A method for constructing an in-vitro three-dimensional lymph node model based on PRI characteristics

Abstract

The application provides a method for constructing an in-vitro three-dimensional lymph node model based on PRI characteristics, and belongs to the technical field of biomedical engineering and tissue engineering. The method utilizes the super-amphiphobic surface to selectively induce the occurrence of plato-rayleigh instability (PRI) phenomenon, forms cell bead structure, and self-assembles a three-dimensional model simulating the key structure of a lymph node on the hydrogel fiber by injecting different types of cell suspensions in different zones. The model construction process has high repeatability, and parameters such as cell concentration and fiber spacing can be flexibly regulated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and tissue engineering technology, specifically relating to a method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women and a leading cause of cancer death. Axillary lymph node metastasis is the earliest and most common route of metastasis for breast cancer, directly leading to tumor spread and disease progression, and determining tumor stage, treatment options, and patient prognosis. As a core peripheral immune organ, lymph nodes are not only the first stop for tumor cell metastasis but also the core site for T lymphocytes to migrate in a directed manner, perform immune surveillance, and mediate anti-tumor immune responses.

[0003] Under physiological conditions, lymph nodes exhibit a characteristic tandem bean-shaped structure with high internal compartmentalization. The paracortical region, rich in T lymphocytes, is supported by a fibrous network of extracellular matrix secreted by fibroblasts (FRCs). This network surrounds high endothelial venules (HEVs), forming unique perivenous channels (PVCS). After entering the lymph node from the blood via the HEV, T lymphocytes migrate directionally along the fibrous network, forming beaded cell clusters on the fiber surface. This core structure of "fibrous scaffold support - beaded cell distribution" is fundamental for signal communication and immune responses among T lymphocytes. Simultaneously, chemokines CCL19, CCL21, and CXCL12 secreted by FRCs bind to CCR7 / CXCR4 receptors on the surface of T lymphocytes. [1] This creates a chemical gradient that precisely guides T lymphocytes to migrate along the fibrous network. To elucidate the mechanism of lymphocyte migration, and to provide an experimental basis for the development of early breast cancer diagnosis technologies, optimization of immunotherapy, and precise intervention in metastasis, thereby improving patient survival and reducing treatment side effects, it is necessary to construct an in vitro lymph node model.

[0004] However, current mainstream in vitro lymph node model construction techniques have the following insurmountable shortcomings: Microfluidic technology, although it can simulate the fluid dynamics of the lymphatic system (such as interstitial flow and lymphatic circulation) through microchannel design, and accurately recreate the functional areas of lymph nodes by combining three-dimensional cell / tissue culture, still suffers from these limitations. [2] However, it relies on complex processing techniques and high technical barriers, and the equipment is expensive. 3D bioprinting technology uses bio-ink as raw material and prints layer by layer according to a preset lymph node topology (such as FRCs mesh scaffolds, HEV channels, PVCS gaps) using a 3D bioprinter. Although the structure is highly controllable and can achieve precise zone printing, it still offers advantages. [3]However, this technology suffers from low printing resolution and limited cell viability. Hydrogel co-culture technology, which involves seeding FRCs, HUVECs, and T / B cells in natural hydrogels (such as Matrigel or type I collagen) or synthetic hydrogels (such as PEGDA), utilizes the three-dimensional space of the hydrogel to support cell growth. However, it only forms loose, disordered three-dimensional cell aggregates and is the most basic in vitro lymph node simulation technology. [4] Lacking the fibrous scaffold structure and beaded cell distribution characteristic of lymph nodes, random cell mixing prevents the formation of functional zones and fails to simulate the directional migration process of T lymphocytes, resulting in low physiological relevance.

[0005] More importantly, existing technologies cannot solve the core challenge of "how to achieve directional arrangement of fiber scaffolds, uniform size of cell clusters, and precise partitioning of different functional cells at low cost and with low operational barriers."

[0006] [1] Lu Xiangchan, Tan Yi. The relationship between lymph node structure and its structural changes and the pathogenesis of acquired immunodeficiency syndrome. Chinese Journal of Clinical New Medicine. 2010;3(6):601-605.

[0007] [2] German SV, Abalymov AA, Kurochkin MA, et al. Plug-and-Play LymphNode-on-Chip: Secondary Tumor Modeling by the Combination of Cell Spheroid, Collagen Sponge and T-Cells. Int J Mol Sci. 2023;24(4):3183.

[0008] [3] Kang HJ, Lee JH, Jin YX, et al. Therapeutic effects of 3D-bioprinted mesenchymal stem cell-based artificial lymph nodes on lymphedema. Int J Bioprint. 2025;11(3):457-474.

[0009] [4] Morrison AI, Kuipers JE, Roest HP, et al. Functional organotypichuman lymph node model with native immune cells benefits from fibroblasticreticular cell enrichment. Sci Rep. 2025;15:12233. Summary of the Invention

[0010] To address the problems existing in the background technology, the purpose of this invention is to provide a method for constructing an in vitro three-dimensional lymph node model based on the properties of PRI (Prato-Rayleigh instability). This method utilizes a superhydrophobic surface to selectively induce Prato-Rayleigh instability (PRI), forming a beaded cell structure. By injecting different types of cell suspensions into specific areas, a three-dimensional model simulating key lymph node structures self-assembles on hydrogel fibers.

[0011] To achieve the above objectives, the technical solution of the present invention is as follows:

[0012] A method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics includes the following steps:

[0013] Step 1. Prepare a superhydrophobic surface;

[0014] Step 2. Orientation of hydrogel fibers:

[0015] Hydrogel fibers were arranged into a one-dimensional parallel fiber array on a superhydrophobic substrate, and the structural parameters of the fiber array simulated the fiber network characteristics of physiological lymph nodes; then the fiber array was cleaned, dried with nitrogen, and sterilized by ultraviolet irradiation.

[0016] Step 3. Prepare a lymph node model:

[0017] On the surface of a superhydrophobic substrate with a fiber array, different cell suspensions corresponding to different functional areas of the lymph node are injected vertically in sections. During the injection process, the cell suspension forms a cell suspension column, with the injection direction of the cell suspension column perpendicular to the extension direction of the hydrogel fiber. At the moment the cell suspension column contacts the substrate, it spontaneously breaks into uniformly sized cell suspension beads based on the Prato-Rayleigh instability (PRI) of the superhydrophobic surface. Each cell suspension bead attaches to the surface of the adjacent fiber, forming a "cell suspension bead-hydrogel fiber" lymph node bead precursor structure.

[0018] Step 4. Transfer the superdihydrophobic substrate carrying the beaded precursor structure of the lymph node to an incubator with a suitable and stable growth environment and culture it for a period of time. This allows the cell clusters in the cell suspension beads to aggregate into spheres and attach to the hydrogel fibers, forming a beaded three-dimensional lymph node model that simulates the "fiber scaffold-cell cluster" of physiological lymph nodes.

[0019] Furthermore, in step 2, the hydrogel fiber is preferably any biocompatible hydrogel with spinnability, and is preferably a hydrogel fiber obtained by polymerizing polyacrylic acid (PAA) and polyvinyl alcohol (PVA).

[0020] Furthermore, in step 2, the hydrogel fibers need to have a porous network structure with a pore size range of 500 nm to 50 μm, which ensures nutrient exchange and cell activity, and accurately replicates the natural microchannels of the lymph node FRCs mesh scaffold.

[0021] Furthermore, the diameter of the hydrogel fibers is determined by the diameter of the high endothelial venule channels in physiological lymph nodes, and the spacing between adjacent fibers simulates the spacing of the fiber network in the paracortical region of lymph nodes.

[0022] Furthermore, the diameter of the hydrogel fiber and the spacing between adjacent fibers are adjusted by the rotation speed of the rotary motor and the forward speed of the stepper motor of the spinning machine. The faster the rotation speed of the rotary motor, the finer the fiber diameter; the faster the forward speed of the stepper motor, the wider the spacing between adjacent fibers.

[0023] Furthermore, in step 2, the diameter of the parallel hydrogel fiber array is 100-300 μm, and the spacing between adjacent fibers is 5-20 mm.

[0024] Furthermore, the spacing between adjacent cell suspension beads is 0.5-1 cm, which ensures that the cell spheres grow independently and form effective intercellular communication.

[0025] Furthermore, the cell suspension required for constructing the lymph node model in step 3 includes a T lymphocyte suspension, a HUVEC human umbilical vein endothelial cell suspension, and a FRCS fibroblast reticulum cell suspension; among which,

[0026] FRCS fibroblasts construct a paracortical region rich in T lymphocytes, while HUVEC human umbilical vein endothelial cells construct microvenous channels (HEVs) for T lymphocyte migration from the T lymphocyte inlet to the T lymphocyte region (paracortical region). These cells are injected into hydrogel fibers in sections according to actual needs. HUVEC human umbilical vein endothelial cells grow along the hydrogel fibers to form microvenous channels. T lymphocyte suspension and FRCS fibroblast suspension are respectively placed at both ends of the microvenous channels (HEVs) formed by HUVEC human umbilical vein endothelial cells. Chemokines CCL19, CCL21, and CXCL12 secreted by FRCS fibroblasts bind to CCR7 / CXCR4 receptors on the surface of T lymphocytes, inducing T lymphocyte directional migration.

[0027] Furthermore, the preparation process of the cell suspension in step 3:

[0028] The desired cells were subjected to cell proliferation treatment. When the cells adhered to the wall and occupied a certain area of ​​the bottom surface, trypsin was added to the cell culture medium to digest the cells. After a period of time, complete culture medium was added to stop the digestion. The cells were collected in a centrifuge tube, and the supernatant was discarded after centrifugation. Polymer solution and complete culture medium were added to the remaining cell pellet at the bottom to dilute the cell concentration to a certain level. The cells were then pipetted until they were completely dispersed to obtain a uniformly dispersed cell suspension.

[0029] The polymer solution is preferably a biocompatible polymer system with low viscosity (1-500 mPa·s) and filamentation capability to ensure that the cell suspension can be injected into the superhydrophobic surface in the form of a liquid column and then uniformly break apart due to the PRI phenomenon. The viscosity and filamentation capability are to prevent the deformation of the cell suspension column and ensure that the cell suspension column remains columnar and does not splash when injected by the injection pump. It contacts the substrate in columnar shape and then uniformly breaks apart due to the PRI phenomenon. The low viscosity is controlled to satisfy both the occurrence of the PRI phenomenon and the spheroidization resistance that the cells need to overcome.

[0030] Furthermore, the cell concentration in the T lymphocyte suspension and the FRCs fibroblast reticulocyte suspension mentioned in step 3 is 1×10⁻⁶. 6 -5×10 6 cells / mL, cell concentration of HUVEC human umbilical vein endothelial cell suspension 2×10 6 -8×10 6 cells / mL.

[0031] Furthermore, in step 3, the injection rate of the cell suspension is 10–30 mL / min, and the needle specification is 19–27 G.

[0032] Furthermore, the volume of the spontaneously broken cell suspension beads in step 3 is 10-50 µL.

[0033] The mechanism of this invention is as follows:

[0034] The paracortical area of ​​physiological lymph nodes (the core region of T cell immune response) is characterized by a structure where "fibroblasts (FRCs) form a framework, with T cells forming discrete beaded clusters along the fibers." Fibroblasts (FRCs) secrete extracellular matrix to form an interwoven three-dimensional network, simultaneously surrounding high endothelial venules (HEVs) and creating PVCS (channels for T lymphocyte migration). After entering the lymph node from the HEVs, T lymphocytes do not diffuse randomly but migrate and aggregate along the fibers, forming discrete clusters that are distributed in a "beaded" pattern.

[0035] Prato-Rayleigh instability (PRI) refers to the physical phenomenon where, under the influence of surface tension, a liquid column or droplet tends to reduce its surface energy, thus forming smaller and more uniform droplets. PRI originates from the competition between interfacial tension and inertial / viscous forces. Interfacial tension tends to disrupt the fluid jet, minimizing the surface area. Conversely, inertial / viscous forces tend to maintain the original shape of the fluid jet. This invention utilizes the physical property of superhydrophobic surfaces to induce the PRI phenomenon. By controlling the viscosity and surface tension of the cell suspension, due to the ultra-low adhesion force between the cell suspension and the superhydrophobic surface, the cell suspension beads adhere more easily to the hydrogel fibers. After the cell suspension column ruptures on the superhydrophobic surface, the cell suspension beads spontaneously adhere to the hydrogel fibers, forming a high-throughput array of cell beads.

[0036] A paracortical region rich in T lymphocytes was constructed using FRCs (fibroblast reticulocytes); a microvenous channel (HEV) was constructed using HUVECs (human umbilical vein endothelial cells) to facilitate T lymphocyte migration from the T lymphocyte inlet to the T lymphocyte region (paracortical region). These cells were then injected into hydrogel fibers in sections according to specific needs. The HUVECs grew along the hydrogel fibers to form the microvenous channel. T lymphocyte suspensions and FRCs suspensions were positioned at opposite ends of the HEV formed by the HUVECs. Chemokines CCL19, CCL21, and CXCL12 secreted by the FRCs bound to CCR7 / CXCR4 receptors on the surface of T lymphocytes, inducing directional migration of T lymphocytes. This targeted assembly method can precisely partition key structural regions of the lymph node, avoiding the chaotic cell distribution problems of traditional random three-dimensional culture, and maximally reproducing the directional migration path of T lymphocytes within the lymph node, providing an ideal in vitro model for studying the migration mechanism of T lymphocytes. In addition, the porous network structure of hydrogel fibers not only ensures nutrient exchange and cell activity during model culture, but also accurately replicates the natural microchannels of the lymph node FRCs mesh scaffold, making the migration behavior of T lymphocytes closer to the physiological state, which facilitates the precise exploration of their directional migration mechanism.

[0037] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0038] (1) By utilizing the low adhesion properties of the superhydrophobic substrate and the PRI phenomenon, precise partitioning and beading of cell suspensions can be achieved. The model construction process is highly reproducible, and parameters such as cell concentration and fiber spacing can be flexibly adjusted. It can be used for the study of lymph node metastasis mechanism of tumors such as breast cancer, screening of immunotherapy drugs (such as chemokine inhibitors and immune checkpoint inhibitors), development of early tumor diagnosis technology, and at the same time, it provides a general technical solution for the construction of peripheral immune organ models.

[0039] (2) In the model, FRCs cells can stably secrete chemokines and HUVEC cells can form lymphocyte transfer microvenous channels (HEVs), which can realistically reproduce the T lymphocyte directional migration process mediated by CCR7-CCL19 / CCL21, providing a physiologically relevant in vitro platform for elucidating the T lymphocyte migration mechanism.

[0040] (3) Based on fluorescently labeled T lymphocytes and transparent hydrogel materials, the migration trajectory of T lymphocytes can be tracked in real time by confocal microscopy, realizing dynamic monitoring of cell behavior. Attached Figure Description

[0041] Figure 1 This is a SEM image of the superhydrophobic surface.

[0042] Figure 2 This is a graph showing the long-term contact angle measurement results on a superhydrophobic surface.

[0043] Figure 3 This is a schematic diagram illustrating the process by which a cell suspension column on a superhydrophobic surface breaks into cell suspension beads due to the PRI phenomenon.

[0044] Figure 4 This is a schematic diagram illustrating the construction process of the cell suspension bead-hydrogel fiber bead structure.

[0045] Figure 5 The image shows the results of the biocompatibility test of hydrogel fibers.

[0046] Figure 6 Optical image of the lymph node bead precursor structure of the "cell suspension bead-hydrogel fiber" constructed in this invention. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0048] Prato-Rayleigh instability (PRI) is a fundamental phenomenon in fluid mechanics, where a cylindrical liquid column spontaneously breaks into a series of uniformly sized droplets under surface tension. Utilizing the PRI principle on superhydrophobic surfaces to fabricate three-dimensional lymph node structures allows a cylindrical liquid column perpendicular to a fiber array to spontaneously break along the fiber axis into a series of uniformly sized spherical droplets under the drive of surface tension. The resulting structure closely matches the lymph node model of "oriented fibers – beaded cell clusters arranged along the fibers." Based on this technological advantage, this invention proposes a novel method for constructing lymph node models to overcome the shortcomings of existing technologies.

[0049] Example 1

[0050] A method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics includes the following steps:

[0051] Step 1: Preparation of superhydrophobic and aphtholyzed surfaces:

[0052] A uniform layer of black candle soot was generated on a glass slide by holding and moving it in the flame of a burning candle for 1 min. The soot-coated slide was then placed in a desiccator with two other slides containing 3 mL of tetraethyl orthosilicate and ammonia solution, respectively. A Stöber reaction was initiated by vacuum desiccation and continued for 24 hours, resulting in the chemical vapor deposition of silica onto the candle soot surface of the glass slide. The sample was then annealed at 550 °C for 2 hours to remove the candle soot. Finally, the calcined sample and 300 μL of 1H,1H,2H,2H-perfluorodecyltrichlorosilane were placed in a vacuum desiccator for 2 hours at room temperature.

[0053] Step 2: Orienting hydrogel fibers:

[0054] Using a Janus spinning machine, biomimetic spider silk hydrogel fibers (obtained by polymerization of polyacrylic acid and polyvinyl alcohol) were spun into a one-dimensional parallel fiber array with a diameter of 100 µm and an adjacent spacing of 6 mm on a superhydrophobic substrate; then the fibers were washed three times with deionized water and physiological saline, dried with nitrogen, and sterilized by ultraviolet irradiation for 40 minutes.

[0055] Step 3: Prepare a lymph node model:

[0056] The superhydrophobic substrate with hydrogel fibers was rotated 90° around a central axis perpendicular to the substrate plane, so that the extension direction of the hydrogel fibers was perpendicular to the subsequent injection direction. Using a moving platform coupled with a microinjection pump, with an injection rate set at 15 mL / min and a 22 G needle, T lymphocyte suspension, HUVEC human umbilical vein endothelial cell suspension, and FRCS fibroblast network cell suspension were sequentially injected onto the surface of the superhydrophobic substrate with the fiber array, along a direction perpendicular to the hydrogel fibers. During injection, the cell suspension formed a cell suspension column, which broke upon contact with the substrate into fragments of approximately 20 mL in volume. µL cell suspension beads, each cell suspension bead specifically attaching to the surface of adjacent fibers to form a "cell suspension bead-hydrogel fiber" precursor structure resembling a beaded lymph node. The preparation process for the three different cell suspensions was as follows: The three types of cells were subjected to cell proliferation treatment. When the cells adhered and occupied 80% of the bottom surface area, trypsin was added to the cell culture medium to digest the cells. After 1 minute, complete culture medium was added to stop the digestion. The cells were collected in centrifuge tubes, and the supernatant was discarded after centrifugation. Polymer solution and complete culture medium were added to the remaining cell pellet at the bottom to dilute the cell concentration to a certain level. The cells were then pipetted until completely dispersed to obtain a uniformly dispersed cell suspension. The cell concentrations of the T lymphocyte suspension and the FRCS fibroblast network cell suspension were 2 × 10⁻⁶ cells / mL. 6 The cell concentration of the HUVEC human umbilical vein endothelial cell suspension was 4 × 10⁻⁶ cells / mL. 6 cells / mL; any commercially available complete culture medium can be used, such as Gibco's DMEM medium.

[0057] Step 4. Transfer the superdihydrophobic substrate carrying the beaded precursor structure of the lymph node to an incubator with a suitable and stable growth environment and culture it for a period of time. This allows the cell clusters in the cell suspension beads to aggregate into spheres and attach to the hydrogel fibers, forming a beaded three-dimensional lymph node model that simulates the "fiber scaffold-cell cluster" of physiological lymph nodes.

[0058] Example 2

[0059] A method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics includes the following steps:

[0060] Step 1: Preparation of superhydrophobic and aphtholyzed surfaces:

[0061] A uniform layer of black candle soot was generated on a glass slide by holding and moving it in the flame of a burning candle for 1.5 min. The soot-coated slide was then placed in a desiccator with two other slides containing 4 mL of tetraethyl orthosilicate and ammonia solution, respectively. A Stöber reaction was initiated by vacuum desiccation and continued for 24 hours, resulting in the chemical vapor deposition of silica onto the candle soot surface of the glass slide. The sample was then annealed at 550 °C for 2 hours to remove the candle soot. Finally, the calcined sample and 400 μL of 1H,1H,2H,2H-perfluorodecyltrichlorosilane were placed in a vacuum desiccator for 2 hours at room temperature.

[0062] Step 2: Orienting hydrogel fibers:

[0063] Using a Janus spinning machine, biomimetic spider silk hydrogel fibers were spun into a one-dimensional parallel fiber array with a diameter of 150 µm and an adjacent spacing of 10 mm on a superhydrophobic substrate. The fibers were then washed three times with deionized water and physiological saline, dried with nitrogen, and sterilized by ultraviolet irradiation for more than 30 minutes.

[0064] Step 3: Prepare a lymph node model:

[0065] The superhydrophobic substrate with hydrogel fibers was rotated 90° around a central axis perpendicular to the substrate plane, so that the extension direction of the hydrogel fibers was perpendicular to the subsequent injection direction. Using a moving platform coupled with a microinjection pump, the injection rate was set to 10 mL / min, and the needle size was 24 G. Along the direction perpendicular to the hydrogel fibers, T lymphocyte suspension, HUVEC human umbilical vein endothelial cell suspension, and FRCS fibroblast network cell suspension were injected in sections onto the surface of the superhydrophobic substrate with fiber arrays. During the injection, the cell suspension formed cell suspension columns. The cell suspension columns broke into cell suspension beads with a volume of about 10 µL upon contact with the substrate. Each cell suspension bead specifically attached to the surface of the adjacent fiber to form a "cell suspension bead-hydrogel fiber" lymph node beaded precursor structure.

[0066] Step 4. Transfer the superdihydrophobic substrate carrying the beaded precursor structure of the lymph node to an incubator with a suitable and stable growth environment and culture it for a period of time. This allows the cell clusters in the cell suspension beads to aggregate into spheres and attach to the hydrogel fibers, forming a beaded three-dimensional lymph node model that simulates the "fiber scaffold-cell cluster" of physiological lymph nodes.

[0067] Example 3

[0068] A method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics includes the following steps:

[0069] Step 1: Preparation of superhydrophobic and aphtholyzed surfaces:

[0070] A uniform layer of black candle soot was generated on a glass slide by holding and moving it in the flame of a burning candle for 2 minutes. The soot-coated slide was then placed in a desiccator with two other slides containing 5 mL of tetraethyl orthosilicate and ammonia solution, respectively. A Stöber reaction was initiated by vacuum desiccation and continued for 24 hours, resulting in the chemical vapor deposition of silica onto the candle soot surface of the glass slide. The sample was then annealed at 550 °C for 2 hours to remove the candle soot. Finally, the calcined sample and 500 μL of 1H,1H,2H,2H-perfluorodecyltrichlorosilane were placed in a vacuum desiccator for 2 hours at room temperature.

[0071] Step 2: Orienting hydrogel fibers:

[0072] Using a Janus spinning machine, biomimetic spider silk hydrogel fibers were spun into a one-dimensional parallel fiber array with a diameter of 300 µm and an adjacent spacing of 10 mm on a superhydrophobic substrate. The fibers were then washed three times with deionized water and physiological saline, dried with nitrogen, and sterilized by ultraviolet irradiation for more than 30 minutes.

[0073] Step 3: Prepare a lymph node model:

[0074] The superhydrophobic substrate with hydrogel fibers was rotated 90° around a central axis perpendicular to the substrate plane, so that the extension direction of the hydrogel fibers was perpendicular to the subsequent injection direction. Using a moving platform coupled with a microinjection pump, the injection rate was set to 15 mL / min, and the needle size was 20 G. T lymphocyte suspension, HUVEC human umbilical vein endothelial cell suspension, and FRCS fibroblast network cell suspension were injected in sections onto the surface of the superhydrophobic substrate with fiber arrays along the direction perpendicular to the hydrogel fibers. During the injection, the cell suspension formed cell suspension columns. The cell suspension columns broke into cell suspension beads with a volume of about 30 µL upon contact with the substrate. Each cell suspension bead specifically attached to the surface of the adjacent fiber to form a "cell suspension bead-hydrogel fiber" lymph node beaded precursor structure.

[0075] Step 4. Transfer the superdihydrophobic substrate carrying the beaded precursor structure of the lymph node to an incubator with a suitable and stable growth environment and culture it for a period of time. This allows the cell clusters in the cell suspension beads to aggregate into spheres and attach to the hydrogel fibers, forming a beaded three-dimensional lymph node model that simulates the "fiber scaffold-cell cluster" of physiological lymph nodes.

[0076] Figure 1 This is a SEM image of the superhydrophobic surface. Figure 1 (a) and Figure 1 (b) shows the morphology of the obtained soot template; Figure 1 (c) and Figure 1(d) shows the morphology of silica deposited by chemical vapor deposition on the surface of the soot template; Figure 1 (e) and Figure 1 (f) shows the morphology of the obtained superhydrophobic surface. As shown in the figure, the superhydrophobic surface exhibits a cauliflower-like morphology with a micro-nano dual-scale structure. This micro-nano morphology causes the liquid to exhibit a Cassie-Baxter state on its surface, minimizing the solid-liquid contact area.

[0077] Figure 2 This image shows the long-term contact angle measurement results on the superhydrophobic surface. The prepared superhydrophobic substrate was immersed in cell culture medium (e.g., ...). Figure 2 (a) and placed in a 37°C cell culture incubator for several days (e.g. Figure 2 (b)). From Figure 2 (c) and Figure 2 As shown in (d), the superhydrophobic substrate, after five days of soaking, still maintained excellent liquid repellency, and the cell suspension exhibited a high apparent contact angle (>150°) and a low sliding angle (<10°). Figure 2 As shown in (e).

[0078] Figure 3 This diagram illustrates the process of a cell suspension column breaking into cell droplets via the PRI phenomenon on a superhydrophobic surface. The synergistic effect of the micro / nano-scale roughness of the superhydrophobic surface and the perfluorosiloxane-derived chemistry results in an extremely low contact area between the cell suspension column and the substrate, with the solid-liquid interfacial tension value approaching the ideal maximum liquid-gas interfacial tension. This maximizes the growth rate of PRI perturbation and optimizes the cell droplet formation efficiency. As shown in the figure, the cell suspension column can break into uniformly sized cell droplets within 3 seconds.

[0079] Figure 4 This diagram illustrates the construction process of the "cell suspension bead-hydrogel fiber" lymph node bead precursor structure. First, a hydrogel fiber array is spun on a superhydrophobic substrate using a Janus spinneret. The substrate is then rotated 90°, and a moving platform and injection pump are used to inject cell suspensions into the surface of the superhydrophobic substrate with the spun fiber arrays. The cell suspension columns break down into individual cell suspension beads through the PRI phenomenon and spontaneously assemble onto the hydrogel fibers, forming the "cell suspension bead-hydrogel fiber" lymph node bead precursor structure.

[0080] Figure 5The figure shows the results of the biocompatibility test of the hydrogel fibers. The hydrogel fibers were extracted with cell culture medium, and the cells were incubated with the extract for 24 h. The blank control group was incubated with cell culture medium. Cell viability was characterized by fluorescent staining: live cells were labeled with Calcien-AM green fluorescence, and dead cells were labeled with PI red fluorescence. As shown in the figure, the hydrogel fibers used to construct the lymph node-like structure of cell suspension beads-hydrogel fibers exhibit good biocompatibility.

[0081] Figure 6 Optical images of the in vitro three-dimensional lymph node model constructed in this invention. T lymphocyte suspension beads, HUVEC human umbilical vein endothelial cell suspension beads, and FRCS fibroblast network cell suspension beads are precisely partitioned and assembled on hydrogel fibers, providing a physiologically relevant in vitro model for elucidating the migration mechanism of T lymphocytes.

[0082] The above description is merely a specific embodiment of the present invention. Any feature disclosed in this specification may be replaced by other equivalent or similar features unless otherwise specified. All disclosed features, or steps in all methods or processes, may be combined in any way except for mutually exclusive features and / or steps.

Claims

1. A method for constructing an in vitro three-dimensional lymph node model based on PRI characteristics, characterized in that, Includes the following steps: Step 1. Prepare a superhydrophobic surface; Step 2. Orientation of hydrogel fibers: Hydrogel fibers are arranged into a one-dimensional parallel fiber array on a superhydrophobic substrate; the arranged fiber array is cleaned, dried with nitrogen, and then sterilized by ultraviolet irradiation; the hydrogel fibers are hydrogel water fibers obtained by polymerization of polyacrylic acid and polyvinyl alcohol. Step 3. Prepare a lymph node model: On the surface of a superhydrophobic substrate with a fiber array, different cell suspensions corresponding to different functional areas of the lymph node are injected vertically in sections. During the injection process, the cell suspension forms a cell suspension column, with the spray direction of the cell suspension column perpendicular to the extension direction of the hydrogel fiber. At the moment the cell suspension column comes into contact with the superhydrophobic substrate, the Prato-Rayleigh instability PRI based on the superhydrophobic surface spontaneously breaks into uniformly sized cell suspension beads. Each cell suspension bead attaches to the surface of the adjacent hydrogel fiber, forming a "cell suspension bead-hydrogel fiber" lymph node beaded precursor structure. Step 4. Transfer the superhydrophobic substrate carrying the beaded precursor structure of the lymph node to an incubator with a suitable and stable growth environment and culture it for a period of time. Allow the cell clusters in the cell suspension beads to aggregate into spheres and attach to the hydrogel fibers to form a beaded three-dimensional lymph node model that simulates the "fiber scaffold-cell cluster" of physiological lymph nodes.

2. The method as described in claim 1, characterized in that, In step 2, the hydrogel fibers need to have a porous network structure with a pore size ranging from 500 nm to 50 μm; the diameter of the hydrogel fibers is determined by the diameter of the high endothelial venule channel in the physiological lymph node; the spacing between adjacent fibers simulates the spacing of the fiber network in the paracortical area of ​​the lymph node.

3. The method as described in claim 2, characterized in that, In step 2, the diameter of the parallel hydrogel fibers is 100-300 μm, and the spacing between adjacent fibers is 5-20 mm.

4. The method as described in claim 1, characterized in that, In step 3, the spacing between adjacent cell suspension beads with different functions on the same hydrogel fiber is 0.5-1 cm.

5. The method as described in claim 1, characterized in that, The cell suspensions required to construct the lymph node model in step 3 include T lymphocyte suspension, HUVEC human umbilical vein endothelial cell suspension, and FRCs fibroblast reticulocyte suspension.

6. The method as described in claim 1, characterized in that, The preparation process of the cell suspension in step 3: The desired cells were subjected to cell proliferation treatment. When the cells adhered to the wall and occupied a certain area of ​​the bottom surface, trypsin was added to the cell culture medium to digest the cells. After a period of time, complete culture medium was added to stop the digestion. The cells were collected in a centrifuge tube, and the supernatant was discarded after centrifugation. Polymer solution and complete culture medium were added to the remaining cell pellet at the bottom to dilute the cell concentration to a certain level. The cells were then pipetted until they were completely dispersed to obtain a uniformly dispersed cell suspension. The polymer solution has a viscosity of 1-500 mPa·s and is capable of forming lines.

7. The method as described in claim 5, characterized in that, The cell concentration in the T lymphocyte suspension and FRCs fibroblast suspension mentioned in step 3 is 1×10⁻⁶. 6 -5×10 6 cells / mL, cell concentration of HUVEC human umbilical vein endothelial cell suspension 2×10 6 -8×10 6 cells / mL.

8. The method as described in claim 1, characterized in that, In step 3, the cell suspension is injected at a rate of 10–30 mL / min, and the needle size is 19–27 G; the volume of spontaneously broken cell suspension beads is 10–50 µL.

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