A tumor microtissue-vascular co-culture microfluidic chip and application thereof in immune drug screening

Abstract

This invention relates to the field of microfluidics, and discloses a tumor microtissue-vascular co-culture microfluidic chip and its application in immunotherapy screening. The chip includes a micropore anchoring layer and a microchannel layer. The micropore anchoring layer has multiple rows of micropore arrays, each micropore unit containing a large circular cavity and at least two anchoring small circular cavities (larger at the top and smaller at the bottom) located at its bottom. Micro-slits are formed at the connection points between the large circular cavity and the microchannels connected in series with each unit. The microchannel layer has multiple rows of independent perfusion channels, the number of which corresponds one-to-one with the number of rows in the micropore array. Each row of channels has an independent sample inlet and a waste outlet at both ends, forming an independent fluid loop. In application, tumor microtissue is in situ loaded and anchored within the small circular cavities through two-phase pre-wetting and stepped flow rate drive; vascular-like structures are constructed within the microchannels; long-term co-culture and parallel immunotherapy are performed in a perfusion medium containing the lactate dehydrogenase inhibitor GSK2837808A. This invention achieves long-term stable fixation and crosstalk-free parallel screening of tumor microtissues under dynamic perfusion, maintaining their original immune and matrix microenvironment for up to 14 days, providing a high-fidelity platform for in vitro prediction and personalized drug evaluation of immunotherapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to a tumor microtissue-blood vessel co-culture microfluidic chip and its application in immunotherapy drug screening. Background Technology

[0002] Currently, cancer treatment has entered the era of precision medicine, represented by targeted therapy, immunotherapy, and cell therapy. However, many challenges remain in clinical practice. On the one hand, while gene-based targeted therapy is instructive, the testing cycle is long and the beneficiary population is limited. On the other hand, the efficacy of tumor immunotherapy is highly dependent on the complex tumor microenvironment (TME), and existing genomic sequencing technologies cannot fully analyze the dynamic characteristics of the TME, resulting in insufficient predictive ability for treatment efficacy.

[0003] In the preclinical research stage, commonly used drug screening models, such as tumor cell lines, organoids, and patient-derived xenograft (PDX) models, all have significant limitations. Cell line models lack tumor heterogeneity; while organoid models can partially preserve tumor structure, their construction cycle is long, success rates vary, and they are difficult to completely preserve the original immune and matrix components; PDX models face problems such as long construction cycles, high costs, and difficulties in humanizing the immune system, limiting their widespread application in immunotherapy research.

[0004] Patient-derived tumor microtissue (PDM) models have shown great potential in tumor immunology research due to their ability to preserve the original tumor's tissue structure, immune cell infiltration, and stromal microenvironment to the greatest extent possible. However, traditional static culture models of PDM are difficult to maintain long-term (usually no more than 7 days). Insufficient nutrient and oxygen supply and accumulation of metabolic waste lead to a rapid loss of the activity and phenotype of immune cells and stromal cells, which cannot meet the needs of long-term studies of tumor-immune interactions.

[0005] Microfluidic chip technology, with its miniaturization, high throughput, and controllable fluid dynamics, provides a new platform for constructing more biomimetic tumor models in vitro. By simulating the in vivo microenvironment on the chip, dynamic perfusion culture of cells and organoids can be achieved. However, when applying it to the screening of tumor immunotherapy drugs, effectively maintaining the original immune and matrix components in the tumor microtissue remains a core challenge. Existing technologies mainly suffer from the following bottlenecks: 1. Micro-tissue fixation challenge: The size of tumor micro-tissues from patients is uneven (usually 70~150µm). Under the continuous perfusion shear force, they are prone to displacement, detachment, or even being washed into the flow channel, resulting in inconsistent culture conditions and experimental failure.

[0006] 2. Parallel processing interference: Most high-throughput chips use a common distribution channel for liquid supply. When multiple conditions are processed in parallel (such as different drugs or dosages), cross-contamination or backflow between channels is easily caused by pressure fluctuations, which seriously affects the accuracy and repeatability of drug screening results.

[0007] 3. Difficulty in maintaining the microenvironment: During long-term (7-14 days) in vitro culture, the accumulation of metabolites (such as lactic acid) in tumor microtissues can lead to microenvironment acidification, which in turn causes immune cell dysfunction and loss of matrix phenotype, making it extremely difficult to conduct stable efficacy assessment within a sufficient time window.

[0008] Therefore, there is an urgent need to develop a microfluidic platform that integrates microtissue stabilization and anchoring, independent parallel fluid control, and long-term microenvironment maintenance functions to enable long-term culture of patient-derived tumor microtissue under dynamic perfusion and reliable high-throughput immunotherapy evaluation. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a tumor microtissue-vascular co-culture microfluidic chip and its application in immunotherapy screening, aiming to maintain the original immunity and matrix microenvironment of patient-derived tumor microtissue in vitro in a long-term and stable manner, and to achieve high-throughput and high-accuracy parallel evaluation of immunotherapy efficacy.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: A tumor microtissue-vascular co-culture microfluidic chip includes a microporous anchoring layer and a microchannel layer attached thereto, which together form a sealed structure; The upper surface of the microporous anchoring layer is provided with multiple rows of microporous arrays; each row of microporous arrays includes multiple microporous units arranged along the row direction and microchannels connected in series with each microporous unit. Each microporous unit includes a large circular cavity and at least two anchoring small circular cavities disposed at the bottom of the large circular cavity; the anchoring small circular cavities are a contraction structure that is larger at the top and smaller at the bottom, used to fix tumor microtissue; microslits are opened at the connection between the large circular cavity and the microchannels to facilitate the exchange of substances between the culture medium and the tumor microtissue in the cavity under perfusion conditions, while restricting the entry of tumor microtissue into the microchannels; The lower surface of the microchannel layer is provided with multiple rows of independent perfusion channels, the number of which is the same as the number of rows of the micropore array and corresponds one-to-one; each row of perfusion channels covers all micropore units of the corresponding row, and each end is provided with an independent sample inlet and a waste liquid outlet, forming an independent fluid loop.

[0011] In the above scheme, the micropore array is arranged in 12 rows, with 8 large circular cavities in each row; each large circular cavity has a diameter of 1000µm and a depth of 100µm; each large circular cavity is provided with 4 anchoring small circular cavities, the top layer of the anchoring small circular cavities has a diameter of 300µm, the bottom layer has a diameter of 180µm, and the depth from the inside of the large circular cavity downwards is 300µm; the distance between adjacent large circular cavities is 400µm, and the distance between adjacent anchoring small circular cavities is 50µm.

[0012] In the above scheme, the opening size of the microslit is 20µm; the microchannel is used to form a blood vessel-like structure under perfusion conditions.

[0013] In the above scheme, there is no common distribution channel or common confluence channel between different irrigation and drainage channels, and each irrigation and drainage channel is connected to the external irrigation drive device through an independent pipeline.

[0014] Application of a tumor microtissue-vascular co-culture microfluidic chip as described above in immunotherapy drug screening.

[0015] The above solution includes the following specific steps: S1: The tumor tissue from the patient was mechanically sheared and sieved to obtain tumor micro-tissue with a diameter of 70-150µm, and then mixed with matrix gel to obtain a tumor micro-tissue-matrix mixed suspension; S2: Perform two-phase pre-wetting and degassing treatment on the microfluidic chip, fill the perfusion channel, the microchannel and the micropore unit with oil phase, remove air bubbles, and form a stable two-phase interface at the micro gap; S3: Inject the tumor microtissue-stromal gel mixed suspension at a set flow rate, so that it selectively enters and remains in the anchored small round cavity under the physical retention effect of surface tension and anchored small round cavity; S4: Inject the oil phase and drive it with a stepped or pulsed flow rate to form discrete droplets of the tumor micro-tissue-matrix mixture suspension in each anchored small circular cavity, and discharge the residual suspension in the flow channel, thereby realizing the in-situ loading of tumor micro-tissue. S5: Place the chip in a 37°C culture environment to allow the matrix adhesive to cross-link and solidify, thus stabilizing the tumor micro-tissue within the anchoring small round cavity. S6: Replace the oil phase with an aqueous culture medium and enter continuous perfusion; inject photocrosslinkable composite hydrogel into the perfusion channel and microchannel, and after crosslinking, selectively remove it to the center of the channel by perfusion shear while retaining it on the channel wall and microchannel wall to form a wall hydrogel layer. S7: Introduce a suspension of vascular endothelial cells into the perfusion channel and microchannel, causing them to adhere to the hydrogel layer on the wall and form a vascular-like structure; S8: After the vascular-like structure stabilizes, DMEM / F12 perfusion medium is introduced for continuous perfusion culture; S9: The immunotherapeutic drug to be tested was added to the perfusion medium for perfusion treatment, and the efficacy of the drug was evaluated by detecting tumor microtissue survival rate, immune cell infiltration and matrix-related phenotypic changes.

[0016] In a further technical solution, the oil phase in step S2 is a fluorinated oil and contains 2% emulsifying surfactant; in step S4, the oil phase flow rate is driven in a stepwise increase manner of 0.1→0.5→1µL / min.

[0017] In a further technical solution, the composite hydrogel mentioned in step S6 is a compound system of methacrylamide gelatin (GelMA) and Collagen IV, with a mass or volume ratio of 4:6.

[0018] In a further technical solution, the perfusion culture medium contains sodium bicarbonate, L-glutamine, serum, antibiotics, and lactate dehydrogenase inhibitor GSK2837808A.

[0019] In a further technical solution, in step S9, multiple test immunotherapies and / or different doses or concentrations of the same immunotherapy are added to the perfusion culture medium corresponding to different drainage channels, and parallel perfusion treatment is performed under the condition that the drainage channels are not interconnected; after the treatment, the tumor microtissue corresponding to each drainage channel is detected, and the detection includes at least one of the following: tumor microtissue activity, expression of immune cell activation markers, expression of matrix-related markers and / or cytokine secretion levels, thereby obtaining the efficacy evaluation results of the immunotherapies.

[0020] Through the above technical solution, the tumor microtissue-vascular co-culture microfluidic chip provided by the present invention and its application in immunotherapy drug screening have the following significant beneficial effects: 1. Achieving efficient anchoring and erosion-resistant stable culture of micro-tissues This invention constructs a dual fixation mechanism of physical retention and size exclusion by setting up a funnel-shaped anchoring cavity that is wider at the top and narrower at the bottom, combined with a connecting microslit with an opening size of only 20µm. This design can stably retain patient-derived tumor microtissue with a diameter of 70-150µm within the cavity, effectively resisting the shear force erosion generated by long-term perfusion (0.8-1.2µL / min), avoiding microtissue displacement, detachment, or loss, significantly improving the stability and consistency of long-term culture, and laying a reliable foundation for subsequent precise drug delivery.

[0021] 2. Eliminate cross-contamination in parallel processing to improve drug screening throughput and accuracy. This invention employs a row-to-row independent perfusion channel design, with each row having its own independent sample inlet and waste outlet, and no common connections or distribution channels between rows. This "row-level independent fluid loop" structure fundamentally suppresses the risks of cross-contamination and backflow between different experimental conditions (such as different drugs or different doses), enabling parallel, high-throughput perfusion processing of up to 12 different conditions on the same chip. This not only significantly improves the efficiency of drug screening but also ensures the independence, reproducibility, and statistical reliability of data from each experimental group.

[0022] 3. Biomimetic construction of a vascularized microenvironment maintains immune and matrix function for up to 14 days. This invention constructs a physiologically functional vascular-like structure within a perfusion channel by combining a GelMA / Collagen IV composite hydrogel layer retained in the channel wall with HUVEC seeding. This structure provides a continuous nutrient supply and metabolic waste removal pathway for tumor microtissues under dynamic perfusion, mimicking the in vivo material exchange process. Combined with a perfusion medium containing the lactate dehydrogenase inhibitor GSK2837808A to regulate the metabolic microenvironment, it effectively inhibits microenvironmental acidification caused by lactate accumulation. Thus, over a culture period of 7–14 days, it stably maintains the activity of immune cells (such as CD8+ T cells), the phenotype of stromal cells (such as α-SMA+ fibroblasts), and vascular structures (CD31+) in tumor microtissues, overcoming the bottleneck of traditional models that struggle to maintain the original microenvironment long-term.

[0023] 4. Preserving the heterogeneity of the original tumor enhances the clinical relevance of immunotherapy efficacy assessment. This invention directly utilizes primary tumor microtissue from patients, preserving to the maximum extent the original tumor's tissue structure, heterogeneity, and complex immune matrix microenvironment. Compared to cell lines or organoid models, the co-culture platform provided by this invention can more realistically reflect the tumor's in vivo response mechanism to immunotherapy drugs. By detecting tumor microtissue survival rate, immune cell infiltration, and phenotypic changes, in vitro evaluation results highly correlated with clinical efficacy can be obtained, providing a high-fidelity technical platform for the in vitro predictive screening of immunotherapeutic drugs and the selection of personalized treatment regimens.

[0024] 5. Controllable operation and strong process compatibility This invention employs a two-phase pre-wetting and in-pore discrete loading method, utilizing surface tension and stepped flow rate to achieve in-situ, controllable, and uniform single-pore discrete loading of tumor microtissues with uneven sizes, avoiding mechanical damage and aggregation of the microtissues. Simultaneously, the construction process of retaining the hydrogel layer on the wall is simple and controllable, ensuring both the adhesion substrate for endothelial cells and maintaining the patency of the main flow channels. It exhibits strong process compatibility, facilitating industrial production and widespread application. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0026] Figure 1 This is a schematic diagram of an overall microfluidic chip for tumor microtissue-blood vessel co-culture disclosed in an embodiment of the present invention; Figure 2 The diagram shows the microchannel layer and the micropore anchoring layer, where (a) is the microchannel layer and (b) is the micropore anchoring layer. Figure 3 Here are schematic diagrams of microporous units; where (a) is a schematic diagram of a large circular cavity and (b) is a three-dimensional view of an anchored small circular cavity; Figure 4 This is a physical image of tumor microtissue within the small circular cavity of a microchip. Figure 5 Immunofluorescence image of the vascular structure formed by the chip; Figure 6 Immunofluorescence images of tumor microtissue at baseline and after continuous culture on a microfluidic chip system for 7 and 14 days; scale bar 50 μm. (a) Immunofluorescence image of PanCK / CD45 / DAPI on day D0; (b) Immunofluorescence image of CD8 / Ki-67 / DAPI on day D0; (c) Immunofluorescence image of CD31 / α-SMA / DAPI on day D0; (d) Immunofluorescence image of PanCK / CD45 / DAPI on day D7; (e) Immunofluorescence image of CD8 / Ki-67 / DAPI on day D7; (f) Immunofluorescence image of CD31 / α-SMA / DAPI on day D7; (g) Immunofluorescence image of PanCK / CD45 / DAPI on day D14; (h) Immunofluorescence image of CD8 / Ki-67 / DAPI on day D14; (i) Immunofluorescence image of CD31 / α-SMA / DAPI on day D14. Figure 7Immunofluorescence staining images of specific markers of immune cells and stromal cells in tumor microtissue cultured for 14 days under ±GSK2837808a component medium; scale bar 50 μm. (a) Immunofluorescence image of CD8 / DAPI in medium containing GSK2837808a component; (b) Immunofluorescence image of α-SMA / / DAPI in medium containing GSK2837808a component; (c) Immunofluorescence image of CD31 / / DAPI in medium containing GSK2837808a component; (d) Immunofluorescence image of CD8 / DAPI in medium without GSK2837808a component; (e) Immunofluorescence image of α-SMA / / DAPI in medium without GSK2837808a component; (f) Immunofluorescence image of CD31 / / DAPI in medium without GSK2837808a component. Figure 8 This diagram illustrates the consistency analysis between the drug screening process for tumor microtissue in a microfluidic chip and the clinical efficacy in patients.

[0027] In the figure, 1. Microchannel layer; 2. Irrigation channel; 3. Sample inlet; 4. Waste liquid outlet; 5. Micropore anchoring layer; 6. Large circular cavity; 7. Anchoring small circular cavity; 8. Microchannel; 9. Microslit. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0029] Example 1: Structural Design and Fabrication of Microfluidic Chips This invention provides a specific implementation of a tumor microtissue-blood vessel co-culture microfluidic chip.

[0030] like Figure 1 and Figure 2 As shown, the microfluidic chip includes a microporous anchoring layer 5 and a microchannel layer 1 bonded thereon. The two are aligned and bonded after oxygen plasma treatment to form a sealed structure. The chip material is preferably polydimethylsiloxane (PDMS) because it has good biocompatibility, gas permeability and optical transparency.

[0031] Microporous anchoring layer 5: Its upper surface is provided with multiple rows of micropore arrays. As a preferred embodiment, such as... Figure 2 As shown in (b), the micropore array is arranged in 12 rows × 8 columns to meet the requirements of high-throughput screening. Each row of the micropore array contains multiple micropore units arranged along the row direction and microchannels 8 connecting the micropore units in series.

[0032] Combination Figure 3Each micropore unit includes a large circular cavity 6 and at least two anchored small circular cavities 7 disposed at the bottom of the large circular cavity 6. In this embodiment, four anchored small circular cavities 7 are disposed within each large circular cavity 6 to maximize space utilization and increase throughput. Specific dimensional parameters are as follows: the diameter of the large circular cavity 6 is 1000µm, and its depth is 100µm; the anchored small circular cavities 7 are designed as a tapering structure (such as a trumpet shape or a stepped shape) with a top layer 10 having a diameter of 300µm, a bottom layer 11 having a diameter of 180µm, and a depth of 300µm from the inside of the large circular cavity 6. The spacing between adjacent large circular cavities 6 is 400µm, and the spacing between adjacent anchored small circular cavities 7 is 50µm. This multi-level porous structure design allows patient-derived tumor microtissue with a diameter of 70–150 µm to fall smoothly into the large circular cavity 6, and under the action of gravity and subsequent fluid, it is selectively retained in the anchoring small circular cavity 7, which is larger at the top and smaller at the bottom, thus achieving physical anchoring.

[0033] A microslit 9 is formed at the connection between the large circular cavity 6 and the microchannel 8. In this embodiment, the opening size of the microslit 9 is 20µm. The microslit 9 has a dual function: firstly, it serves as a material exchange window, allowing the culture medium in the perfusion channel 2 to enter the large circular cavity 6 through the microslit 9 and exchange nutrients and metabolic waste with the tumor microtissue fixed in the anchoring small circular cavity 7; secondly, it serves as a physical restraint structure, using its narrow 20µm opening to effectively restrict the entry of tumor microtissue (diameter ≥70µm) from the large circular cavity 6 into the microchannel 8, preventing the microtissue from being washed away during the perfusion process.

[0034] Microchannel layer 1: such as Figure 2 As shown in (a), its lower surface is provided with multiple rows of independent irrigation channels 2. The number of rows of irrigation channels 2 is the same as the number of rows of the micropore array (12 rows in this embodiment) and they correspond one-to-one. Each row of irrigation channels 2 covers all 8 micropore units of the corresponding row and together with the microchannels 8 below, forms an irrigation circuit. Figure 1 As shown, each row of irrigation channels 2 has an independent sample inlet 3 and a waste outlet 4 at both ends, forming a completely independent fluid loop. Crucially, there are no common distribution or confluence channels between different rows of irrigation channels 2; each row of irrigation channels 2 is connected to an external injection pump or other irrigation drive device via independent piping. This "row-level independence" design fundamentally eliminates the risk of cross-contamination between different channels during parallel experiments under multiple conditions, ensuring the accuracy of drug screening.

[0035] Example 2: In situ loading and fixation of tumor microtissue This embodiment provides a method for loading tumor microtissue.

[0036] First, tumor microtissue preparation: (1) The malignant tumor tissue from the patient was placed in the primary tissue transport and preservation solution, stored in an ice bath at 4°C, and transported to the laboratory within 24 hours for separation in a biosafety cabinet.

[0037] (2) Place the tumor tissue in a cell culture dish and wash it three times with PBS at 4°C. The PBS contains 100µg / mL of broad-spectrum antibacterial agent Primocin, 100U / mL of penicillin-streptomycin and 0.1% BSA. Remove necrotic areas and adipose tissue as much as possible.

[0038] (3) The tumor tissue was placed in DMEM / F12 complete medium containing 100µg / mL Primocin, 0.1% BSA, 10µM ROCK inhibitor Y-27632 and 200µg / mL DNase I. The tissue was cut into tumor micro-tissues with a diameter of less than 150µm on ice using aseptic cutting.

[0039] (4) Use a 150µm mesh sieve for primary screening and collect the sieve material; then use a 70µm cell sieve for secondary screening and collect the precipitate left on the 70µm filter screen to obtain tumor micro-tissue with a diameter of 70-150µm.

[0040] (5) The tumor microtissue was rinsed from the filter screen into the culture dish and the culture medium was recovered. The mixture was centrifuged at 1500 rpm for 5 min, the supernatant was discarded and the microtissue was resuspended in 50 µL of DMEM / F12 complete culture medium to obtain the tumor microtissue. Then, the tumor microtissue was mixed with matrix gel to form a tumor microtissue-matrix mixed suspension.

[0041] Subsequently, following step S2, the chip undergoes a two-phase pre-wetting treatment. Fluorinated oil (such as Novec 7500) containing 2% emulsified surfactant is injected into the perfusion channel 2 through the injection port 3, ensuring the oil phase fills the entire perfusion channel 2, microchannels 8, and micropore units (including large circular pores 6 and anchored small circular pores 7). The oil phase effectively eliminates all air bubbles within the chip and forms stable oil-gas or oil-solid interfaces at the hydrophilic micro-gap 9, preparing for subsequent aqueous suspension loading.

[0042] Following step S3, the tumor microtissue-stromal gel suspension was injected through inlet 3 at a low flow rate of 0.5 µL / min. Due to the hydrophobic effect of the oil phase and the surface tension within the microchannels, the aqueous suspension preferentially enters the hydrophilic micropore region. Under the combined action of gravity settling and the physical retention of the anchoring small circular cavity 7, the tumor microtissue is selectively captured and retained within the anchoring small circular cavity 7.

[0043] To ensure that each small circular cavity contains only one or more discrete micro-organisms and to drain excess suspension from the flow channel, fluorinated oil is reinjected at a stepped flow rate (e.g., 0.1 µL / min → 0.5 µL / min → 1 µL / min) following step S4. This stepped upflow gently shears the continuous aqueous phase within the flow channel, causing it to break into discrete droplets within the anchored small circular cavity 7. It also flushes out excess, unanchored micro-organisms and suspension from the flow channel, achieving "in-orifice discretization" loading.

[0044] Finally, following step S5, the loaded chip was placed in a 37°C incubator and left to stand for 30 minutes to allow the matrix adhesive to cross-link and solidify, thereby stably fixing the tumor microtissue within the anchoring small circular cavity 7, forming a structure similar to... Figure 4 The physical state shown.

[0045] Example 3: Construction of a vascularized co-culture system This embodiment provides a method for vascularization construction.

[0046] After fixation of the tumor microtissue, following step S6, degreasing and initial perfusion were performed first. Preheated aqueous culture medium (e.g., DMEM / F12, containing: 1.1% sodium bicarbonate, 2 mM L-glutamine, 10% fetal bovine serum (FBS), 100 µg / mL Primocin, 1% penicillin-streptomycin, and 10 µM lactate dehydrogenase inhibitor GSK2837808A) was pumped at a flow rate of 1 µL / min for 5–10 minutes to remove residual fluorinated oil from the flow channels and provide initial nutrition to the tumor microtissue.

[0047] Next, the wall hydrogel layer is constructed. A prepolymerized composite hydrogel solution is injected into the irrigation channel 2 and microchannel 8. In this embodiment, the composite hydrogel is a mixture of methacrylamide gelatin (GelMA) and type IV collagen (Collagen IV) with a mass / volume ratio of 4:6. After filling, it is accelerated at 37°C for 10 minutes, and then sprayed with 25 mW / cm² hydrogel. 2 Irradiation with ultraviolet light for 40 seconds allows for full cross-linking, forming a hydrogel layer with an elastic modulus of approximately 5-8 kPa. After cross-linking, the culture medium is again introduced at a high flow rate (e.g., 2-5 µL / min). The perfusion shear force washes away any unbound or poorly bound hydrogel in the central region of the channels, leaving only a thin layer of hydrogel on the channel walls and microchannel 8 walls. This "wall retention" process provides a matrix for subsequent cell adhesion while ensuring the patency of the main channels.

[0048] Subsequently, following step S7, a suspension of human umbilical vein endothelial cells (HUVECs) (with a density of approximately 1 × 10⁻⁶) was introduced into the perfusion channel 2 and the microchannel 8. 7HUVECs were allowed to stand for 1-2 hours to allow them to fully adhere to the hydrogel layer on the wall. After adhesion, low-rate perfusion (0.5 µL / min) was resumed. Under continuous fluid shear stimulation, HUVECs grew and proliferated along the channel wall, eventually forming cells like... Figure 5 The image shows a vascular-like structure with a complete lumen.

[0049] Example 4: Validation of long-term maintenance of the immune and matrix microenvironment This embodiment verifies the effectiveness of the chip system of the present invention in maintaining the tumor microenvironment during long-term culture.

[0050] Following step S8, after the vascular-like structures stabilized, DMEM / F12 perfusion medium containing specific additives was introduced. The perfusion medium contained: 1.1% sodium bicarbonate, 2 mM L-glutamine, 10% fetal bovine serum (FBS), 100 µg / mL primorcinol, 1% penicillin-streptomycin, and 10 µM lactate dehydrogenase inhibitor GSK2837808A. Continuous perfusion culture was performed using a syringe pump at a constant flow rate of 1 µL / min.

[0051] Tumor microtissue was recovered at baseline (D0), on day 7 (D7), and day 14 (D14) of continuous culture. After fixation with 4% paraformaldehyde, the tissue was embedded, sectioned, and subjected to immunofluorescence staining analysis to assess the maintenance of tumor cells, immune cells, matrix, and vascular-related components. The specific steps included: (1) After fixing, dehydrating, embedding in paraffin, and sectioning the tumor microtissue, antigen retrieval was performed, followed by blocking with 3% BSA for 30 min.

[0052] (2) Add primary antibodies and incubate overnight at 4°C. The primary antibodies include epithelial cell marker PanCK, immune cell markers CD45 and CD8, proliferation marker Ki67, fibroblast marker α-SMA, and vascular endothelial cell marker CD31.

[0053] (3) The next day, after washing with PBS, add the corresponding fluorescent secondary antibody and incubate in the dark for 50 min. After washing, mount with DAPI-containing mounting medium.

[0054] (4) Imaging analysis was performed using a confocal microscopy system.

[0055] The results are as follows Figure 6As shown, compared to D0, PanCK+ tumor cells, CD45+ and CD8+ immune cells, α-SMA+ stromal cells, and CD31+ vascular structures were consistently present in the tumor microtissues of D7 and D14, and the Ki67 proliferation index remained stable. This indicates that, under the vascularized co-culture and dynamic perfusion system provided by this invention, the original immune and stromal microenvironment components of patient-derived tumor microtissues can be stably maintained for up to 14 days.

[0056] To further verify the effect of GSK2837808A, a control experiment was set up: the control group used culture medium without GSK2837808A, while all other culture conditions were exactly the same as the experimental group. Immunofluorescence detection was performed after 14 days of culture, and the results are as follows: Figure 7 As shown. Compared to the experimental group with added GSK2837808A ( Figure 7 (a)-(c)), control group ( Figure 7 The fluorescence signals of CD8+ T cells, α-SMA+ fibroblasts, and CD31+ vascular structures in (d)-(f) were all significantly reduced. This indicates that the lactate dehydrogenase inhibitor GSK2837808A plays a synergistic role in the vascularized perfusion system of this invention by regulating the metabolic microenvironment, and is crucial for maintaining the long-term activity of immune cells and matrix phenotype.

[0057] Example 5: Parallel screening and efficacy evaluation of immunotherapeutic drugs This embodiment demonstrates how to use the chip of the present invention for screening immunotherapies.

[0058] Following the method described in the foregoing embodiments, tumor microtissues from the same patient were loaded and cultured in all 96 wells of the chip (12 rows × 8 columns), and a vascularized co-culture system was constructed.

[0059] Based on clinical needs, eight different immunotherapy drugs (or eight different concentrations of the same drug) were designed, with four rows reserved as controls (such as negative controls and vector controls). Utilizing the completely independent 12-row perfusion channels 2 of the chip, culture media containing the eight test drugs and two control conditions were connected to the corresponding 10-row injection ports 3 via a syringe pump. The remaining two rows can be used to set up other controls as needed.

[0060] Following step S9, after the vascular structure stabilized, parallel perfusion treatment with drug-containing culture medium was initiated. After 72 hours of treatment, live / dead cell staining (Calcein-AM / PI) was performed, and images were acquired using a microscopic imaging system. The activity of the drug-treated tumor microtissue was assessed using a live / dead cell viability and toxicity assay reagent. Green and red fluorescence images were acquired, and the green area of ​​live tissue and the red area of ​​dead tissue were quantitatively analyzed using image analysis software. The calculation method is as follows: Dlive = DG / (DG + DR) Clive = CG / (CG + CR) Wherein, DG represents the green fluorescence area of ​​the drug-treated group, DR represents the red fluorescence area of ​​the drug-treated group, and Dlive represents the activity of the drug-treated group; CG represents the green fluorescence area of ​​the control group, CR represents the red fluorescence area of ​​the control group, and Clive represents the activity of the control group.

[0061] The tumor micro-tissue viability corresponding to each row was quantitatively calculated using image analysis software. The formula for calculating the tumor inhibition rate (TIR) ​​is as follows: TIR = (1 - mean viability of treatment group / mean viability of control group) × 100%.

[0062] TIR ≥ 30% is used as the reference threshold for in vitro sensitivity.

[0063] Subsequently, the in vitro screening results were analyzed for consistency with the corresponding patients' clinical efficacy. The results were as follows: Figure 8 As shown, several drug combinations assessed as sensitive on the chip also showed good objective response rate (ORR) and progression-free survival (PFS) benefits in the corresponding patients' clinical treatment, demonstrating that the chip system of the present invention has good clinical predictive value.

[0064] In summary, this invention, by combining microporous anchoring structures, tiered independent perfusion circuits, wall-preserving vascularization construction technology, and metabolic regulatory factors, successfully solves the key technical challenges in long-term in vitro culture and drug screening of patient-derived tumor microtissues, providing an efficient and reliable high-throughput platform for tumor immunotherapy research and personalized medicine.

[0065] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microfluidic chip for tumor microtissue-vascular co-culture, characterized in that, It includes a microporous anchoring layer and a microchannel layer attached thereto, which together form a sealed structure; The upper surface of the microporous anchoring layer is provided with multiple rows of microporous arrays; each row of microporous arrays includes multiple microporous units arranged along the row direction and microchannels connected in series with each microporous unit. Each microporous unit includes a large circular cavity and at least two anchoring small circular cavities disposed at the bottom of the large circular cavity; the anchoring small circular cavities are a contraction structure that is larger at the top and smaller at the bottom, used to fix tumor microtissue; microslits are opened at the connection between the large circular cavity and the microchannels to facilitate the exchange of substances between the culture medium and the tumor microtissue in the cavity under perfusion conditions, while restricting the entry of tumor microtissue into the microchannels; The lower surface of the microchannel layer is provided with multiple rows of independent perfusion channels, the number of which is the same as the number of rows of the micropore array and corresponds one-to-one; each row of perfusion channels covers all micropore units of the corresponding row, and each end is provided with an independent sample inlet and a waste liquid outlet, forming an independent fluid loop.

2. The tumor microtissue-vascular co-culture microfluidic chip according to claim 1, characterized in that, The micropore array is arranged in 12 rows, with 8 large circular cavities in each row; each large circular cavity has a diameter of 1000µm and a depth of 100µm; each large circular cavity has 4 anchoring small circular cavities, with the top layer of the anchoring small circular cavities having a diameter of 300µm, the bottom layer having a diameter of 180µm, and a depth of 300µm from the inside of the large circular cavity; the distance between adjacent large circular cavities is 400µm, and the distance between adjacent anchoring small circular cavities is 50µm.

3. The tumor microtissue-vascular co-culture microfluidic chip according to claim 1, characterized in that, The microslit has an opening size of 20µm; the microchannel is used to form a blood vessel-like structure under perfusion conditions.

4. The tumor microtissue-vascular co-culture microfluidic chip according to claim 1, characterized in that, There is no common distribution channel or common confluence channel between different irrigation and drainage channels. Each irrigation and drainage channel is connected to the external irrigation drive device through an independent pipeline.

5. The application of a tumor microtissue-vascular co-culture microfluidic chip as described in any one of claims 1-4 in the screening of immunotherapeutic drugs.

6. The application according to claim 5, characterized in that, Includes the following steps: S1: The tumor tissue from the patient was mechanically sheared and sieved to obtain tumor micro-tissue with a diameter of 70-150µm, and then mixed with matrix gel to obtain a tumor micro-tissue-matrix gel suspension. S2: Perform two-phase pre-wetting and degassing treatment on the microfluidic chip, fill the perfusion channel, the microchannel and the micropore unit with oil phase, remove air bubbles, and form a stable two-phase interface at the micro gap; S3: Inject the tumor microtissue-stromal gel mixed suspension at a set flow rate, so that it selectively enters and remains in the anchored small round cavity under the physical retention effect of surface tension and anchored small round cavity; S4: Inject the oil phase and drive it with a stepped or pulsed flow rate to form discrete droplets of the tumor micro-tissue-matrix mixture suspension in each anchored small circular cavity, and discharge the residual suspension in the flow channel, thereby realizing the in-situ loading of tumor micro-tissue. S5: Place the chip in a 37°C culture environment to allow the matrix adhesive to cross-link and solidify, thus stabilizing the tumor micro-tissue within the anchoring small round cavity. S6: Replace the oil phase with an aqueous culture medium and enter continuous perfusion; inject photocrosslinkable composite hydrogel into the perfusion channel and microchannel, and after crosslinking, selectively remove it to the center of the channel by perfusion shear while retaining it on the channel wall and microchannel wall to form a wall hydrogel layer. S7: Introduce a suspension of vascular endothelial cells into the perfusion channel and microchannel, causing them to adhere to the hydrogel layer on the wall and form a vascular-like structure; S8: After the vascular-like structure stabilizes, DMEM / F12 perfusion medium is introduced for continuous perfusion culture; S9: The immunotherapeutic drug to be tested was added to the perfusion medium for perfusion treatment, and the efficacy of the drug was evaluated by detecting tumor microtissue survival rate, immune cell infiltration and matrix-related phenotypic changes.

7. The application according to claim 6, characterized in that, The oil phase in step S2 is a fluorinated oil and contains 2% emulsifying surfactant; the oil phase flow rate in step S4 is driven by a stepwise increase of 0.1→0.5→1µL / min.

8. The application according to claim 6, characterized in that, The composite hydrogel mentioned in step S6 is a compound system of methacrylamide gelatin (GelMA) and Collagen IV, with a mass or volume ratio of 4:

6.

9. The application according to claim 6, characterized in that, The perfusion medium contains sodium bicarbonate, L-glutamine, serum, antibiotics, and lactate dehydrogenase inhibitor GSK2837808A.

10. The application according to claim 6, characterized in that, In step S9, multiple immunotherapies to be tested and / or different doses or concentrations of the same immunotherapy are added to the perfusion culture medium corresponding to different drainage channels, and parallel perfusion treatment is performed under the condition that the drainage channels are not interconnected. After the treatment, the tumor microtissue corresponding to each drainage channel is detected, and the detection includes at least one of the following: tumor microtissue activity, expression of immune cell activation markers, expression of matrix-related markers and / or cytokine secretion levels, so as to obtain the efficacy evaluation results of the immunotherapies.

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