A tumor immune microphysiological system, a construction method thereof and application thereof

Abstract

The present application relates to a kind of tumor immune microphysiological system chip, comprising: microfluidic chip ontology, and integrated in the chip ontology tumor culture unit, lymph culture unit and blood vessel connecting unit;The tumor culture unit includes tumor organoid flow channel and tumor culture medium flow channel;The lymph culture unit includes lymph organoid flow channel and lymph culture medium flow channel;The blood vessel connecting unit includes blood vessel flow channel.The blood vessel flow channel is in fluid communication with the tumor organoid flow channel and the lymph organoid flow channel respectively by microstructure array, to connect the tumor culture unit with the lymph culture unit.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of tumor immunology and microfluidics, specifically to a tumor immune microphysiological system, its construction method and its application, and more specifically to a tumor immune microphysiological system based on a microfluidic chip, its construction method and its application. Background Technology

[0002] The tumor immune microenvironment is the core site regulating tumor occurrence, development, and treatment response. It comprises various components, including tumor cells, immune cells (such as T cells, B cells, dendritic cells, and macrophages), stromal cells (such as cancer-associated fibroblasts), and vascular networks. These components, through complex physical interactions and signal exchange, collectively determine the trajectory and efficacy of the immune response. Currently, in-depth research into tumor immune mechanisms, the screening and optimization of anti-tumor drugs, and the evaluation and validation of personalized treatment regimens all heavily rely on in vitro models. However, existing in vitro models have significant limitations: traditional two-dimensional cell culture models cannot replicate the three-dimensional spatial structure of cells in vivo, intercellular interactions, and the physiologically relevant biomechanical microenvironment, making it difficult to simulate key immune processes such as T cell activation, transvascular migration of immune cells, and tumor invasion. While animal models provide a certain in vivo environment, they suffer from significant species differences, long experimental cycles, high costs, and ethical constraints. Their immune response mechanisms differ fundamentally from those of humans, leading to significant deviations in the clinical translation of research findings.

[0003] Microphysiological systems, through the deep integration of microfluidics and three-dimensional cell culture, can construct organ physiological microenvironments in vitro that closely resemble those in vivo, providing a feasible path to overcome the bottlenecks of traditional in vitro models. While existing microfluidic tumor immune model technologies have overcome some limitations of traditional models to a certain extent, they still have many shortcomings. Most models focus only on simulating a single function of tumor or immunity, lacking a systematic design that integrates "tumor modules," "immune modules," and a functional "vascular module," failing to reproduce the complete cycle of synergistic effects among "tumor-immunity-vascular" in vivo. Even when multiple modules exist, the connections between them are often limited to molecular diffusion, lacking functional vascular barriers that can guide the directional migration of immune cells, and failing to simulate the crucial steps of immune cells homing from lymphoid tissue to the tumor site. Furthermore, the immune cell composition in existing models is often singular, often lacking key antigen-presenting cells (such as dendritic cells) and stromal cells, and failing to form key functional structures such as germinal centers, leading to distorted simulation of immune responses. The lack of clarity regarding core construction parameters (such as bio-ink formulation, cell ratio, and hydrodynamic conditions) makes it difficult to compare and reproduce results between different laboratories, severely restricting its application in drug development and precision medicine.

[0004] Shah, S.; D'Souza, GGM Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis. Cells 2025, 14, 732. https: / / doi.org / 10.3390 / cells14100732 This review summarizes the current application status of multicellular tumor spheroids as in vitro models of the tumor microenvironment, pointing out that traditional two-dimensional cell culture cannot reproduce the three-dimensional structure, cellular heterogeneity, oxygen gradient, and cell-matrix interactions of tumor tissues, leading to low clinical translation efficiency in drug screening; while animal models have problems such as species differences, high costs, and ethical restrictions. This paper proposes that the multicellular tumor sphere model can simulate the spatial structure of tumors, such as the proliferative zone, quiescent zone, and hypoxic core zone, and can integrate matrix components such as fibroblasts and endothelial cells, thus reproducing the tumor-matrix cross-linking effect to a certain extent. However, it also clarifies its limitations: it lacks a functional vascular network and a complete lymphoid immune module, and cannot simulate the physiological process of immune cells migrating across the vascular barrier to the tumor site after activation from lymphoid tissue. The immune cell composition is simple and it is difficult to form functional structures such as germinal centers, resulting in a distortion of the immune response simulation. Furthermore, the core construction parameters are vague and the reproducibility is poor, making it difficult to meet the needs of tumor immune mechanism research, immunotherapy drug screening, and personalized treatment plan evaluation.

[0005] CN120888400A discloses a microfluidic chip and model construction method that enables precise localization of organoids and temporal interaction with the tumor microenvironment. The chip is equipped with vascular channels, a central channel, neural and immune cell channels, and culture medium channels. Precise localization of tumor organoids is achieved through open pores and a thermosensitive hydrogel, and temporal interaction is simulated by dynamic shear force regulation. However, it still has significant limitations: neural and immune cells are mixed in the same channel, failing to construct a complete lymphoid organoid module containing B-cell, T-cell, and lymph node stromal cells, thus failing to reproduce the functional structure of immune cell activation and proliferation; the vascular channel only achieves endothelial cell spreading and budding, failing to form a biomimetic vascular network with barrier function, making it difficult to simulate the key process of immune cell migration across blood vessels; and core parameters such as cell ratio and bio-ink formulation are not clearly defined, lacking the ability to warn of safety risks such as cytokine storms caused by immunotherapies, thus failing to meet the needs of tumor immune mechanism research and personalized treatment evaluation.

[0006] In view of this, there is an urgent need to construct a multi-component synergistic, parameter-defined, and highly biomimetic tumor immune microphysiological system to bridge the gap between existing in vitro models and the in vivo physiological environment, and to provide a reliable tool for basic research and clinical translation in the field of oncology.

[0007] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a tumor immune microphysiological system, its construction method, and its application. More specifically, it provides a tumor immune microphysiological system based on a microfluidic chip, its construction method, and its application, which can be used for research on tumor immune mechanisms, screening of anti-tumor drugs, and formulation of personalized tumor treatment plans.

[0009] The first aspect of this invention provides a tumor immune microphysiological system chip, comprising a microfluidic chip body, and a tumor culture unit, a lymphatic culture unit, and a vascular connection unit integrated within the chip body; the tumor culture unit includes a tumor organoid channel and a tumor culture medium channel; the lymphatic culture unit includes a lymphatic organoid channel and a lymphatic culture medium channel; the vascular connection unit includes a vascular channel; the vascular channel is fluidly connected to the tumor organoid channel and the lymphatic organoid channel respectively through a microstructure array, thereby connecting the tumor culture unit and the lymphatic culture unit. Preferably, the width of the tumor organoid channel is 500-1500 μm, the width of the tumor culture medium channel is 1100-1600 μm, the width of the lymphatic organoid channel is 300-700 μm, the width of the lymphatic culture medium channel is 1100-1600 μm, the width of the vascular channel is 1100-1600 μm, and the channel height is 200-650 μm.

[0010] The cross-sectional shape of the microstructure is an irregular closed free curve profile; the maximum height of the circumscribed rectangle of the cross-section (the maximum span along the specified direction) is 150-350μm, and the corresponding width (the maximum span perpendicular to the above height direction) is 100-300μm; the gap size between adjacent microstructure cross-sectional profiles is set in the range of 70-120μm.

[0011] According to a preferred embodiment, the microfluidic chip body is preferably a composite substrate structure, comprising a reservoir layer, a cell culture layer and a substrate layer stacked sequentially; preferably, the reservoir layer is made of PMMA, PS or PC plastic; preferably, the cell culture layer is made of PDMS; preferably, the substrate layer is made of glass.

[0012] According to a preferred embodiment, the tumor organoid channel is loaded with tumor organoids and microvascular gel; preferably, the tumor organoids comprise tumor cells and tumor-associated fibroblasts; preferably, the microvascular gel comprises vascular endothelial cells and fibroblasts.

[0013] According to a preferred embodiment, the lymphoid organoid channels are loaded with lymphoid organoids; preferably, the lymphoid organoids comprise B cell spheres and non-B cell populations; preferably, the B cell spheres comprise B cells and lymph node stromal cells. The cell sources of the lymphoid organoids are not limited to peripheral blood and immune organs such as tonsils, lymph nodes, and spleen.

[0014] According to a preferred embodiment, the non-B cell population comprises dendritic cells and / or macrophages.

[0015] According to a preferred embodiment, the inner wall of the vascular channel is lined with an endothelial cell layer, preferably forming a biomimetic vascular network.

[0016] According to a preferred embodiment, the diameter of the tumor organoid sphere is 200 μm ± 20 μm. This diameter range is consistent with the size of small tumor foci in vivo (100-300 μm) and can ensure the nutritional supply of cells within the sphere (cell viability in the core area ≥ 80%). If the diameter is greater than 220 μm, it will lead to hypoxic necrosis of the core cells. If the diameter is less than 180 μm, it is impossible to simulate the three-dimensional structure of the tumor microenvironment.

[0017] According to a preferred embodiment, the spacing of the microstructure array is 80-120 μm. This spacing range ensures the free diffusion of cytokines and nutrients (diffusion rate ≥ 1 × 10⁻⁶). -5 (cm / s) can effectively block the flow of tumor organoids (200μm±20μm in diameter) and lymphoid organoids (200μm±20μm in diameter). If the distance is less than 80μm, the exchange of substances will be blocked. If the distance is greater than 120μm, the flow of cells will be blocked, which will destroy the functional independence of each unit.

[0018] According to a preferred embodiment, the chip body includes a plurality of tumor culture units, lymphatic culture units, and vascular connection units arranged in an array.

[0019] A second aspect of the present invention provides a method for preparing the tumor immune microphysiological system chip, comprising the following steps: fabricating a microfluidic chip body; loading tumor organoids and microvascular gels into the tumor organoid channels and perfusing tumor culture medium into the tumor culture medium channels; loading lymphoid organs into the lymphoid channels and perfusing lymph culture medium into the lymphoid culture medium channels; and constructing a vascular network in the vascular channels.

[0020] According to a preferred embodiment, the step of loading the tumor organoids and microvascular gel includes: preparing tumor organoid spheres containing tumor cells and cancer-associated fibroblasts; mixing vascular endothelial cells, fibroblasts and the tumor organoid spheres in vascularized bio-ink, injecting into the tumor organoid channels and gelling.

[0021] According to a preferred embodiment, the step of loading the lymphoid organoid includes: preparing B cell spheres containing B cells and lymph node stromal cells; mixing the B cell spheres with non-B cell populations in lymphoid organoid bio-ink; injecting the mixture into the lymphoid organoid channel and solidifying it.

[0022] According to a preferred embodiment, the step of constructing the vascular network includes: coating the vascular channels with an extracellular matrix protein solution; injecting an endothelial cell suspension into the coated vascular channels and allowing them to stand to adhere and form an endothelial cell layer. Preferably, this microphysiological system integrates multiple chip units, enabling high-throughput rapid drug evaluation and detection.

[0023] According to a preferred embodiment, during the cultivation process, the chip is placed on a programmable shaker for cultivation, and the shaking angle, interval time, and chip orientation are adjusted to promote fluid exchange and vascular network maturation. Preferably, the shaker parameters are adjusted as follows: initially, the chip flow channel is parallel to the shaking direction (7°, 8-minute interval) to promote substance exchange; when a liquid level difference appears after 6 days of cultivation, the chip is rotated 90° (the flow channel is perpendicular to the shaking direction), and adjusted to a 12-hour interval and a 10° angle; cultivation lasts for 10 days.

[0024] According to a preferred embodiment, the cells of the tumor organoids and / or lymphoids are derived from the autologous cells of the same patient.

[0025] The present invention also provides the application of the tumor immune microphysiological system chip in screening anti-tumor drugs.

[0026] The present invention also provides the application of the aforementioned tumor immune microphysiological system chip in evaluating individualized tumor treatment plans.

[0027] The present invention also provides the application of the aforementioned tumor immune microphysiological system chip in the study of tumor immune mechanisms.

[0028] This invention achieves multi-dimensional technological breakthroughs in in vitro tumor immune models through systematic and innovative design, and its technical effects are significantly superior to existing technologies, as detailed below: Multi-component deep synergy, biomimicry closely approximating the physiological state in vivo: This invention innovatively integrates tumor cells, cancer-associated fibroblasts (CAF), lymph node stromal cells and core functional cells such as T cells, dendritic cells (DC), and macrophages to construct a complete three-in-one system of "tumor module - lymph module - vascular module", accurately replicating the physiological closed loop of "tumor occurrence - immune activation - vascular migration" in vivo. By defining the optimal cell ratio of tumor cells to CAF 3:1 and vascular endothelial cells to CAF 4:1, and combining it with a "Matrix Gel / GelMA / Collagen" composite bio-ink and a special "Vascularization Bio-ink I+II" formula, tumor organoids are formed into three-dimensional structures with complete microvascular networks. Lymphoid organoids differentiate into functionally defined B-cell and T-cell regions, and vascular connector units construct a biomimetic vascular endothelial layer with barrier function. This can realistically simulate key physiological processes such as immune cell migration across blood vessels, tumor antigen presentation, immune cell activation, and tumor infiltration. Its biomimicry not only far exceeds that of traditional two-dimensional culture models and single-module microfluidic models, but also solves the core pain point of incomplete simulation of "tumor-immunity-vascular" interaction in existing technologies, providing a "miniaturized physiological system" that closely resembles the in vivo tumor immune microenvironment for in vitro research.

[0029] Parameter standardization and process standardization significantly improve repeatability and stability: This invention precisely defines key parameters throughout the entire chip fabrication process, forming a standardized technical system. This includes the component ratios and cell concentrations of bio-inks (e.g., the total cell concentration of 3×10⁻⁶ in tumor organoid bio-inks). 7 -4×10 7 / mL, HUVEC inoculation concentration 2×10 7 The parameters, including flow rate ( / mL), channel structure parameters (micropillar array spacing 80-120 μm), hydrodynamic conditions (water / oil phase flow rate, liquid exchange volume and frequency), and culture environment parameters (shaker oscillation angle, interval time, temperature, and CO2 concentration), are all clearly quantified. Simultaneously, the operational procedures for cell resuscitation, cell spheroid preparation, channel perfusion, and culture maintenance are standardized, specifying key operational nodes such as injecting the mixed suspension into the channel within 1 min and collagen coating time of 2-6 h, as well as abnormal handling protocols. This standardized design ensures that the coefficient of variation of experimental results between different batches and different laboratories is ≤10%, completely solving the industry problem of poor reproducibility and difficulty in comparing results caused by the ambiguity of core parameters in existing tumor immunology microfluidic models, laying a solid foundation for technology promotion and industrial application.

[0030] The functional system is comprehensively covered, realizing integrated evaluation of "efficacy-safety-mechanism research": This invention breaks through the limitations of existing models with single functions and constructs a multi-dimensional functional evaluation system: At the drug screening level, it can be evaluated through tumor organoid apoptosis rate, immune cell activation rate (CD8+), etc. + T cell CD69 + / GranzymeB + Percentage, CD80 in DC cells + / CD86 + This system uses indicators such as proportion and vascular integrity to accurately assess the efficacy of various anti-tumor drugs, including immune checkpoint inhibitors and anti-angiogenic drugs. In terms of safety assessment, by detecting the concentrations of cytokines such as IL-6, IL-1β, and TNF-α, it can effectively predict serious adverse reactions such as cytokine release syndrome (CRS) induced by immunotherapies, filling the gap in existing in vitro models that cannot simultaneously assess efficacy and safety. At the mechanistic research level, using laser confocal dynamic observation, cytokine spectrum analysis, and immune cell migration trajectory tracking, it can deeply analyze core mechanisms such as tumor immune escape, immune cell cross-barrier migration, and tumor-stromal cell signaling interaction. Furthermore, the system supports personalized construction based on the patient's own cells. By adjusting the cell ratio and experimental protocol, it can achieve quantitative evaluation of various treatment regimens, including single-drug, dual-drug, and radiotherapy-chemotherapy combinations, providing precise references for the development of individualized clinical treatment plans. Its application scenarios cover the entire chain of basic tumor immunology research, preclinical drug screening, and personalized clinical medicine, with extremely broad application prospects.

[0031] The invention boasts outstanding technical adaptability and practicality, balancing high-throughput and precision requirements: The chip itself employs a composite substrate structure of PMMA / PS / PC+PDMS+glass, ensuring structural stability, cell compatibility, and optical observation capabilities, supporting various microscopic detection techniques such as laser confocal microscopy. The chip's design incorporates multiple arrayed functional units, meeting high-throughput drug screening needs and significantly improving experimental efficiency. Furthermore, its cell separation, culture, and chip construction processes are highly compatible with clinical sample processing procedures, enabling efficient preparation of autologous cell models from patient samples such as surgical tumor tissue, peripheral blood, and lymph node tissue. The culture cycle is short (vascular maturation requires only 10 days, and drug evaluation takes 72 hours), operation is convenient, and costs less than animal models, offering significant advantages in clinical translational applications. Compared to traditional animal models, this invention avoids clinical translational biases caused by species differences and has no ethical restrictions. Compared to traditional organoid models, it supplements immune and vascular modules, more realistically reflecting in vivo treatment responses, providing a more efficient and precise integrated platform tool for basic research and clinical translation in the field of oncology.

[0032] The CN120888400A patented chip design is mainly used for research on the interaction between nerve cells, neuroimmune cells, and tumor cells, especially brain neurotumors. The chip adopts a 4-channel design. In addition to two culture medium channels, the remaining two channels simulate the interaction between brain tumors (such as gliomas) and the surrounding microenvironment (including nerve cells and brain immune cells, microglia). It is limited to local immune responses and lacks a design for systemic immune responses.

[0033] This application employs a 5-channel, 3-zone (tumor zone - vascular zone - lymphatic zone) design, primarily aimed at reproducing the in vitro immune process of solid tumors. It recreates the process from antigen release to antigen presentation to immune organs (lymph nodes), immune activation, and the infiltration and killing of tumor cells by activated immune cells via blood vessels, enabling precise pharmacological and pharmacodynamic evaluation of anti-tumor immunotherapies. Its differentiated advantages also include: 1) The tumor zone not only includes tumor cells / tumor organoids but also tumor microenvironment cells (including endothelial microvascular networks, fibroblasts, and immune cells); 2) Multi-level blood vessels are achieved through mechanical channel design and endothelial cell self-assembly. The endothelium of the vascular channels acts as a barrier, connecting with the capillary-like network formed in the tumor zone, facilitating the vascular delivery of antibodies, cells, and other biological drugs; 3) The lymphatic zone is a unique design used to reproduce the core function of lymph nodes as the immune command and control hub of tumor immunity; 4) Furthermore, to achieve high-throughput drug screening, the chip adopts an integrated design, allowing for simultaneous multi-group experimental detection and verification. Figure 4 ). Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the flow control chip for the lung cancer immune microphysiological system described in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the flow channel structure of the lung cancer immune microphysiological system flow control chip described in Embodiment 1 of the present invention; Figure 3 This is a partially enlarged schematic diagram of the flow channel structure of the lung cancer immune microphysiological system flow control chip described in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the lung cancer immune microphysiological system flow control chip described in Embodiment 1 of the present invention, which includes multiple structural units; Figure 5 This is a schematic diagram of the lung cancer immune microphysiological system flow control chip described in Embodiment 1 of the present invention being cultured; Figure 6 This is a fluorescence image of dextran perfusion in microvessels as described in Example 1 of the present invention (EGFP represents vascular endothelial cells, and RFP represents dextran). Figure 7This is a fluorescence staining image of tumor organoids co-cultured with microvascular gel as described in Example 1 of the present invention (EpCAM represents tumor cells, and CD31 represents vascular endothelial cells). Figure 8 This is an immunofluorescence staining image of the lymphoid organoid flow tract described in Embodiment 1 of the present invention (CD19 represents B cells, and CD3 represents T cells).

[0035] List of reference numerals 1: Storage layer; 2: Cell culture layer; 3: Basal layer; 4: Inlet; 5: Outlet; 6: Tumor culture medium channel; 7: Tumor organoid channel; 8: Vascular channel; 9: Lymphoid organoid channel; 10: Lymphoid culture medium channel; 11: Microcolumn array; 12: Tumor culture medium; 13: Tumor organoid; 14: Microvessels; 15: Endothelial cell culture medium; 16: B cell spheres; 17: T cell area; 18: Lymphoid culture medium. Detailed Implementation

[0036] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Without conflict, the technical features in the embodiments of the present invention can be combined with each other.

[0037] Unless otherwise specified, all chemical reagents and biological materials used in this invention are commercially available and processed and applied in accordance with standard operating procedures; all experimental steps are carried out under normal laboratory conditions (37°C, 5% CO2) to ensure the repeatability and reliability of the technical solution.

[0038] To overcome the shortcomings of existing technologies, this invention provides a tumor immune microphysiological system based on microfluidic chips, its construction method, and its application. By integrating three core modules—tumor, immunity, and blood vessels—and clarifying key parameters, it achieves precise simulation of the tumor immune microenvironment in vivo.

[0039] The technical solution adopted by this invention to solve its technical problem is: (I) Tumor Immune Microphysiological System Based on Microfluidic Chip 1. Microfluidic chip body A composite substrate of "plastic (PMMA / PS / PC) + PDMS + glass" is used to balance structural stability, cell compatibility, and optical observation requirements. The liquid storage layer is made of PMMA / PS / PC plastic with a thickness of 5-10mm. It has through-hole units with a diameter of 5-10mm corresponding to the inlet / outlet of the cell culture layer to store the culture medium and ensure a stable liquid supply.

[0040] Cell culture layer: made of PDMS (thickness 0.2-1mm), with inlet and outlet ports of 1.5-2mm in diameter. As the core culture area for cells and organoids, the high permeability of PDMS can ensure long-term aerobic cell culture.

[0041] Substrate: Made of glass (0.1-1mm thick), with good optical transparency, supporting microscopic observation techniques such as laser confocal microscopy.

[0042] The flow channel structure is designed as a five-channel system, including tumor culture medium channel, lymphatic culture medium channel, blood vessel channel, tumor organoid channel, and lymphatic organoid channel. Each channel is separated by a micropillar array (100μm±20μm spacing) - the micropillar array can realize the exchange of substances (such as cytokines and nutrients) between channels, while avoiding cell cross-flow and ensuring the functional independence of each unit.

[0043] The chip pretreatment is performed by high temperature and high pressure sterilization (121℃, 0.1MPa, 20min). After cooling, the flow channels are rinsed twice with sterile calcium and magnesium-free DPBS (Gibco, catalog number 14190144) to eliminate the risk of contamination. The chip body can integrate multiple structural units (array distribution) to support high-throughput experimental needs.

[0044] 2. Integrated structural unit The structural units include a tumor culture unit, a lymphatic culture unit, and a vascular connection unit, which are connected by a micropillar array to achieve dynamic interaction of "tumor cells-immune cells-vascular network".

[0045] (1) Tumor culture unit Composed of tumor organoid channels and tumor culture medium channels, the core of which is to construct tumor organoids containing microvessels to simulate the in situ growth microenvironment of tumors. Tumor organoid flow channel: loaded with tumor organoid spheres and microvascular gel, the two work together to achieve three-dimensional growth and vascularization of tumor cells.

[0046] Tumor organoid spheres: Based on the proportion of clinical tumor tissue cells, tumor cells and CAF cells (cancer-associated fibroblasts) are mixed in a ratio of 1:1 to 10:1 and embedded in a "Matrix Gel / GelMA / HAMA / collagen" composite bio-ink (preferably 15% Matrix Gel (Biocoat, catalog number 354262) + 5% GelMA (EFL, catalog number GM-60) + 5% Gelatin (SIGMA, catalog number G7041)) to form organoid spheres with a diameter of 200μm±20μm. The total cell concentration in the bio-ink is controlled at 3×10⁻⁶. 7 -4×10 7 / mL, ensuring close contact and signal transduction between cells.

[0047] Microvascular gel: To mimic tumor angiogenesis, vascular endothelial cells (6×10⁻⁶) are deposited... 6 -8×10 6 / mL) and fibroblasts (1.5×10 6 -4×10 6 Mix the two components (in 2:1 to 4:1 ratio) and embed them in the "Vascularized Bio-Ink I+II" composite system (mixed and cured at a 4:1 volume ratio). Ink I contains 7.5-12.5 mg / ml fibrinogen (SIGMA, catalog number F3879) and 0.2-1 mg / mL I-collagen (Corning, catalog number 354249), while Ink II contains 5-25 U / ml thrombin (SIGMA, catalog number T7009). The two components can be quickly cured to form a supporting gel after mixing, while promoting the formation of endothelial cell tubular structures.

[0048] Abnormalities: For issues related to bio-ink coagulation and cell viability, if coagulation is too rapid or cell damage occurs, reduce the fibrinogen / thrombin ratio; if clot degradation is too rapid, moderately increase 0.15-0.2 U / ml aprotinin (SIGMA, catalog number Y0001154) (avoid excessive levels that could affect clot formation); if endothelial cells lack tubular structures, check cell viability (if viability is low, reactivate the cells), increase collagen concentration, or supplement with 20-100 ng / ml hEGF (Novoprotein, catalog number C029) and 10-50 ng / ml hFGF (Novoprotein, catalog number C751) to promote angiogenesis.

[0049] Tumor culture medium flow channel: Loaded with a special tumor organoid culture medium, based on Advanced DMEM / F12 (Gibco, catalog number 12634010), with added cytokines such as Wnt-3a, R-spondin1, and hEGF. After sterilization through a 0.22μm filter membrane, it can maintain the stemness and proliferative capacity of tumor organoids and ensure long-term culture stability.

[0050] (2) Lymphatic culture unit Composed of lymphoid organoid channels and lymphoid culture medium channels, its core function is to construct functional lymphoid organoids, simulating the microenvironment for lymphocyte activation and migration in vivo. Lymphoid organoid channels: Loaded with lymphoid organoids (B cell spheres + non-B cells), embedded in lymphoid organoid bio-ink (preferably 0.3% HAMA (EFL, catalog number HAMA-150K) + 5% gelatin (SIGMA, catalog number G7041) + 0.5 mg / mL I-collagen (Corning, catalog number 354249)) to ensure the three-dimensional growth and immune activity of lymphocytes.

[0051] B-cell spheres: B cells sorted by anti-CD19 magnetic beads according to the proportion of cells in the lymph node (viability ≥85%, concentration 2×10⁻⁶). 7 -4×10 7 / mL) and lymph node stromal cells (purity ≥95%, concentration 1×10 6 -3×10 6 Mix ( / mL) at a ratio of 5:1 to 12:1 to prepare cell spheres with a diameter of 200μm±20μm. Lymph node stromal cells can secrete chemokines, inducing B cell aggregation and activation.

[0052] Non-B cells: Residual cells after PBMC sorting (viability ≥85%, concentration 2×10⁻⁶) 7 -4×10 7 / mL), can replenish autologous DC cells (dendritic cells, 1×10 6 -3×10 6 / mL) and macrophages (1×10 6 -3×10 6 / mL) - DC cells are responsible for antigen presentation, macrophages participate in tumor killing, and the two work together to improve lymphatic immune function.

[0053] Abnormal handling: If the diameter of B cell spheroids deviates from 200μm±20μm, correct it by adjusting the flow rate of the microfluidic oil phase (increase the flow rate if >220μm, decrease the flow rate if <180μm); if the viable cell rate is <85%, re-sort the cells or optimize the induction conditions.

[0054] Lymphocyte culture medium channels: Loaded with a dedicated lymphocyte culture medium, based on AdvancedRPMI-1640 (Gibco, catalog number 12633020), supplemented with immune cell activating factors such as IL-2, IL-4, and IL-21 to maintain lymphocyte activity and immune function. Specifically, the lymphocyte organoid channels are loaded with lymphocyte organoids; the lymphocyte organoids include B cell spheres and non-B cell populations; the B cell spheres include B cells and lymph node stromal cells. The non-B cell populations include dendritic cells and / or macrophages.

[0055] (3) Vascular connection unit With blood vessels as the core, a functional vascular network is constructed to separate tumors from lymphatic units, serving as a "bridge" for material exchange and cell migration. Pretreatment and inoculation of vascular channels: First, inject 100-500 μg / mL type I collagen coating solution (Corning, catalog number 354249), and incubate at 37°C in a humidified chamber for 2-6 hours to enhance the adhesion ability of HUVECs (human umbilical vein endothelial cells); then resuspend the HUVECs to 2×10⁻⁶. 7 -4×10 7 / mL, take 1-2μL and inject into the flow channel, let stand at 37℃ and 5% CO2 for 30-60min for adhesion, then add 600μL of endothelial cell culture medium (Scien Cell, catalog number 1001).

[0056] Connection relationship: One side of the vascular channel is connected to the tumor organoid channel through a microcolumn array, and the other side is connected to the lymphoid organoid channel, which can realize the migration of antigens secreted by tumor cells and lymphoid immune cells to the tumor area, as well as the delivery of drugs to the target area through blood vessels.

[0057] Abnormal handling: If the HUVEC adhesion rate is <80%, extend the coating time or increase the collagen concentration; if cells detach during shake culture, check the coating solution concentration and replenish it.

[0058] (II) Construction method of microfluidic tumor immune microphysiological system 1. Preliminary preparations (1-2 days before construction) The core task is to complete cell resuscitation and pre-culture to ensure that all cell types are in optimal viability: HUVECs were revived in endothelial cell culture medium and cultured to a cobblestone morphology; human lung fibroblasts (HLF) were revived in medium containing 1×LSGS at a density of 10⁶ and cultured to a spindle shape.

[0059] Tumor cells and CAF cells were revived using their respective culture media and cultured to the logarithmic growth phase (when they proliferate vigorously and have high activity).

[0060] Lymph node stromal cells were cultured in a special medium (containing FGF-2) to the logarithmic growth phase, and CD31 was selected by flow cytometry. - α-SMA + Cells (purity ≥ 95%).

[0061] Macrophages were obtained by induced differentiation of PBMCs with 40-50 ng / mL M-CSF for 7 days, and dendritic cells were obtained by induced differentiation of PBMCs with 500 U / ml IL-4 + 500-1000 U / ml GM-CSF until maturity (CD80). + CD86 + ≥70%).

[0062] 2. Cell spheroid preparation and immune cell processing (Day 1) Tumor sphere preparation: Digest logarithmic phase tumor cells and CAF cells (room temperature, tryple express, 3 min), centrifuge at 300×g for 5 min, and then mix them in tumor organoid bio-ink in proportion; spheres are formed using a T-type / cross microfluidic chip (aqueous phase flow rate 5-10 μL / min, oil phase flow rate 20-60 μL / min), and tumor spheres of 200 μm ± 20 μm are collected. Only those with a viable cell rate of ≥85% as detected by Calcein-AM / PI staining can be used.

[0063] Immunotherapy: PBMCs were sorted using anti-CD19 magnetic beads (Mitteni, catalog number 130-050-301) to obtain B cells and non-B cells. B cells were mixed with lymph node stromal cells to prepare 200μm±20μm B cell spheres, which were then cultured in B cell culture medium. Non-B cells were cultured in a culture medium containing IL-2, CD3, and CD28 to maintain immune activity.

[0064] Abnormal handling: If the diameter of tumor spheroids / B-cell spheroids is abnormal, adjust the oil phase flow rate; if the viable cell rate is <85%, shorten the digestion time or replace with fresh cells.

[0065] 3. Flow channel perfusion and cell seeding (Day 2) Perform the procedures in the following order: "chip pretreatment - lymphatic drainage - tumor drainage - vascular drainage" to avoid cross-contamination and drainage blockage. Chip pretreatment: After sterilization, rinse the channels twice with sterile calcium- and magnesium-free DPBS and blot dry any residual liquid; if the channels are blocked, backwash with 200 μL DPBS (flow rate ≤10 μL / min); if the channels are still blocked, replace the chip.

[0066] Lymphatic channel perfusion: Non-B cells (DCs / macrophages can be added) and B cell spheres were resuspended separately in lymphoid organoid bio-ink, mixed and slowly injected into the lymphoid organoid channel (flow rate ≤2μL / min), cured with 30mW UV for 15s, and then 400μL of lymphoid culture medium was added.

[0067] Tumor channel perfusion: Tumor spheres were resuspended in vascularized bio-ink I, mixed with vascular endothelial cells and CAF cells, and then mixed with ink II at a ratio of 4:1. The mixture was injected into the tumor organoid channel within 1 min (avoiding coagulation), allowed to stand at 37°C for 10 min to solidify, and then 400 μL of tumor culture medium was added.

[0068] Vascular channel preparation: Inject collagen coating solution and incubate. After discarding the solution, inoculate HUVECs. After adhesion, add 600 μL of endothelial cell culture medium.

[0069] 4. Cultivation and Maintenance Medium change: Change the medium every 2 days (to avoid nutrient depletion and accumulation of metabolic waste, and to create a pressure difference to promote angiogenesis). On Day 2, add 600 μL of endothelial culture medium to the vascular channel and 400 μL to each of the lymphatic / tumor channels. On Day 4, reverse the volume (400 μL for vascular channels and 600 μL for lymphatic / tumor channels). When changing the medium, slowly aspirate the liquid along the channel wall to avoid cell shedding.

[0070] Shaking parameters adjustment: Initially, the chip flow channel is parallel to the shaking direction (7°, 8min interval) to promote material exchange; when a liquid level difference appears after 6 days of culture, rotate the chip 90° (the flow channel is perpendicular to the shaking direction) and adjust to a 12h interval and 10° angle; after 10 days of culture, confirm vascular maturity (tubular structure ratio > 60%) by CD31 staining, stop shaking and enter the detection stage.

[0071] Abnormal handling: If a large number of cells detach after changing the medium, reduce the rate of adding medium; if the culture medium is turbid (suspected contamination), replace it immediately and add 1× antibiotic. If there is no improvement after 24 hours, terminate the culture.

[0072] (III) Application of Microfluidic Tumor Immune Microphysiological System This system can be widely used in tumor immune mechanism research, anti-tumor drug screening, and personalized treatment plan evaluation, achieving precise analysis through multi-dimensional detection.

[0073] 1. Application in tumor immune mechanism research The core objective is to dynamically observe the interaction process between "tumor-lymphatic system-blood vessel" and reveal the mechanisms of immune regulation. Dynamic observation: Tumor cells, vascular endothelial cells and non-B cells were labeled with Cell Tracker. On Day 3, Day 7 and Day 10, the proliferation of tumor organoids (diameter changes), formation of microvascular lumen (number of tubular structures >50μm) and migration trajectory of immune cells were observed (photographed every 2 hours for 8 consecutive hours).

[0074] Cell viability assay: Samples were taken on Day 3, Day 7, and Day 10, and Calcein-AM / PI double staining was used to detect cell viability and assess cell viability at different stages.

[0075] Cytokine concentration detection: The concentrations of cytokines such as IFN-γ, TNF-α, and IL-10 were detected using ELISA or flow cytometry (ABplex Human Cytokine 12-Plex Assay Kit, catalog number RK04296) to analyze the degree of immune activation. The detection limit of ELISA is 0.5 pg / mL, while flow cytometry can detect 12 cytokines simultaneously, which is more efficient.

[0076] Handling abnormalities: If the fluorescence signal is weak, extend the incubation time of the Cell Tracker or increase the concentration; if the ELISA reading is below the detection limit, increase the sample loading amount or extend the incubation time of the secondary antibody.

[0077] 2. Application in antitumor drug screening For immune checkpoint inhibitors (such as PD-1 inhibitors) and anti-angiogenic drugs (such as VEGF inhibitors), the efficacy and safety of these drugs are evaluated through concentration gradient testing and multi-indicator detection. Drug treatment: After the blood vessels matured on Day 10 of culture, drugs with concentration gradients of 0.01-10 μM were added to the blood vessel channels and cultured at 37°C and 5% CO2 for 72 h.

[0078] Multi-indicator detection: Tumor organoid apoptosis rate: Calcein-AM / PI staining, an apoptosis rate >30% was considered an effective concentration.

[0079] Immune cell activation: Immunofluorescence detection of CD8 + T cells (CD69) + / GranzymeB + ), DC cells (CD80) + / CD86 + ), macrophages (CD86) + / CD206 + If the percentage of active ingredients is greater than 50%, it is considered to be effectively activated.

[0080] Vascular integrity: CD31 / ZO1 fluorescent staining, a tubular structure breakage rate of <20% is considered to meet safety standards.

[0081] 3. Application in the evaluation of individualized treatment plans Personalized systems are built based on patient samples to screen for optimal treatment plans, achieving "precision medicine": Patient sample processing: surgical tumor tissue (digestion and separation of tumor cells and CAF cells), peripheral blood (separation of PBMCs), and lymph node tissue (separation of lymph node stromal cells) were collected and induced to differentiate into autologous macrophages and dendritic cells.

[0082] Personalized system construction: The ratio of tumor cells to CAF cells is adjusted according to the patient's tumor tissue pathology results, and autologous DC / macrophages are supplemented for non-B cells.

[0083] Treatment regimen testing: Design single-drug, dual-drug combination, and radiotherapy-chemotherapy combination regimens (3 doses each), and culture the blood vessels for 72 hours after adding the drugs.

[0084] Multidimensional detection and treatment plan screening: Detecting tumor killing efficiency (≥60%) and immune response intensity (CD69).+ ≥40%), cytokine storm risk (IL-6 / IL-1β / TNF-α <100pg / mL), and selected the recommended regimens based on the weighted scores of "tumor killing (40%) + immune response (30%) + risk (30%)".

[0085] In one embodiment, the microfluidic chip body comprises a reservoir layer 1, a cell culture layer 2, and a base layer 3, which are sequentially stacked and bonded. The reservoir layer 1 provides a stable liquid supply to the five-channel system of the cell culture layer 2 through an inlet 4 and an outlet 5. The five-channel system includes a tumor culture medium channel 6, a tumor organoid channel 7, a blood vessel channel 8, a lymphoid organoid channel 9, and a lymphoid culture medium channel 10. Adjacent channels are separated by a micropillar array 11 to form an "exchangeable and barrier" interface. The micropillar array 11 allows cytokines and nutrients to cross the channels while maintaining the independence of the channels within the cell culture layer 2. The diffusion and inhibition of cell crossflow allow tumor organoids 13 and their surrounding microvessels 14 to be in a three-dimensional culture and vascular reconstruction environment within the tumor organoid channel 7 when the tumor culture medium 12 is perfused in the tumor culture medium channel 6. When the lymph culture medium 18 is perfused in the lymph culture medium channel 10, lymphoid organoids containing B cell spheres 16 and T cell regions 17 maintain immune activity within the lymphoid organoid channel 9. The tumor culture unit, lymph culture unit, and vascular connection unit are structurally coupled into a "tumor-vascular-lymphatic" transmission link through the spacing and connection relationship of the micropillar array 11.

[0086] Furthermore, the vascular connection unit, with vascular channel 8 as its core, is used to construct a functional vascular barrier from the endothelial cell layer supported by endothelial cell culture medium 15. When vascular channel 8 is pretreated and inoculated with endothelial cells, it is in a working position where it is fluidly connected to tumor organoid channel 7 and lymphoid organoid channel 9 via micropillar array 11. This allows signaling molecules released by tumor organoid 13 to enter vascular channel 8 via micropillar array 11 and further influence immune activation on the lymphoid organoid side. This is necessary to induce immune cells to cross the barrier. In the case of barrier migration, the vascular channel 8 is in a "bridging" position so that the non-B cell population from lymphoid organs can migrate directionally through the endothelial barrier corresponding to the vascular channel 8 and enter the tumor organoid channel 7 to interact with the tumor microenvironment while maintaining its activity and function. The non-B cell population may include dendritic cells and / or macrophages to undertake the synergistic role of antigen presentation and tumor killing, thereby enabling the tumor culture unit, lymphoid culture unit and vascular connection unit within the same chip to form a closed loop coupling in structure and realize the continuous delivery of the immune link in function.

[0087] Example 1: Construction of a Tumor Immune Microphysiological System Based on a T-shaped Microfluidic Chip I. Core Materials and Equipment 1. Cells: Human lung cancer A549, lung cancer CAF, HUVEC, HLF, healthy human PBMCs; macrophages (PBMCs induced by M-CSF for 7 days), DC cells (PBMCs induced by IL-4 / GM-CSF for 6 days, maturation rate 85%).

[0088] 2. Key reagents: tumor / lymphoid organoid bio-ink, vascularization bio-ink (I+II), 50 μg / mL type I collagen coating solution, anti-CD19 magnetic bead sorting kit.

[0089] 3. Chips and Equipment: Microfluidic chips (PMMA+PDMS+glass), syringe pumps, UV crosslinkers, programmable shakers.

[0090] Figure 1 This diagram illustrates the overall structure of the microfluidic chip for the lung cancer immune microphysiology system. The diagram shows the three-layer composite substrate structure of the microfluidic chip of this invention, consisting of a reservoir layer, a cell culture layer, and a substrate layer from top to bottom. The spatial relationships and core functional areas of each layer are labeled. The diagram clearly shows the corresponding connections between the inlet, outlet, and each flow channel, presenting the overall layout design of the chip.

[0091] Figure 2 This diagram illustrates the flow channel structure of a fluid control chip for the lung cancer immune microphysiology system. The diagram details the distribution and connectivity of the five-channel system within the chip, including channels for tumor culture medium, tumor organoids, blood vessels, lymphoid organs, and lymphoid culture medium. Each channel is separated by an array of micropillars, clearly defining their arrangement and spatial location. This visually reflects the fluid connectivity design of the three major modules: tumor, blood vessels, and lymphatic system, and demonstrates the structural basis for material exchange and directed cell migration between these modules.

[0092] Figure 3 This is a magnified schematic diagram of a portion of the flow channel structure. The diagram shows a magnified view of the micropillar array and the connection points of the flow channels, labeling the morphology and spacing (80-120 μm) of the micropillars and the local dimensional characteristics of the flow channels. The magnified view demonstrates how the micropillar array achieves its core function of "allowing material exchange and preventing cell cross-flow," clarifies the isolation and connectivity mechanisms between the flow channels, and explains the key structural design that ensures the functional independence of each culture unit and the microscopic working principle of the chip.

[0093] Figure 4This is a schematic diagram of a chip structure containing multiple structural units. The diagram illustrates the design of integrating multiple arrayed tumor culture units, lymphocyte culture units, and vascular connection units into the chip body. The arrangement and overall layout of the multiple units are clearly shown, highlighting the chip's core advantage of supporting high-throughput experiments. This demonstrates that the design can simultaneously conduct multiple parallel experiments, meeting the efficient experimental needs of scenarios such as drug screening and mechanism research, and intuitively reflecting the chip's potential for industrial application.

[0094] Figure 5 This diagram illustrates the chip culture process. It dynamically displays the chip's operational status during culture, labeling the core components within each channel (tumor culture medium, tumor organoids, microvessels, endothelial cell culture medium, B-cell spheroids, lymphatic culture medium, etc.) and the direction of fluid flow. It also shows the interaction between the chip and the culture equipment, intuitively reflecting the culture mode of "periodic medium change" and "dynamic fluid exchange," and demonstrating the dynamic mechanisms of nutrient supply, metabolic waste removal, and cell interaction during chip culture.

[0095] Figure 6 Fluorescence imaging of vascular endothelial cells co-labeled with perfused dextran. This image shows the fluorescence tracer results, clearly revealing the spatial distribution relationship between the vascular wall structure and the perfused material within the vascular lumen through the fluorescence signals of EGFP (a marker of vascular endothelial cells) and RFP (an intraluminal dextran tracer). The image shows that EGFP-labeled endothelial cells form a continuous and complete vascular network structure, while RFP-labeled dextran completely fills the vascular lumen. The signals of the two components highly overlap, directly verifying the integrity of the vascular structure and the success of the perfusion operation, demonstrating that this imaging method can reproduce the structure and perfusion state of blood vessels in vivo.

[0096] Figure 7 This image shows the fluorescence staining results of tumor organoids co-cultured with microvascular gel. The immunofluorescence staining clearly reveals the spatial distribution relationship between tumor organoids and microvessels through the fluorescence signals of EpCAM (tumor cell marker) and CD31 (vascular endothelial cell marker). The image shows a microvascular network tightly surrounding the tumor organoids, with intact tubular structures forming functional connections with the tumor organoids. This directly verifies the successful co-construction of the "tumor-blood vessel" within the tumor organoid channels, demonstrating that this system can reproduce the in vivo tumor vascularization microenvironment.

[0097] Figure 8This image shows the immunofluorescence staining results of lymphoid organoids. The fluorescence signals of CD19 (a B-cell marker) and CD3 (a T-cell marker) reveal the clearly defined regional structures of B-cell and T-cell regions within the lymphoid organoids. The staining results show that B cells and T cells form functionally specialized regions within the lymphoid organoids, consistent with the cell distribution characteristics of lymph nodes in vivo. This validates the core design goal of lymphoid organoids—"mimicking the structure of the in vivo immune system"—and demonstrates that this system can reproduce the functional regionalization and activation microenvironment of immune cells.

[0098] II. Construction Steps 1. Preliminary preparation: Prepare 50μm gelatin spheres for T-type chip (8μL / min aqueous phase, 40μL / min oil phase); revive each cell to the logarithmic growth phase / functional maturity.

[0099] 2. Day 1: Processing of Cell Spheroids and Immune Cells: A549 and CAF were mixed at a ratio of 3:1 to prepare 200 μm tumor spheres (85% viable cell rate). PBMCs were sorted to obtain B cells (88% viability) and non-B cells. B cells were combined with lymph node stromal cells to produce 200μm diameter B cell spheres (86% viability). Non-B cells were supplemented with DCs / macrophages (90% viability).

[0100] 3. Day 2: Flow channel injection: Inject immune cell suspension into the lymphatic drainage channel, solidify with 30mW UV for 15s, and add 400μL of lymphatic culture medium.

[0101] Tumor spheres were injected into the tumor flow channel with vascularized bio-ink (I:II=4:1), cured at 37℃ for 10 min, and then 400 μL of tumor culture medium was added.

[0102] After 4 hours of collagen coating of the vascular channels, HUVECs were inoculated (adhesion rate 85%) and 600 μL of endothelial culture medium was added.

[0103] 4. Culture and maintenance: Replace the medium every 2 days, and adjust the shaker parameters according to the stages. Day 10: Vessels mature (tubular structures account for 68%).

[0104] III. Comparison of System Representation and Traditional Models 1. System Characterization Microvascularization: Observation using laser confocal microscopy Figure 6 CD31 antibody staining showed that the microvascular network tightly surrounded the tumor organoid spheres.

[0105] Tumor-vascular interaction: observation by laser confocal microscopy ( Figure 7 CD31 antibody staining showed that the microvascular network tightly surrounded the tumor organoid spheres.

[0106] Lymphoid unit structure: observation of lymphoid organoid pathways ( Figure 8 The CD19 antibody-labeled B cells and CD3 antibody-labeled T cells form distinct partitions, consistent with the distribution characteristics of lymph node cells in vivo.

[0107] 2. Comparison with traditional tumor organoid models IV. Conclusion This invention integrates the "tumor-lymphatic-vascular" three-unit system through a microfluidic chip, which solves the defects of traditional tumor organoid models that "lack vascular network and immune function". It can reproduce the core structure and cell interaction of the tumor immune microenvironment in vivo, providing a high-fidelity in vitro model for subsequent mechanism research and drug screening.

[0108] Example 2: Construction and Application of the System in Personalized Lung Cancer Immune Microphysiological System I. Core Design (1) To ensure the biomimicry and clinical relevance of the personalized tumor immune microphysiological system, it is necessary to efficiently isolate tumor cells, CAF cells, PBMCs, B cells, lymph node stromal cells, autologous macrophages, and autologous DC cells from patient samples. The specific operation method is as follows: Isolation and culture of tumor cells and CAF cells Sample pretreatment: In a biosafety cabinet, the surgically removed tumor tissue from the patient was immersed in DPBS containing 10× penicillin-streptomycin-amphoteric B for 15 min, gently agitated 3 times during the process. Then, it was washed 3 times with sterile DPBS (5 min each time) to remove blood and impurities from the tissue surface. The washed tumor tissue was separated into tissue blocks of about 1×1 mm using a sterile scalpel and transferred to sterile centrifuge tubes.

[0109] Tissue digestion: Add digestion solution pre-warmed to 37°C to centrifuge tubes (formula: Advanced DMEM / F-12 medium + 1% FBS + 10mM HEPES buffer + 1×GlutaMAX supplement + 100μg / ml primary cell antibiotic Primocin + 10μM Y-27632 + 2mg / ml collagenase I + 2mg / ml collagenase IV + 1mg / ml hyaluronidase + 100μg / ml DNAI enzyme + 1× penicillin-streptomycin-amphoteric acid B), tighten the cap, and place on a 37°C horizontal shaker. Digest at 60 rpm for 1.5 hours. Take samples every 30 minutes for microscopic examination to observe the dispersion of tissue blocks.

[0110] Cell purification: After digestion, centrifuge tubes were placed at 4°C and centrifuged at 300×g for 5 min, discarding the supernatant; the cell pellet was resuspended in 10 ml of serum-free DMEM medium and centrifuged at 4°C for 300×g for 5 min. This process was repeated twice to thoroughly remove residual digestive enzymes; pre-cooled matrix gel was added to the final cell pellet and gently pipetted to mix (adjusting the cell concentration to 1×10⁻⁶). 6 / mL).

[0111] Organoid culture: Suspend the matrix gel-cell mixture and drop it into the center of each well of a 24-well plate (50 μL per well). Carefully transfer to a 37°C, 5% CO2 incubator and incubate for 20-30 minutes until the matrix gel is completely cured. Add 500 μL of organoid culture medium to each well (formulation: based on Advanced DMEM / F12, supplemented with 10 mM HEPES, 1×GlutaMAX, 100 ng / ml Wnt-3a, 100 ng / ml R-spondin1, 1×B27 Supplement, 10 mM Nicotinamide, 1.25 mM N-acetylcysteine, 100 ng / ml hNoggin, 50 ng / ml hEGF, 100 ng / ml hFGF-10, 500 nM A83-01, and 10.5 μM... Y-27632 (after mixing all components, it is filtered through a 0.22μm sterile filter membrane for sterilization) and then placed in an incubator for 7-10 days.

[0112] Cell sorting: After organoid formation, aspirate the culture medium from the wells, add ice-cold DMEM medium, and resuspend the matrix gel by pipetting. Transfer to 15ml centrifuge tubes. Wash the well plates twice with ice-cold DMEM medium to ensure that all remaining organoids are collected. Add ice-cold DMEM to the centrifuge tubes to 12ml, place on ice, and gently pipette 15 times with a 1ml pipette tip. Centrifuge at 4℃, 300×g for 5min. Discard the supernatant, resuspend the cell pellet in DPBS, and filter through a 40μm cell filter. Centrifuge the filtered single-cell suspension at 4℃, 300×g for 5min, resuspend in CAF-specific medium (formulation: DMEM / F12 + 10% FBS + 5ng / ml TGF-β + 1× penicillin-streptomycin), seed into culture flasks, and culture at 37℃, 5% CO2 to obtain CAF cells. Centrifuge unfiltered cell spheres (50-100μm in diameter) at 4℃, 300×g for 5min, resuspend in tumor organoid medium, and continue culturing to obtain tumor organoids.

[0113] PBMC separation Collect peripheral blood from the patient (heparin anticoagulated) and dilute the blood sample with PBS at a ratio of at least 1:1 in a conical tube.

[0114] Add an equal volume of Ficoll-Paque® to the diluted sample.

[0115] Centrifuge at 400×g for 20 minutes at room temperature.

[0116] Collect the PBMCs at the junction of the PBS and Ficoll-Paque® layers into a new test tube.

[0117] Add PBS to the test tube and wash the cells.

[0118] Centrifuge at 300-400×g for 4-5 minutes at 2-8°C and discard the supernatant.

[0119] Resuspend the cells in an appropriate amount of flow cytometry staining buffer or a dedicated buffer, and then perform cell counting and viability analysis.

[0120] Repeat step 6 to centrifuge the cells, then resuspend them in an appropriate amount of RPMI 1640 complete medium to achieve a final cell concentration of 2 × 10⁻⁶. 6 Cells / mL.

[0121] Isolation and culture of B cells and lymph node stromal cells Sample processing: In a biosafety cabinet, the patient's lymph node tissue (surgically removed or punctured sample) was washed three times (5 min each time) with DPBS containing 2× penicillin-streptomycin-amphoteric B, placed on a 40 μm cell filter, and gently ground with a sterile grinding rod. The filtered cell suspension was collected.

[0122] Red blood cell removal: Transfer the cell suspension to a centrifuge tube, centrifuge at 400×g for 5 min at 4°C, and discard the supernatant; add ACK red blood cell lysis buffer (at 1×10⁻⁶). 7 Add 1 ml of lysis buffer to the cells and incubate at room temperature for 3 min, gently inverting the cells twice during the incubation period. Add 5 times the volume of sterile DPBS to terminate the lysis, centrifuge at 400×g for 5 min at 4°C, discard the supernatant, and wash twice with DPBS.

[0123] B cell sorting: The cell pellet was resuspended in DPBS containing 1% BSA (cell concentration 1×10⁻⁶). 8 Add CD19 magnetic beads (magnetic beads: cells = 1:10) to the cell culture medium ( / mL), incubate at 4℃ for 15 min; place in a magnetic field for separation, the adsorbed cells are B cells, wash and resuspend in B cell culture medium; the unadsorbed cells are non-B cells, which are collected for later use.

[0124] Lymph node stromal cell culture: Collect the tissue residue that did not pass through a 400μm filter after grinding, add 2mg / ml collagenase (dissolved in DMEM / F12 medium), and digest at 37℃ with shaking for 30min; centrifuge at 4℃, 300×g for 5min, and discard the supernatant; resuspend the cell pellet in lymph node stromal cell-specific medium (formulation: DMEM / F12, 10% FB, +1× penicillin-streptomycin-amphoteric B, 2.5μg / ml LFGF-2), seed into culture flasks, and culture at 37℃, 5% CO2, changing the medium every 2 days, and obtain lymph node stromal cells after 7-10 days of culture.

[0125] Induced differentiation of autologous macrophages PBMCs were isolated from whole blood under aseptic conditions.

[0126] Preparation of RPMI 1640 complete medium: Add fetal bovine serum (final concentration 10%) and 2 mM L-glutamine to RPMI 1640 medium (if using medium not currently supplemented with GlutaMAX). Bring the medium temperature to 37°C. Optional: Add 1% penicillin-streptomycin (5000 units / mL).

[0127] Cells were resuspended in RPMI 1640 complete medium to achieve a cell concentration of 2 × 10⁻⁶. 6 Cells / mL.

[0128] Transfer the cell solution to a cell culture dish.

[0129] The mononuclear cells were cultured for 24 hours in an incubator at 37°C and 5% CO2 to allow them to adhere to the culture vessel.

[0130] Prepare RPMI 1640 complete medium containing 40-50 ng / mL M-CSF in sterile conical tubes. Optional: Add 20 ng / mL IL-4.

[0131] Replace the culture medium in the petri dish with a medium containing M-CSF and IL-4.

[0132] Culture the cells in a 37°C, 5% CO2 incubator for 6 days. During these 6 days, supplement with RPMI 1640 complete medium containing 40–50 ng / mL M-CSF every 3–4 days (optional: add 20 ng / mL IL-4). Examine cell health and growth density under a microscope.

[0133] Cells can be collected when many granules appear in the cytoplasm and the cells have slightly elongated. Furthermore, a large number of cells should adhere to the culture plate. When collecting cells, discard the old culture medium and rinse the culture dish twice with 1×PBS, discarding the PBS after each rinse.

[0134] Add 10 mL of 10 mM EDTA to each culture dish and let it stand at room temperature for 10 minutes or until the cells no longer adhere to the culture dish.

[0135] Collect the cells in a 50 mL conical tube and centrifuge at 300–400 × g for 4–5 minutes at room temperature.

[0136] Discard the supernatant and rinse the cells with 1×PBS.

[0137] Centrifuge at 300–400 × g for 4–5 minutes.

[0138] Discard the supernatant and resuspend the cells in flow cytometry staining buffer or culture medium.

[0139] Isolation and purification of autologous DC cells Prepare fresh peripheral blood mononuclear cells (PBMCs) at a concentration of 1-2×10⁻⁶. 5 cells / cm 2 Seed the cells at a density of 1000 mg / L into culture flasks, add 25 mL of RPMI 1640 or CTS™ AIM-V medium, and incubate in a humidified environment of 37°C and 5% CO2 for 2-3 hours; collect non-adherent cells, and wash adherent cells (mainly CD14 cells) with calcium- and magnesium-free CTS™ PBS. + Mononuclear cells were cultured three times, with the addition of culture medium containing 50-100 ng / mL recombinant human IL-4 and 50-100 ng / mL GM-CSF. The cells were then cultured at 37°C in a humid environment with 5% CO2 until day 3.

[0140] Transfer the old culture medium from the culture flask to a sterile centrifuge tube and centrifuge at 200×g for 5-10 minutes to collect non-adherent or slightly adherent cells. Discard the supernatant and gently resuspend the cell pellet in an equal volume of pre-warmed fresh culture medium containing 500 U / ml IL-4 and 500 U-1000 U / ml GM-CSF. Transfer the resuspended cell suspension back to the original culture flask and co-culture it with the adherent cells in the flask.

[0141] On day 6 of culture, 50 ng / mL tumor necrosis factor-α (TNF-α) was added to the culture medium to induce induction for 48 hours.

[0142] During the isolation and culture of the patient-derived cells, all operations must comply with aseptic procedures, and all reagents used must be sterilized by filtration through a 0.22μm filter membrane. The cell culture environment must be strictly controlled for temperature (37±0.5℃), CO2 concentration (5±0.5%), and humidity (≥95%) to ensure cell viability and functional stability, providing high-quality cellular raw materials for the construction of personalized tumor immune microphysiological systems.

[0143] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A tumor immune microphysiological system chip, characterized in that, include: The microfluidic chip body, and the tumor culture unit, lymphatic culture unit and vascular connection unit integrated within the chip body; The tumor culture unit includes a tumor organoid flow channel and a tumor culture medium flow channel; The lymphoid culture unit includes lymphoid organoid channels and lymphoid culture medium channels; The vascular connection unit includes a vascular flow channel; The vascular flow channel is fluidly connected to the tumor organoid flow channel and the lymphoid organoid flow channel respectively through a microstructure array, thereby connecting the tumor culture unit and the lymphoid culture unit.

2. The tumor immune microphysiological system chip according to claim 1, characterized in that, The microfluidic chip body comprises a reservoir layer, a cell culture layer, and a substrate layer stacked sequentially, bonded by oxygen plasma surface activation followed by direct pressing; preferably, the reservoir layer is made of PMMA, PS, or PC plastic; preferably, the cell culture layer is made of PDMS; preferably, the substrate layer is made of glass.

3. The tumor immune microphysiological system chip according to claim 1, characterized in that, The tumor organoid channels are filled with cell-loaded hydrogel, and the cell types include tumor cells / organoids / assemblies and tumor-associated fibroblasts, vascular endothelial cells, immune cells and other tumor microenvironment cells.

4. The tumor immune microphysiological system chip according to claim 1, characterized in that, The lymphoid organoid channels are filled with lymphoid organs; the lymphoid organs preferably include B cell spheres and non-B cell populations, preferably, the non-B cell populations include dendritic cells and / or macrophages, T cells, lymph node fibroblast reticulocytes (FRCs); the B cell spheres preferably include B cells and their subsets and stromal cells such as fibroblast reticulocytes.

5. The tumor immune microphysiological system chip according to claim 1, characterized in that, The inner wall of the blood vessel channel is lined with an endothelial cell layer, forming a biomimetic blood vessel network.

6. A method for preparing a tumor immune microphysiological system chip as described in any one of claims 1-5, characterized in that, Includes the following steps: The microfluidic chip body is fabricated by casting the liquid storage layer through processing an acrylic mold, and the cell culture layer is obtained by processing an SU-8 mold and then casting it using photolithography. A composite hydrogel containing tumor models and microvascular microenvironment cells is loaded into the tumor organoid flow channel, and tumor culture medium is perfused into the tumor culture medium flow channel; Lymphoid organs are loaded into the lymphoid organoid channels, and lymphoid culture medium is infused into the lymphoid culture medium channels; A vascular network was constructed in the vascular channel and perfused with endothelial cell culture medium.

7. The method according to claim 6, characterized in that, The steps of loading the tumor organoids and microvascular gels include: mixing a tumor model, vascular endothelial cells, and fibroblasts with vascularized bio-ink, injecting the mixture into the channels of the tumor organoids, and performing in-situ gelation; wherein the tumor model is selected from one or more combinations of tumor cells, tumor organoids, and tumor assemblies; the vascularized bio-ink is a gellifiable biocompatible matrix that assists in the formation of microvessels in the tumor area, and the microvessels are permeable and perfusionable; The gelatable biocompatible matrix is ​​selected from: bio-derived hydrogels, synthetic hydrogels, or combinations thereof; wherein the bio-derived hydrogels include proteins, polysaccharides, or derivatives thereof; and the synthetic hydrogels include polyethylene glycol, polyacrylates, peptides, or derivatives thereof. The bio-derived hydrogel is selected from: fibrin, collagen, gelatin, elastin, laminin, alginate, hyaluronic acid, chondroitin sulfate, chitosan, matrix gel, decellularized extracellular matrix, or any combination thereof; The gelatable biocompatible matrix comprises a combination system of fibrinogen and thrombin; The gelatable biocompatible matrix also contains type I collagen and / or matrix gel. Preferably, the final concentration of fibrinogen in the bio-ink is 2-10 mg / mL, the final concentration of thrombin is 1 IU / mL, the final concentration of type I collagen is 0.2-1 mg / mL, and the concentration of matrix gel is 1-15%.

8. The method according to claim 6, characterized in that, The steps of loading the lymphoid organoids include: preparing B cell spheres containing B cells and lymph node stromal cells; mixing the B cell spheres with non-B cell populations in lymphoid organoid bio-ink; injecting the mixture into the lymphoid organoid channels and solidifying it.

9. The method according to claim 6, characterized in that, The steps for constructing the vascular network include: coating the vascular channels with an extracellular matrix protein solution; injecting an endothelial cell suspension into the coated vascular channels and allowing them to stand to adhere to the walls and form an endothelial cell layer.

10. The application of the tumor immune microphysiological system chip as described in any one of claims 1-5 in screening anti-tumor drugs, especially antibody drugs, vaccines, cell therapy and other biological drugs, or evaluating personalized tumor treatment plans, or studying tumor immune mechanisms.

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