A perfusable tumor organoid vascularization model and a method of constructing the same

Abstract

The application provides a perfusable tumor organoid vascularization model and a construction method thereof, and relates to the technical field of organoid culture. The application uses temperature-sensitive degradable materials and photo-crosslinking modified temperature-sensitive materials, prints a composite scaffold through a double-channel, combines a multi-component gel mask partition solidification process, improves the stability of a perfusion pipeline, accurately constructs a partitioned highly bionic complex microenvironment in a macro three-dimensional space, and constructs a perfusable tumor organoid vascularization model. The model has high bionics, stable structure, can be applied to deepening the research on cell and microenvironment interaction and screening of tumor-related drugs, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a perfusionable tumor organoid vascularization model and its construction method. Background Technology

[0002] In research on constructing perfusionable tissue structures based on extrusion-based 3D bioprinting technology, the sacrificial template method is a mainstream strategy. The core of this method lies in using an "escape" or "sacrificial" ink that can be selectively removed under specific conditions to pre-print the target channel network structure, which is then encapsulated in a matrix material (i.e., bio-ink) that carries cells. After the matrix solidifies, the sacrificial template is removed, thereby forming hollow, perfusionable microchannels.

[0003] Currently, the typical technical approach widely adopted in this field involves using the temperature-sensitive hydrogel material Pluronic F127 as a sacrificial ink, alternating or compounding it with biocompatible hydrogels encapsulating living cells (such as gelatin, methacrylamide gelatin, fibrin, and their complexes with extracellular matrix components) to construct three-dimensional tubular tissue structures at the millimeter to centimeter scale with embedded simple vascular networks. This method provides a preliminary model for exploring basic nutrient delivery and vascularization prototypes in tissue engineering. For example, patent CN106163581A discloses a method for forming tissue constructs with embedded vascular systems by printing multilayer structures containing sacrificial materials. Subsequent research (such as CN1079211781A and CN107427537A) has extended this approach to more complex tissue types, attempting to construct structures embedded with epithelial or neural tissue networks.

[0004] However, existing technologies based on extrusion printing and sacrificial perfusion structures face a series of key bottlenecks in promoting the application of complex tissues, especially pathological models simulating the tumor microenvironment:

[0005] (1) Insufficient stability of the perfusion structure: The tubular structure formed by the printing of single sacrificial materials, such as F127, has limited mechanical strength and structural fidelity during subsequent encapsulation, curing and perfusion culture of bio-ink. It is easy to deform or even collapse, making it difficult to maintain complex, long-term and reliable fluid perfusion, which limits the feasibility of long-term dynamic research.

[0006] (2) Limited functional integration of bio-inks: Although biomaterials such as methacrylamide gelatin (GelMA) possess good biocompatibility and certain biomimetic properties, current technologies typically use them as relatively homogeneous cell carriers. When constructing biomimetic microenvironments, the precise integration and structured distribution of various functionalized biomimetic gels (such as those with different mechanical properties and biochemical components) in three-dimensional space has not been effectively achieved. Furthermore, there is a lack of effective strategies for the controllable loading and spatiotemporally specific release of bioactive factors (such as growth factors and chemokines) in printed structures.

[0007] (3) Lack of ability to simulate complex microenvironments and partitioned culture: Most existing methods focus on constructing single or homogeneous tissue regions and have not made full use of the advantages of printing technology to spatially program multiple materials. By finely controlling the synergistic effect between biomimetic gel (as a cell carrier and microenvironment matrix) and biocompatible mask or barrier structure, a partitioned co-culture system with different physical and biochemical microenvironments dominated by "organoids" (simulating solid tumors or specific tissue units) and "functionalized vascular networks" can be realized on a three-dimensional scale.

[0008] (4) Challenges in constructing large-scale biomimetic models: The aforementioned limitations make it difficult for existing technologies to construct and maintain a complete tumor microenvironment containing heterogeneous cell communities (tumor cells, stromal cells, etc.), functional perfusion vascular networks, and complex extracellular matrix components in centimeter-scale (e.g., cube) large-scale three-dimensional models with high fidelity. This limits long-term, dynamic, and physiologically close three-dimensional research on key "tumor-vascularization interactions" (such as angiogenesis, tumor cell invasion, drug delivery barriers, etc.) in tumor development and progression.

[0009] Meanwhile, another important platform for microphysiological system research—organ-on-a-chip technology (as described in patent CN116042391A and the literature Adv. Funct. Mater. 2023, 34, 2306676)—uses microfluidic technology to construct chamber structures (upper and lower layers or side-by-side) containing endothelial barriers on a chip, achieving precise simulation of the microphysiological environment of specific tissue interfaces (such as the vascular barrier). This type of technology focuses on the mechanistic study of key local structures and functions, and has high controllability and high throughput potential. However, its structures are usually relatively planar or limited to the microchannel scale, posing inherent challenges in constructing highly biomimetic three-dimensional tissue microenvironments with centimeter-scale three-dimensional volume, especially in simulating complex three-dimensional pathological structures such as solid tumors.

[0010] In summary, existing extrusion-based bioprinting techniques combined with sacrificial templates still have significant limitations in constructing stable, multifunctional, partitioned, and large-scale three-dimensional biomimetic tissue models. There is an urgent need to develop a new technological strategy that can improve the stability of perfusion channels, achieve the controllable integration of various biomimetic gels and biological factors, and ultimately precisely construct partitioned, highly biomimetic complex microenvironments (such as the tumor microenvironment) in macroscopic three-dimensional space. This will deepen our understanding of cell-microenvironment interactions during tissue development, disease mechanisms, and drug responses. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a method for constructing a perfusionable vascularized tumor organoid model. The method involves fabricating a scaffold with a multi-layered mesh structure using dual-channel extrusion printing. The first support layer is a thermosensitive biodegradable material, and the second support layer is a photocrosslinked modified thermosensitive material. This scaffold is then assembled into a chip culture chamber using a baffled reservoir. Suspensions of tumor organoids, endothelial cells, and tumor-associated fibroblasts are prepared. A biogel containing matrix components, peptides, and cytokines, or two types of biomimetic gel precursor solutions, are prepared. The biogel is directly cured under ultraviolet light, while the biomimetic gel undergoes central drip-curing and side-masking partitioned curing. After removing the scaffold at low temperature, the vascularized culture medium is replaced for dynamic co-culture, ultimately yielding a structurally stable, highly biomimetic, and perfusion-capable vascularized tumor organoid model.

[0012] The first objective of this invention is to provide a method for constructing a perfusionable tumor organoid vascularization model, comprising the following steps:

[0013] Step S1: Prepare the chip, bracket, and liquid reservoir;

[0014] The scaffold is fabricated using a dual-channel extrusion printing process, which includes sequentially and alternately printing multiple layers of first support layer and multiple layers of second support layer. The projections of the first support layer and the second support layer in the height direction of the scaffold are both mesh structures. The first support layer uses a thermosensitive biodegradable material, and the second support layer uses a photocrosslinked modified thermosensitive material.

[0015] The liquid storage tank includes a liquid storage tank body and a baffle. The liquid storage tank body has an open cavity structure with a through hole at the bottom of the cavity. The baffle is adapted to cover the opening of the liquid storage tank body.

[0016] The chip is provided with a culture chamber and liquid inlet and outlet ports at both ends. The liquid storage tank and the support are set in the culture chamber. The liquid storage tanks are assembled at both ends of the support. The liquid storage tanks are adapted to the liquid inlet and outlet ports of the chip.

[0017] Step S2: Prepare tumor organoids, cell suspensions, and extracellular matrix precursor solutions;

[0018] The cell suspension includes an endothelial cell suspension and a tumor-associated fibroblast suspension;

[0019] The extracellular matrix precursor solution is a biogel precursor solution or a biomimetic gel precursor solution, wherein the biomimetic gel precursor solution includes a first biomimetic gel precursor solution and a second biomimetic gel precursor solution.

[0020] The biogel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, matrix gel, fibrinogen and collagen.

[0021] The first biomimetic gel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, norbornene-modified heparin, norbornene-modified gelatin, mercapto-tetra-arm polyethylene glycol, dextran, GFOGER, cytokines and photoinitiators.

[0022] The second biomimetic gel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, norbornene-modified heparin, norbornene-modified gelatin, mercapto-tetra-arm polyethylene glycol, dextran, matrix metalloproteinase mimic peptide, fibrinogen and photoinitiator.

[0023] Step S3: Model assembly and infusion;

[0024] An extracellular matrix precursor solution is added to the culture chamber of the chip and cured by ultraviolet light. Pre-cooled culture medium is added to the culture chamber through the chip's inlet and outlet ports. The sacrificial scaffold is allowed to liquefy by being allowed to stand at low temperature. After confirming that the tubing is unobstructed, it is replaced with preheated tumor organoid vascularization culture medium and placed in an incubator for dynamic co-culture.

[0025] Furthermore, in step S1, the bracket also includes a plurality of printed support columns, which penetrate multiple layers of the first support layer and multiple layers of the second support layer along the height direction of the bracket.

[0026] Furthermore, the density of the tumor organoids is 5 × 10⁻⁶. 3 / mL-2×10 4 / mL, the cell density of the endothelial cell suspension is 5×10 6 / mL-1.5×10 7 / mL, the cell density of the tumor-associated fibroblast suspension is 5×10⁶ cells / mL. 5 / mL-2.5×10 6 / mL.

[0027] In one embodiment of the present invention, the density of the tumor organoid is 5 × 10⁻⁶. 3 / mL, the cell density of the endothelial cell suspension is 1×10 7 / mL, the cell density of the tumor-associated fibroblast suspension is 5×10⁶ cells / mL. 6 / mL.

[0028] Further, in step S2, the cytokine is one or more of R-spinal plate specific protein 1, vascular endothelial growth factor, basic fibroblast growth factor 2, and platelet-derived growth factor.

[0029] Further, in step S2, the GFOGER comprises the amino acid sequence shown in SEQ ID NO.1, and the matrix metalloproteinase mimic peptide comprises the amino acid sequence shown in SEQ ID NO.2.

[0030] Further, in step S2, the concentration of norbornene-modified heparin is 0.2-2% (w / v), the concentration of norbornene-modified gelatin is 0.5-5% (w / v), the concentration of mercapto-tetraarm polyethylene glycol is 0.2-5% (w / v), the concentration of dextran is 0.1-5% (w / v), and the concentration of photoinitiator is 0.01-0.1% (w / v).

[0031] Furthermore, in step S3, when the extracellular matrix precursor solution is a biomimetic gel precursor solution, the step of preparing a mask is also included;

[0032] The mask includes a substrate and a cutout area. The cutout area is formed on the substrate and includes an outer frame cutout portion and an inner protrusion portion. The inner protrusion portion is connected to the inner wall of the outer frame cutout portion through at least one connecting portion to divide the outer frame cutout portion into multiple independent sub-cutout areas. The inner protrusion portion, the connecting portion and the substrate are an integral structure, and the outline of the cutout area matches the outline of the target partition photocuring area.

[0033] Further, in step S3, the first biomimetic gel precursor solution is dropped onto the culture scaffold area in the center of the model chamber, cured by ultraviolet light exposure, and then placed in an incubator for inverted incubation; the model is taken out, and the second biomimetic gel precursor solution is dropped onto both sides of the culture scaffold, covered with a mask, cured by ultraviolet light exposure, and then placed in an incubator for inverted incubation.

[0034] Furthermore, step S3 also includes the step of adding growth factors, wherein the growth factors are vascular endothelial growth factor and fibroblast growth factor 2.

[0035] In one embodiment of the present invention, the final concentration of the vascular endothelial growth factor and fibroblast growth factor 2 is 50 ng / mL.

[0036] Furthermore, in step S3, the perfusion flow rate is controlled at 5-20 μL / min during dynamic co-culture.

[0037] In one embodiment of the present invention, the perfusion flow rate is 10 μL / min.

[0038] A second objective of this invention is to provide a perfusionable tumor organoid vascularization model obtained by the above-described construction method.

[0039] The beneficial effects of this invention are:

[0040] (1) The stent adopts a multi-layer mesh structure printed by dual channels, combined with thermo-sensitive biodegradable and photo-crosslinked modified materials, taking into account both molding stability and precise removal, and efficiently constructing a through-filling channel; the baffle design of the liquid storage tank further optimizes the filling effect and avoids air bubble interference.

[0041] (2) The extracellular matrix precursor solution has a rich formulation containing GFOGER, matrix metalloproteinase mimic peptides and a variety of cytokines, which accurately simulate the tumor microenvironment and promote the synergistic growth of tumor organoids and vascularized structures.

[0042] (3) The biomimetic gel adaptable mask partitioning curing process enables precise division of different gel regions, improves the heterogeneity and biomimeticity of the model structure, and provides a more in vivo experimental carrier for the study of tumor vascularization mechanism.

[0043] (4) The model has stable perfusion characteristics and the dynamic co-culture mode maintains cell activity and functional integrity. It can be widely used in scenarios such as tumor drug sensitivity detection and angiogenesis inhibitor screening, and has strong practicality. Attached Figure Description

[0044] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0045] Figure 1 This is a schematic diagram of the perfusionable tumor organoid vascularization model provided by the present invention;

[0046] Figure 2 This is a schematic diagram of the structure of the support and the liquid storage tank in Embodiment 1 of the present invention;

[0047] Figure 3 The modulus measurement results of biomimetic gel I in Example 1 of the present invention are shown, where A is the storage modulus and loss modulus, and B is the normalized shear stress.

[0048] Figure 4 The modulus measurement results of biomimetic gel II in Example 1 of the present invention are shown, where A is the storage modulus and loss modulus, and B is the normalized shear stress.

[0049] Figure 5 This is a schematic diagram of the mask in Embodiment 1 of the present invention, where A is a side view of the mask structure and B is a physical image of the mask;

[0050] Figure 6 This is a schematic diagram of the infusion process of the model in Embodiment 1 of the present invention;

[0051] Figure 7 This is a physical diagram of the model infusion process in Embodiment 1 of the present invention;

[0052] Figure 8 This is an imaging image of the biomimetic gel system liver cancer vascularization microphysiological model in Example 1 of the present invention, with a scale bar of 100 μm;

[0053] Figure 9 This is an imaging image of the biomimetic gel system liver cancer vascularization microphysiological model in Example 1 of the present invention, with a scale bar of 200 μm;

[0054] Figure 10 This is an imaging image of the blank control group without lung cancer in Example 2 of the present invention, with a scale bar of 200 μm;

[0055] Figure 11 This is an imaging image of the vascularization model of lung cancer organoids in the biomimetic gel system in Example 2 of the present invention, with a scale bar of 200 μm;

[0056] Figure 12 This is an imaging image of the lung cancer vascularization microphysiological model in the biogel system of Example 3 of the present invention, with a scale bar of 500 μm;

[0057] Figure 13 This invention provides statistical and quantitative characterization of vascular and organoid parameters in the lung cancer vascularization microphysiological model using the biogel system in Example 3 of this invention.

[0058] Figure 14 This is a schematic diagram of the 3D printing support structure of different strategies in Comparative Example 1 of the present invention;

[0059] Figure 15 The images show the lumen structures obtained by removing the sacrificial scaffold using different strategies in Comparative Example 1 of this invention.

[0060] Figure 16 The figure shows the rheological test results of different biomimetic gel systems in Comparative Example 2 of this invention;

[0061] Figure 17 The images show liver cancer organoids of different biomimetic gel systems in Comparative Example 2 of this invention.

[0062] Figure 18 This is an imaging image of the vascular structure in the static culture group of Comparative Example 3 of the present invention;

[0063] Figure 19 This is an imaging image of the vascular structure in the dynamic culture group of Comparative Example 3 of the present invention. Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0065] The materials involved in the following embodiments are as follows:

[0066]

[0067] The polypeptide sequences involved in the following examples are shown below:

[0068]

[0069] Example 1: Construction and characterization of a biomimetic gel-liver cancer organoid vascularization model

[0070] I. Model Cell Culture Chamber

[0071] (1) Frame printing

[0072] 40% Pluronic F127 thermosensitive hydrogel was pre-mixed in barrel 1. ® F-127 (w / v, refers to 40 g added per 100 mL of solution, the same below) and 30% Pluronic F127-DA were prepared in container 2. ® F-127-DA was stored at 4°C. It was used with an extrusion-based bio-3D printer (Genevo Bio-Architect). ® The SR printing stand has two printheads with an outer diameter of 0.8 mm and an inner diameter of 0.5 mm. The two printheads print sequentially, F127-DA (green) and F127 (white), with each layer having a height of 0.4 mm. The first two layers are printed sequentially, repeated three times, for a total of six layers. The stand volume is 300 mm². 3 Support structure such as Figure 2 As shown in (1), the projection of the first support layer and the second support layer in the height direction of the bracket is a mesh structure, and also includes multiple 3D printed support columns, which penetrate multiple layers of the first support layer and multiple layers of the second support layer along the height direction of the bracket.

[0073] (2) Printing of liquid storage tank

[0074] Using Mofang Precision nanoArch ®The P150 micro-nano 3D printer, in conjunction with Mofang biocompatible resin, prints a liquid reservoir and a baffle that acts as a bubble trap (used to capture, retain, and remove air bubbles in a liquid). The reservoir measures 9 mm × 3 mm × 4 mm, and the baffle measures 7 mm × 1.25 mm × 0.2 mm. The structure of the reservoir and baffle is as follows: Figure 2 As shown in (2) in the figure. The main body of the liquid storage tank has an open cavity structure with a through hole at the bottom of the cavity and a baffle adapted to cover the opening of the main body of the liquid storage tank.

[0075] (3) Chip preparation

[0076] The chip (general-purpose polystyrene) was immersed in 75% ethanol solution for 10 min, then rinsed three times with sterile water, dried and sterilized under ultraviolet light for 30 min. The liquid storage tank and support obtained in the above steps were constructed in the chip chamber.

[0077] II. Cell Preparation

[0078] After resuscitation, RFP-labeled HUVECs (human umbilical vein endothelial cells) and LI-CAFs (hepatocellular carcinoma-associated fibroblasts) were expanded and cultured until 80% of the cell volume was reached, then passaged at a cell ratio of 1:2. After further expansion and culture until 80% coverage, the cells were digested and resuspended in fresh culture medium. The cell densities of both cell types were measured using a cell counter. Cells were centrifuged at 300 g for 5 min, and 1×10⁶ cells were prepared using ECM medium and tumor-associated fibroblast culture medium, respectively. 7 HUVEC density / mL and 5×10 6 / mL of LI-CAF cell suspension.

[0079] Passaged HUVEC and LI-CAF cells were digested and resuspended in ECM medium and tumor-associated fibroblast culture medium, respectively. Cell density was measured using a cell counter. The two cell types were diluted to 2 × 10⁻⁶ cells / mL with culture medium. 4 / mL. 100 μL of HUVEC and LI-CAF cell suspensions were respectively added to a 96-well plate with a low-adsorption U-shaped bottom. After mixing well, the cells were centrifuged at 300 g for 5 min to induce cell aggregation. After static culture for 48 h, vascular cell spheres were formed.

[0080] Human hepatocellular carcinoma organoids were cultured in Maxiglium until they reached a size of 150 μm. The Maxiglium was then broken up by pipetting in pre-chilled F12 medium (4°C), collected in centrifuge tubes, and incubated at 4°C for 15 min. After removing the Maxiglium, the organs were centrifuged at 300 g for 5 min, the supernatant was discarded, and the organs were resuspended in human hepatocellular carcinoma organoid culture medium and counted. A suitable amount of hepatocellular carcinoma organoids was taken, and pre-warmed TryPLE digestion solution (trypsin-alternative enzyme solution) at 37°C was added. The organs were gently pipetted 15 times to obtain hepatocellular carcinoma organoids with a size of 70 μm. The density of the hepatocellular carcinoma organoids was adjusted to 5 × 10⁻⁶ using human hepatocellular carcinoma organoid culture medium. 3 / mL.

[0081] III. Formulation of Bionic Gel

[0082] A vascularized tumor-encapsulating system was constructed using an extracellular matrix biomimetic gel (heparin biomimetic gel). The internal tumor organoid-dominant growth part used a heparin biomimetic gel (biomimetic gel I) that allows organoid growth and maintains the stability of the permeable structure, while the surrounding vascularized dominant growth part used a heparin biomimetic gel (biomimetic gel II) that allows capillary growth.

[0083] (1) Preparation of biomimetic gel I for culturing tumor organoid dominant cells (CN120173883A Photocurable biomimetic gel for tumor organoid culture that can bind cytokines and its application)

[0084] Deacetylated Hep-NB (norbornene-modified heparin), GFOGER peptide (amino acid sequence as shown in SEQ ID NO. 1), R-Spondin 1 (R-spinal plate-specific protein 1), Gel-NB (norbornene-modified gelatin), 4-PEG-SH (thiol tetraarm polyethylene glycol), dextran, and photoinitiator LAP were dissolved in PBS (phosphate buffer) and the solutions were thoroughly mixed. The concentrations of Hep-NB, GFOGER, R-Spondin 1 (5 μg / mL), Gel-NB (3.8%), 4-PEG-SH (1.2%), dextran (1%), and photoinitiator LAP (0.06%) were mixed at a concentration of 3 × 10⁻⁶. 3 / mL of liver cancer organoids (150 μm) and 1×10 2 A biomimetic gel I precursor solution was prepared by using vascular cell spheres at a concentration of / mL.

[0085] (2) Preparation of biomimetic gel II for culturing vascularized dominant cells (Patent on application of heparin biomimetic gel for vascularization, application number 2025110349869)

[0086] Deacetylated Hep-NB, GFOGER, VEGF (vascular endothelial growth factor), FGF2 (basic fibroblast growth factor 2), PDGF-BB (platelet-derived growth factor), Gel-NB, 4-PEG-SH, MMP (matrix metalloproteinase mimic peptide, amino acid sequence as shown in SEQ ID NO. 2), fibrinogen, and photoinitiator LAP were dissolved in PBS solution, and the above component solutions were thoroughly mixed. The concentrations of Hep-NB (0.35%), GFOGER (0.06%), VEGF (2 μg / mL), FGF2 (2 μg / mL), PDGF-BB (2 μg / mL), Gel-NB (2.5%), 4-PEG-SH (0.5%), MMP (0.08%), fibrinogen (0.1%), and photoinitiator LAP (0.05%) were mixed to a concentration of 1 × 10⁻⁶. 6 / mL of RFP-HUVEC cell suspension, 1×10 5 / mL of LI-CAF cell suspension and 5×10 2 A biomimetic gel II precursor solution was prepared from liver cancer organoids (70 μm) at a concentration of 1 / mL.

[0087] The modulus of the biomimetic gel was tested using a rheometer. 600 μL of the biomimetic gel precursor solution was dropped into a 2 cm polytetrafluoroethylene mold, and the sample was tested under light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 Biomimetic gel discs were prepared by irradiating the sample under ultraviolet light for 5 min. The samples were then placed on the rheometer stage for rheological testing. The storage modulus and loss modulus of biomimetic gel I are shown below. Figure 3 As shown in A, the storage modulus and loss modulus of biomimetic gel II are as follows: Figure 4 As shown in Figure A, at different frequencies, the storage modulus (G') of biomimetic gel I is around 200 Pa, and the storage modulus (G') of biomimetic gel II is around 80 Pa. Moreover, G' is greater than the loss modulus (G''), indicating that both biomimetic gel I and biomimetic gel II form a stable three-dimensional network structure under ultraviolet light irradiation.

[0088] The stress relaxation properties of the biomimetic gel were tested using a rheometer, and the results are as follows: Figure 3 B and Figure 4 As shown in Figure B, the half-stress relaxation time of biomimetic gel I is about 22 min, and the half-stress relaxation time of biomimetic gel II is about 5 min, indicating that the biomimetic gels form a three-dimensional network structure with cell migration under ultraviolet light.

[0089] IV. Mask Preparation

[0090] Draw a top view of the mask and import it into the corresponding software for the laser cutting machine. Select an opaque acrylic sheet with a thickness of 1.5 mm to fabricate the mask. The mask can block the light source to achieve zoned curing of the biomimetic gel within the model. The side view and actual image of the mask structure are shown below. Figure 5 As shown, the structure includes a substrate and a hollowed-out area. The substrate has a plate-like structure, and the hollowed-out area is formed on the substrate. It includes an outer frame hollowed-out portion and an inner protrusion. The inner protrusion is connected to the inner wall of the outer frame hollowed-out portion through at least one connecting portion to divide the outer frame hollowed-out portion into multiple independent sub-hollowed-out areas. The inner protrusion, the connecting portion and the substrate are an integrated structure, and the outline of the hollowed-out area matches the outline of the target partition photocuring area.

[0091] V. Model Assembly and Pouring

[0092] A schematic diagram of the model assembly and infusion process is shown below. Figure 6 As shown, a sacrificial scaffold (A) was extruded and printed within the model chamber. Liquid reservoirs (B) were assembled at both ends of the scaffold. 90 μL of biomimetic gel I was dropped into the scaffold in the center of the model culture chamber. The culture was conducted under light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 Irradiate the sample under ultraviolet light for 5 min, then incubate it upside down in a 37 ℃ incubator for 5 min (C). Then, drop 120 μL of biomimetic gel II onto both sides of biomimetic gel I, cover the top of the model with a mask, and incubate under light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 The model was irradiated under ultraviolet light for 5 min, then inverted in a 37 ℃ incubator for 10 min (D). Next, the model was placed in a low-temperature environment (4 ℃) for cooling (E). After observing complete liquefaction of the sacrificial scaffold in the model, the perfusion rate was set to 10 μL / min, and the syringe pump was started to perfuse the colored liquid (F). Figure 7 As shown, air bubbles appear at the reservoir position at the beginning of the infusion process. During the infusion process, these air bubbles are blocked outside the internal channel by the air bubble trap structure of the reservoir. At the end of the infusion, the colored liquid fills the channel, indicating that this model has an infusion function.

[0093] Two 10 mL syringes were used, each containing an appropriate amount of pre-chilled PBS solution at 4°C. These syringes were connected to the inlet and outlet ports of the model via Teflon tubing. 400 μL of pre-chilled culture medium solution at 4°C was added to the model chamber, and the syringes were placed on ice for 15 min. The perfusion flow rate was controlled at 10 μL / min using a syringe pump, and the model was incubated on ice for 20 min. After confirming F127 removal and perfusion tubing patency, the solution in the syringes and model chamber was replaced with pre-warmed hepatocellular carcinoma organoid vascularization culture medium at 37°C. This medium was prepared by mixing endothelial cell culture medium and hepatocellular carcinoma organoid culture medium at a 1:1 volume ratio, with the addition of VEGF and FGF2 at a final concentration of 50 ng / mL. The hepatocellular carcinoma organoid vascularization microphysiological model was placed in an incubator for dynamic co-culture at a flow rate of 10 μL / min. After 24 h, the perfusion flow rate was adjusted to 5 μL / min, and the culture medium in the syringes was replaced with fresh hepatocellular carcinoma organoid vascularization culture medium every 48 h.

[0094] VI. Immunofluorescence staining and confocal characterization

[0095] After 5 days of dynamic co-culture, microphysiological model samples were collected, the culture medium in the chamber was gently aspirated, and the samples were washed with DPBS (Dulbecco's Phosphate Buffered Saline). 400 μL of 4% paraformaldehyde fixative was added and the samples were incubated overnight at 4°C. After washing with DPBS, 400 μL of 0.5% Triton X-100 permeabilization buffer was added and the samples were incubated at room temperature for 30 min at 40 rpm on a shaker.

[0096] After washing with DPBS, add 400 μL of goat serum blocking solution and incubate at room temperature for 1 h on a shaker at 40 rpm. Prepare anti-CD31 antibody (anti-CD3) and anti-pan-CK antibody (anti-pan CK) with primary antibody dilution buffer according to the recommended ratio, and add 400 μL of each to different chip chambers and incubate overnight at 4°C.

[0097] After recovering the primary antibody and washing with DPBS, 400 μL of Alexa Fluor was added. ® The 488-labeled secondary antibody solution was incubated at room temperature in the dark for 2 h. After washing three times with DPBS, 400 μL of DAPI (4,6-diamidinyl-2-phenylindole dihydrochloride) staining solution was added, and the mixture was incubated at room temperature in the dark for 20 min. After washing again, 300 μL of DPBS was added to the chip chamber, and the chip was stored at 4°C. The images were then captured and recorded under a confocal microscope.

[0098] like Figure 8 and Figure 9As shown, endothelial cells and fibroblasts can self-assemble to form a complete vascular network structure, and liver cancer organoids maintain a normal growth state. Endothelial cells, labeled with red fluorescent protein, can normally express CD31, while liver cancer organoids normally express panCK.

[0099] Example 2: Construction and characterization of a biomimetic gel-induced vascularization model of lung cancer organoids

[0100] (1) Chip preparation and bracket printing

[0101] The chip was immersed in 75% ethanol solution for 10 min, then rinsed three times with sterile water, dried, and sterilized under ultraviolet light for 30 min. Following the scaffold and reservoir printing method in Example 1, a reservoir and a three-dimensional culture scaffold were constructed within the chip chamber.

[0102] (2) Cell preparation

[0103] After resuscitation, HUVECs (human umbilical vein endothelial cells) and LU-CAFs (lung cancer-associated fibroblasts) labeled with RFP (red fluorescent protein) were expanded and cultured until they covered 80% of the bottom of the flask. The cells were then passaged at a 1:2 ratio. After further expansion and culture until 80% coverage, the cells were digested and resuspended in ECM medium and tumor-associated fibroblast medium, respectively. Cell densities for both types of cells were measured using a cell counter. The cells were centrifuged at 300 g for 5 min, and 1×10⁻⁶ cells were prepared using the two media mentioned above. 7 HUVEC density / mL and 5×10 6 / mL of LU-CAF cell suspension.

[0104] Passaged HUVEC and LU-CAF cells were digested and resuspended in ECM medium and tumor-associated fibroblast medium, respectively. Cell density was measured using a cell counter. The two cell types were diluted to 4 × 10⁴ / mL using the aforementioned media. 50 μL of each HUVEC and LU-CAF cell suspension was added to a 96-well plate with a low-adsorption U-shaped bottom. After thorough mixing, the cells were centrifuged at 300 g for 5 min to induce cell aggregation. After static culture for 48 h, vascular cell spheroids were formed.

[0105] Lung cancer organoids were cultured in Maxiglium until they reached a size of 120 μm. The Maxiglium was then broken up by pipetting in pre-cooled F12 medium at 4 °C, collected in centrifuge tubes, and maintained at 4 °C for 15 min. After removing the Maxiglium, the tubes were centrifuged at 300 g for 5 min, the supernatant was discarded, and the organoids were resuspended in human lung cancer organoid culture medium and counted. A suitable amount of organoids was taken and repeatedly pipetted 25 times to obtain lung cancer organoids with a size of 60 μm. The density of the lung cancer organoids was adjusted to 5 × 10⁻⁶ using human lung cancer organoid culture medium.3 / mL.

[0106] (3) Biomimetic gel preparation and cell loading

[0107] Acetylated Hep-NB, GFOGER, R-Spondin 1, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in PBS solution and thoroughly mixed. The concentrations of Hep-NB (0.8%), GFOGER (0.06%), R-Spondin 1 (5 μg / mL), Gel-NB (3.8%), 4-PEG-SH (2%), dextran (1%), and photoinitiator LAP (0.06%) were mixed. The mixture was then 4 × 10⁻⁶ ppm. 3 Lung cancer organoids (120 μm) per mL and 1×10 2 A biomimetic gel I precursor solution was prepared by using vascular cell spheres at a concentration of / mL.

[0108] Deacetylated Hep-NB, GFOGER, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were dissolved in PBS solution. The solutions were thoroughly mixed, with the following concentrations: Hep-NB 0.35%, GFOGER 0.06%, VEGF 2 μg / mL, FGF2 2 μg / mL, PDGF-BB 2 μg / mL, Gel-NB 2.5%, 4-PEG-SH 0.5%, MMP 0.08%, fibrinogen 0.1%, and photoinitiator LAP 0.05%. A mixture of these components was prepared at a concentration of 1 × 10⁻⁶. 6 / mL of RFP-HUVEC cell suspension, 1×10 5 / mL of CAF cell suspension and 5×10 2 A biomimetic gel II precursor solution was prepared from lung cancer organoids (60 μm) at a concentration of 1 / mL.

[0109] (4) Mask preparation

[0110] Refer to the method in Example 1.

[0111] (5) Model assembly and grouting

[0112] Using a pipette, 90 μL of the biomimetic gel I precursor solution was dropped into the culture scaffold area in the center of the model chamber, at a flow rate of 5 mW / cm². 2Expose the sample to ultraviolet light at a certain intensity for 5 min, then invert it in a 37℃ incubator for 5 min. After removal, add 120 μL of biomimetic gel II precursor solution to each side of the culture scaffold within the model chamber. Cover the top of the model with a mask and apply UV light at 5 mW / cm². 2 Expose to ultraviolet light intensity for 5 min, then invert in a 37 ℃ incubator for 10 min.

[0113] Two 10 mL syringes were used, each containing an appropriate amount of pre-chilled PBS solution at 4°C. These syringes were connected to the inlet and outlet ports of the model via Teflon tubing. 400 μL of pre-chilled culture medium solution at 4°C was added to the model chamber, and the syringes were placed on ice for 15 min. The perfusion flow rate was controlled at 10 μL / min using a syringe pump, and the model was incubated on ice for 20 min. After confirming F127 removal and perfusion tubing patency, the solution in the syringes and model chamber was replaced with pre-warmed lung cancer organoid vascularization culture medium at 37°C. This medium was prepared by mixing endothelial cell culture medium and lung cancer organoid culture medium at a 1:1 volume ratio, with the addition of VEGF and FGF2 at a final concentration of 50 ng / mL. The lung cancer organoid vascularization microphysiological model was placed in an incubator and dynamically cultured at a flow rate of 10 μL / min. After 24 h, the perfusion flow rate was adjusted to 5 μL / min, and the culture medium in the syringes was replaced with fresh lung cancer organoid vascularization culture medium every 48 h. The blank control group, which did not contain lung cancer organoids, used endothelial cell culture medium throughout the entire process.

[0114] (6) Characterization of lung cancer organoids and angiogenesis

[0115] Immunofluorescence staining and confocal characterization were performed according to the method in Example 1.

[0116] After 48 hours of dynamic co-culture, the overall structure of the central scaffold in the chip chamber was observed under a stereomicroscope. Every 48 hours, the vascularization process and the growth status of lung cancer organoids were observed under a fluorescence microscope, and the differences were compared with those of the blank control group without lung cancer organoids.

[0117] like Figure 10 As shown in the imaging results on day 4 of culture, endothelial cells were labeled with red fluorescent protein. Even without lung cancer organoids in the system, endothelial cells and fibroblasts were able to self-assemble into a complete vascular network structure. Figure 11 As shown, under the condition of co-culturing with lung cancer organoids, endothelial cells migrate to the lung cancer organoids, forming blood vessels that encapsulate the lung cancer organoids. At the same time, the integrity of the blood vessel structure is disrupted, which is consistent with the characteristics of vascular dysfunction in the tumor microenvironment in the human body.

[0118] Example 3: Construction and characterization of a bio-matrix-lung cancer organoid vascularization model

[0119] (1) Chip preparation and bracket printing

[0120] The chip was immersed in 75% ethanol solution for 10 min, then rinsed three times with sterile water, dried, and sterilized under ultraviolet light for 30 min. Following the scaffold and reservoir printing method in Example 1, a reservoir and a three-dimensional culture scaffold were constructed within the chip chamber.

[0121] (2) Cell preparation

[0122] After resuscitation, HUVECs (human umbilical vein endothelial cells) and LU-CAFs (lung cancer-associated fibroblasts) labeled with RFP (red fluorescent protein) were expanded and cultured until they covered 80% of the bottom of the flask, and then passaged at a cell ratio of 1:2. After expanding and cultured again until they covered 80% of the bottom of the flask, the cells were digested and resuspended in fresh culture medium, and the density of the two cell types was measured using a cell counter.

[0123] Passaged HUVEC and LU-CAF cells were digested, resuspended in fresh culture medium, and cell density was measured using a cell counter. The two cell lines were then diluted with culture medium to a concentration of 4 × 10⁶ cells / mL. 4 / mL. Take 50 μL of HUVEC and LU-CAF cell suspensions respectively and add them to a 96-well plate with a low-adsorption U-shaped bottom. After mixing well, centrifuge at 300 g for 5 min to induce cell aggregation. After static culture for 48 h, vascular cell spheres are formed.

[0124] Lung cancer organoids were cultured in Matrigel until they reached a size of 100 μm. The Matrigel was then broken up by pipetting in pre-cooled F12 medium at 4°C, collected in centrifuge tubes, and kept at 4°C for 15 min. After removing the Matrigel, the tubes were centrifuged at 300 g for 5 min, the supernatant was discarded, and the organs were resuspended in fresh medium and counted.

[0125] (3) Preparation of bio-matrix gel and cell loading

[0126] 2×10 5 HUVEC cells, 7×10 4 LU-CAF cells, 500 lung cancer organoids, and 10 vascular cell spheres were resuspended in 20 μL of culture medium and mixed with 27 μL of type I collagen (pH adjusted to neutral), 27 μL of Matrigel, 27 μL of fibrinogen, and 0.5 μL of 100 U / mL thrombin to prepare a biogel precursor solution.

[0127] (4) Construction of dynamic co-cultivation system

[0128] After rapidly mixing the gel precursor solution with a pipette, add it dropwise to the culture scaffold area in the center of the chip chamber. Incubate at 37°C and invert for 30 min. Remove the chip chamber and add 700 μL of a mixed solution of 10% GelMA and 0.5% LAP at a flow rate of 10 mW / cm². 2 Expose to ultraviolet light intensity for 2 minutes, then place in a 37℃ incubator and let stand for 5 minutes.

[0129] Take two 10 mL syringes, draw an appropriate amount of 4°C pre-cooled PBS solution, and connect them to the inlet and outlet ports at both ends of the chip through Teflon tubing. Add 200 μL of 4°C pre-cooled culture medium solution to the chip chamber and incubate on ice for 15 min. Use a syringe pump to control the perfusion flow rate at 10 μL / min and maintain incubation on ice for 20 min. After confirming that F127 has been removed and the perfusion tubing is unobstructed, replace the solution in the syringes and chip chamber with 37°C pre-warmed lung cancer organoid vascularization culture medium. Place the chip in an incubator for dynamic culture at a flow rate of 10 μL / min. Replace the culture medium in the syringes with fresh lung cancer organoid vascularization culture medium every 48 h.

[0130] (5) Quantitative characterization and assessment of organoid vascularization in lung cancer

[0131] The vascularization process and lung cancer organoid growth status were observed under a microscope every 48 hours. Dynamic co-culture continued until day 7, at which point microarray samples were collected and imaged using fluorescence microscopy. ImageJ software was used to statistically analyze the size, vessel length, and number of intersections of the lung cancer organoids, thereby assessing organoid growth and vascularization progress.

[0132] Figure 12 The image shows the imaging results on day 7 of culture. Endothelial cells, labeled with red fluorescent protein, self-assembled in the biogel system to form a dense vascular network and were able to migrate to lung cancer organoids, forming vascular structures that encapsulate tumor spheres. Three parallel experiments were set up, with images taken from random locations in each group. Statistical analysis was performed using ImageJ software. The results are as follows: Figure 13 As shown, the average number of vascular network intersections per unit area was 546, the average total length was 60930 μm, and the average vascular area ratio was 64.8%, indicating a good vascularization process. The average diameter of lung cancer organoids was 125 μm, and the average roundness was 0.94, indicating that lung cancer organoids can maintain normal morphology and growth status.

[0133] Comparative Example 1: 3D Printing Stand - F127 Stand and F127 / F127DA Dual-Channel Printing Stand

[0134] (1) F127 bracket

[0135] Prepare 40% Pluronic F127 (w / v, 40 g per 100 mL of solution, the same below) in advance and store at 4℃. Immerse the chip in 75% ethanol solution for 10 min, remove and rinse three times with sterile water, dry and sterilize under UV light for 30 min. Use Genofi Bio-Architect 3D printer. ® SR prints a substrate in a chip chamber. The printhead has an outer diameter of 0.8 mm and an inner diameter of 0.5 mm. Each layer is 0.4 mm high, and 6 layers are printed. The substrate volume is 280 mm². 3 .

[0136] (2) F127 / F127DA Dual-Channel Printer Stand

[0137] Pre-prepared solutions of 40% Pluronic F127 and 30% Pluronic F127-DA (polyether F127 diacrylate) were stored at 4°C. The chips were immersed in a 75% ethanol solution for 10 min, then rinsed three times with sterile water, dried, and sterilized under UV light for 30 min. The Genentech Bio-Architect 3D printer was used. ® SR prints a scaffold within the chip chamber. The printhead has an outer diameter of 0.8 mm and an inner diameter of 0.5 mm. Pluronic F127 and Pluronic F127-DA are printed sequentially, with each layer having a height of 0.4 mm. Six layers are printed, resulting in a scaffold volume of 300 mm². 3 After printing, place the chip in a UV lamp box at 10 mW / cm². 2 Expose to ultraviolet light for 2 minutes. The structure of the F127 holder and the F127 / F127DA dual-channel printing holder is as follows. Figure 14 As shown.

[0138] (3) Formulation of biomimetic gel

[0139] Acetylated Hep-NB, GFOGER, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in PBS solution, and the solutions were thoroughly mixed. The concentrations of Hep-NB (0.8%), GFOGER (0.06%), Gel-NB (3.8%), 4-PEG-SH (2%), dextran (1%), and photoinitiator LAP (0.06%) were used to prepare the biomimetic gel I precursor solution.

[0140] Deacetylated Hep-NB, GFOGER, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were dissolved in PBS solution, and the above component solutions were thoroughly mixed. The concentrations of Hep-NB were 0.35%, GFOGER was 0.06%, Gel-NB was 2.5%, 4-PEG-SH was 0.5%, MMP was 0.08%, fibrinogen was 0.1%, and photoinitiator LAP was 0.05%, thus preparing the biomimetic gel II precursor solution.

[0141] (4) Sacrificial stent removal and perfusion test

[0142] Using a pipette, 90 μL of the biomimetic gel I precursor solution was dropped into the scaffold region in the center of the model chamber, at a flow rate of 5 mW / cm². 2 Expose the model to ultraviolet light at a specific intensity for 5 min. Add 120 μL of biomimetic gel II precursor solution to each side of the support within the model chamber. Cover the top of the model with a mask and apply ultraviolet light at 5 mW / cm². 2 Expose to ultraviolet light intensity for 5 min, then place in a 37 ℃ incubator and let stand for 10 min.

[0143] Take two 10 mL syringes, draw an appropriate amount of 4 °C pre-cooled PBS solution, and connect them to the inlet and outlet ports at both ends of the model through Teflon tubing. Add 400 μL of 4 °C pre-cooled culture medium solution to the model chamber and incubate on ice for 15 min. Use a syringe pump to control the perfusion flow rate at 10 μL / min and maintain incubation on ice for 20 min. After confirming that F127 has been removed and the perfusion tubing is unobstructed, place the model in an incubator and perform perfusion testing at a flow rate of 10 μL / min.

[0144] (5) Imaging characterization and comparison

[0145] After maintaining a flow rate of 10 μL / min for 72 h, the changes in the lumen structure after sacrificial removal of the two types of stents were observed under a microscope, and the differences between the two were compared.

[0146] like Figure 15 As shown, after removing the sacrificial layer material, the tubular structure formed by the F127 printed scaffold underwent significant deformation after 72 hours of perfusion, making it difficult to maintain long-term perfusion culture. The F127 / F127DA dual-channel printed scaffold forms a support structure after photopolymerization, and the tubular structure obtained after removing the sacrificial layer has high fidelity and can maintain structural stability for a long time.

[0147] Comparative Example 2: Hepatocellular Carcinoma Vascularization Model - Biomimetic Gel Strength

[0148] The chip was immersed in 75% ethanol solution for 10 min, then rinsed three times with sterile water, dried, and sterilized under ultraviolet light for 30 min. Following the scaffold and reservoir printing method in Example 1, a reservoir and a three-dimensional culture scaffold were constructed within the chip chamber.

[0149] Passaged HUVEC and LI-CAF cells were digested, resuspended in fresh culture medium, and cell density was measured using a cell counter. The two cell lines were then diluted with culture medium to a concentration of 2 × 10⁶ cells / mL. 4 / mL. 100 μL of HUVEC and LI-CAF cell suspensions were respectively added to a 96-well plate with a low-adsorption U-shaped bottom. After mixing well, the cells were centrifuged at 300 g for 5 min to induce cell aggregation. After static culture for 48 h, vascular cell spheres were formed.

[0150] Hepatocellular carcinoma organoids were cultured in Matrigel until they reached a size of 150 μm. The Matrigel was then broken up by pipetting in pre-cooled F12 medium at 4°C, collected in centrifuge tubes, and kept at 4°C for 15 min. After removing the Matrigel, the tubes were centrifuged at 300 g for 5 min, the supernatant was discarded, and the tubes were resuspended in fresh medium and counted.

[0151] Deacetylated Hep-NB, GFOGER, R-Spondin 1, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in PBS solution and thoroughly mixed. The concentrations of Hep-NB (0.35%), GFOGER (0.06%), R-Spondin 1 (5 μg / mL), Gel-NB (3.8%), 4-PEG-SH (2%), dextran (1%), and photoinitiator LAP (0.06%) were mixed. The mixture was then 3 × 10⁻⁶ ppm. 3 / mL of liver cancer organoids and 1×10 2 Biomimetic gel precursor solution A was prepared by using vascular cell spheres at a concentration of / mL.

[0152] Deacetylated Hep-NB, GFOGER, R-Spondin 1, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in PBS solution and thoroughly mixed. The concentrations of Hep-NB (0.35%), GFOGER (0.06%), R-Spondin 1 (5 μg / mL), Gel-NB (3.8%), 4-PEG-SH (1.2%), dextran (1%), and photoinitiator LAP (0.06%) were mixed. The mixture was then 3 × 10⁻⁶ ppm. 3 / mL of liver cancer organoids and 1×10 2Biomimetic gel precursor solution B was prepared by using vascular cell spheres at a concentration of / mL.

[0153] Using a pipette, 90 μL of biomimetic gel precursor solution A was added to the culture scaffold area in the center of the model chamber at a flow rate of 5 mW / cm². 2 Expose the sample to ultraviolet light at a certain intensity for 5 min, then invert it in a 37℃ incubator for 10 min to obtain co-culture model A. Use a pipette to add 90 μL of biomimetic gel precursor solution B to the culture scaffold area in the center of the model chamber at a concentration of 5 mW / cm². 2 Expose the sample to ultraviolet light intensity for 5 min, then invert it in a 37℃ incubator for 10 min to obtain co-culture model B.

[0154] Take two 10 mL syringes, draw an appropriate amount of 4°C pre-cooled PBS solution, and connect them to the inlet and outlet ports at both ends of the model via Teflon tubing. Add 400 μL of 4°C pre-cooled culture medium solution to the model chamber and incubate on ice for 15 min. Use a syringe pump to control the perfusion flow rate at 10 μL / min and maintain incubation on ice for 20 min. After confirming that F127 has been removed and the perfusion tubing is open, replace the solution in the syringes and model chamber with 37°C pre-warmed hepatocellular carcinoma organoid vascularization culture medium. Place the hepatocellular carcinoma organoid vascularization microphysiological model in an incubator and dynamically culture it at a flow rate of 10 μL / min. After 24 h, adjust the perfusion flow rate to 5 μL / min, and replace the culture medium in the syringes with fresh hepatocellular carcinoma organoid vascularization culture medium every 48 h.

[0155] The modulus of the biomimetic gel was tested using a rheometer. 600 μL of cell-free biomimetic gel precursor solution A or B was dropped into a 2 cm polytetrafluoroethylene mold, and the sample was tested under light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 Biomimetic gel discs were prepared by irradiating the samples under ultraviolet light for 5 min. The samples were then placed on the rheometer stage for rheological testing. After maintaining dynamic co-culture for 3 days, co-culture models A and B were observed under a microscope to compare the morphological differences of liver cancer organoids in the two systems.

[0156] like Figure 16 and Figure 17 As shown, the content of 4-PEG-SH in the biomimetic gel system is related to the gel strength. In system A, the content of 4-PEG-SH is 2%, and the storage modulus of the gel reaches 250 Pa, causing the liver cancer organoids to be compressed and deformed, and even fused. In system B, the content of 4-PEG-SH is 1.2%, and the storage modulus of the gel is around 100 Pa, allowing the liver cancer organoids to maintain normal morphology and growth.

[0157] Comparative Example 3: Lung Cancer Vasification Model - Static Culture and Dynamic Culture

[0158] After resuscitation, HUVECs (human umbilical vein endothelial cells) and LU-CAFs (lung cancer-associated fibroblasts) labeled with RFP (red fluorescent protein) were expanded and cultured until they covered 80% of the bottom of the flask, and then passaged at a cell ratio of 1:2. After further expansion and culture until 80% coverage, the cells were digested and resuspended in fresh culture medium, and the density of both cell types was measured using a cell counter. The cells were centrifuged at 300 g for 5 min, and 1×10⁻⁶ cells were prepared separately with culture medium. 7 HUVEC density / mL and 5×10 6 / mL of LU-CAF cell suspension.

[0159] Passaged HUVEC and LU-CAF cells were digested, resuspended in fresh culture medium, and cell density was measured using a cell counter. The two cell lines were then diluted with culture medium to a concentration of 4 × 10⁶ cells / mL. 4 / mL. Take 50 μL of HUVEC and LU-CAF cell suspensions respectively and add them to a 96-well plate with a low-adsorption U-shaped bottom. After mixing well, centrifuge at 300 g for 5 min to induce cell aggregation. After static culture for 48 h, vascular cell spheres are formed.

[0160] Lung cancer organoids were cultured in Matrigel until they reached a size of 120 μm. The Matrigel was then broken up by pipetting in pre-cooled F12 medium at 4°C, collected in centrifuge tubes, and kept at 4°C for 15 min. After removing the Matrigel, the tubes were centrifuged at 300 g for 5 min, the supernatant was discarded, and the organoids were resuspended in fresh medium. The tubes were repeatedly pipetted 25 times to obtain lung cancer organoids with a size of 60 μm. The density of the lung cancer organoids was adjusted to 5 × 10⁻⁶ using culture medium. 3 / mL.

[0161] Deacetylated Hep-NB, GFOGER, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were dissolved in PBS solution, and the above component solutions were thoroughly mixed. The concentrations of Hep-NB (0.35%), GFOGER (0.06%), VEGF (2 μg / mL), FGF2 (2 μg / mL), PDGF-BB (2 μg / mL), Gel-NB (2.5%), 4-PEG-SH (0.5%), MMP (0.08%), fibrinogen (0.1%), and photoinitiator LAP (0.05%) were mixed to a concentration of 1 × 10⁻⁶. 6 / mL of RFP-HUVEC cell suspension, 1×10 5 / mL of CAF cell suspension and 5×10 2A biomimetic gel precursor solution was prepared from lung cancer organoids at a concentration of / mL.

[0162] Use a pipette to draw 35 μL of the biomimetic gel precursor solution and add it dropwise into a 24-well low-adsorption culture plate at a concentration of 5 mW / cm². 2 Expose the samples to ultraviolet light for 5 min, then invert them in a 37°C incubator for 10 min. After removing them, add 500 μL of lung cancer organoid vascularization culture medium to each well, return them to the incubator for static culture, and replace the medium with fresh medium every 48 h.

[0163] A dynamic culture model of lung cancer vascularization was constructed following the steps in Example 2.

[0164] On day 5 of culture, lung cancer organoid vascularization models under static and dynamic culture systems were observed under a microscope, and imaging results were recorded to compare the vascularization process under static and dynamic culture conditions.

[0165] like Figure 18 and Figure 19 As shown, although endothelial cells in the static culture group could self-assemble to form blood vessel-like structures, the overall structure was discontinuous and failed to form a complete network. In the dynamic culture group, due to the more abundant supply of nutrients and the effect of fluid shear forces, a more complete vascular network structure could be formed, and the stability of the structure could be maintained for a longer culture time.

[0166] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a perfusionable tumor organoid vascularization model, characterized in that, Includes the following steps: Step S1: Prepare the chip, bracket, and liquid reservoir; The scaffold is fabricated using a dual-channel extrusion printing process, which includes sequentially and alternately printing multiple layers of first support layer and multiple layers of second support layer. The projections of the first support layer and the second support layer in the height direction of the scaffold are both mesh structures. The first support layer uses a thermosensitive biodegradable material, and the second support layer uses a photocrosslinked modified thermosensitive material. The liquid storage tank includes a liquid storage tank body and a baffle. The liquid storage tank body has an open cavity structure with a through hole at the bottom of the cavity. The baffle is adapted to cover the opening of the liquid storage tank body. The chip is provided with a culture chamber and liquid inlet and outlet ports at both ends. The liquid storage tank and the support are set in the culture chamber. The liquid storage tanks are assembled at both ends of the support. The liquid storage tanks are adapted to the liquid inlet and outlet ports of the chip. Step S2: Prepare tumor organoids, cell suspensions, and extracellular matrix precursor solutions; The cell suspension includes an endothelial cell suspension and a tumor-associated fibroblast suspension; The extracellular matrix precursor solution is a biogel precursor solution or a biomimetic gel precursor solution, wherein the biomimetic gel precursor solution includes a first biomimetic gel precursor solution and a second biomimetic gel precursor solution. The biogel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, matrix gel, fibrinogen and collagen. The first biomimetic gel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, norbornene-modified heparin, norbornene-modified gelatin, mercapto-tetra-arm polyethylene glycol, dextran, GFOGER, cytokines and photoinitiator, wherein the cytokines are one or more of R-spinal plate specific protein 1, vascular endothelial growth factor, basic fibroblast growth factor 2 and platelet-derived growth factor, and the amino acid sequence of GFOGER is shown in SEQ ID NO.1; The second biomimetic gel precursor solution is obtained by mixing endothelial cell suspension, tumor-associated fibroblast suspension, tumor organoids, norbornene-modified heparin, norbornene-modified gelatin, thiol-tetra-arm polyethylene glycol, dextran, matrix metalloproteinase mimic peptide, fibrinogen, and a photoinitiator. The concentration of norbornene-modified heparin is 0.2-2% w / v, the concentration of norbornene-modified gelatin is 0.5-5% w / v, the concentration of thiol-tetra-arm polyethylene glycol is 0.2-5% w / v, the concentration of dextran is 0.1-5% w / v, and the concentration of the photoinitiator is 0.01-0.1% w / v. The amino acid sequence of the matrix metalloproteinase mimic peptide is shown in SEQ ID NO.

2. Step S3: Model assembly and infusion; An extracellular matrix precursor solution is added to the culture chamber of the chip and cured by ultraviolet light. Pre-cooled culture medium is added to the culture chamber through the chip's inlet and outlet ports. The sacrificial scaffold is allowed to liquefy by being allowed to stand at low temperature. After confirming that the tubing is unobstructed, it is replaced with preheated tumor organoid vascularization culture medium and placed in an incubator for dynamic co-culture.

2. The construction method according to claim 1, characterized in that: In step S1, the bracket further includes multiple printed support columns, which penetrate multiple layers of the first support layer and multiple layers of the second support layer along the height direction of the bracket.

3. The construction method according to claim 1, characterized in that: The density of the tumor organoids is 5 × 10⁻⁶. 3- 2×10 4 / mL / mL, the cell density of the endothelial cell suspension is 5×10 6 / mL-1.5×10 7 / mL, the cell density of the tumor-associated fibroblast suspension is 5×10⁶ cells / mL. 5 / mL -2.5×10 6 / mL.

4. The construction method according to claim 1, characterized in that: In step S3, when the extracellular matrix precursor solution is a biomimetic gel precursor solution, the step of preparing a mask is also included. The mask includes a substrate and a cutout area. The cutout area is formed on the substrate and includes an outer frame cutout portion and an inner protrusion portion. The inner protrusion portion is connected to the inner wall of the outer frame cutout portion through at least one connecting portion to divide the outer frame cutout portion into multiple independent sub-cutout areas. The inner protrusion portion, the connecting portion and the substrate are an integral structure, and the outline of the cutout area matches the outline of the target partition photocuring area.

5. The construction method according to claim 4, characterized in that: In step S3, the first biomimetic gel precursor solution is dropped onto the culture scaffold area in the center of the model chamber, cured by ultraviolet light exposure, and then placed in an incubator for inverted incubation. The model is then removed, and the second biomimetic gel precursor solution is dropped onto both sides of the culture scaffold. After covering with a mask, it is cured by ultraviolet light exposure and then placed in an incubator for inverted incubation.

6. The construction method according to claim 1, characterized in that: In step S3, the perfusion flow rate is controlled at 5-20 μL / min during dynamic co-culture.

Images