A bionic lymph node organoid, a construction method and application thereof

Abstract

The present application relates to a kind of bionic lymph node organoids and its construction method, the organoids include in three-dimensional space: one B cell-rich core region;And one T cell-rich region, it is at least partially wrapped in the core region in space;Wherein, the core region can be differentiated to form the light area and dark area with structural difference in response to external stimulation.The method includes preparing first bioink and second bioink, the first bioink contains B lymphocyte, and the second bioink contains T lymphocyte;Using the first and second bioink, the organoid prototype with the spatial structure of T cell-rich region wrapping B cell-rich region is formed by three-dimensional construction technology;The organoid prototype is solidified to stabilize its structure;External stimulation is applied to the structure after solidification, and the B cell-rich region is differentiated to form light area and dark area.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and organoid technology, specifically to a biomimetic lymph node organoid, its construction method, and its application. Background Technology

[0002] Lymph nodes are core functional units of the human adaptive immune system, composed of T-cell regions, B-cell regions, and germinal centers. Through T / B cell collaboration, B-cell proliferation and differentiation into plasma cells, and the secretion of specific antibodies, they achieve precise immune responses against pathogens. Current in vitro immunology research mainly relies on two-dimensional cell culture or animal models, but these have significant limitations: two-dimensional culture systems lack the three-dimensional spatial structure and intercellular microenvironment of in vivo cells, making it impossible to reproduce key immune processes such as T / B cell collaboration and germinal center formation, leading to distorted simulations of immune responses; while animal models can partially simulate in vivo immune responses, they suffer from significant species differences (e.g., different activation mechanisms of immune cells in mice and humans), long experimental cycles (usually 1-2 months), strict ethical restrictions, and high costs, making it difficult to meet the needs of high-throughput research and personalized medicine.

[0003] The rise of organoid technology has provided a new direction for simulating the function of lymph nodes in vitro, but it suffers from several key drawbacks: First, its cellular composition is singular, failing to simulate the multi-cell synergistic immune microenvironment of T / B / APC / FRC cells; second, it lacks precise spatial topological control, making it impossible to form the crucial "T-zone enveloping B-zone" structure essential for germinal center function; third, its functional validation system is incomplete, generally lacking monitoring of key safety indicators such as cytokine storms; and fourth, the neglect of the cell adaptation phase in the construction process leads to low cell activity and poor differentiation efficiency. Therefore, developing a construction method that simultaneously addresses the three major challenges of spatial biomimicry, functional integrity, and safety control is a critical technological bottleneck that urgently needs to be overcome in this field.

[0004] Bogoslowski, Ania, et al. "Organoid Models of Lymphoid Tissues." Organoids 4.2 (2025): 7. This paper summarizes the research progress of organoid models of lymphoid tissues, systematically describes the current status of organoid construction for primary lymphoid tissues such as bone marrow and thymus, and secondary lymphoid tissues such as lymph nodes and spleen, and analyzes the advantages of organoids in simulating the three-dimensional microstructure of lymphoid organs, achieving scalable culture, and personalized construction. It also points out the key problems existing in the current lymphoid tissue organoid models, including immature cell phenotypes, incomplete stromal cell populations, heterogeneity in structure and function, and the lack of robust organoid models for some lymphoid organs such as the spleen.

[0005] Zhong, Zhe, et al. "Human immune organoids to decode B cell response in healthy donors and patients with lymphoma." Nature Materials 24.2 (2025):297-311. A synthetic hydrogel simulating the lymphoid tissue microenvironment is disclosed, capable of constructing GCs derived from human tonsils and B cells derived from peripheral blood mononuclear cells. Compared to immune organoids derived from tonsils, immune organoids derived from peripheral blood mononuclear cells can maintain the survival of GCs, B cells, and plasma cells for a longer period and exhibit unique programmed B cell characteristics, including GC compartments, high-frequency somatic mutations, immunoglobulin class switching, and B cell cloning. Chemical inhibition of transcriptional and epigenetic processes can enhance plasma cell formation. Integrating polarized CXCL12 protein into the lymphoid organoid microarray can modulate the germinal center response of healthy donor B cells, but is ineffective for B cells derived from lymphoma patients. This system can rapidly and controllably simulate immune responses and B cell diseases. However, the hydrogel based on chemical reactions in this technical solution has poor controllability and cannot achieve spatial topological reconstruction of lymphoid organoids. In addition, the self-assembly of immune cells in this paper has drawbacks such as poor stability and lack of spatial structure.

[0006] CN111032687A discloses a method for constructing three-dimensional lymphoid organoids using multiphoton laser bioprinting technology. The core of this method involves mixing a cell population, including T cells, B cells, and antigen-presenting cells, with a polymer precursor, and then inducing polymerization through energy beam projection to form lymphoid organoids containing a cellular matrix. This method can be used in scenarios such as immune protein production, vaccine evaluation, and cytokine storm prediction. This paper identifies key problems in the construction of lymphoid organoids in existing technologies: traditional two-dimensional culture cannot reproduce functional interactions between cells, while early three-dimensional models lack precise control over cell spatial partitioning and struggle to balance structural stability and cell viability. Furthermore, they have limitations in high-throughput applications and clinical translation adaptability. The core conclusion is that by using computer-controlled energy beam projection technology to achieve precise spatial positioning of immune cells, combined with the use of biocompatible polymer matrices, lymphoid organoids that can simulate the immune response process can be constructed, providing a new technical path for in vitro immunology research and drug development. However, this technology does not involve the physiological spatial topological design of "T region wrapping B region", nor does it achieve the directional induction of differentiation between the light and dark zones of the germinal center, and there is still room for improvement in biomimicry and functional integrity.

[0007] In summary, existing in vitro lymphoid models suffer from core problems such as low structural biomimicry, insufficient precision in functional regulation, and difficulty in forming germinal centers, failing to meet the needs of vaccine development, immune mechanism research, and other fields for highly biomimetic lymphoid nodule models. Therefore, developing a biomimetic lymphoid nodule organoid construction technology that can accurately reproduce the spatial structure of natural lymphoid nodules and achieve stable formation of functional germinal centers has significant scientific research value and application prospects.

[0008] 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

[0009] To address the shortcomings of existing technologies, this invention provides a biomimetic lymphoid organoid, its construction method, and its applications. This invention is applicable to in vitro simulation of lymphatic immune responses, development of in vitro humanized antibodies, screening of immunomodulatory drugs, and evaluation of vaccine immunogenicity and safety. It can also serve as a precise in vitro immune model, providing technical support for research on lymphatic immune mechanisms and the formulation of clinical immunotherapy protocols.

[0010] The first aspect of this invention provides a biomimetic lymph node organoid. The organoid comprises, in three-dimensional space: a core region enriched with B cells; and a T cell-rich region that at least partially encloses the core region; wherein the core region is capable of differentiating into structurally distinct light and dark regions in response to external stimuli.

[0011] According to a preferred embodiment, the core region further includes one or more of lymph node stromal cells, macrophages, and dendritic cells.

[0012] According to a preferred embodiment, the T-cell enrichment region further comprises one or more of lymph node stromal cells, macrophages, and dendritic cells.

[0013] According to a preferred embodiment, the organoid is composed of a biocompatible hydrogel matrix; the hydrogel matrix comprises one or more of HAMA, GelMA, AlgMA, SilMA, collagen, matrix gel, gelatin, and nanoclay.

[0014] According to a preferred embodiment, the dark area and the light area are distinguished by differences in B cell distribution density, proliferative activity, and / or expression of cell surface markers. The dark area is rich in Ki-67. + Highly proliferating B cells and / or CXCR4 + B cells, with the bright area rich in CD83+ B cells.

[0015] A second aspect of this invention provides a method for constructing biomimetic lymph node organoids, comprising the following steps: (1) Prepare a first bio-ink and a second bio-ink, wherein the first bio-ink contains B lymphocytes and the second bio-ink contains T lymphocytes; (2) Using the first and second bio-inks, an organoid prototype with a spatial structure of T-cell enrichment region enclosing B-cell enrichment region was formed by three-dimensional construction technology; (3) Solidify the organoid prototype to stabilize its structure; (4) Apply external stimulation to the solidified structure to induce B cell enrichment differentiation to form light and dark areas.

[0016] According to a preferred embodiment, the three-dimensional construction technology is bio-3D printing technology or microfluidic chip technology.

[0017] According to a preferred embodiment, the first bio-ink and the second bio-ink comprise a biocompatible hydrogel material. The hydrogel material is selected from HAMA, GelMA, AlgMA, SilMA, collagen, matrix gelatin, gelatin, nanoclay, and any combination thereof.

[0018] According to a preferred embodiment, the external stimulus is selected from antigenic substances including gradient chemical signals, viruses, and vaccines. Preferably, the chemical signal is provided by the CXCL12 chemokine. Preferably, the organoid produces antigen-specific antibodies upon antigen stimulation.

[0019] The third aspect of this invention provides the application of the aforementioned biomimetic lymph node organoid or the aforementioned method for constructing biomimetic lymph node organoids in any of the following aspects: (1) In vitro study of lymphoid immune response mechanisms; (2) Development of in vitro humanized antibodies; (3) Evaluate the immunogenicity or safety of the vaccine or biological drug selection; (4) Construct disease-related complex immune microphysiological models.

[0020] The biomimetic lymph node organoid and its construction method provided by this invention have at least the following beneficial technical effects: 1. High biomimicry and complete process: The "T-zone enveloping B-zone" structure is precisely constructed using bio-3D printing or microfluidic chips, replicating the spatial topology of in vivo lymphoid nodules. A 1-2 day adaptation culture is set up to reduce cell stress, combined with CXCL12 gradient induction to efficiently form germinal center light and dark zones (8-12 days), completely replicating the structure of in vivo lymphoid nodules and the immune response process. Multi-dimensional functional validation ensures that the organoid can stably achieve core functions such as T / B cell collaboration and antibody secretion, with a functional maintenance period of 2-4 weeks, overcoming the shortcomings of existing methods such as low biomimicry and low differentiation efficiency.

[0021] 2. Excellent technical controllability and safety: Clearly defined quantitative parameters for each step (such as printing rate, channel flow rate, and culture time), with ≥90% repeatability across different laboratories according to standard operating procedures; For the first time, cytokine storm detection is incorporated into the validation system, and safety risks in organoid applications are avoided through monitoring the concentration of key inflammatory factors and cytotoxicity. It is particularly suitable for the simultaneous evaluation of "efficacy-safety" of vaccines and immunotherapies, making up for the lack of safety assessment in existing technologies.

[0022] 3. Wide range of applications and significant translational value: It can be used for immune mechanism research (visualizing dynamic processes such as T / B cell collaboration and germinal center formation), high-throughput immunomodulatory drug screening (single round screening completed in 3-7 days, much shorter than the 1-2 months of animal models), personalized immunotherapy guidance (using patients' own cells to construct organoids and test drug response), while reducing reliance on animal experiments, lowering research costs and ethical controversies.

[0023] 4. Good biocompatibility and applicability: The biological materials used meet the requirements of cell compatibility and there is no risk of contamination by animal-derived pathogens; the organoid culture conditions are mild and can be co-cultured with mucosal organs such as lung and intestine, expanding to the field of mucosal immunity research, and the scope of application is far greater than that of traditional lymphoid organoid models. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the biomimetic lymph node organoid construction process of the present invention, wherein "1" represents the cell-biomaterial complex preparation step, "2" represents the "T region encapsulates B region" structure construction step, "3" represents the 2-day adaptation culture step, "4" represents the -CXCL12 concentration gradient induction step, and "5" represents the multi-dimensional functional verification step.

[0025] Figure 2 This is an immunofluorescence staining image of a patient's lymph node section.

[0026] Figure 3 This is an immunofluorescence staining image of an "outer T, inner B" structure generated by the Trident microfluidic chip in this invention.

[0027] Figure 4This is a diagram of the "outer T, inner B" structure generated by the present invention using a bio-3D printer.

[0028] Figure 5 This is a flow cytometry comparison of primary lymphocyte 2D culture and T cells (CD3+) and B cells (CD19+) on day 7 of the culture of the biomimetic lymphoid organoid of this invention.

[0029] Figure 6 This invention presents flow cytometry statistics of biomimetic lymph node organoids and 2D cultured immune cell subtypes. The left side shows the statistics of T cell subtypes, and the right side shows the statistics of B cell subtypes.

[0030] Figure 7 This is a flow cytometry result of the biomimetic lymph node organoid B cell subtype of the present invention.

[0031] Figure 8 This is an immunofluorescence staining image of the light and dark partitions of B cells in the biomimetic lymph node organoid of this invention.

[0032] Figure 9 This is a statistical graph of the flow cytometry results of B cell subtypes in the CXCL12 gradient-induced and uninduced groups of the biomimetic lymph node organoid of this invention.

[0033] Figure 10 This is a flow cytometry result of T cell subtypes in the biomimetic lymph node organoid of this invention under vaccine stimulation.

[0034] Figure 11 This is a flow cytometry result of B cell subtypes in the biomimetic lymph node organoid of this invention under vaccine stimulation.

[0035] Figure 12 This is a bright-field image of the biomimetic lymph node organoid and 2D cultured cells of the present invention under conditions of no vaccine stimulation.

[0036] Figure 13 This is a statistical graph showing the ELISpot detection results of antibody-secreting cells produced by the biomimetic lymph node organoids and 2D cultured cells under vaccine stimulation according to the present invention.

[0037] Figure 14 The results show the live and dead cell staining at 0, 7, and 12 days and their statistical graphs (green represents live cells, and red represents dead cells).

[0038] Figure 15 The bar chart shows the percentage of Naïve B cells in CD19+ B cells under different culture conditions.

[0039] Figure 16 The bar chart shows the proportion of plasmablasts and plasma cells in CD19+ B cells under different culture conditions.

[0040] Figure 17The bar chart shows the proportions of CD8+ and CD4+ in CD3+ T cells under different culture conditions. Detailed Implementation

[0041] The following is a detailed explanation with reference to the accompanying drawings.

[0042] Figure 1 This is a schematic diagram illustrating the construction process of a biomimetic lymph node organoid, demonstrating the complete technical chain of this invention from raw material preparation to functional verification. The process consists of 5 core steps: Step 1: Preparation of cell-biomaterial composites The first and second bio-inks were prepared by mixing the sorted and purified B cell population and T cell population with biocompatible hydrogel to form uniformly dispersed B-region complex and T-region complex, providing functional raw materials for subsequent structure construction.

[0043] Step 2: Construction of the "T-zone wrapping B-zone" structure By using bio-3D printing (layer-by-layer printing of ring-shaped T-regions and cylindrical B-regions) or microfluidic chips (forming a shell-core structure), the spatial encapsulation of T-cell-rich regions on B-cell-rich regions can be achieved, thus reproducing the macroscopic topological structure of lymph node nodules in vivo.

[0044] Step 3: Adaptation Culture The solidified structure was cultured in a special culture medium containing cytokines such as IL-2, IL-4, and IL-21 to reduce stress damage to cells caused by the structure construction, ensure cell viability ≥85%, and lay the foundation for subsequent differentiation.

[0045] Step 4: CXCL12 concentration gradient induction A concentration gradient of 0-100 ng / mL CXCL12 chemokine was applied using a concentration gradient device to induce self-organization and differentiation of B cell enrichment areas into dark areas (CXCL4). + High-growth zone and bright zone (CD83) + (Mature zone), reproducing the formation process of germinal centers in the body.

[0046] Step 5: Multi-dimensional functional verification By using flow cytometry to detect T / B cell subtypes and activation status, ELISA / ELISpot to detect antibody secretion, and cytokine detection to assess safety, we can ensure that organoid functions are intact and controllable.

[0047] Example 1: Construction of biomimetic lymph node organoids based on Trident microfluidic chip technology 1. Materials and Cell Preparation Biomaterials: A mixture of 8% PEG (8 kDa) / 0.3% HAMA (150K) / 5% Gelatin / 0.5 mg / mL I-collagen (T-zone bio-ink) and 8% DEX (500 kDa) / 0.3% HAMA / 5% Gelatin / 0.5 mg / mL I-collagen (B-zone bio-ink) were selected. Both exhibit photosensitivity (405 nm UV response) and thermosensitivity (4℃ gelation). Preliminary gelation occurred at 4℃ for 1 minute, and under 405 nm UV light (10 mW / cm²), the gel was successfully applied. 2 Complete cross-linking is achieved after 15 seconds of irradiation.

[0048] Cell preparation: Lymph nodes from lung cancer patients were surgically obtained and transported at 4°C in pre-cooled tissue preservation solution. Cells were washed three times with DPBS containing 2× penicillin-streptomycin-amphoteric B in a biosafety cabinet, ground through a 40μm filter, and the cell suspension was collected. The suspension was centrifuged at 4°C and 400×g for 5 minutes, the supernatant was discarded, and ACK erythrocyte lysis buffer was added for incubation for 3 minutes (to remove erythrocytes). After centrifugation again, the cells were washed twice with DPBS. B cells were sorted using CD19 magnetic beads (Mitteni, catalog number 130-050-301), and T cells were sorted using CD3 magnetic beads (Mitteni, catalog number 130-097-043). Tissue that did not pass through the filter was collected, digested with 2 mg / ml collagenase at 37°C for 30 minutes, and the digested cells were collected. After centrifugation and washing, the cells were cultured in FRC-specific medium to obtain FRC cells.

[0049] Magnetic bead sorting includes: 1. Determine the number of cells.

[0050] 2. Centrifugation and washing: Centrifuge the cell suspension at 300×g for 10 minutes. Thoroughly aspirate the supernatant. This step is to remove serum (which may interfere with magnetic bead binding) and other components from the original culture medium.

[0051] 3. Resuspension of cells: at a rate of 10 million (10 7 Resuspend cells in 80 µL of buffer at a ratio of 1:1. The buffer consists of PBS (pH 7.2), EDTA (2 mM), fetal bovine serum (0.5%), and (optional) glucose (2-5 mM). Pre-cool to 4°C and filter sterilize using a 0.22 µm filter membrane before use.

[0052] 4. Add magnetic beads: at a rate of 10 per 10 million (10 7 Add 20 µL of magnetic beads per cell.

[0053] Note: Cell suspension volume (80 µL): magnetic bead volume (20 µL) = 4:1. This means that ultimately every 10 7Each cell was labeled in a total volume of 100 µL.

[0054] 5. Mixing and Incubation: Mix thoroughly by pipetting or vortexing to ensure full contact between the cells and magnetic beads. Incubate in a refrigerator (4°C) for 15 minutes.

[0055] 6. Washing to remove free magnetic beads: Add a large amount of buffer solution (per 10) 7 Dilute the cells with 1-2 mL of water. Centrifuge again at 300×g for 10 minutes. Discard the supernatant thoroughly. This step is to wash away any excess magnetic beads that have not bound to the cells, preventing them from clogging the subsequent sorting column.

[0056] 7. Resuspension of cells: up to 100 million (10 8 10 cells were resuspended in 500 µL buffer.

[0057] 8. Prepare the column: Insert the column into the magnetic rack. Moisten the column with buffer solution and allow it to air dry. Connect the waste liquid tube at the bottom.

[0058] 9. Sample Loading and Flow-through (Collection of Target Cells): Add the cell suspension to the column. Let it flow down naturally. The flowing liquid (flow-through solution) contains the target cells. Collect it in a clean test tube.

[0059] 10. Washing (to improve recovery rate): Add sufficient buffer to the column for washing, allowing it to drain naturally each time. Combine all the flow-through solution with the flow-through solution from step 9. This contains the enriched target cells.

[0060] 11. Removal from the magnetic field and elution (removal of impurities): Remove the entire column from the magnetic rack. Place it on a new collection tube (as a waste tube). Add an appropriate amount of buffer solution. Push the stopcock firmly and quickly all the way down to expel all the liquid. The expelled liquid will contain other cells labeled with magnetic beads; proceed to the next step.

[0061] Antibody type and fluorescent label type for flow cytometry sorting (if used): CD3 Monoclonal Antibody (SK7), PE-Cyanine5.5, eBioscience™ - 35-0036-42; CD19 Monoclonal Antibody (HIB19), PE, eBioscience™ - 12-0199-42; Fixed Viability Dye eFluor™ 506, eBioscience™ - 65-0866-18; The flow cytometer used was the CytoFLEX SRT cell sorter.

[0062] Figure 2 Immunofluorescence staining images of human lymph node sections reveal the microstructural characteristics of natural lymph nodes in vivo, serving as a reference standard for the biomimetic design of this invention. The staining results show a clear spatial distribution of "T cell regions enclosing B cell regions" within natural lymph nodes. CD3-labeled (red fluorescence) T cells are concentrated in the peripheral region of the lymph nodes, forming a ring-like enclosing structure; CD19-labeled (green fluorescence) B cells are enriched in the central region, and density differences due to B cell proliferation and maturation can be observed within the central region (corresponding to the dark and light areas of the germinal center in vivo); simultaneously, CD31-labeled (blue fluorescence) vascular structures surround the lymph nodes, providing nutrition and signal support to immune cells.

[0063] The organoids constructed in Examples 1-4 of this invention have a macroscopic structure of "outer T, inner B" (such as the microfluidic shell-core structure in Example 1 and the 3D-printed layered structure in Example 2) that is highly consistent with the spatial topology of natural lymph nodes, and the B cell light and dark partitions induced by CXCL12 ( Figure 8 This further replicates the functional structure of natural hair regrowth centers. Figure 2 This provides in vivo control evidence for evaluating the biomimicry of the organoids of the present invention, proving that the present invention can accurately simulate the core structural features of natural lymph nodes.

[0064] Induction of macrophage differentiation: PBMCs were isolated from whole blood of the patient or a healthy person under sterile conditions.

[0065] Preparation of RPMI 1640 complete medium: Add fetal bovine serum (final concentration 10%) and 2 mM L-glutamine to RPMI 1640 medium (Note: the medium is not supplemented with GlutaMAX). Set the medium temperature to 37°C. Optionally, 1% penicillin-streptomycin (5000 units / mL) may be added.

[0066] Cells were resuspended in RPMI 1640 complete medium to achieve a cell concentration of 2 × 10⁻⁶. 6 Cells / mL.

[0067] Transfer the cell solution to a cell culture dish.

[0068] 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.

[0069] Prepare RPMI 1640 complete medium containing 40–50 ng / mL M-CSF in sterile conical tubes. Optionally, 20 ng / mL IL-4 may be added.

[0070] Replace the culture medium in the petri dish with a medium containing M-CSF and IL-4.

[0071] Cells were cultured for 6 days in an incubator at 37°C and 5% CO2. During these 6 days, every 3–4 days, RPMI 1640 complete medium containing 40–50 ng / mL M-CSF was supplemented, with optional addition of 20 ng / mL IL-4. Cell health and growth density were examined under a microscope.

[0072] 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.

[0073] 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.

[0074] Collect the cells in a 50 mL conical tube and centrifuge at 300–400 × g for 4–5 minutes at room temperature.

[0075] Discard the supernatant and rinse the cells with 1×PBS.

[0076] Centrifuge at 300–400 × g for 4–5 minutes.

[0077] Discard the supernatant and resuspend the cells in flow cytometry staining buffer or culture medium.

[0078] Isolation and purification of DC cells: Prepare fresh peripheral blood mononuclear cells (PBMCs) or lymph nodes from autologous or healthy individuals, at a concentration of 1-2 × 10⁻⁶. 5 cells / cm 2 Seed the cells at a suitable density into culture flasks, add 25 mL of RPMI 1640 or CTS™ AIM-V medium, and incubate in a humidified environment at 37°C and 5% CO2 for 2-3 hours; collect non-adherent cells, and wash adherent cells (mainly CD14) 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, and cultured at 37°C in a humid environment of 5% CO2 until day 3.

[0079] 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.

[0080] On day 6 of culture, 50 ng / mL tumor necrosis factor-α (TNF-α) was added to the culture medium for induction for 48 hours. All cell types were separated into live cells using magnetic beads before use.

[0081] Adjust cell concentration: B cells 3 × 10 7 cells / mL, T cells 1×10 7 cells / mL, 1×10⁶ FRC cells 6 Cells / mL, macrophages and DCs 3×10⁶ each 5 per mL.

[0082] Note: When using PBMCs and lymph nodes to isolate and purify DC cells, the isolation and purification steps were the same, and no differences were found in the obtained DC cells.

[0083] Preparation of cell-biomaterial complexes: B cells, FRCs, macrophages, DCs and B-zone bio-ink were mixed in proportion and gently pipetted with a 1000 μL pipette (avoiding the formation of air bubbles) to form B-zone complexes; using the same method, T cells were replaced with B cells to prepare T-zone complexes, ensuring uniform cell dispersion.

[0084] 2. Trident microfluidic chip constructs a shell-core structure Chip debugging: A Trident microfluidic chip was used to connect the injection pump to the chip channel. The central channel pressure was set to 150 mbar (to deliver the B-zone complex), the outer channels on both sides were set to 100 mbar (to deliver the T-zone complex), and the shear channel pressure was set to 150 mbar (to deliver mineral oil containing 10% SPAN80). The entire chip was placed on a 37°C constant temperature stage for 30 minutes to preheat (to maintain the fluidity of the bio-ink).

[0085] Core-shell structure formation: The B-region complex was injected into the central channel injection pump, and the T-region complex was injected into the peripheral channel injection pump. The injection pumps were started, forming an "outer T, inner B" core-shell structure in the chip confluence region. The EP tube was placed in an ice bath environment to collect the structure, and it was initially cured at 4°C for 1 minute, followed by UV crosslinking (405nm, 10mW / cm). 2Irradiate for 15 seconds to fully solidify; add pre-cooled lymphocyte immune cell culture medium, centrifuge at 4°C and 400×g for 5 minutes, discard the supernatant, wash twice with pre-cooled culture medium to remove uncured bio-ink and mineral oil, and transfer to a 24-well plate.

[0086] 3. Adaptation and Cultivation Adaptation culture: Add 1 mL of lymphocyte immune cell culture medium to each well of a 24-well plate and incubate at 37°C, 5% CO2, 20% O2 for 2 days; during culture, observe under an inverted microscope, the organoid structure is intact, and the T / B cell partitioning is obvious. Figure 3 After the culture was completed, organoids from 3 wells were stained with trypan blue. The average cell viability was 85%, which met the requirements for subsequent experiments.

[0087] Example 2: Construction of biomimetic lymph node organoids based on bio-3D printing technology 1. Materials and Cell Preparation Biomaterials: A HAMA / Gelatin / I-collagen mixture was selected, and preliminary gelation was performed at 4℃ for 1 minute. The gel was then analyzed under 405nm UV light (10mW / cm²). 2 After 15 seconds of irradiation, complete cross-linking was achieved, with a Young's modulus of 634.69±17.93 Pa, matching the mechanical properties of human lymphatic tissue.

[0088] Cell preparation and complex preparation: Same as in Example 1, cell viability after sorting is greater than 95%, complex is uniformly dispersed and free of bubbles.

[0089] 2. 3D Printing Structure Construction Printing parameter settings: Extrusion-type bio 3D printer, 20G nozzle installed, printing platform temperature 4℃, nozzle temperature 18℃, printing speed 15mm / s, extrusion speed 0.04mm. 3 / s.

[0090] Layered printing: First, the T-region complex is printed to form a ring-shaped outer layer (200 μm wide), then the B-region complex is printed to form a central core (2600 μm in size), and finally the top layer of the T-region complex (100 μm thick) is printed, resulting in an overall organoid height of 200 μm. After printing, it is illuminated with 405 nm ultraviolet light (10 mW / cm²). 2 Irradiation for 15 seconds completely cures the structure, forming a "T-zone enveloping B-zone" structure. Figure 4 ).

[0091] 3. Adaptation, induction, and functional verification Adaptation culture: Same as in Example 1, cell viability after culture was 85%, and the structure remained intact.

[0092] CXCL12 gradient induction: Organoids were transferred to the central channel of a concentration gradient microfluidic chip. Culture medium containing 100 ng / ml CXCL12 was introduced into one peripheral channel of the chip, while culture medium without CXCL12 was introduced into the other, forming a linear gradient of 0-100 ng / ml. The cells were cultured at 37℃ and 5% CO2 for 10 days, with continuous perfusion to maintain the gradient. On day 8, immunofluorescence staining revealed the formation of dark areas (CXCR4+) and bright areas (CD83+) in the B cell region. Figure 8 Germinal centers were successfully constructed. Flow cytometry analysis showed that, compared to the 2D group and the group without CXCL12, the addition of CXCL12 promoted GC B cell survival. Figure 9 ).

[0093] Functionality verification: B / T cell segmentation: Flow cytometry (Beckman CytoFLEX SRT) showed CD3+ in 2D culture. + Cells accounted for 87.9%, CD19 + Cells accounted for 6.27%; in the organoids of this invention, CD3 + Cells accounted for 70.8%, CD19 + Cells accounted for 22.1% ( Figure 5 CD3 in 2D culture + CD4 + Cells accounted for 7.97%, CD3 + CD8 + Cells accounted for 2.17%; in the organoids of this invention, CD3... + CD4 + Cells accounted for 40.66%, CD3 + CD8 + Cells accounted for 4.47%; in the organoid CD19 of this invention + The proportion of Naïve cells increased by 21.84% (22.84% vs 1.00%) compared to 2D culture, and the proportion of Transitional cells increased by 15.54% (17.84% vs 2.3%). Figure 6 ).

[0094] B cell function: Flow cytometry showed a complete range of B cell subtypes. Figure 7 Seven days after antigen stimulation, ELISApot analysis showed 35 antibody-secreting cells per 10-10. 5 Cells, proving that B cells can secrete antibodies.

[0095] CXCL12 gradient induction: Organoids were transferred to the central channel of a concentration gradient microfluidic chip. Culture medium containing 100 ng / ml CXCL12 was introduced into one peripheral channel of the chip, while culture medium without CXCL12 was introduced into the other, forming a linear gradient of 0-100 ng / ml. The cells were cultured at 37℃ and 5% CO2 for 10 days, with continuous perfusion to maintain the gradient. On day 8, immunofluorescence staining revealed the formation of dark areas (CXCR4+) and bright areas (CD83+) in the B cell region. Figure 8 Germinal centers were successfully constructed. Flow cytometry analysis showed that, compared to the 2D group and the group without CXCL12, the addition of CXCL12 promoted GC B cell survival. Figure 9 ).

[0096] Figure 14 The results show the live and dead cell staining and statistical graphs at 0, 7, and 12 days (green for live cells, red for dead cells). The printing process ensured that the loaded cells maintained high viability after printing. The cell viability was 81% ± 6%, with a slight decrease after 12 days (67.67% ± 4.33%).

[0097] Figure 15 The bar chart shows the proportion of Naïve B cells in CD19+ B cells under different culture conditions, demonstrating the difference in the proportion of naïve B cells in CD19+ B cells under "normal culture (2D)" and "organoid culture (3D)" conditions. This indicates that organoid (3D) culture is more effective than normal 2D culture in maintaining / expanding naïve B cells.

[0098] Figure 16 The bar chart shows the proportion of plasmablasts and plasma cells in CD19+ B cells under different culture conditions. During culture, the proportion of plasmablasts significantly decreased, with plasma cells becoming the main terminal cells of differentiation. Organoid culture is more effective than conventional 2D culture in maintaining / expanding plasma cells.

[0099] Figure 17 The bar chart shows the proportions of CD8+ and CD4+ cells in CD3+ T cells under different culture conditions. Both 2D and 3D cultures increased the proportions of CD4+ and CD8+ cells in CD3+ T cells; among them, 3D culture had a more significant effect on the expansion of CD4+ cells, making them the dominant subpopulation in the CD3+ T cell population.

[0100] Example 3: Detection of CAR-T cell cytokine storm using biomimetic lymph node organoids 1. Experimental Design Organoid preparation: The organoids constructed in Example 2 were cultured until day 12 (germinal center maturation stage), with cell viability ≥85%.

[0101] Experimental groups: A blank control group (organoids only + non-targeted T cells) and a CAR-T cell treatment group (organoids + CD19-targeted CAR-T cells) were set up, with 3 replicate wells in each group; 100 μL of CAR-T cell suspension (effect-to-target ratio 1:5) was added to each well of the CAR-T cell treatment group, and an equal amount of non-targeted T cells was added to the blank control group. Both groups were added in batches at 0h, 3 days, and 6 days with an equal amount of tumor target cells.

[0102] 2. Experimental Procedure Co-culture and sampling: The cells were co-cultured at 37℃ and 5% CO2 for 7 days. The supernatant was collected at 6 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days and 9 days for cytokine detection.

[0103] 3. Results Analysis Cytokine concentrations: As shown in Table 1, on day 7, the concentrations of IL-6 (216.55 pg / mL) and TNF-α (116.37 pg / mL) in the CAR-T cell treatment group were significantly higher than those in the blank control group (28.56 pg / mL and 23.05 pg / mL, respectively), while the concentration of IL-2 (23.99 pg / mL) was significantly lower than that in the blank control group (316.25 pg / mL), consistent with the characteristics of a cytokine storm; the concentration of IL-10 (1000.98 pg / mL) was lower than that in the blank control group (1723.00 pg / mL), suggesting an imbalance in inflammatory regulation.

[0104] Table 1 Immune cell activation: In the CAR-T cell treatment group, CD45 + CD3 + CD69 + T cell activation rate reached 65%, CD45 + CD11b + The macrophage activation rate reached 58%, which was significantly higher than that of the blank control group (T cell activation rate 12%, macrophage activation rate 8%), demonstrating that CAR-T cells can activate innate immune cells in organoids and amplify the inflammatory response.

[0105] Conclusion: This organoid can mimic the in vivo immune microenvironment and accurately capture the dynamic changes of CAR-T cell-induced cytokine storm, providing an in vitro model for optimizing CAR-T therapy dosage and screening for combined anti-inflammatory drugs.

[0106] Example 4: Application of biomimetic lymph node organoids in vaccine immunogenicity evaluation 1. Experimental Design Vaccine and grouping: Inactivated influenza A virus solution (H1N1 subtype) was selected as the test antigen. Vaccine group (organoid + 3 μL inactivated influenza A virus solution) and control group (organoid only) were set up, with 3 replicate wells in each group; the organoid constructed in Example 2 was used for evaluation.

[0107] 2. Experimental Procedure Stimulation and culture: The vaccine antigen was added to the organoid culture system and cultured at 37°C and 5% CO2 for 2 days; the control group was added with only an equal volume of culture medium. The antigen-containing culture medium was removed and replaced with normal culture medium, and cultured for another 5 days.

[0108] 3. Results Analysis Cell morphology and aggregation state: Figure 12 This is a bright-field comparison image of biomimetic lymph node organoids (3D group) and 2D cultured cells (2D group) under stimulation with and without vaccine (inactivated influenza A virus solution). The image visually demonstrates the immune response advantages of the 3D biomimetic structure in terms of cell morphology and aggregation state. Specific characteristics are as follows: In the non-vaccine stimulation group (left column): the cells in the 2D group were distributed in a single layer and had a uniform morphology (mostly round or short spindle-shaped) with no obvious cell aggregation; the organoids in the 3D group had a spherical three-dimensional structure with tightly aggregated cells and clear boundaries. Density stratification due to differences in cell type could be observed inside (corresponding to the spatial partitioning of the T and B regions), which reflects the ability of the 3D structure to simulate the cellular microenvironment. Vaccine-stimulated group (right column): Although a small amount of cells in the 2D group showed aggregation, they were still mainly in a dispersed state, and there was no obvious change in cell morphology; the organoid volume in the 3D group increased significantly, the internal cell aggregation degree further increased, and local transparent areas could be observed under bright field (corresponding to the bright area of ​​the germinal center, because the cytoplasm of B cells is transparent after maturation), suggesting that functional aggregation and differentiation of cells in the 3D structure occurred under vaccine stimulation. Furthermore, comparing the survival status of the two groups of cells: 7 days after vaccine stimulation, the 2D group showed obvious cell debris (dark spots) and decreased cell density; the 3D group's organoid structure remained intact without obvious debris, demonstrating that the 3D biomimetic microenvironment can enhance cell tolerance to vaccine stimulation and provide structural support for a sustained immune response. This figure morphologically demonstrates the structural and functional advantages of the organoids of this invention compared to the 2D culture system in simulating the in vivo immune response process.

[0109] T cell activation: Flow cytometry showed that CD3 in the vaccine group + CD4 + Cells accounted for 85.03%, CD3 + CD8 + Cells accounted for 11.19% ( Figure 10 CD3 + CD4 + CD69+ The proportion of activated T cells was 28%, significantly higher than that of the control group (10%), proving that the vaccine can activate T cells.

[0110] B-cell differentiation: Flow cytometry analysis showed that the vaccine group had CD19 + CD20 + CD27 + CD38 + Germ center B cells (2.41%), CD19 + CD20 + CD27 + CD38 - Memory B cells (18.08%), CD19 + CD20 - CD27 + CD38 + The ratio of plasmablasts to plasma cells (7.03%) was significantly higher than that in the control group. Figure 11 The B cell differentiation and maturation process is normal.

[0111] Antibody secretion: ELISApot assay showed that the vaccine group of this invention had 200 antibody-secreting cells per 1010 5 The percentage increase in cells relative to the unstimulated group was significantly higher than that in the 2D culture group, thus improving detection sensitivity. Figure 13 The concentration of influenza A-specific IgG in the supernatant detected by ELISA was 1.8 μg / mL, which was significantly higher than that in the control group (0.1 μg / mL), proving that the vaccine can induce organoids to produce a specific humoral immune response.

[0112] Cytokine balance: As shown in Table 2, the concentrations of IL-6 (853.92 pg / mL), TNF-α (43.13 pg / mL), and IL-1β (4479.46 pg / mL) in the vaccine group were higher than those in the control group, but all were within the safety threshold and there was no risk of cytokine storm, demonstrating that the vaccine has good immunogenicity and safety.

[0113] Table 2 In summary, this invention solves the core defects of existing lymphoid organoids through precise spatial structure construction, complete culture process and multi-dimensional functional verification. It can be widely used in immunological research, drug screening and clinical translation, and has important technical value and application prospects.

[0114] 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 biomimetic lymph node organoid, characterized in that, The organoids comprise in three-dimensional space: A core area enriched with B cells; and A T-cell enrichment region that spatially at least partially encloses the core region; Within the core region, light and dark regions with different structures can differentiate in response to external stimuli.

2. The biomimetic lymph node organoid according to claim 1, characterized in that, The core region also includes one or more of lymph node stromal cells, macrophages, and dendritic cells.

3. The biomimetic lymph node organoid according to claim 1 or 2, characterized in that, The T-cell enrichment area also includes one or more of lymph node stromal cells, macrophages, and dendritic cells.

4. The biomimetic lymph node organoid according to any one of claims 1 to 3, characterized in that, The organoids are composed of a biocompatible hydrogel matrix; the hydrogel matrix comprises one or more of HAMA, GelMA, AlgMA, SilMA, collagen, matrix gel, gelatin, and nanoclay.

5. The biomimetic lymph node organoid according to any one of claims 1 to 4, characterized in that, The dark area and the light area are distinguished by differences in B cell distribution density, proliferation activity, and / or expression of cell surface markers; the dark area is rich in Ki-67. + Highly proliferating B cells and / or CXCR4 + B cells, the bright region is rich in CD83 + B cells.

6. A method for constructing biomimetic lymph node organoids, characterized in that, Includes the following steps: (1) Prepare a first bio-ink and a second bio-ink, wherein the first bio-ink contains B lymphocytes and the second bio-ink contains T lymphocytes; (2) Using the first and second bio-inks, an organoid prototype with a spatial structure of T-cell enrichment region enclosing B-cell enrichment region is formed by three-dimensional construction technology; (3) The organoid prototype is solidified to stabilize its structure; (4) Apply external stimulation to the solidified structure to induce the enrichment differentiation of the B cells to form light and dark areas.

7. The construction method according to claim 6, characterized in that, The three-dimensional construction technology is either bio-3D printing technology or microfluidic chip technology.

8. The construction method according to claim 6 or 7, characterized in that, The first and second bio-inks contain biocompatible hydrogel materials; the hydrogel materials are selected from HAMA, GelMA, AlgMA, SilMA, collagen, matrix gel, gelatin, nanoclay, and any combination thereof.

9. The construction method according to any one of claims 6 to 8, characterized in that, The external stimulus is selected from antigenic substances including gradient chemical signals, viruses, and vaccines; preferably, the chemical signal is provided by CXCL12 chemokine. Preferably, the organoid produces antigen-specific antibodies upon antigen stimulation.

10. The application of the biomimetic lymph node organoid according to any one of claims 1 to 5 or the method for constructing the biomimetic lymph node organoid according to any one of claims 6 to 9 in any of the following aspects: (1) In vitro study of lymphoid immune response mechanisms; (2) Development of in vitro humanized antibodies; (3) Evaluate the immunogenicity or safety of the vaccine or biological drug selection; (4) Construct disease-related complex immune microphysiological models.

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