Method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel and its use

Abstract

This invention provides a method for constructing an esophageal cancer biomimetic organ model based on a complex hydrogel and its use, belonging to the field of medical biomimetic organ models. The biomimetic organ model is manufactured using a hydrogel as its framework. This invention constructs an in vitro biomimetic organ model of esophageal cancer with bioactivity, better simulates Pg infection in esophageal cancer patients, and finally introduces commonly used chemotherapy drugs to confirm the relationship between Pg and malignant changes in esophageal cancer, verify the drug screening function of the model, and compare it with clinical treatment effects.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomimetic organ models, and specifically, to a method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel and its use.

Background Art

[0002] Cancer biological research tends to construct innovative in vitro 3D culture models because the in vivo cancer biological behavior cannot be reproduced in conventional and current 2D cell cultures. People usually focus on developing effective methods for treating cancer under the dual tube alignment in in vitro and in vivo model systems.

[0003] However, these still cannot simulate the pathophysiology of cancer. Two-dimensional cancer cell culture on polystyrene (TCP) is easy, but they cannot represent the physiological culture system and often lead to deceptive conclusions. Even if the 2D plate is wrapped with extracellular matrix (ECM) proteins (e.g., laminin, collagen, and fibrin), the lack of specific physiological patterns suppresses the adhesion, proliferation, signal transduction, migration, and response to treatment stimuli of cells in space.

[0004] Existing 3D biomimetic organ models are constructed through a non-cell-supported skeleton. For example, in the method for biomimetic construction of an artificial organ with the authorized announcement number CN104382670B, a resin model is constructed using a 3D model, and then a metal thin film is built, and a micro-nano fiber structure layer is deposited to remove the resin to construct the organ skeleton. Then, in the way of coating the thin film of the biomimetic organ model, an artificial biomimetic organ model is formed, and its physiological model is similar to a 2D thin film. The differences in the obtained experimental and test results are not large, and a culture system with true physiological significance could not be formed. That is, the biomimetic organ model also suppresses the adhesion, proliferation, signal transduction, migration, and response to treatment stimuli of cells in space.

[0005] On the other hand, using mouse models and xenotransplantation is an extremely expensive method, requiring high economic and time costs, expertise and skills in manipulating mice, and inevitably raising several moral and ethical issues. Furthermore, there are objective differences in the genotypes and biological characteristics of mouse and human stromal cells. In recent years, tissue engineering has made remarkable progress in the production of 3D in vitro models. These models can better simulate the complexity of the micro and coarse internal environment within the body, reconstruct the microecology of protoesophageal cancer, and improve tumor growth and dry markers. Such biomimetic models are likely to help predict patient recovery and provide constructive insights for fundamentally improving treatment.

[0006] In vivo models of esophageal cancer are difficult to develop because the carcinogen induction model relies heavily on the genetic background of the mice, making it difficult to simulate the tumor formation process in patients, and the cost of raising severely immunodeficient mice is high.

[0007] The background for genetically engineered mouse models is clear: tumors are in their original location in the esophagus, but they are costly, have too long a reproductive cycle, and esophageal cancer cell cultures are monolithic in type. During the process of cell permanence, genetic information shifts, and certain chromosomal fragments are lost or increased.

[0008] Biomimetic organ model cultures can highly replicate the microenvironment and behavior of in vivo tumor growth under low economic and time-consuming conditions. [Overview of the Initiative]

[0009] In view of this, the present invention provides a method for constructing an esophageal cancer biomimetic organ model based on a complex hydrogel and its use, for constructing an in vitro biomimetic organ model of esophageal cancer with bioactivity, for better simulating Pg infection in esophageal cancer patients, and finally introducing commonly used chemotherapy drugs to confirm the relationship between Pg and malignant changes in esophageal cancer, verify the drug screening function of the model and compare it with clinical treatment effects.

[0010] To solve the above technical problems, the present invention provides a method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel manufactured using a hydrogel as the framework of the biomimetic organ model. The components and mass percentage concentrations of the hydrogel are as follows: The methacrylic anhydride gelatin (GelMa) content is 5-15%. The polyethylene glycol diacrylate (PEGDA) content is 2-4%. The concentration of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) is 0.1-0.5%. The ultrapure water content is 80.5-92.9%.

[0011] The method for constructing the esophageal cancer biomimetic organ model is as follows: Step S1: The hydrogel preparation involves swirling together methacrylate anhydride gelatin, polyethylene glycol diacrylate, phenyl-2,4,6-trimethylbenzoylphosphinate lithium, and pure water to obtain the hydrogel. Step S2 involves constructing the plastic glass unit by coaxially arranging two coarse hollow glass tubes and a fine hollow glass tube of different diameters, thereby forming an inner and outer lumen on both the inside and outside of the fine hollow glass tube, and connecting the inner and outer lumens using two injection needles. Step S3, the construction of the microflow control unit, involves connecting the injection needle and syringe using a hose, connecting the syringe to the microflow pump, placing the internal phase escape material into the syringe connected to the lumen, and placing the hydrogel external phase into the syringe connected to the outer lumen. Step S4 involves the formation of a biomimetic organ model skeleton and cell loading, which includes driving a microflow pump to form a tubular fluid with an inner phase escape material and a hydrogel outer phase by the inner and outer lumens, further irradiating the tubular fluid with an ultraviolet curing lamp to obtain a solid hydrogel skeleton, culturing tumor cells to adhere to the inner wall of the solid hydrogel skeleton, culturing vascular endothelial cells to adhere to the outer wall to obtain an esophageal cancer biomimetic organ model, and having an inner phase flow velocity to outer phase flow velocity ratio of 1:2 to 10.

[0012] In this invention, a preform formed from a composite component of 2-4% polyethylene glycol diacrylate (PEGDA) and 5-15% methacrylic anhydride-gelatin (GelMA) is physically crosslinked under the catalytic effect of a 0.1% photoinitiator (Lap) to form a supramolecular crosslinked network.

[0013] PEGDA was obtained by polymerizing ethylene glycol monomer into polyethylene glycol (PEG), and then modifying both ends of the PEG polymer chain with acrylic molecules. PEGDA has more carbon-carbon double bond structures than the original PEG molecule. Under the action of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Lap), a specific photoinitiator, Lap splits into two radicals under photoexcitation of an appropriate wavelength, catalyzing the carbon-carbon double bond groups to generate intermolecular crosslinking, forming a supramolecular interpenetrating network, converting the preform from a liquid to a solid state and enabling photocuring. After crosslinking, PEGDA of different concentrations has different water absorption and mechanical hardness; higher concentrations result in greater hardness, lower gel water content, and smaller pores. In the process of inventing this invention, the inventors discovered that when the PEGDA concentration is less than 2%, the mechanical strength after the hydrogel is completely crosslinked is low, making it impossible to print continuous-length fibers by microflow-controlled coaxial printing. When the PEGDA concentration exceeds 4%, the hardness after the hydrogel crosslinking is too high, and the surface structure is too dense, which is unfavorable for sustained cell adhesion and free exchange of bioactive components.

[0014] Gelatin was prepared by chemically modifying it with methacrylic anhydride (MA). Gelatin is a readily available natural extracellular matrix that is widely present in animal tissues such as skin and fascia, possesses good biocompatibility, and can be used as a culture substrate for various stem cells such as neural stem cells (nSCs) and mesenchymal stem cells (mSCs). In the process of inventing this invention, the inventors discovered that when the GelMA concentration was less than 5%, the preform viscosity was too low, the fluidity was too high, it could not maintain stability before UV curing, and it dispersed easily in the collection solution. When the GelMA concentration exceeded 15%, the preform viscosity was too high, it was sensitive to changes in ambient temperature, it solidified easily when the room temperature was below 25°C, it clogged the equipment, and its fluidity and printability decreased.

[0015] GelMA molecules can also form composite hydrogels by catalyzing themselves with photoinitiators or by crosslinking with PEGDA or other carbon-carbon double bond-containing polymers.

[0016] As a photoinitiator, Lap's concentration is crucial to the final degree of crosslinking of the two photocurable hydrogels, and the degree of crosslinking of the hydrogels determines the porosity and water content of the forming material. In the process of the present invention, the inventors found that when the Lap concentration is less than 0.1%, the hydrogels cannot crosslink sufficiently and quickly, and cannot form continuous helical fibrous threads descending into the collection liquid, or the formed fibers have low mechanical strength and the hollow passages collapse and deform. When the Lap concentration exceeds 0.5%, the degree of crosslinking of the hydrogels is too high and crosslinking is too fast, making them prone to crosslinking at the outlet of the apparatus, clogging the conduits, wasting the photoinitiator, and increasing costs.

[0017] Furthermore, in step S2, the diameter ratio of the coarse hollow glass tube to the fine hollow glass tube is 3:1, the ratio of the inner diameter to the outer diameter of the coarse hollow glass tube is 1:2, and the ratio of the inner diameter to the outer diameter of the fine hollow glass tube is 1:1.2.

[0018] Furthermore, the internal phase escape material in step S3 is a polyvinyl alcohol solution, and the mass percentage concentration of the polyvinyl alcohol solution is 10%.

[0019] Furthermore, the ambient temperature of the hydrogel skeleton in step S4 is 25-28°C.

[0020] Furthermore, the tumor cells are located on the inner wall of the hydrogel skeleton, and the vascular endothelial cells are located on the outer wall of the hydrogel skeleton.

[0021] Furthermore, the wavelength of the ultraviolet curing lamp in step S4 is 365 nm, the power is 100 W, and the irradiation time of the ultraviolet curing lamp is 20 s.

[0022] Furthermore, both the tumor cells and the vascular endothelial cells in step S4 are cell suspensions, and the requirements for the adherent growth of the tumor cells and the vascular endothelial cells are as follows: When the tumor cells are in the logarithmic growth phase in a T75 culture flask, they are digested using pancreatic enzymes, the medium is resuspended, counted, and the cell concentration of the tumor cells is 1×10 7 cells / mL, When the vascular endothelial cells are in the logarithmic growth phase in a T75 culture flask, they are digested using pancreatic enzymes, the medium is resuspended, counted, and the cell concentration of the vascular endothelial cells is 5×10 5 cells / mL.

[0023] Furthermore, the method for the adherent growth of tumor cells on the inner wall of the hydrogel skeleton is as follows: Using a self-made needle with a tip of 100 μm, inject the cell suspension of tumor cells into the inside of the conduit of the hydrogel skeleton, then transfer the hydrogel skeleton into a 6-well plate, place it in an incubator at 37°C, incubate, and after 4 h, the tumor cells will adhere to the wall of the fiber passage.

[0024] Furthermore, the method for the adherent growth of vascular endothelial cells on the outer wall of the hydrogel skeleton is as follows: Drop a small amount of the cell suspension of high-concentration vascular endothelial cells onto the outer surface of the fiber several times using a syringe, gently transfer it to an incubator, incubate and culture, and after 4 h, the vascular endothelial cells will adhere to the outer surface of the hydrogel skeleton.

[0025] Another object of the present invention is a method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel, which is used when constructing the biomimetic organ model.

Brief Description of the Drawings

[0026] [Figure 1] It is a SEM photograph forming a hydrogel skeleton with different pore sizes at different crosslinking degrees in the present invention. [Figure 2] It is a stereoscopic optical microscope photograph of the hydrogel skeleton when forming vortex flow, laminar flow, and turbulent flow in the present invention. [Figure 3] It is an inverted optical microscope photograph of the influence of different passage inner diameters on cell distribution density and adhesion state in the present invention. [Figure 4] It is an inverted optical microscope bright field photograph with ESCC and HUVEC respectively supported on the inner and outer surfaces of the hydrogel skeleton in the present invention. [Figure 5] It is a field emission scanning electron microscope image of cells supported on the hydrogel skeleton in the present invention. [Figure 6] It is a schematic diagram of a plastic glass unit in the present invention.

Modes for Carrying Out the Invention

[0027] To make the objects, technical means, and advantages of the embodiments of the present invention clearer, hereinafter, referring to FIGS. 1 to 6 of the embodiments of the present invention, the technical means of the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are not all embodiments of the present invention, but some embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art belong to the protection scope of the present invention.

[0028] Methacrylated gelatin is GelMa, polyethylene glycol diacrylate is PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate is LAP.

Examples

[0029] In this example, tests and demonstrations were conducted regarding the proportions of methacrylic anhydride gelatin, polyethylene glycol diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and ultrapure water. The specific implementation procedure is as follows: Step 1: The hydrogel and outer phase are prepared by weighing the GelMa solid and placing it in a 10% mass percent first solution using ultrapure water. Then, PEGDA solution is added to the first solution to obtain a second solution, where the volume concentration of PEGDA solution in the second solution is 2.5%. Subsequently, LAP is added to the second solution to obtain a third solution, where the mass percentage concentration of LAP in the third solution is 0.1%. The third solution is vortex-kneaded at 26°C to obtain the hydrogel outer phase. PVA-205 is weighed and placed in an inner phase escape material with ultrapure water at 26°C to a 10% mass percent concentration. Step 2: Two rough hollow glass tubes were used, with an inner diameter of 580 μm and an outer diameter of 1.00 mm. One of the rough hollow glass tubes was removed, and one end was pulled out from the tapered tip using a tube stretcher. Then, the tapered tip was polished from a flat opening with a diameter of 400-500 μm using sandpaper. The other rough hollow glass tube was removed, fired and heat-stretched using a portable spray gun, and then cut into a thin hollow glass tube approximately 5 cm long at the appropriate position using a stone engraving pen. The inner diameter of the thin hollow glass tube was 100-150 μm. The two hollow glass tubes were immersed in anhydrous ethanol and treated in an ultrasonic cleaner for 3-5 minutes to remove electrostatically adsorbed dust and glass residue from polishing inside the tubes. After cleaning and drying the hollow glass tubes, the thicker one was used as the outer tube and the thinner one as the inner tube. They were arranged coaxially on a glass slide and fixed with epoxy resin, with the distance between the thinner hollow glass tube and the flat end controlled to approximately 200 μm, and the space in the center. Two flat-ended syringe needles were taken and cut with a blade to create one and two slits respectively, and placed at the entrances of the inner and outer tubes. They were then fixed in place with epoxy resin, and placed in a ventilated drying area for approximately 6 hours to ensure good airtightness, thus completing the construction of the microflow control device. Step 3: Using two 30cm plastic hoses, connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hoses is 1.5mm and the inner diameter is 1.02mm. Place the fluid passage of the hydrogel outer phase in a five-light environment, fix the syringe to a microflow pump, open the microflow pump, set the internal phase flow rate to 0.8mL / h and the external phase flow rate to 2mL / h, and allow the hydrogel outer phase and internal phase escape material to flow. Microflow-controlled spinning is performed, and both are synthesized into a single-layer hollow hydrogel skeleton. As shown in Figure 2, left 2, the two-phase fluid forms a laminar flow, generating a hydrogel skeleton with smooth passages. Before microflow-controlled spinning, the internal and external phase passages are completely filled with ultrapure water to maintain passage lubrication.

[0030] Using PBS as the collection solution, the hydrogel skeleton is collected. The PBS collection solution and hydrogel tube are received in a 5 cm tall glass bottle, and the eluted hydrogel skeleton is directly exposed to UV light for 20 seconds using a UV lamp curer. If the state is stable, a spirally descending fibrous hydrogel skeleton can be seen. The outer hydrogel phase transitions to a solidified state under UV irradiation conditions, and the inner PVA-205 solution disperses and overflows into the collection solution, forming hollow fibers with smooth passages.

[0031] Step 4: The hydrogel skeleton obtained by microflow spinning was washed 3-4 times with PBS, then immersed in 75% alcohol, irradiated under ultraviolet light for 1 hour, washed the alcohol from the hydrogel skeleton with sterile PBS, and then immersed overnight in 1640 culture medium.

[0032] Tumor cells were cultured in T75 culture bottles until the logarithmic growth phase, then digested with pancreatic enzymes, resuspended in the culture medium, and counted. The density was 1 × 10⁻⁶. 7 A cell suspension of cells / mL was aspirated using a 1mL syringe, and the tumor cell suspension was injected into the hydrogel skeleton tubing using a custom-made needle with a 100μm tip. The hydrogel skeleton was then transferred to a 6-well plate and incubated in a 37°C chamber. After 4 hours, the tumor cells adhered to the fibrous passages, and the hydrogel skeleton was transferred to another well.

[0033] Endothelial cells were cultured in T75 culture bottles until the logarithmic growth phase, digested with pancreatic enzymes, resuspended in the culture medium, and counted to 5 × 10⁶. 5 The cells were diluted to 1 / mL. A high-concentration suspension of vascular endothelial cells was dropped in small amounts onto the outer surface of the fibers several times using a syringe, gently transferred to a warming chamber, incubated, and after 4 hours, the vascular endothelial cells attached a wall to the outer surface of the hydrogel skeleton, obtaining a biomimetic organ model.

[0034] The above biomimetic organ model was scanned with an electron microscope and observed as shown in the upper right of Figure 1. [Examples]

[0035] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 5%, 2.5%, and 0.1%, respectively, with the remaining component being ultrapure water. [Examples]

[0036] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 15%, 2.5%, and 0.1%, respectively, with the remaining component being ultrapure water. [Examples]

[0037] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2%, and 0.1%, respectively, with the remaining component being ultrapure water. [Examples]

[0038] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 4%, and 0.1%, respectively, with the remaining component being ultrapure water. [Examples]

[0039] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 0.3%, respectively, with the remaining component being ultrapure water. [Examples]

[0040] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 0.5%, respectively, with the remaining component being ultrapure water. [Examples]

[0041] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 8%, 3.5%, and 0.2%, respectively, with the remaining component being ultrapure water. [Examples]

[0042] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 12%, 3.5%, and 0.3%, respectively, with the remaining component being ultrapure water. [Examples]

[0043] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 12%, 3%, and 0.4%, respectively, with the remaining component being ultrapure water. [Examples]

[0044] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 14%, 2.5%, and 0.4%, respectively, with the remaining component being ultrapure water. [Examples]

[0045] The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 7%, 3%, and 0.35%, respectively, with the remaining component being ultrapure water.

[0046] <Comparative Example 1> The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 5%, and 0.1%, respectively, with the remaining component being ultrapure water. The biomimetic organ models produced in the above proportions were scanned with an electron microscope and observed as shown in the upper left of Figure 1.

[0047] <Comparative Example 2> The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 1%, respectively, with the remaining component being ultrapure water. The biomimetic organ models produced in the above proportions were scanned with an electron microscope and observed as shown in the lower left of Figure 1.

[0048] <Comparative Example 3> The difference between this example and Example 1 is that the concentrations of three substances—GelMa (methacrylic anhydride gelatin), PEGDA (polyethylene glycol diacrylate), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)—were adjusted to obtain biomimetic organ models with different hardness and pores. The specific concentrations were: The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 0%, and 0.1%, respectively, with the remaining component being ultrapure water. The biomimetic organ models produced in the above proportions were scanned with an electron microscope and observed as shown in the lower right of Figure 1.

[0049] <Comparative Example 4> The difference between this embodiment and Embodiment 1 is that this embodiment involves related verification and testing to control the relationship between the internal and external phase flow velocities of the flow pump.

[0050] The difference between this embodiment and Embodiment 1 lies in the third step, specifically, Step 3: Using two 30cm plastic hoses, connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hoses is 1.5mm and the inner diameter is 1.02mm. Place the fluid passage of the hydrogel outer phase in a five-light environment, fix the syringe to a microflow pump, open the microflow pump, set the internal phase flow velocity to 0.1mL / h and the external phase flow velocity to 2mL / h, and allow the hydrogel outer phase and internal phase escape material to flow. Microflow-controlled spinning is performed, and both are synthesized into a single-layer hollow hydrogel skeleton. As shown in Figure 2, left 1, the two-phase fluid forms a vortex flow, generating a hydrogel skeleton with locally poreless or narrowed curved passages. Before microflow-controlled spinning, the internal and external phase passages are completely filled with ultrapure water to maintain passage lubrication.

[0051] <Comparative Example 5> The difference between this embodiment and Embodiment 1 is that this embodiment involves related verification and testing to control the relationship between the internal and external phase flow velocities of the flow pump.

[0052] The difference between this embodiment and Embodiment 1 lies in the third step, specifically, Step 3: Using two 30cm plastic hoses, connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hoses is 1.5mm and the inner diameter is 1.02mm. Place the fluid passage of the hydrogel outer phase in a five-light environment, fix the syringe to a microflow pump, open the microflow pump, set the internal phase flow rate to 0.4mL / h and the external phase flow rate to 0.4mL / h, and allow the hydrogel outer phase and internal phase escape material to flow. Microflow-controlled spinning is performed, and both are synthesized into a single-layer hollow hydrogel skeleton. As shown in Figure 2, left 3, the two-phase fluid forms turbulence, generating a hydrogel skeleton with locally wrinkled passages. Before microflow-controlled spinning, the internal and external phase passages are all filled with ultrapure water to maintain passage lubrication.

[0053] <Functionality Verification> The esophageal cancer biomimetic model, in which a hydrogel skeleton supports cells, is an open system to the culture medium. Transfer and administration to the overall model are achieved by adding a specific proportion of P. gingivalis (P. gland) solution or a certain concentration of drug to the medium.

[0054] Pg recovery and subculturing: Gram staining is used to observe the purity and condition of the bacterial suspension, and the OD value is measured to calculate the bacterial suspension concentration. A 1 mL Pg bacterial suspension with an OD value of 1 was collected, centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the suspension was resuspended in sterile PBS. The bacterial-to-cell ratio was 10:1, and the cells were infected for 24 hours. Changes in cell growth behavior before and after bacterial infection were observed. Using Sg (Streptococcus Gordon) as a negative control group, infection of cells at the same concentration for the same duration did not significantly affect cell proliferation and activity within the fibers.

[0055] Drug solutions with different concentration gradients were dissolved in serum-free medium, with armycin at concentrations of 0.5–64 μg / mL and 5-fluorouracil at concentrations of 10–1000 μg / mL, creating 10 concentrations for each group. A 24-hour solution exchange treatment was then performed.

[0056] The levels of asmycin were measured, and the half-lethal doses of 5-fluorouracil were 1.5 μg / mL and 160 μg / mL, respectively.

[0057] Immunofluorescence and immunogrouping of tissue samples are performed on a biomimetic esophageal model. Cell layers on the hydrogel surface are digested by pancreatic enzymes, and mixed cell lines are collected from the bracket surface. Functional tests such as fluid cell sorting, Western blotting, and PCR are then performed. This novel in vitro platform allows us to characterize the effects of gingival porphyrin monospores on the malignant progression of esophageal cancer, as well as the application response and prognosis of commonly used chemotherapy drugs in a new model. analysis

[0058] From the electron microscope scan results of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, it can be seen in Figure 1 that, when the GelMA concentration remains at 10%, the greater the ratio of PEGDA1 to LAP, the higher the degree of crosslinking of the hydrogel and the smaller the pores.

[0059] By mixing GelMA and PEGDA and performing microflow-controlled spinning, the mechanical strength of the cured material is ensured, and the mechanical modulus of the cured material can be adjusted by changing the ratio of PEGDA and LAP, thereby obtaining biomaterials with different hardness and porosity as shown in Figure 1.

[0060] Simultaneously, the incorporation of GelMA provides good biocompatibility to the entire material, and in this way, hydrogels of a specific form can carry cells outside the body and form bioresponsive microtissues and organic matter.

[0061] Observations of the photographs of the optical microscope in Example 1, Comparative Example 4, and Comparative Example 5 show that when the external phase flow velocity is controlled to 2 mL / h, the inner diameter of the passage increases as the internal phase flow velocity increases, and when the internal phase flow velocity is fixed at 0.4 mL / h, the inner diameter of the passage decreases as the external phase flow velocity increases.

[0062] When the flow velocity ratio between the outer and inner phases changes between 2 and 10, a stable laminar flow can be formed to coaxially print a hydrogel framework with smooth passages, as shown in Figure 2, left 2.

[0063] However, if the outer phase velocity is too high and the inner phase velocity is relatively too low, causing the two-phase fluid to form a vortex, a hydrogel framework that is either locally pore-less or narrows the curved passage will be printed, as shown in Figure 2, left 1.

[0064] When the external phase velocity is too low and the internal phase velocity is relatively too high, causing the two-phase fluid to form turbulence, a hydrogel skeleton with localized wrinkled passages is printed, as shown in Figure 2, left 3.

[0065] As shown in Figure 3, when cells are loaded onto hydrogel skeletons with different inner diameters, the narrow inner diameter of the passage makes injection into the hollow passages of cells inconvenient, reducing operability. Furthermore, the cells tend to aggregate and cluster because they cannot expand and adhere sufficiently.

[0066] If the diameter of the passage increases, cells that do not adhere to the wall after being injected into the passage will also fall off during operations such as fluid exchange and spill into the free medium, reducing the cell carrying efficiency of the fiber bracket.

[0067] Considering the stability of the internal and external phase fluids in the coaxial apparatus and the surface adhesion of cells in different passage diameters, we chose to set the external phase flow rate to 2 mL / h and the internal phase flow rate to 0.8 mL / h.

[0068] The above is a preferred embodiment of the present invention, and those skilled in the art can make several improvements and finishes without departing from the principles of the present invention, and these improvements and finishes should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel manufactured using a hydrogel as the framework of the biomimetic organ model, The components and mass percentage concentrations of the hydrogel are as follows: The methacrylic anhydride gelatin content is 5-15%. The polyethylene glycol diacrylate content is 2-4%. The concentration of lithium phenyl-2,4,6-trimethylbenzoylphosphinate is 0.1-0.5%. The ultrapure water content is 80.5-92.9%. The method for constructing the esophageal cancer biomimetic organ model is as follows: Step S1: The hydrogel is prepared by swirling together methacrylate anhydride gelatin, polyethylene glycol diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and pure water to obtain the hydrogel. Step S2: The construction of the plastic glass unit involves coaxially arranging two coarse hollow glass tubes and a fine hollow glass tube of different diameters, thereby forming an inner and outer lumen on both the inside and outside of the fine hollow glass tube, and connecting the inner and outer lumens using two injection needles. Step S3 involves constructing the microflow control unit by connecting the injection needle and syringe using a hose, connecting the syringe to the microflow pump, placing the internal phase escape material into the syringe connected to the lumen, and placing the hydrogel external phase into the syringe connected to the outer lumen. Step S4: Formation of a biomimetic organ skeleton and cell support is performed by driving a microflow pump to form a tubular fluid with an inner phase escape material and a hydrogel outer phase by the inner and outer lumens, further irradiating the tubular fluid with an ultraviolet curing lamp to obtain a solid hydrogel skeleton, culturing tumor cells to adhere to the inner wall of the solid hydrogel skeleton, culturing vascular endothelial cells to adhere to the outer wall to obtain an esophageal cancer biomimetic organ model, and the ratio of the inner phase flow velocity to the outer phase flow velocity is 1:2 to 10, characterized in that the method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel is characterized in that the inner phase flow velocity is 1:2 to 10.

2. A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that the diameter ratio of the coarse hollow glass tube to the fine hollow glass tube in step S2 is 3:1, the ratio of the inner diameter to the outer diameter of the coarse hollow glass tube is 1:2, and the ratio of the inner diameter to the outer diameter of the fine hollow glass tube is 1:1.

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3. The method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that the internal phase escape material in step S3 is a polyvinyl alcohol solution, and the mass percentage concentration of the polyvinyl alcohol solution is 10%.

4. A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that the ambient temperature of the hydrogel skeleton in step S4 is 25 to 28°C.

5. A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that the tumor cells are located on the inner wall of the hydrogel skeleton and the vascular endothelial cells are located on the outer wall of the hydrogel skeleton.

6. A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that the wavelength of the ultraviolet curing lamp in step S4 is 365 nm, the power is 100 W, and the irradiation time of the ultraviolet curing lamp is 20 s.

7. In step S4, both the tumor cells and vascular endothelial cells are cell suspensions, and the requirements for wall-attaching growth of the tumor cells and vascular endothelial cells are, When tumor cells are in the logarithmic growth phase in a T75 culture bottle, they are digested with pancreatic enzymes, the culture medium is resuspended, and the cells are counted. The cell concentration of the tumor cells is 1 × 10⁻⁶. ７ It is ⸪ / mL, When vascular endothelial cells are in the logarithmic growth phase in a T75 culture bottle, they are digested with pancreatic enzymes, the culture medium is resuspended, and the cells are counted. The cell concentration of the vascular endothelial cells is 5 × 10⁻⁶. ５ A method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel according to claim 1, characterized in that the concentration is 1 / mL.

8. The method for allowing tumor cells to adhere to and grow on the inner wall of the aforementioned hydrogel skeleton is: A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel, characterized in that a cell suspension of tumor cells is injected into the tubules of a hydrogel skeleton using a self-made needle with a 100 μm tip, the hydrogel skeleton is then transferred to a 6-well plate, incubated in a 37°C warming chamber, and after 4 hours, tumor cells adhere to the walls of the fibrous passages.

9. The method for performing wall-adhesion growth of vascular endothelial cells on the outer wall of the aforementioned hydrogel skeleton is: A method for constructing a biomimetic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that a high concentration of vascular endothelial cell suspension is dropped in small amounts several times onto the outer surface of a fiber using a syringe, gently transferred to a warming box, incubated, and after 4 hours, the vascular endothelial cells attach a wall to the outer surface of the hydrogel skeleton.

10. The method for constructing an esophageal cancer biomimetic organ model based on a composite hydrogel according to any one of claims 1 to 9 is characterized by being used when constructing the biomimetic organ model.

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