Preparation method of nerve and tumor interaction model, nerve and tumor interaction model and application

Abstract

The invention provides a preparation method of a nerve and tumor interaction model, the nerve and tumor interaction model and application, and relates to the technical field of bio-manufacturing, the preparation method of the nerve and tumor interaction model comprises the following steps: simulating extension nerve axons in ganglions in vivo according to the biological characteristics of nerve and tumor interaction, creating a three-dimensional structure corresponding to the 3D culture model of the neurons and the tumors according to the biological behaviors of the neurons and the tumor tissues and the spatial position relationship between the neurons and the tumor tissues; adding neuronal cells, tumor cells, tumor microenvironment cells and growth factors required by cell growth into the matrix to obtain the bio-ink; and according to the three-dimensional structure corresponding to the 3D culture model of the neurons and the tumors, simulating the spatial position relationship between the neurons and tumor tissues, and carrying out 3D model printing by using the bio-ink to obtain a nerve and tumor interaction model.

Description

Technical Field

[0001] The present invention relates to the field of biomanufacturing technology, and in particular to a preparation method of a nerve-tumor interaction model, a nerve-tumor interaction model and applications thereof. Background Art

[0002] This section is intended to provide a background or context for embodiments of the present invention. No description herein is admitted to be prior art by virtue of its inclusion in this section.

[0003] In recent years, the potential role of the nervous system in the occurrence and development of tumors has gradually attracted the attention of researchers, and an emerging field, tumor neurology, has gradually emerged. However, most current studies on the interaction between nerves and tumors are still based on experimental studies at the 2D cell level or in vivo level in animals (e.g., mice), and there are few in vitro bionic models of nerve-tumor interactions. The main reason is that existing research cannot construct highly bionic in vitro models of nerves and tumors.

[0004] Currently, in vitro construction of tumor organoids primarily focuses on 3D cell culture, without involving biomimetic design of different tissues or cellular spatial structures. Furthermore, the nervous system has not been innovatively incorporated into tumor model construction, thus failing to simulate the influence of nerves on tumors. Meanwhile, research areas or inventions combining 3D printing technology with organoid construction are currently focused on two main areas: the use of 3D printing to create single tumor models; and the use of 3D printing solely to create tumor organoids or the molds required for organoids, without involving cell-laden printing.

[0005] In summary, there is an urgent need for a technical solution that can overcome the above-mentioned defects and construct a highly biomimetic in vitro model of nerves and tumors. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention proposes a preparation method, a nerve-tumor interaction model and its application; the present invention adopts 3D bioprinting to repeatedly obtain an in vitro model with good consistency, construct a highly biomimetic nerve-tumor interaction model, and realize effective recording of the dynamic interaction of the microenvironment.

[0007] In a first aspect of an embodiment of the present invention, a method for preparing a nerve-tumor interaction model is proposed, the method comprising:

[0008] Step 1: Based on the biological characteristics of the interaction between nerves and tumors, the three-dimensional structure of the neuron-tumor culture model is created, which mimics the biological behavior of nerve axons extending from ganglia in the body to the tumor site, as well as the spatial relationship between neurons and tumor tissue.

[0009] Step 2: adding neurons, tumor cells, tumor microenvironment cells, and growth factors required for cell growth to a matrix to obtain a bio-ink; wherein the matrix is ​​made of a degradable biocompatible material, and the growth factors are used to support the growth and migration of neurons and tumor cells;

[0010] Step 3: Based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, simulate the spatial position relationship between neurons and tumor tissues, use the bio-ink to print the 3D model, and obtain a nerve-tumor interaction model.

[0011] In a second aspect of an embodiment of the present invention, a nerve-tumor interaction model is proposed, and the nerve-tumor interaction model is prepared by the above method.

[0012] In a third aspect of an embodiment of the present invention, an application of the above-mentioned method and / or the above-mentioned nerve-tumor interaction model in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs or evaluating the therapeutic effect of nerve blocking on tumors is proposed.

[0013] The present invention provides a method for preparing a nerve-tumor interaction model, a nerve-tumor interaction model, and the use of this method and / or the nerve-tumor interaction model in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs, or evaluating the therapeutic effect of nerve blockade on tumors. The main process of model preparation is as follows: based on the biological characteristics of nerve-tumor interaction, the biological behavior of nerve axons extending from ganglia in the body to the location of tumor occurrence, and the spatial positional relationship between neurons and tumor tissue, a three-dimensional structure corresponding to a 3D culture model of neurons and tumors is created; neurons, tumor cells, tumor microenvironment cells, and growth factors required for cell growth are added to the matrix to obtain a bio-ink; wherein the matrix is ​​made of a degradable biocompatible material, and the growth factors are used to support the growth and migration of neurons and tumor cells; based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, the spatial positional relationship between neurons and tumor tissue is simulated, and a 3D model is printed using the bio-ink to obtain a nerve-tumor interaction model. The model constructed by the present invention through biological 3D printing technology can accurately simulate the spatial position relationship and microenvironment of the interaction between nerves and tumors, providing an experimental platform closer to the physiological state for studying the mechanism of tumor invasion; the model prepared by the present invention has high spatial resolution, can achieve the precise distribution of nerve fibers and tumor tissues, and facilitates the observation of dynamic interactions between cells; based on this model, cell behavior can be monitored in real time, providing an important tool for screening anti-tumor drugs and studying treatment strategies; the model preparation method of the present invention is simple, efficient, and highly repeatable, and is suitable for large-scale production and preclinical research. Through the present invention, it can replace 2D cell level and in vivo animal experiments, with high efficiency and low cost, providing strong technical support for the study of nerve-tumor interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0015] Figure 1 1 is a flow chart of a method for preparing a nerve-tumor interaction model according to an embodiment of the present invention.

[0016] Figure 2 FIG2 is a flow chart of a method for preparing a nerve-tumor interaction model according to another embodiment of the present invention.

[0017] Figure 3 It is a schematic diagram of a bionic model of a specific embodiment of the present invention.

[0018] Figure 4 It is a schematic flow chart of a model preparation method according to a specific embodiment of the present invention.

[0019] Figure 5 Schematic diagram of the construction and identification of organoids derived from pancreatic cancer clinical samples according to a specific embodiment of the present invention.

[0020] Figure 6 This is a diagram of a 4x imaging model of nerve-tumor interaction using biological 3D printing according to a specific embodiment of the present invention.

[0021] Figure 7 This is a 10x imaging diagram of a nerve-tumor interaction model of biological 3D printing according to a specific embodiment of the present invention.

[0022] Figure 8 This is a bright field image of a 3D bio-printed nerve (GRG)-tumor interaction model according to a specific embodiment of the present invention.

[0023] Figure 9 This is a bright field image of a 3D bio-printed nerve (SH-SY5Y) tumor interaction model according to a specific embodiment of the present invention.

[0024] Figure 10 3D printing is a flow chart of a method for preparing biological ink for constructing a nerve-tumor interaction model according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0025] The principles and spirit of the present invention will be described below with reference to several exemplary embodiments. It should be understood that these embodiments are provided solely to enable those skilled in the art to better understand and implement the present invention, and are not intended to limit the scope of the present invention in any way. Rather, these embodiments are provided to make this disclosure more thorough and complete, and to fully convey the scope of the present disclosure to those skilled in the art.

[0026] Those skilled in the art will appreciate that the embodiments of the present invention may be implemented as a system, apparatus, device, method, or computer program product. Therefore, the present disclosure may be implemented in the following forms: entirely in hardware, entirely in software (including firmware, resident software, microcode, etc.), or in a combination of hardware and software.

[0027] According to an embodiment of the present invention, a preparation method of a nerve-tumor interaction model, a nerve-tumor interaction model and its application are proposed, which relate to the field of biomanufacturing technology. The various cells in the model of the present invention are all constructed into an in vitro 3D model of nerves and an in vitro 3D model of tumors by means of biomanufacturing (biological 3D printing technology), and the spatial position relationship between nerves and tumors in vivo is simulated to obtain a biomimetic nerve-tumor interaction model. The model contains multiple types of cells: neuronal cells (for example, SH-SY5Y cell line, mouse dorsal root ganglion cells or iPSC-induced differentiated neurons), tumor cells (for example, tumor cell lines or tumor cells derived from clinical samples) and their microenvironment cells. This model can be used to study the mechanism of nerve-tumor interaction, screen anti-tumor drugs and evaluate the effect of nerve blocking on tumor treatment.

[0028] The principles and spirit of the present invention are explained in detail below with reference to several representative embodiments of the present invention.

[0029] Figure 1 FIG. 1 is a flow chart of a method for preparing a nerve-tumor interaction model according to an embodiment of the present invention. Figure 1 As shown, the method includes:

[0030] Step 1: Based on the biological characteristics of the interaction between nerves and tumors, the three-dimensional structure corresponding to the 3D culture model of neurons and tumors is created by imitating the biological behavior of nerve axons extending from ganglia in the body to the location where tumors occur, as well as the spatial positional relationship between neurons and tumor tissues.

[0031] The present invention designs a three-dimensional structure with a bionic spatial position relationship by imitating the biological behavior of the interaction between nerves and tumors in the body, providing a model blueprint for subsequent 3D printing.

[0032] Step 2: Adding neurons, tumor cells, tumor microenvironment cells, and growth factors required for cell growth to a matrix to produce a bio-ink. The matrix is ​​made of a degradable, biocompatible material, and the growth factors are used to support the growth and migration of neurons and tumor cells.

[0033] The present invention mixes cells with biomaterials to form printable functional ink, which supports cell survival, growth and spatial structure maintenance.

[0034] Step 3: Based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, simulate the spatial position relationship between neurons and tumor tissues, use the bio-ink to print the 3D model, and obtain a nerve-tumor interaction model.

[0035] The present invention is based on three-dimensional structural design and uses biological 3D printing technology to accurately locate cell distribution and form a solid model with biomimetic spatial relationships.

[0036] refer to Figure 2 In another embodiment of the present invention, the method further comprises:

[0037] Step 4: placing the nerve-tumor interaction model in a simulated in vivo environment, culturing it with a culture medium, and recording the dynamic process of the nerve-tumor interaction.

[0038] The present invention simulates the in vivo microenvironment culture model and monitors the dynamic interaction process between nerves and tumor cells in real time, providing data support for mechanism research.

[0039] In order to explain the preparation method of the above-mentioned nerve-tumor interaction model more clearly, each step is described in detail below.

[0040] In one embodiment, for step 1, based on the biological characteristics of the interaction between nerves and tumors, the biological behavior of extending nerve axons in ganglia in the body and extending to the location where the tumor occurs, as well as the spatial positional relationship between neurons and tumor tissues, is simulated to create a three-dimensional structure corresponding to a 3D culture model of neurons and tumors.

[0041] Specifically, for biological characteristics, the researchers simulated the behavior of in vivo ganglia (such as dorsal root ganglia) extending axons into tumor tissue, recreating the anatomical characteristics of nerve fibers invading the tumor microenvironment. Reference was made to the distribution patterns of nerve fibers in clinical tumor tissue (such as the spatial proximity of nerves and tumor cells in neuroinvasive tumors such as pancreatic cancer and glioma).

[0042] When designing three-dimensional structures, it mainly includes neuron models and tumor models.

[0043] Neuron model: Construct neural modules of a set density (high density) and place them around the tumor model to simulate the axons of neurons in the spinal cord in the body extending to the vicinity of the tumor organ, wrapping around or invading the tumor (such as radiating from the "ganglion core" to the surrounding area).

[0044] Tumor model: An organoid structure containing tumor cells and microenvironment cells (e.g., fibroblasts, immune cells, etc.) is constructed, located in the middle of multiple neural modules, to simulate the organ tissue where tumors occur.

[0045] Spatial relationship between the neuron model and the tumor model: Define the relative position of the nerves and the tumor (for example, the nerve model is located around the tumor model and extends toward its center, or is interspersed within the tumor tissue) to ensure that the nerve axons can directly contact the tumor cells after printing.

[0046] Regarding design tools and methods, biological modeling software (e.g., Mimics, 3DSlicer) can be used to construct digital models based on clinical images (MRI or tissue sections) or biological data (neuronal axon growth trajectories), thereby obtaining the corresponding three-dimensional structures of 3D culture models of neurons and tumors. In combination with in vivo experimental data (e.g., nerve fiber distribution density in mouse tumor models), three-dimensional structural parameters (such as nerve bundle diameter and tumor organoid size) can be optimized.

[0047] In one embodiment, for step 2, neuronal cells, tumor cells, tumor microenvironment cells, and growth factors required for cell growth are added to a matrix to obtain a bio-ink, wherein the matrix is ​​made of a degradable biocompatible material, and the growth factors are used to support the growth and migration of neuronal cells and tumor cells. The specific method includes:

[0048] For 3D tumor models, the excised primary tumor tissue is digested into single cells. The digested single cells and growth factors required for cell growth are added to the matrix, and the mixture is mixed to obtain a bio-ink containing tumor cells and tumor microenvironment cells.

[0049] For the 3D model of neurons, extracted mouse or rat neuronal cells, neural cell lines, or iPSC cells (induced pluripotent stem cells, iPS cells) induced differentiated neuronal cells are digested into single cells. The digested single cells and growth factors required for cell growth are added to the matrix, and the mixture is mixed to obtain a bio-ink containing neurons.

[0050] Specifically, the matrix is ​​a biomaterial prepared by mixing natural hydrogel (eg, Matrigel) and synthetic hydrogel (eg, GelMA, porous hydrogel) in different proportions.

[0051] The concentration of natural hydrogel is 5-20%, and the mass concentration of synthetic hydrogel is 5-15%.

[0052] Synthetic hydrogels (e.g., GelMA) can provide mechanical support and cell adhesion sites, and the pore size (micrometer level) allows nutrient diffusion and nerve axon penetration.

[0053] Natural hydrogels (e.g., Matrigel) can mimic the extracellular matrix (ECM), promote tumor cell sphering and neuronal axon extension, and enhance ink viscosity to maintain the stability of printed structures.

[0054] For growth factors required for cell growth, NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), FGF (fibroblast growth factor), etc. can be added to support neuron survival, tumor cell proliferation and intercellular signaling.

[0055] During the cell processing and mixing process, for tumor cell components, primary tumor tissues (such as pancreatic cancer and glioma tissues) are treated with digestive enzymes (pancreatin and collagenase) to form a single-cell suspension, which contains tumor cells and natural microenvironment cells (such as tumor-associated fibroblasts and macrophages); tumor cell lines (such as SH-SY5Y neuroblastoma cells and A549 lung cancer cells) can also be used to mix with microenvironment cells.

[0056] For neuronal components, mouse or rat dorsal root ganglion (DRG) cells, or iPSC-induced differentiated neurons and SH-SY5Y neuron-like cells were extracted and digested into single cells.

[0057] When mixing, cell density control needs to be considered. For example, the concentration of tumor cells and neuronal cells is 1×10 6 -5×10 6 cells / mL, to avoid excessively high density causing printing blockage or excessively low density affecting structural strength.

[0058] Low-temperature (0-4°C) pre-curing is performed first, and then light curing is performed after printing is completed; low-temperature operation (0-4°C) can prevent the natural hydrogel (Matrigel) from solidifying prematurely, ensuring that the cells are evenly dispersed and their activity is not damaged.

[0059] In one embodiment, for step 3, based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, the spatial positional relationship between neurons and tumor tissue is simulated, and the 3D model is printed using the bio-ink to obtain a nerve-tumor interaction model. The specific method includes:

[0060] For a 3D model of a tumor, a bio-ink containing tumor cells and tumor microenvironment cells is used for 3D model printing. During the printing process, the model is pre-cured at a first temperature and then photocured by ultraviolet irradiation after printing. The 3D model is then cultured in situ using a growth factor-containing culture medium to generate an in vitro 3D model of the tumor.

[0061] For a 3D model of a neuron, a bio-ink containing the neuron is used for 3D model printing; during the printing process, pre-curing is performed at a first temperature environment, and after printing is completed, light curing is performed by ultraviolet irradiation; the 3D model is cultured in situ using a culture medium containing growth factors to generate an in vitro 3D model of the neuron;

[0062] When printing the two 3D models, the 3D models are printed according to the three-dimensional structures corresponding to the 3D culture models of neurons and tumors, so as to obtain a bionic model of the interaction between nerves and tumors in the spatial position relationship within the body.

[0063] Specifically, the temperature range of the first temperature environment is 0-4°C.

[0064] In actual application scenarios, the printing method can be extrusion printing or suspension printing.

[0065] Extrusion printing is suitable for high-viscosity biological inks. The ink is squeezed out of the nozzle through air pressure or mechanical pressure and then stacked layer by layer to form a biomaterial.

[0066] Suspension printing uses a support bath (such as agarose gel) to fix the ink structure to avoid gravity deformation and is suitable for complex three-dimensional structures.

[0067] Printing precision is at the micron level (50-200μm), ensuring the restoration of the fine structure of nerve fibers and the tumor microenvironment. The nozzle diameter can be selected based on cell size and structural accuracy (for example, a 25G nozzle corresponds to a 75μm aperture).

[0068] During the printing process, low-temperature pre-curing is first performed, and after printing is completed, light curing is performed by ultraviolet irradiation. Pre-curing is to maintain a low temperature (0-4°C) to initially solidify the natural hydrogel (Matrigel) and maintain the shape stability of the printed lines. Light curing is to use ultraviolet light (365nm, 5-10mW / cm 2 ) for 1-5 minutes to trigger the photocrosslinking reaction of the synthetic hydrogel (GelMA) to form a high-strength hydrogel scaffold.

[0069] In one embodiment, layered printing can be employed, first printing the neural model (e.g., peripheral nerve bundles) and then the tumor model (filling the gaps between or around the neural structure), ensuring precise spatial alignment of the two. The printed model can be placed in a conventional culture dish and observed under a microscope. Scanning electron microscopy (SEM) or confocal microscopy can be used to observe the orientation of the printed nerve fibers and the integrity of the tumor organoids.

[0070] It should be noted that although the operations of the method of the present invention are described in a specific order in the above embodiments and drawings, this does not require or imply that these operations must be performed in this specific order, or that all illustrated operations must be performed to achieve the desired results. Additionally or alternatively, certain steps may be omitted, multiple steps may be combined into one step, and / or one step may be broken down into multiple steps.

[0071] In one embodiment, the present invention further proposes a nerve-tumor interaction model, which is prepared by the above-mentioned preparation method.

[0072] In one embodiment, the present invention also proposes an application based on the above-mentioned preparation method and / or nerve-tumor interaction model in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs or evaluating the therapeutic effect of blocking nerves on tumors.

[0073] The preparation method of the nerve-tumor interaction model, the nerve-tumor interaction model and applications of the present invention are described below with reference to specific embodiments. Specific embodiment one:

[0075] Using extrusion-type biological 3D printing, an in vitro nerve-tumor interaction model was constructed. Figure 3 As shown, reference Figure 4 , the processing process is:

[0076] Step 101: Isolate primary tumor tissue single cells and primary rat dorsal root ganglion single cells.

[0077] Tumor tissue was minced, digested in a water bath with digestive fluid for 10 to 40 minutes until the tumor tissue was completely digested into a single-cell mixture, including tumor cells, tumor fibroblasts, and immune cells. At the same time, rat dorsal root ganglia were obtained, digested in a water bath with digestive fluid for 15-20 minutes to obtain a rat dorsal root ganglion cell suspension.

[0078] In step 102 , cells are mixed with synthetic hydrogel (GelMA) and natural hydrogel (Matrigel) to prepare bio-ink.

[0079] The single cell suspension described in step 101 is resuspended with the GelMA+Matrigel mixture prepared in advance in proportion and mixed evenly.

[0080] Step 103: biological 3D printing (extrusion printing or suspension printing); cross-linking and curing (low-temperature curing, ultraviolet cross-linking), in vitro culture; dynamic monitoring, and real-time microscopic imaging observation.

[0081] The cell-laden bio-ink obtained in step 102 is injected into a bio-3D printer (injected into the print head of an extrusion printer) and printed according to a preset program. The cells are cultured in vitro, dynamically monitored, and observed using real-time microscopic imaging. Specific embodiment two:

[0083] Using suspended biological 3D printing method, an in vitro nerve-tumor interaction model is constructed. The bionic model is as follows Figure 3 As shown, reference Figure 4 , the processing process is:

[0084] Step 101: Isolate primary tumor tissue single cells and primary rat dorsal root ganglion single cells.

[0085] Tumor tissue was minced, digested in a water bath with digestive fluid for 10 to 40 minutes until the tumor tissue was completely digested into a single-cell mixture, including tumor cells, tumor fibroblasts, and immune cells. At the same time, rat dorsal root ganglia were obtained, digested in a water bath with digestive fluid for 15-20 minutes to obtain a rat dorsal root ganglion cell suspension.

[0086] In step 102 , cells are mixed with synthetic hydrogel (GelMA) and natural hydrogel (Matrigel) to prepare bio-ink.

[0087] The single cell suspension described in step 101 is resuspended with the GelMA+Matrigel mixture prepared in advance in proportion and mixed evenly.

[0088] Step 103: biological 3D printing (extrusion printing or suspension printing); cross-linking and curing (low-temperature curing, ultraviolet cross-linking), in vitro culture; dynamic monitoring, and real-time microscopic imaging observation.

[0089] The cell-laden bio-ink obtained in step 102 is injected into the bio-3D printer (injected into the print head of the suspension printer), and printing is performed according to a pre-set program.

[0090] refer to Figures 5 to 9 Schematic diagram for explanation. Figure 5 Schematic diagram of the construction and identification of organoids derived from pancreatic cancer clinical samples according to one embodiment of the present invention. Figure 5As shown in the figure, multimodal imaging of cells is shown, where Phase represents phase contrast imaging (bright field image), which presents the morphology and outline of the cells and is used to observe the overall appearance structure of the cells. CK19 represents the fluorescent marker of cytokeratin 19 (red). CK19 is a marker for epithelial cells or certain tumor cells, used to indicate the distribution and location of specific cell types. DAPI is a fluorescent dye (blue) that binds to DNA, marking the cell nucleus and showing the position and number of cell nuclei. Merge is a merged image that superimposes the red fluorescence of CK19, the blue fluorescence of DAPI and the Phase bright field image to comprehensively display the cell morphology, the distribution of specific markers and the position of the cell nucleus, facilitating a comprehensive analysis of cell characteristics. Figure 5 The dimensions are 100 μm.

[0091] Figure 6 This is a 4x imaging model of nerve-tumor interaction using 3D bioprinting according to a specific embodiment of the present invention. Figure 6 As shown, Phase represents phase contrast imaging (bright field), which is used to demonstrate the morphological outlines of cells or models. SH-SY5Y is a neuroblastoma cell, labeled with blue fluorescence in the figure, representing the neural cell portion. Organoid represents organoids, and the green fluorescence in the figure represents the tumor organoid portion. Figure 6 The spatial distribution of nerves (SH-SY5Y) and tumor organoids was demonstrated by multimodal imaging. Figure 6 The dimensions are 100 μm.

[0092] Figure 7 This is a 10x imaging model of the interaction between nerves and tumors using 3D bioprinting according to a specific embodiment of the present invention. Figure 7 As shown, high-magnification (10×) fluorescence imaging can more clearly present the fluorescent labeling details of SH-SY5Y (blue fluorescence) and tumor organoids (green fluorescence), making it easier to observe the fluorescent signal distribution and interaction between nerves and tumors. Figure 7 The dimension mark 50 μm is indicated in the figure.

[0093] Figure 8 This is a bright field image of a biological 3D printed nerve (GRG)-tumor interaction model according to a specific embodiment of the present invention. Figure 8 As shown, DRG stands for dorsal root ganglion. The left side of the figure shows the mouse dorsal root ganglion, and the right side shows the pancreatic cancer-like assembly. Bright field imaging shows the morphology and spatial position relationship between the nerve (DRG) and tumor-like assembly. Figure 8 The dimensions are 200 μm.

[0094] Figure 9This is a bright field image of a biological 3D printed nerve (SH-SY5Y) tumor interaction model according to a specific embodiment of the present invention. Figure 9 As shown, the left side of the figure is the SH-SY5Y model, and the right side is the pancreatic cancer assembly. Bright field imaging is used to observe the appearance structure and relative position of the nerve (SH-SY5Y) model and the tumor assembly. Figure 9 The dimensions are 200 μm. Specific embodiment three:

[0096] refer to Figure 10 , for the biological 3D printing method of preparing biological ink for constructing a nerve-tumor interaction model, the specific process is as follows:

[0097] In step 201, tumor tissue is minced, digested in a water bath with digestive fluid, and digested for 10-40 minutes until the tumor tissue is completely digested into a single-cell mixture, wherein the single-cell mixture includes tumor cells, tumor fibroblasts, and immune cells. Simultaneously, rat dorsal root ganglia are obtained, digested in a water bath with digestive fluid, and digested for 15-20 minutes to obtain a rat dorsal root ganglion cell suspension.

[0098] In step 202, a bio-ink prepared with 10% synthetic hydrogel (GelMA) + 20% natural hydrogel (Matrigel) is used to resuspend tumor tissue single cells and rat dorsal root ganglion single cells, and the cells are transferred into 5 mL injection syringes to obtain a cell-laden nerve-tumor interaction model bio-printing ink. Specific embodiment four:

[0100] The present invention proposes a method for preparing the nerve-tumor interaction model, and / or the application of the nerve-tumor interaction model in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs, or evaluating the therapeutic effect of nerve blocking on tumors.

[0101] Based on this model, cell behavior and signal pathway changes can be monitored in real time, the mechanism of interaction between nerves and tumors can be studied, anti-tumor drugs can be screened, and the effect of nerve blocking on tumor treatment can be evaluated.

[0102] Specifically, the extrusion-printed and / or suspension-printed nerve-tumor interaction model was cultured in vitro, and live-dead cell staining was performed on it. The fluorescence ratio was observed under a fluorescence microscope, and the cell viability of the nerve-tumor interaction model obtained by this method and the cytotoxicity of the materials and processes were analyzed.

[0103] In this example, a neural-tumor interaction model was co-cultured in situ in an incubator. During the culture process, anti-tumor drugs or neural blockade were added to the neural-tumor interaction model culture vessel to assess tumor cell apoptosis within the 3D tumor culture model. The behavior of neural cells and tumor cells was monitored in real time. The neural blockade method included at least gene editing of neural cells, gene editing of neural cell receptors, inhibitors targeting neural cells, or inhibitors targeting neurotransmitter receptors. These methods are not limited to these and can be selected or adjusted based on actual conditions in actual application scenarios.

[0104] The present invention provides a method for preparing a nerve-tumor interaction model, a nerve-tumor interaction model, and the use of this method and / or the nerve-tumor interaction model in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs, or evaluating the therapeutic effect of nerve blockade on tumors. The main process of model preparation is as follows: based on the biological characteristics of nerve-tumor interaction, the biological behavior of nerve axons extending from ganglia in the body to the location of tumor occurrence, and the spatial positional relationship between neurons and tumor tissue, a three-dimensional structure corresponding to a 3D culture model of neurons and tumors is created; neurons, tumor cells, tumor microenvironment cells, and growth factors required for cell growth are added to the matrix to obtain a bio-ink; wherein the matrix is ​​made of a degradable biocompatible material, and the growth factors are used to support the growth and migration of neurons and tumor cells; based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, the spatial positional relationship between neurons and tumor tissue is simulated, and a 3D model is printed using the bio-ink to obtain a nerve-tumor interaction model. The model constructed by the present invention through biological 3D printing technology can accurately simulate the spatial position relationship and microenvironment of the interaction between nerves and tumors, providing an experimental platform closer to the physiological state for studying the mechanism of tumor invasion; the model prepared by the present invention has high spatial resolution, can achieve the precise distribution of nerve fibers and tumor tissues, and facilitates the observation of dynamic interactions between cells; based on this model, cell behavior can be monitored in real time, providing an important tool for screening anti-tumor drugs and studying treatment strategies; the model preparation method of the present invention is simple, efficient, and highly repeatable, and is suitable for large-scale production and preclinical research. Through the present invention, it can replace 2D cell level and in vivo animal experiments, with high efficiency and low cost, providing strong technical support for the study of nerve-tumor interactions.

[0105] The acquisition, storage, use, and processing of data in the technical solution of this application comply with relevant laws and regulations.

[0106] Those skilled in the art will appreciate that embodiments of the present invention may be provided as methods, apparatus, or computer program products. Thus, the present invention may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present invention may take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to magnetic disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0107] The present invention is described with reference to flowcharts and / or block diagrams of methods and computer program products according to embodiments of the present invention. It should be understood that each process and / or block in the flowcharts and / or block diagrams, as well as combinations of processes and / or blocks in the flowcharts and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowcharts and / or block diagrams. Figure 1 a process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0108] These computer program instructions may also be stored in a computer readable memory that can direct a computer or other programmable data processing device to work in a specific manner, so that the instructions stored in the computer readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1 a process or multiple processes and / or boxes Figure 1 The function specified in one or more boxes.

[0109] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operational steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing the instructions executed on the computer or other programmable device for implementing the process. Figure 1 a process or multiple processes and / or boxes Figure 1 The steps for the function specified in one or more boxes.

[0110] Finally, it should be noted that the above-described embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the above-described embodiments, those skilled in the art should understand that any person skilled in the art can modify or easily conceive of changes to the technical solutions described in the above-described embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein with equivalents. Such modifications, changes, or replacements do not deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for preparing a nerve-tumor interaction model, characterized in that: The method includes: Step 1: Based on the biological characteristics of the interaction between nerves and tumors, the three-dimensional structure of the neuron-tumor culture model is created, which mimics the biological behavior of nerve axons extending from ganglia in the body to the tumor site, as well as the spatial relationship between neurons and tumor tissue. Step 2: adding neurons, tumor cells, tumor microenvironment cells, and growth factors required for cell growth to a matrix to obtain a bio-ink; wherein the matrix is ​​made of a degradable biocompatible material, and the growth factors are used to support the growth and migration of neurons and tumor cells; Step 3: Based on the three-dimensional structure corresponding to the 3D culture model of neurons and tumors, simulate the spatial position relationship between neurons and tumor tissues, use the bio-ink to print the 3D model, and obtain a nerve-tumor interaction model.

2. The method for preparing the nerve-tumor interaction model according to claim 1, characterized in that: The method further includes: Step 4: placing the nerve-tumor interaction model in a simulated in vivo environment, culturing it with a culture medium, and recording the dynamic process of the nerve-tumor interaction.

3. The method for preparing the nerve-tumor interaction model according to claim 1, characterized in that: Step 1 includes: For the neuron model, a neural module with a set density is constructed and placed around the tumor model to simulate the neuronal axons in the spinal cord extending to the vicinity of the tumor organ and wrapping around or invading the tumor. For tumor models, we construct organoid structures containing tumor cells and microenvironment cells, which are located in the middle of multiple neural modules to simulate the organ tissue where the tumor occurs; Based on the relative positions of nerves and tumors, the spatial relationship between the neuron model and the tumor model is determined. A digital model is constructed using biological modeling software based on clinical images or biological data to obtain the three-dimensional structure corresponding to the 3D culture model of neurons and tumors. The parameters of the three-dimensional structure are optimized based on experimental data.

4. The method for preparing the nerve-tumor interaction model according to claim 1, characterized in that: Step 2 includes: For 3D tumor models, the excised primary tumor tissue is digested into single cells. The digested single cells and growth factors required for cell growth are added to the matrix, and the mixture is mixed to obtain a bio-ink containing tumor cells and tumor microenvironment cells. For the 3D model of neurons, extracted mouse or rat neuronal cells, neural cell lines, or neuronal cells differentiated from iPS cells are digested into single cells. The digested single cells and growth factors required for cell growth are added to the matrix, and the mixture is mixed to obtain a bio-ink containing neurons.

5. The method for preparing the nerve-tumor interaction model according to claim 4, characterized in that: The matrix is ​​a biological material made by mixing natural hydrogel and synthetic hydrogel.

6. The method for preparing the nerve-tumor interaction model according to claim 4, characterized in that: The concentration of natural hydrogel is 5-20%, and the mass concentration of synthetic hydrogel is 5-15%.

7. The method for preparing the nerve-tumor interaction model according to claim 1, characterized in that: Step 3 includes: For a 3D model of a tumor, a bio-ink containing tumor cells and tumor microenvironment cells is used for 3D model printing. During the printing process, the model is pre-cured at a first temperature and then photocured by ultraviolet irradiation after printing. The 3D model is then cultured in situ using a growth factor-containing culture medium to generate an in vitro 3D model of the tumor. For a 3D model of a neuron, a bio-ink containing the neuron is used for 3D model printing; during the printing process, pre-curing is performed at a first temperature environment, and after printing is completed, light curing is performed by ultraviolet irradiation; the 3D model is cultured in situ using a culture medium containing growth factors to generate an in vitro 3D model of the neuron; When printing the two 3D models, the 3D models are printed according to the three-dimensional structures corresponding to the 3D culture models of neurons and tumors, so as to obtain a bionic model of the interaction between nerves and tumors in the spatial position relationship within the body.

8. The method for preparing the nerve-tumor interaction model according to claim 7, characterized in that: The 3D model is printed using extrusion printing or suspension printing, with a printing accuracy of micron level.

9. The method for preparing the nerve-tumor interaction model according to claim 7, characterized in that: The temperature range of the first temperature environment is 0-4°C.

10. A nerve-tumor interaction model, characterized in that: The nerve-tumor interaction model is prepared by the method according to any one of claims 1-9.

11. Use of the method according to any one of claims 1 to 9 and / or the nerve-tumor interaction model according to claim 10 in analyzing the mechanism of nerve-tumor interaction, screening anti-tumor drugs, or evaluating the therapeutic effect of nerve blocking on tumors.

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