Method for constructing composite hydrogel-based esophageal cancer bionic organ model, and application of method

Abstract

Provided are a method for constructing a composite hydrogel-based esophageal cancer bionic organ model, and an application of the method, relating to the technical field of medical bionic organ models. Hydrogel is used as a backbone of a bionic organ model for preparation. An esophageal cancer in-vitro bionic organ model having biological activity is constructed, thereby better simulating in-vivo infection of Pg of an esophageal cancer patient; and finally, by introducing clinically common chemotherapeutic drugs, the association between Pg and malignant transformation of esophageal cancer is confirmed, the drug screening function of the model is verified, and the comparison with the clinical efficacy is carried out.

Description

Construction method and application of esophageal cancer bionic organ model based on composite hydrogel Technical Field

[0001] The present invention relates to the technical field of medical bionic organ models, and in particular to a method for constructing an esophageal cancer bionic organ model based on a composite hydrogel and applications thereof. Background Art

[0002] Cancer biology research is increasingly trending towards the construction of innovative in vitro 3D culture models, as traditional and current 2D cell cultures cannot reproduce in vivo cancer biological behavior. Researchers are often working to develop effective cancer treatments using a two-pronged approach using both in vitro and in vivo model systems.

[0003] However, these still fail to mimic the pathophysiology of cancer. Two-dimensional cancer cell cultures on polystyrene (TCP) are easy to perform, but they do not represent physiological culture systems and often lead to misleading conclusions. Even if 2D plates are coated with extracellular matrix (ECM) proteins such as laminin, collagen, and fibronectin, the absence of specific physiological patterns can inhibit cell adhesion, proliferation, signal transduction, migration, and response to therapeutic stimuli in space.

[0004] Existing 3D bionic organ models are established through non-cell-supported skeletons. For example, a bionic construction method for artificial organs with authorization announcement number CN104382670B uses a 3D model to construct a resin model, then constructs a metal film, deposits a micro-nano fiber structure layer to remove the resin, and constructs an organ skeleton. The bionic organ model film is then covered to form an artificial bionic organ model. Its physiological pattern is similar to that of a 2D film, and the experimental and test results obtained are not much different. It is impossible to form a truly physiological culture system, that is, the bionic organ model will also inhibit the adhesion, proliferation, signal transduction, migration and response to therapeutic stimuli of cells in space.

[0005] On the other hand, the use of mouse models and xenotransplantation is a very expensive method, requiring high economic and time costs, expertise and skills in operating mice, and inevitably some ethical issues. In addition, there are objective differences in the genotype and biological characteristics of mouse and human stromal cells. In recent years, tissue engineering has achieved remarkable results in the creation of 3D in vitro models. These models can better simulate the complexity of the microscopic and macroscopic in vivo environment to reconstruct the native esophageal cancer microecology and upregulate tumor proliferation and stemness markers. Such biomimetic models are more likely to help predict patient prognosis and provide constructive suggestions for fundamentally improving treatment.

[0006] In vivo models of esophageal cancer: Carcinogen-induced models are largely dependent on the genetic background of the mice and are difficult to simulate tumor formation in patients. The breeding cost of severely immunodeficient mice is high and difficult.

[0007] The background of the genetically engineered mouse model is clear, and the tumor forms in situ in the esophagus, but the cost is high and the reproduction cycle is too long. The esophageal cancer cell line culture is of a single type, and during the process of cell immortalization, the genetic information shifts, and certain specific chromosome segments are lost or added.

[0008] The cultivation of bionic organ models can highly restore the microenvironment and behavior of tumor growth in vivo at low economic and time costs.

[0009] Summary of the Invention

[0010] In view of this, the present invention provides a method for constructing an esophageal cancer bionic organ model based on composite hydrogel and its application, constructing an in vitro bionic organ model of esophageal cancer with biological activity, better simulating the in vivo infection of Pg in esophageal cancer patients, and finally introducing common clinical chemotherapy drugs to confirm the association between Pg and esophageal cancer malignancy, verifying the drug screening function of the model and comparing it with the clinical efficacy.

[0011] To solve the above technical problems, the present invention provides a method for constructing an esophageal cancer bionic organ model based on a composite hydrogel, wherein the hydrogel is used as the skeleton of the bionic organ model. The components and mass percentage concentrations of the hydrogel are as follows:

[0012] The method for constructing the esophageal cancer bionic organ model specifically comprises the following steps:

[0013] S1, preparing a hydrogel, vortex-mixing methacrylic anhydride gelatin, polyethylene glycol diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and pure water to obtain a hydrogel;

[0014] S2. Build the shaped glass unit by placing two thick hollow glass tubes of different diameters coaxially with a thin hollow glass tube, forming an inner cavity and an outer cavity on the inner and outer sides of the thin hollow glass tube, and connecting the inner and outer cavities with two injection needles.

[0015] S3. Construction of a microfluidic control unit: connecting the injection needle to the syringe using a flexible tube, and connecting the syringe to a microfluidic pump; loading the inner phase escaping material into the syringe connected to the inner cavity, and loading the hydrogel outer phase into the syringe connected to the outer cavity;

[0016] S4. Formation of the bionic organ model skeleton and cell loading, driving the microflow pump, so that the inner phase escape material and the hydrogel outer phase form a tubular fluid through the inner and outer cavities, and then irradiating the tubular fluid with a UV curing lamp to obtain a solid hydrogel skeleton. Tumor cells and vascular endothelial cells are grown on the inner and outer walls of the hydrogel skeleton respectively to obtain an esophageal cancer bionic organ model. The ratio of the inner phase flow rate to the outer phase flow rate is 1:2-10.

[0017] The present invention adopts 2-4% polyethylene glycol diacrylate (PEGDA) and 5-15% methacrylic anhydride-gelatin (GelMA). The precast gel formed by the composite components is physically cross-linked under the catalysis of the photoinitiator 0.1% Lap to form a supramolecular cross-linked network.

[0018] PEGDA is obtained by polymerizing ethylene glycol monomers to form polyethylene glycol (PEG), followed by the addition of acrylic acid molecules to both ends of the PEG macromolecular chain. PEGDA has an additional carbon-carbon double bond compared to the original PEG molecule. Under the action of a specific photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Lap), Lap undergoes homolytic cleavage into two free radicals under light excitation of an appropriate wavelength. These radicals catalyze intermolecular crosslinking of the carbon-carbon double bond groups, forming a supramolecular interpenetrating network that transforms the preformed gel from a liquid to a solid state, achieving photocuring. After crosslinking, different PEGDA concentrations exhibit varying water absorption and mechanical hardness. Higher concentrations increase hardness, reduce gel water content, and reduce pore size. During the development of this invention, the inventors discovered that when the PEGDA concentration is below 2%, the mechanical strength of the fully crosslinked hydrogel is low, making it impossible to print continuous-length fibers via microfluidic coaxial printing. When the PEGDA concentration is above 4%, the crosslinked hydrogel becomes too hard and the surface structure is too dense, hindering sustained cell adhesion and the free exchange of bioactive components.

[0019] Gelatin is prepared by chemical modification and addition of methacrylic anhydride (MA). Since gelatin is widely present in animal skin, sarcolemma and other tissues, it is an easily available natural extracellular matrix with good biocompatibility. It can be used as a culture matrix for various stem cells such as neural stem cells (nSC) and mesenchymal stem cells (mSC). During the invention process of the present invention, the inventors found that: when the concentration of GelMA is lower than 5%, the viscosity of the prefabricated adhesive is too low and the fluidity is large. It cannot maintain stability before UV curing and is easily dispersed in the collection liquid. When the concentration of GelMA is higher than 15%, the viscosity of the prefabricated adhesive is too high and it is sensitive to changes in ambient temperature. It is easy to solidify when the room temperature is below 25°C, clogging the device, and reducing the fluidity and printing suitability.

[0020] GelMA molecules can also form composite hydrogels by cross-linking themselves or with other macromolecules containing carbon-carbon double bonds through photoinitiator catalysis.

[0021] As a photoinitiator, Lap's concentration is crucial to the ultimate crosslinking degree of the two photocurable hydrogels, which in turn determines the porosity and water content of the resulting material. During the development of this invention, the inventors discovered that when the Lap concentration is below 0.1%, the hydrogel fails to fully and rapidly crosslink, failing to form continuous spiraling fibers in the collected fluid or resulting fibers exhibit low mechanical strength and collapse and deformation of the hollow channels. When the Lap concentration is above 0.5%, the hydrogel crosslinks excessively and rapidly, easily causing crosslinking at the device outlet, leading to clogging of the catheter, waste of photoinitiator, and increased costs.

[0022] Furthermore, in step S2, the diameter ratio of the thick hollow glass tube to the thin hollow glass tube is 3:1, the ratio of the inner diameter to the outer diameter of the thick hollow glass tube is 1:2, and the ratio of the inner diameter to the outer diameter of the thin hollow glass tube is 1:1.2.

[0023] Furthermore, in step S3, the internal phase escaping material is an aqueous solution of polyvinyl alcohol, and the mass percentage concentration of the polyvinyl alcohol in the aqueous solution is 10%.

[0024] Furthermore, the temperature of the environment in which the hydrogel skeleton is located in step S4 is 25-28°C.

[0025] 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.

[0026] Furthermore, in step S4, the wavelength of the ultraviolet curing lamp is 365 nm, the power is 100 W, and the irradiation time of the ultraviolet curing lamp is 20 s.

[0027] Furthermore, in step S4, the tumor cells and vascular endothelial cells are both cell suspensions, and the requirements for the tumor cells and vascular endothelial cells to adhere to the wall and grow are as follows:

[0028] When the tumor cells were in the logarithmic growth phase in the T75 culture flask, they were digested with trypsin, resuspended in culture medium, and counted. The cell concentration of the tumor cells was 1×10 7 / mL;

[0029] When the vascular endothelial cells were in the logarithmic growth phase in the T75 culture flask, they were digested with trypsin, resuspended in the culture medium, and counted. The cell concentration of the vascular endothelial cells was 5×10 5 pieces / mL.

[0030] Furthermore, the method for growing tumor cells adherently on the inner wall of the hydrogel skeleton is as follows:

[0031] A homemade needle with a 100 μm tip was used to inject the tumor cell suspension into the channels of the hydrogel skeleton. The hydrogel skeleton was then transferred to a 6-well plate and incubated in a 37°C incubator. After 4 hours, the tumor cells adhered to the fiber channel.

[0032] Furthermore, the method for growing vascular endothelial cells adherently on the outer wall of the hydrogel skeleton is as follows:

[0033] A high concentration of vascular endothelial cell suspension was added dropwise to the outer surface of the fiber through a syringe in small amounts multiple times, and then smoothly transferred to an incubator for incubation and culture. After 4 hours, the vascular endothelial cells adhered to the outer surface of the hydrogel skeleton.

[0034] Another object of the present invention is to use the method for constructing an esophageal cancer bionic organ model based on a composite hydrogel in constructing a bionic organ model. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is an SEM photograph of a hydrogel skeleton with different pore sizes formed by different cross-linking degrees of the present invention;

[0036] FIG2 is a stereoscopic optical microscopic photograph of the hydrogel skeleton when eddy flow, laminar flow, and turbulent flow are formed according to the present invention;

[0037] FIG3 is an inverted optical microscope photograph showing the effects of different channel inner diameters on cell distribution density and adhesion status according to the present invention;

[0038] FIG4 is an inverted light microscopic bright field photograph of the inner and outer surfaces of the hydrogel skeleton of the present invention loaded with ESCC and HUVEC, respectively;

[0039] FIG5 is a field emission scanning electron microscopy image of cells loaded on the hydrogel skeleton of the present invention;

[0040] FIG6 is a schematic diagram of a shaping glass unit according to the present invention. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with Figures 1-6 of the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present invention.

[0042] Methacrylated gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP

[0043] Example 1

[0044] This example is a test and demonstration of the ratio of methacrylic anhydride gelatin, polyethylene glycol diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate and ultrapure water. The specific implementation steps are as follows:

[0045] The first step is to configure the hydrogel outer phase. Weigh the GelMa solid and use ultrapure water to configure it into a first solution with a mass percentage concentration of 10%. Add PEGDA solution to the first solution to obtain a second solution, so that the volume concentration of PEGDA solution in the second solution is 2.5%. Then add LAP to the second solution to obtain a third solution, and the mass percentage concentration of LAP in the third solution is 0.1%. Vortex mix the third solution at 26°C to obtain the hydrogel outer phase. Weigh PVA-205 and use ultrapure water to configure it into a 10% mass percentage concentration of the internal phase escape material at 26°C.

[0046] The second step is to use two thick hollow glass tubes, the inner diameter of the thick hollow glass tube is 580μm, and the outer diameter of the thick hollow glass tube is 1.00mm. Take one of the thick hollow glass tubes, use a tube puller to pull out a tapered tip at one end, and then use sandpaper to grind the tapered tip into a flat end with a diameter of 400-500μm; take the other thick hollow glass tube, use a portable spray gun to calcine and hot pull, and use a stone carving pen to cut out a thin hollow glass tube with a length of about 5cm at the appropriate position, and the inner diameter of the thin hollow glass tube is 100-150μm; immerse the two hollow glass tubes in anhydrous ethanol and put them into an ultrasonic Process in a cleaning machine for 3-5 minutes to remove the dust adsorbed by static electricity in the tube and the glass residue produced by grinding. After the hollow glass tube is clean and dry, the thicker one is used as the outer tube and the thinner one is used as the inner tube. They are coaxially arranged on a glass slide and fixed with epoxy resin. The distance between the thin hollow glass tube and the flat end is controlled to be about 200μm, and the space is centered; take two flat-end syringe needles and use a blade to cut one and two notches respectively and place them at the entrance of the inner and outer tubes respectively. They are also fixed with epoxy resin and placed in a ventilated and dry place. The curing time is about 6 hours to ensure good airtightness, and the construction of the microfluidic device is completed.

[0047] The third step is to use two 30cm plastic hoses to connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hose is 1.5mm and the inner diameter is 1.02mm. The fluid channel of the hydrogel outer phase is placed in a five-light environment. The syringe is fixed on the microflow pump and the microflow pump is turned on to make the inner phase flow rate 0.8mL / h and the outer phase flow rate 2mL / h. The hydrogel outer phase and the inner phase escape material flow for microfluidic spinning. The two cooperate to generate a single-layer hollow structure hydrogel skeleton, as shown in the second left in Figure 2. The two-phase fluid forms a laminar flow to produce a hydrogel skeleton with a smooth channel. Before microfluidic spinning, both the inner and outer phase channels must be filled with ultrapure water to keep the channels lubricated.

[0048] PBS was used as the collecting liquid to collect the hydrogel skeleton. A 5-cm-high glass bottle was used to receive the PBS collecting liquid and the hydrogel tube. The outflowing hydrogel skeleton was directly irradiated with a UV lamp curing device for 20 seconds. When the state was stable, a spirally descending fibrous hydrogel skeleton could be seen. The outer phase hydrogel was transformed into a solidified state under UV light conditions, while the inner phase PVA-205 solution overflowed into the collecting liquid, forming a hollow fiber with a smooth channel.

[0049] Step 4: Wash the hydrogel skeleton obtained by microfluidic spinning with PBS 3-4 times, then soak the hydrogel skeleton in 75% alcohol and irradiate it under ultraviolet light for 1 hour. After washing the alcohol in the hydrogel skeleton with sterile PBS, the hydrogel skeleton was immersed in 1640 culture medium overnight.

[0050] When the tumor cells were cultured in a T75 culture flask to the logarithmic growth phase, they were digested with trypsin, resuspended in culture medium, and counted. A 1 ml syringe was used to draw cells at a density of 1×10 7 A cell suspension of 100 μg / mL of tumor cells was injected into the channels of the hydrogel skeleton using a homemade needle with a 100 μm tip. The hydrogel skeleton was then transferred to a 6-well plate and incubated in a 37°C incubator. After 4 hours, the tumor cells adhered to the fiber channels, and the hydrogel skeleton was clamped and transferred to another well.

[0051] Endothelial cells were cultured in T75 culture flasks until the logarithmic growth phase, digested with trypsin, resuspended in culture medium, counted, and diluted to 5 × 10 5 / mL; a high concentration of vascular endothelial cell suspension was added dropwise to the outer surface of the fiber through a syringe in small amounts and multiple times, and then steadily transferred to an incubator for incubation and culture. After 4 hours, the vascular endothelial cells adhered to the outer surface of the hydrogel skeleton to obtain a bionic organ model.

[0052] The above bionic organ model was scanned by electron microscope, and the observation was as shown in the upper right of Figure 1.

[0053] Example 2

[0054] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0055] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 5%, 2.5%, and 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0056] Example 3

[0057] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0058] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 15%, 2.5%, and 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0059] Example 4

[0060] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0061] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2%, and 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0062] Example 5

[0063] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0064] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 4%, and 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0065] Example 6

[0066] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0067] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 0.3%, respectively, and the remaining ingredients are ultrapure water;

[0068] Example 7

[0069] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0070] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 0.5%, respectively, and the remaining ingredients are ultrapure water;

[0071] Example 8

[0072] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0073] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 8%, 3.5%, and 0.2%, respectively, and the remaining ingredients are ultrapure water;

[0074] Embodiment 9

[0075] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0076] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 12%, 3.5%, and 0.3%, respectively, and the remaining ingredients are ultrapure water;

[0077] Example 10

[0078] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0079] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 12%, 3%, and 0.4%, respectively, and the remaining ingredients are ultrapure water;

[0080] Example 11

[0081] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0082] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 14%, 2.5%, and 0.4%, respectively, and the remaining ingredients are ultrapure water;

[0083] Example 12

[0084] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0085] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 7%, 3%, and 0.35%, respectively, and the remaining ingredients are ultrapure water;

[0086] Comparative Example 1

[0087] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0088] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 5%, and 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0089] The bionic organ model made according to the above proportions was scanned by an electron microscope, and the observations were as shown in the upper left of Figure 1.

[0090] Comparative Example 2

[0091] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0092] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%, 2.5%, and 1%, respectively, and the remaining ingredients are ultrapure water;

[0093] The bionic organ model made according to the above proportions was scanned by an electron microscope, and the result was as shown in the lower left of Figure 1.

[0094] Comparative Example 3

[0095] The difference between this embodiment and the first embodiment is that the concentrations of methacrylic anhydride gelatin: GelMa, polyethylene glycol diacrylate: PEGDA, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate: LAP are adjusted to obtain bionic organ models with different hardness and porosity. The specific concentrations are as follows:

[0096] The concentrations of methacrylic anhydride gelatin, polyethylene glycol diacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are 10%: 0%: 0.1%, respectively, and the remaining ingredients are ultrapure water;

[0097] The bionic organ model made according to the above proportions was scanned by an electron microscope, and the result was as shown in the lower right of Figure 1.

[0098] Comparative Example 4

[0099] The difference between this embodiment and the first embodiment is that the relationship between the inner phase flow rate and the outer phase flow rate controlled by the flow pump is verified and tested.

[0100] The difference between this embodiment and the first embodiment lies in the third step, and the specific differences are as follows:

[0101] The third step is to use two 30cm plastic hoses to connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hose is 1.5mm and the inner diameter is 1.02mm. The fluid channel of the hydrogel outer phase is placed in a five-light environment. The syringe is fixed on the micro-flow pump and the micro-flow pump is turned on to make the inner phase flow rate 0.1mL / h and the outer phase flow rate 2mL / h. The hydrogel outer phase and the inner phase escape material flow for microfluidic spinning. The two cooperate to generate a single-layer hollow structure hydrogel skeleton, as shown in the left one in Figure 2. The two-phase fluid forms a vortex, producing a hydrogel skeleton with locally no pores or narrowed and curved channels. Before microfluidic spinning, both the inner and outer phase channels must be filled with ultrapure water to keep the channels lubricated.

[0102] Comparative Example 5

[0103] The difference between this embodiment and the first embodiment is that the relationship between the inner phase flow rate and the outer phase flow rate controlled by the flow pump is verified and tested.

[0104] The difference between this embodiment and the first embodiment lies in the third step, and the specific differences are as follows:

[0105] The third step is to use two 30cm plastic hoses to connect the injection needle to the syringe containing the hydrogel. The outer diameter of the plastic hose is 1.5mm and the inner diameter is 1.02mm. The fluid channel of the hydrogel outer phase is placed in a five-light environment. The syringe is fixed on the microflow pump and the microflow pump is turned on to make the inner phase flow rate 0.4mL / h and the outer phase flow rate 0.4mL / h. The hydrogel outer phase and the inner phase escape material flow and perform microfluidic spinning. The two cooperate to generate a single-layer hollow structure hydrogel skeleton, as shown in the third from the left in Figure 2. The two-phase fluid forms turbulence, producing a hydrogel skeleton with locally wrinkled and tortuous channels. Before microfluidic spinning, both the inner and outer phase channels must be filled with ultrapure water to keep the channels lubricated.

[0106] Functional Verification

[0107] The hydrogel skeleton-loaded cell-based esophageal cancer biomimetic model is an open system relative to the culture medium. A specific proportion of Pg bacterial solution or a certain concentration of drug is added to the culture medium to achieve transfection and medication of the entire model.

[0108] Pg resuscitation and passage; Gram staining to observe its purity and condition; measuring OD value to calculate bacterial concentration;

[0109] Take 1 mL of Pg bacterial solution with an OD value of 1 and centrifuge at 12000 rpm for 10 minutes, discard the supernatant and resuspend in sterile PBS; infect cells with a ratio of bacteria to cells of 10:1 for 24 hours, and observe the changes in cell growth behavior before and after bacterial infection;

[0110] Sg (Streptococcus gordonii) was used as a negative control group. The cells were infected with the same concentration and for the same time, and it was found that it had no significant effect on the cell proliferation and activity in the fibers.

[0111] Serum-free culture medium was used to dissolve and prepare drug solutions with different concentration gradients, including doxorubicin from 0.5-64 μg / mL and 5-fluorouracil from 10-1000 μg / mL. Ten concentrations were set for each group, and the solution was changed for 24 hours.

[0112] The median lethal doses of doxorubicin and 5-fluorouracil were calculated to be 1.5 μg / mL and 160 μg / mL, respectively.

[0113] The bionic esophageal model was subjected to in situ immunofluorescence, immunohistochemistry, and ELISA. The cell layer on the hydrogel surface was digested with trypsin, and the mixed cell lines on the scaffold surface were recovered for flow cytometry sorting, Western Blot, PCR and other functional tests. A new platform was constructed in vitro to characterize the effect of Porphyromonas gingivalis on the malignant progression of esophageal cancer, as well as the application response and prognosis of commonly used clinical chemotherapy drugs in the new model.

[0114] analyze

[0115] From the electron microscopy scanning results of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, it can be seen that in Figure 1, when the GelMA concentration remains unchanged at 10%, the greater the ratio of PEGDA1 to LAP, the higher the cross-linking degree of the hydrogel and the smaller the pores.

[0116] Microfluidic spinning of a mixture of GelMA and PEGDA can not only ensure the mechanical strength of the cured material, but also adjust the mechanical modulus of the cured material by adjusting the ratio of PEGDA to LAP, thus obtaining biomaterials with different hardness and porosity as shown in Figure 1;

[0117] At the same time, the incorporation of GelMA provides better biocompatibility for the material as a whole. In this way, hydrogels with specific morphologies can load cells in vitro to form bioresponsive microtissues and organic-like substances.

[0118] Through observation of the photos of the stereomicroscope of Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that when the external phase flow rate is controlled at 2 mL / h, the inner diameter of the channel increases as the internal phase flow rate increases; the internal phase flow rate is fixed at 0.4 mL / h, and the inner diameter of the channel decreases as the external phase flow rate increases.

[0119] When the flow rate ratio of the outer phase to the inner phase varies between 2 and 10, a stable laminar flow can be formed, and a hydrogel skeleton with smooth channels can be coaxially printed, as shown in Figure 2 (second from the left).

[0120] However, if the external phase flow rate is too high and the internal phase flow rate is relatively too low, the two-phase fluid will form a vortex, and a hydrogel skeleton with local no pores or narrow and curved channels will be printed, as shown in the left side of Figure 2;

[0121] If the external phase flow rate is too low and the internal phase flow rate is relatively too high, the two-phase fluid will form turbulence, and a hydrogel skeleton with local wrinkled and tortuous channels will be printed, as shown in Figure 2 (third from the left).

[0122] By loading cells into hydrogel skeletons with different inner diameters, as shown in Figure 3, when the inner diameter of the channel is too small, it is not convenient to inject cells into the hollow channel, the operability is reduced, and the cells cannot fully stretch and adhere, and tend to aggregate and distribute in clusters.

[0123] When the inner diameter of the channel is too large, cells injected into the channel that are not attached to the wall will fall off and overflow into the free culture medium during operations such as fluid replacement, reducing the cell loading efficiency of the fiber scaffold.

[0124] Considering the stability of the internal and external phase fluids in the coaxial device and the surface adhesion of cells under different channel inner 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.

[0125] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing an esophageal cancer bionic organ model based on a composite hydrogel, characterized in that: The hydrogel is used as the skeleton of the bionic organ model for preparation. The components and mass percentage concentrations of the hydrogel are as follows: The method for constructing the esophageal cancer bionic organ model specifically comprises the following steps: S1, preparing a hydrogel, vortexing methacrylic anhydride gelatin, polyethylene glycol diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate and pure water to obtain a hydrogel; S2. The construction of the shaping glass unit is performed by placing two thick hollow glass tubes and a thin hollow glass tube with different diameters coaxially, so that the inner and outer sides of the thin hollow glass tube form an inner cavity and an outer cavity, and two injection needles are used to connect the inner cavity and the outer cavity respectively; S3, construction of a microfluidic control unit, using a hose to connect the injection needle to the syringe, and connecting the syringe to a microflow pump, loading the inner phase escape material into the syringe connected to the inner cavity, and loading the hydrogel outer phase into the syringe connected to the outer cavity; S4. Formation of the skeleton of a bionic organ model and cell loading, driving a microflow pump so that the inner phase escape material and the hydrogel outer phase form a tubular fluid through the inner and outer cavities, and then using an ultraviolet curing lamp to irradiate the tubular fluid to obtain a solid hydrogel skeleton; culturing tumor cells on the inner wall of the solid hydrogel skeleton and culturing vascular endothelial cells on the outer wall to obtain an esophageal cancer bionic organ model, and the ratio of the inner phase flow rate to the outer phase flow rate is 1:2-10.

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

2.

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

4. The method for constructing a bionic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that: The temperature of the environment where the hydrogel skeleton is located in step S4 is 25-28°C.

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

7. The method for constructing an esophageal cancer bionic organ model based on a composite hydrogel according to claim 1, characterized in that: In step S4, both the tumor cells and the vascular endothelial cells are cell suspensions, and the requirements for the tumor cells and the vascular endothelial cells to adhere to the wall and grow are as follows: When the tumor cells were in the logarithmic growth phase in the T75 culture flask, they were digested with trypsin, resuspended in the culture medium, and counted. The cell concentration of the tumor cells was 1×10 7 Pieces / mL; When the vascular endothelial cells were in the logarithmic growth phase in the T75 culture flask, they were digested with trypsin, resuspended in the culture medium, and counted. The cell concentration of the vascular endothelial cells was 5×10 5 Pieces / mL.

8. The method for constructing an esophageal cancer bionic organ model based on a composite hydrogel according to claim 1, characterized in that: The method for growing tumor cells on the inner wall of the hydrogel skeleton is as follows: A homemade needle with a 100 μm tip was used to inject the cell suspension of tumor cells into the channels of the hydrogel skeleton, and then the hydrogel skeleton was transferred to a 6-well plate and incubated in a 37°C incubator. After 4 hours, the tumor cells adhered to the fiber channel.

9. The method for constructing a bionic organ model of esophageal cancer based on a composite hydrogel according to claim 1, characterized in that The method for growing vascular endothelial cells on the outer wall of the hydrogel skeleton is as follows: A high concentration of vascular endothelial cell suspension is added to the outer surface of the fiber in small amounts and multiple times through a syringe, and then smoothly transferred to an incubator for incubation and culture. After 4 hours, the vascular endothelial cells adhere to the outer surface of the hydrogel skeleton.

10. Use of the method for constructing an esophageal cancer bionic organ model based on a composite hydrogel according to any one of claims 1 to 9 in constructing a bionic organ model.

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