchored in a suitable extracellular matrix (ECM) microenvironment for their growth. This soil-based transplantation recovery strategy avoids direct contact between organoids and external hard materials, as well as the environmental stress caused by secondary resuspension operations. This allows them to rapidly recover their cell cycle and maintain normal self-organization and growth phenotype after transfer to culture plates.

Claims

1. A chip integrating organoid size screening and recovery, characterized in that, include: A rigid substrate and a flexible polymer layer reversibly bonded to the surface of the rigid substrate; a microfluidic channel is formed between the flexible polymer layer and the rigid substrate, the microfluidic channel having an inlet and an outlet; The height of the top surface of the microfluidic channel decreases in a step-like manner along the direction of fluid flow from the inlet to the outlet, forming 4 to 12 steps, and the height of each step of the microfluidic channel is less than the height of the step above. The height of the microfluidic channel at the inlet is 240μm to 800μm, and the height at the outlet is 20μm to 200μm.

2. The chip for integrated organoid size screening and recovery according to claim 1, characterized in that, The height difference between each step of the microfluidic channel and the previous step is 20μm to 150μm; the width of the microfluidic channel is 2.0mm to 3.0mm, and the total length is 30mm to 40mm.

3. The chip for integrated organoid size screening and recovery according to claim 1, characterized in that, The flexible polymer layer is made of polydimethylsiloxane, and is composed of the following raw materials in parts by weight: Basic prepolymer: 8 to 12 parts; Hardener: 0.8 to 1.2 parts; The preparation method of the polymer flexible layer includes the following steps: mixing the basic prepolymer and curing agent and degassing, pouring it onto the surface of a positive mold with a stepped microstructure, heating at 75℃~85℃ for 1.5 hours~2.5 hours, demolding, and punching holes to obtain the final product.

4. The chip for integrated organoid size screening and recovery according to claim 3, characterized in that, The positive mold is prepared using a multilayer photolithography process, which includes the following steps: Photoresist is spin-coated onto the surface of a silicon wafer, and then exposed and developed to form the first basic structure. The spin coating, alignment, exposure and development steps are repeated on the first base structure to stack 4 to 12 photoresist layers, each with a thickness of 20 μm to 150 μm.

5. The chip for integrated organoid size screening and recovery according to claim 1, characterized in that, The inner surface of the microfluidic channel has a biological sealing layer, which is formed by wetting the channel surface with bovine serum albumin buffer at a concentration of 0.5% to 2% (w / v) and incubating for 20 to 40 minutes.

6. The chip for integrated organoid size screening and recovery according to claim 1, characterized in that, This includes a method for screening and recovering organoids by size, which comprises the following steps: S1. Chip pretreatment: Inject culture medium into the chip channel and expel air from the channel; S2. Gravity-driven sorting: Add organoid suspension to the inlet to establish a liquid level difference between the inlet and outlet. The suspension is driven by hydrostatic pressure to flow through the stepped flow channel, so that the organoids are retained at the corresponding height of the stepped steps. S3. In-situ gel fixation: 80μL to 200μL of liquid thermosensitive matrix gel is injected into the flow channel at a temperature of 0℃ to 4℃ to encapsulate the retained organoids. Then the chip is placed in an environment of 36℃ to 38℃ and left to stand for 10 to 20 minutes to allow the thermosensitive matrix gel to turn into a solid state. S4. Physical recycling: The flexible polymer layer is peeled off from the rigid substrate, and the solid gel strip is cut according to the position of the step, separating and collecting gel blocks from different areas.

7. The chip for integrated organoid size screening and recovery according to claim 6, characterized in that, The preparation process of the organoid suspension includes: using dispersing enzymes or collagenases to dissociate the organoids, washing them, and resuspending them in a basal culture medium, adjusting the density of the organoids to 500 organs / mL to 2000 organs / mL.

8. The chip for integrated organoid size screening and recovery according to claim 6, characterized in that, In step S3, the raw material components of the temperature-sensitive matrix adhesive include laminin, type IV collagen, and nestin.

9. A chip for integrated organoid size screening and recovery according to claim 6, characterized in that, In step S4, the physical recycling method is selected from any of the following: Method 1: Cut vertically along the boundary line of different height steps on the gel strip and transfer the cut gel block to the culture container; Method 2: Soak the cut gel blocks in organoid recovery solution at 2℃~6℃ for 30 minutes~50 minutes, and then collect the organoids by centrifugation.

10. A chip for integrated organoid size screening and recovery according to claim 6, characterized in that, In step S1, a pipette tip is inserted into the inlet beforehand, and an initial liquid level difference is established by injecting 100μL to 250μL of liquid. In step S2, the fluid is driven solely by the pressure generated by the liquid level difference, and the sorting process lasts for 5 to 10 minutes.

Images