Vascular organ chip preparation method based on degradable bionic stent

Abstract

The invention discloses a preparation method of a vascular organ chip based on a degradable bionic stent. The degradable biological scaffold (PCL-FBE scaffold for short) is obtained by mixing a swimming bladder extract and polycaprolactone and carrying out electrostatic spinning. On the basis, a vascular organ chip which can be integrated in a micro-fluidic system is further constructed, and a whole process for completing three-dimensional dynamic culture of vascular organs by using the chip is established. According to the present invention, the swim bladder extract and polycaprolactone are compounded to form the electrospinning scaffold, such that the defects of the mechanical property, the degradability and the biocompatibility of the traditional bionic scaffold are successfully overcome, the organoid culture effect is significantly improved, and the efficient and sustainable innovation platform is provided for the tissue engineering and the regenerative medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical engineering, and particularly relates to a preparation method of a blood vessel organ chip based on a degradable biomimetic scaffold. BACKGROUND

[0002] Vessel organ is a microvascular structure model constructed by stem cells, progenitor cells or vascular endothelial cells and pericytes through three-dimensional in vitro culture technology. This kind of organ can simulate the morphology, function and physiological microenvironment of natural blood vessels, and has important value in the fields of regenerative medicine, disease mechanism research (such as atherosclerosis, diabetic vasculopathy), drug screening and toxicity testing. However, the culture of traditional vessel organ often faces challenges, such as insufficient structural complexity, low mechanical strength, difficulty in accurately regulating cell-cell interaction, and lack of long-term stable functional vascular network. These problems limit its application in clinical transformation and complex pathological simulation. Tissue engineering scaffold occupies a very key position in the field of modern medicine and biology research, which aims to provide a suitable microenvironment and structural support for cell growth and tissue regeneration, to promote the repair and functional reconstruction of damaged tissues or organs. With the continuous progress of science and technology, the cross-fusion of multiple disciplines brings unprecedented opportunities and challenges to the design and preparation of tissue engineering scaffolds, which provides an innovative solution for the construction of vessel organ combined with tissue engineering scaffolds. The scaffold is usually made of biocompatible materials (such as gelatin, collagen, polycaprolactone or hydrogel), and its three-dimensional structure and physical and chemical properties can significantly optimize the culture effect of the organ, such as the 3D porous structure of the scaffold can provide physical support for the adhesion, migration and self-organization of vascular cells, guide the formation of lumen structure of endothelial cells, and promote the directional arrangement of pericytes or smooth muscle cells, simulate the layered structure of natural blood vessels, and by adjusting the mechanical properties (such as elastic modulus, stiffness) of the scaffold, the mechanical microenvironment of different blood vessels (such as arteries, veins or capillaries) can be simulated, the mechanical stability of the organ is enhanced, and the deposition and remodeling of extracellular matrix are promoted. At the same time, with the help of microfluidic technology, blood vessel channels with perfusion function can be constructed, the oxygen / nutrient transport efficiency of the organ is improved, and the functional integration with the host vascular system is supported, laying the foundation for survival after transplantation.

[0003] Poly (caprolactone) (PCL) is a synthetic polymer material widely used in biomedical field. It has excellent mechanical properties, strong processability, and can be prepared into various shape and structure of scaffold material. The degradation rate of poly (caprolactone) is relatively controllable, and in the application of tissue engineering, it can be reasonably designed according to the time requirement of different tissue repair. Swim bladder as a kind of natural biomaterial has unique physical and chemical properties. It is rich in collagen and other bioactive ingredients, and has good biocompatibility and biodegradability. The microstructure of swim bladder presents a certain porosity and fibrous network, which is conducive to cell adhesion, growth and exchange of substances in tissue. Integrating swim bladder into poly (caprolactone) tissue engineering scaffold can not only provide natural bioactive signals for cells, but also enhance the overall performance of the scaffold by utilizing its unique structural characteristics, and promote the cell-material interaction in the process of tissue engineering construction.

[0004] In summary, the biomimetic scaffold combining the excellent biological performance of swim bladder and the excellent mechanical performance of poly (caprolactone) can carry retinal organoids in a microfluidic system, which is expected to integrate the advantages of each component and form a new scaffold system with high bionics, precise regulation and good biocompatibility. This innovative microfluidic culture system has great potential application value in drug screening, toxicity testing, disease modeling, retinal tissue repair, eye disease treatment model construction and wider tissue engineering and regenerative medicine field, and is worth further research and exploration. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application aims to provide a preparation method of poly (caprolactone) - swim bladder extract biomimetic scaffold (referred to as PCL-FBE scaffold). On this basis, a blood vessel organ chip that can be integrated into a microfluidic system is further constructed, and a whole process of three-dimensional dynamic culture of blood vessel organ using the chip is established.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows:

[0007] In the first aspect, the present application provides a preparation method of poly (caprolactone) - swim bladder extract biomimetic scaffold, comprising the following steps: dissolving poly (caprolactone) in hexafluoroisopropanol, then adding swim bladder extract, stirring and mixing to obtain a polymer solution; the polymer solution is prepared into electrospun fiber membrane by electrospinning, and the poly (caprolactone) - swim bladder extract biomimetic scaffold is obtained.

[0008] Preferably, the mass ratio of poly (caprolactone) and swim bladder extract is 10:1.

[0009] Preferably, the swim bladder extract is prepared by the following method: removing the swim bladder from fresh silver carp, removing the fat, mucous membrane and blood vessels attached to the swim bladder, and thoroughly washing with physiological saline; after washing the swim bladder tissue in a sterile PBS solution, the tissue is treated with a sodium dodecyl sulfate solution at room temperature; then the swim bladder is rinsed with pre-cooled sterile PBS solution to wash off the residual reagent to obtain the decellularized tissue; the decellularized tissue is dissolved in acetic acid, and the viscous liquid is filtered with a filter screen, then dialyzed and freeze-dried to obtain the swim bladder extract.

[0010] Preferably, the concentration of the sodium dodecyl sulfate solution is 0.5% w / v.

[0011] In a second aspect, the present application provides a polycaprolactone-swim bladder extract biomimetic scaffold prepared by the above preparation method.

[0012] In a third aspect, the present application provides the use of the above-mentioned polycaprolactone-swim bladder extract biomimetic scaffold in the preparation of a vascular organ chip (microfluidic chip).

[0013] In a fourth aspect, the present application provides a method for preparing a vascular organ chip (microfluidic chip) based on a degradable biomimetic scaffold, comprising the following steps:

[0014] S1: bonding of PC cover plate and PDMS film A

[0015] 1) The injection-molded PC cover plate is immersed in an activation reagent, then rinsed and dried;

[0016] 2) A film cutting machine is used to cut a 25x55 mm PDMS film with a thickness of 200 µm into the required hollow flow channel pattern A, i.e. PDMS film A;

[0017] 3) After placing the PDMS film A and the activated PC cover plate in a plasma cleaning machine, they are gently pressed together to obtain the chip;

[0018] 4) A film cutting machine is used to cut a PDMS film with a thickness of 200 µm and a diameter matching the outer shape of the chip to obtain a sealing ring;

[0019] 5) After placing the bonded chip and the sealing ring in a plasma cleaning machine, they are gently pressed together, then baked in an 80℃ constant temperature oven for 30 min for curing to obtain the upper chip;

[0020] S2: bonding of PC bottom plate and PDMS film B

[0021] 6) The injection-molded PC bottom plate is immersed in an activation reagent, then rinsed and dried;

[0022] 7) Use a film cutting machine to cut a 25x55 mm PDMS film with a thickness of 200 μm into the required hollow flow channel pattern B, that is, PDMS film B;

[0023] 8) After placing the PDMS film B and the activated PC bottom plate in the plasma cleaning machine, gently press the bonding, and then bake in a constant temperature oven at 80℃ for 30 min for curing to obtain the lower chip;

[0024] S2: Assemble the complete microfluidic chip

[0025] 9) Align the upper chip and the lower chip, and seal by clamps;

[0026] S4: Addition of electrospun fiber membrane (PCL-FBE scaffold)

[0027] 10) Cut the electrospun membrane into a circular piece with a diameter of 3 mm;

[0028] 11) Place the cut circular piece in the small chamber in the middle of the chip, ensuring that it is located between the PDMS layers of the upper and lower chips;

[0029] 12) Gently press the chip so that the electrospun membrane is tightly attached to the chamber wall, forming a stable support or functional layer.

[0030] In a fifth aspect, the present application provides a vascular organ chip prepared according to the above preparation method.

[0031] In a sixth aspect, the present application provides the use of the above-mentioned vascular organ chip in three-dimensional culture of vascular organs.

[0032] In a seventh aspect, the present application provides a method for three-dimensional culture of vascular organs, comprising the following steps: placing the vascular organ chip into a cell culture box, and injecting cell suspension into the chamber, so as to culture the vascular organ in a microfluidic environment with an electrospun membrane.

[0033] The present application constructs a degradable, high-bionic and function-enhanced biological scaffold by compounding fish swim bladder extract (FBE) and polycaprolactone (PCL) electrospinning, and integrates it into a microfluidic culture system for three-dimensional dynamic culture of vascular organs / retinal organs. Compared with traditional scaffolds, the present application has the following beneficial effects:

[0034] 1. Significantly improve the mechanical properties of the scaffold: after compounding the fish swim bladder extract and polycaprolactone in a specific ratio, the elastic modulus and elongation at break of the scaffold are significantly improved, overcoming the defect of "poor mechanical properties" of traditional bionic scaffolds.

[0035] 2. Achieving biodegradability: By introducing natural swim bladder extract and maintaining the biodegradable properties of polycaprolactone, the scaffold can degrade in a controlled time in the body, avoiding secondary damage caused by long-term residue, and achieving the "biodegradable" requirement.

[0036] 3. Significantly enhancing biocompatibility: The swim bladder extract is rich in bioactive ingredients such as collagen, elastin and polysaccharides, as well as trace elements such as calcium, potassium and magnesium, which can reduce immunogenicity, improve cell adhesion, proliferation and differentiation efficiency, and significantly improve the biocompatibility of the scaffold.

[0037] 4. Promoting the survival and functionalization of organoids: In the microfluidic system, the addition of a vascular organoid culture platform containing the scaffold successfully simulated the key functional characteristics of the retinal vascular microenvironment, especially for the study of blood-retinal barrier mechanism and anti-angiogenic therapy.

[0038] 5. Multifunctional application prospect: The scaffold system has mechanical support, biodegradability and high biocompatibility, and can be widely used in drug screening, toxicity testing, disease model construction, tissue repair (especially retinal and vascular tissues), and broader tissue engineering and regenerative medicine fields, with high translation value.

[0039] 6. Efficient use of resources: Fish swim bladder, as a waste byproduct of fish processing, is high-value utilization, reducing raw material costs and achieving waste recycling, in line with the concept of green and sustainable development.

[0040] In summary, the electrospun scaffold prepared by compounding fish swim bladder extract and polycaprolactone successfully overcomes the shortcomings of traditional biomimetic scaffolds in mechanical properties, degradability and biocompatibility, significantly improves the effectiveness of organoid culture, and provides an efficient and sustainable innovative platform for tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 : Fish swim bladder extract freeze-dried physical map.

[0042] Figure 2 : H&E staining, Masson's trichrome staining, Safranin O staining and VVG staining of fish swim bladder tissue before and after decellularization.

[0043] Figure 3 : Collagen content determination of fish swim bladder tissue before and after decellularization, ns indicates no difference.

[0044] Figure 4 : DNA content determination of fish swim bladder tissue before and after decellularization, ** p < 0.01.

[0045] Figure 5SEM characterization of PCL-FBE.

[0046] Figure 6 Fiber diameter (a), porosity (b) and water contact angle (c) of PCL-FBE, * p < 0.05.

[0047] Figure 7 Stress-strain curve (a), elastic modulus (b), ultimate tensile strength (c) and elongation at break (d) of PCL-FBE, *** p < 0.001, **** p < 0.0001, ns means no difference.

[0048] Figure 8 CCK8 proliferation test of RPE1 and L929 cells, ** p < 0.01, ns means no difference.

[0049] Figure 9 SEM images of RPE1 proliferation on PCL and PCL-1%FBE.

[0050] Figure 10 Nuclei staining, ZO-1 staining (a-f) and cell number quantification (g) of RPE1 on PCL and PCL-1%FBE.

[0051] Figure 11 Nuclei staining, BEST-1 staining (h-m) and cell number quantification (n) of RPE1 on PCL and PCL-1%FBE.

[0052] Figure 12 Nuclei staining, PEDF staining (o-t) and cell number quantification (u) of RPE1 on PCL and PCL-1%FBE.

[0053] Figure 13 Fluorescence quantification of ZO-1, BEST-1 and PEDF staining of RPE1 on PCL and PCL-1%FBE, ** p < 0.01.

[0054] Figure 14 Macroscopic morphology of PCL-FBE.

[0055] Figure 15 SEM images of PCL-FBE.

[0056] Figure 16 Weight change of PCL-FBE degradation.

[0057] Figure 17 Schematic diagram of microfluidic system.

[0058] Figure 18 : Microfluidic chip and system physical map.

[0059] Figure 19 : iPSCs were seeded at a density of 1.25 x 10 4 cells / well into a V-bottom 96-well plate.

[0060] Figure 20 : iPSCs formed aggregates with smooth boundaries.

[0061] Figure 21 : iPSCs were induced to form vascular organoids.

[0062] Figure 22 : Immunofluorescence staining and data statistics of vascular organoids. (a) Immunofluorescence staining images of vascular organoids, (b) BEST-1 staining mean fluorescence intensity statistics and (c) PEDF staining mean fluorescence intensity statistics. DETAILED DESCRIPTION

[0063] The following examples are further illustrations of the application and are not intended to limit the same.

[0064] Example 1 Preparation of PCL-FBE

[0065] 1. Preparation of swim bladder extract (FBE)

[0066] Swim bladders were removed from fresh silver carp, and the fat, mucous membrane and blood vessels attached to the swim bladders were removed, and then washed thoroughly with normal saline. Subsequently, the swim bladder tissue was washed in sterile phosphate buffer solution (PBS, pH = 7.4) for 4 hours. Then, the tissue was treated with 0.5% (w / v) sodium dodecyl sulfate (SDS) solution at room temperature with gentle shaking. After that, the swim bladder was washed with sterile PBS at 4°C to wash away the residual reagents to obtain the decellularized tissue. The decellularized tissue was dissolved in acetic acid for 72 h, and the viscous liquid was filtered with gauze and dialyzed for 4 days, with water changed every 4 h. The sample obtained after dialysis was frozen in a freeze dryer until the sample was completely dried (about 24 h) and stored at -20°C. Figure 1

[0067] The swim bladder tissue before and after decellularization was subjected to H&E staining, Masson's trichrome staining, Safranin O staining and Verhoeff-Van Gieson staining Figure 2 , collagen content determination Figure 3 and DNA content determination Figure 4 .

[0068] ​First, H&E staining, running water for 5 min, hematoxylin staining for 4 min, running water for 3 min, hydrochloric acid ethanol differentiation solution for 2 s, running water for 5 min, eosin staining for 20 s, running water for 2 min, 80%, 90%, 95% ethanol each for 2 times, anhydrous ethanol I, II each for 5 min, xylene I, II each for 5 min, appropriate drying in the fume hood, neutral resin sealing, and then observed under a general optical microscope.

[0069] Second, Masson's trichrome staining, prepare weak acid aqueous solution: mix weak acid and pure water according to 1:5, prepare 5 mL, aniline blue aqueous solution: mix aniline blue and pure water according to 1:5, prepare 1 mL, hematoxylin staining solution: mix reagents A and B according to 1:1. Running water for 3 min, Weigert iron hematoxylin staining for 5 min, acid ethanol differentiation solution for 1 s, running water for 1 min, Masson blue solution for 30 s, running water for 1 min, ponceau red staining solution for 6 min, weak acid working solution for 1 min, phosphomolybdic acid solution for 1-2 min, 5-fold diluted aniline blue for 10 s, weak acid working solution for 2 min, 95% ethanol rapid dehydration for 2 min, anhydrous ethanol dehydration for 5 min, xylene transparency for 5 min, neutral resin sealing after 3 min in the fume hood.

[0070] Third, safranin O staining, safranin staining solution drop staining for 20 min, running water for 5 min, xylene transparency for 5 min, 50% neutral resin sealing, and observed under a general optical microscope.

[0071] Then, Verhoeff-Van Gieson staining, hematoxylin nuclear staining for 5 min, VG staining solution for 1 min, 95% alcohol rapid differentiation for a few seconds, anhydrous ethanol dehydration for 5 s, xylene transparency for 5 min, neutral resin sealing, and then observed. The results of tissue sections show that the number of nuclei in the swim bladder tissue before and after decellularization is significantly reduced.

[0072] Finally, the collagen content of the swim bladder tissue was determined. A sample with a wet weight of 20-50 mg was weighed, added to 1 mL of physiological saline, and crushed. The sample was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The strips were equilibrated at room temperature for 20 min before use. The remaining sample was sealed in a self-sealing bag and returned to the refrigerator. Standard wells and sample wells were prepared. 50 μL of different concentrations (0, 2.5, 5, 10, 20, 40 ng / mL) of standard were added to each standard well. Except for the blank wells, 10 μL of sample was added to each sample well, followed by 40 μL of sample diluent to dilute 5 times. Except for the blank wells, 100 μL of horseradish peroxidase-labeled detection antibody was added to each standard and sample well. The reaction wells were sealed with sealing film and incubated at 37℃ in a water bath or incubator for 60 min. The liquid in the wells was discarded, and the sample was patted dry on absorbent paper. Each well was filled with washing buffer and allowed to stand for 1 minute. After washing for 1 minute, remove the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 50 μL of substrate A and 50 μL of substrate B to each well, incubate at 37°C in the dark for 15 minutes, add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 minutes.

[0073] For DNA content determination in fish swim bladder tissue, weigh samples with a wet weight ≤25 mg (n=3), add 180 μL of Buffer ATL, cut into small pieces, add 20 μL of proteinase K, vortex, incubate at 56℃ for 20 min until completely dissolved, vortex for 15 s, add 200 μL of Buffer AL, mix well, add 200 μL of anhydrous ethanol, vortex, transfer the mixture to a DNA mini-collection column, attach a 2 mL centrifuge tube to the collection column, centrifuge at 6000 g for 1 min, discard the supernatant, replace with a new 2 mL centrifuge tube, add 500 μL of Buffer AW1, centrifuge at 6000 g for 1 min, discard the supernatant, replace with a new 2 mL centrifuge tube, add 500 μL of Buffer AW2, centrifuge at 20000 g for 3 min, discard the supernatant and centrifuge tube, transfer the collection column to a new 1.5–2 mL microcentrifuge tube, add 500 μL of Buffer AE, incubate at room temperature for 1 min, centrifuge at 6000 g for 1 min, add 200 μL of Buffer AE, incubate at room temperature for 1 min, centrifuge at 6000 g for 1 min to obtain DNA extraction solution. Prepare standard curves using DNA standards of 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0 μg / mL. Take 20 μL of DNA extraction solution and DNA standard into a 96-well plate, add Houchst33258 buffer, incubate in the dark for 5 min, and measure the fluorescence intensity with a fluorescence spectrophotometer. The excitation wavelength is 356 nm and the emission wavelength is 492 nm.

[0074] H&E staining results showed that the nuclei of the decellularized swim bladder tissue were almost completely removed, leaving only a few fragments; the extracellular matrix was light pink, and the collagen fiber framework was more prominent, with a lack of cellular components overall. Masson's trichrome staining results showed that the blue collagen fibers remained in the decellularized swim bladder tissue, but the red cytoplasm and black nuclei were significantly reduced, the collagen fibers were more loosely arranged, and the blue background of the extracellular matrix was more prominent. Safranin O staining results showed that the red staining intensity of the decellularized swim bladder tissue was reduced, and the distribution became loose, indicating that part of the glycosaminoglycans (GAGs) were removed during the decellularization process, and the residual GAGs still existed but the density was reduced. Verhoeff-Van Gieson staining results showed that the black elastic fibers remained in the decellularized swim bladder tissue, and red collagen was still visible, but the tissue structure was more loose, the arrangement of elastic fibers had increased gaps, and the fiber framework was more obvious. Figure 2 These results indicate that the decellularization process successfully removed the cellular components while retaining key extracellular matrix components, providing a good scaffold foundation for subsequent tissue engineering applications.

[0075] Collagen quantification results showed that there was no significant change in collagen content in the swim bladder tissue before and after decellularization, indicating that collagen was not lost during the decellularization process. Figure 3 DNA quantification results showed that the DNA content of the decellularized swim bladder tissue was significantly reduced, indicating the success of decellularization. Figure 4

[0076] 2. Preparation of electrospun poly-caprolactone-swim bladder extract scaffolds (PCL-FBE)

[0077] Prior to electrospinning, PCL-FBE solutions composed of different contents of swim bladder extract were prepared. First, a 10% (w / v) PCL solution in hexafluoroisopropanol (HFIP) was prepared and stirred for 24 hours. Second, FBE was added to the PCL solution at concentrations of 0, 0.5, 1, and 5% (w / v) in the total system, and then stirred to prepare a uniform solution. Different volume percentages of FBE samples were labeled as PCL, PCL-0.5%FBE, PCL-1%FBE, and PCL-5%FBE, respectively. The polymer solution was loaded into a 10 mL disposable syringe, a 21-gauge needle was used for the needle tip, and the syringe pump was controlled at a rate of 1 mL / h. The distance between the needle tip and the collector was adjusted to 18 cm. The charged solution jet was sprayed towards the collector, during which the solvent evaporated, and the fibers were deposited on the surface of the collector to form a flat fiber membrane. The thickness of the electrospun membrane was controlled by the processing time. The fibers were dried in a vacuum desiccator at 25°C for 72 hours to dissipate the remaining solvent before characterization.

[0078] ​The electrospun fiber membrane prepared by electrospinning was observed by scanning electron microscope to observe the morphology of the membrane and the continuity of the fibers Figure 5 ), and the fiber diameter, porosity size and water contact angle size were analyzed by imageJ software Figure 6 ). The SEM image showed that the electrospun fiber membrane had good fiber uniformity, moderate porosity and high continuity, and FBE could be uniformly blended with PCL without adversely affecting the overall morphology of the fibers. This structural feature is conducive to subsequent biomedical or functional material applications, such as tissue engineering scaffolds Figure 5 ). ImageJ quantitative analysis results showed that the fiber diameter of the electrospun fiber membrane did not change significantly, the appropriate porosity provided a channel for material transport and a large cell attachment area, and the water contact angle increased with the increase of the concentration of fish mucus extract (FBE), indicating the hydrophilicity of fish mucus Figure 6 .

[0079] 3. Mechanical property test of PCL-FBE

[0080] The mechanical properties of the membranes were evaluated by tensile test using a linear tensile testing machine (LIGAO, China) at an elongation rate of 10 mm / min. The samples were cut into 1.0x7.0 cm rectangles in two directions. The parameters (ultimate tensile strength, elongation at break and elastic modulus) were calculated according to the stress-strain curve. The addition of fish mucus extract increased the hardness of PCL-FBE, and too high a concentration increased the elastic modulus, which reduced the mechanical properties of PCL-5%FBE Figure 7 ).

[0081] Example 2 Effect of PCL-FBE on promoting RPE1 adhesion and proliferation

[0082] 1. CCK8 cell proliferation test

[0083] The electrospun membrane was cut into 21 mm round pieces and ultraviolet sterilized. The membrane was placed in a 12-well plate and pressed around with a steel ring in a clean bench. Logarithmic growth RPE1 and L929 cells were taken out, the old culture medium was discarded, 2 mL PBS was added and washed three times, 2 mL trypsin was added for 1 min, 4 mL complete culture medium was added to terminate digestion, the cells were mixed and transferred to a 15 mL centrifuge tube, centrifuged, 1 mL culture medium was added to resuspend, counted, and the cell concentration was diluted according to the counting data, so that the density was 1.2x10 5The cells were inoculated into 12-well plates, and a circle of PBS was added around the 12-well plates, 1 mL per well, and the cells were treated for 24 hours. 600 μL of CCK8 solution was mixed with 6 mL of culture medium, 1 mL of mixed solution was added to each well, and the CCK8 amount added was 1:10 of the initial culture volume. The mixed solution was added in the form of medium replacement. The cells were incubated in the cell incubator for 3 hours. The supernatant in the well plate was aspirated and added to a 96-well plate, five replicate wells were set for each sample. The supernatant was mixed before adding the liquid. The absorbance value at 450 nm was determined by an enzyme-labeled instrument. The results showed that the scaffold added with the swim bladder extract had no toxic effect on the cells, and the cell survival rate of PCL-1%FBE was the highest. Figure 8 .

[0084] 2. SEM characterization of RPE1 proliferation on PCL-FBE

[0085] RPE1 cells were inoculated on PCL-FBE and cultured for 1, 3, and 7 days, respectively. The cells were fixed with 2.5% glutaraldehyde for 30 min, and then dehydrated with 50%, 70%, 80%, 90%, and 95% ethanol gradient. After surface gold plating, the cells were observed by scanning electron microscopy. The cells adhered to the surface after one day of proliferation on PCL-FBE, gradually expanded outward after three days, and covered the entire membrane surface after seven days. Figure 9 This indicates that the PCL-FBE electrospun scaffold can quickly promote RPE1 cell adhesion, proliferation, and formation of a complete cell coverage layer, demonstrating its excellent biocompatibility and application prospects as a cell support matrix.

[0086] 3. Immunofluorescence staining of RPE1 proliferation on PCL-FBE

[0087] RPE1 cells were cultured for three days at 60000 cells per well, 1 mL preheated 1 × PBS was used to wash the cells for 3 times, 10 min each time, the cells grown on the membrane were fixed with 4% PFA for 20 min, 1 mL 1 × PBS was used to wash for 3 times, 10 min each time, permeabilized in 0.5% Triton X-100 in PBS for 10 min at room temperature, 400 μL 5% normal goat serum was added to block for 30 min at room temperature, the primary antibody: 400 μL rabbit anti-ZO-1 (1:1000, diluted with PBS), rabbit anti-PEDF (1:200), mouse anti-BEST-1 (1:200) were added to the cell culture well, incubated overnight at 4℃, 1 mL 1 × PBS was used to wash for 3 times, 10 min each time, the secondary antibody: Alexa Fluor conjugated goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were diluted with PBS at 1:200, 400 μL was added to the cell culture well, incubated for 1 h in the dark, 1 mL 1 × PBS was used to wash for 3 times, 10 min each time, DAPI mounting medium was added to the glass slide, the fibrous membrane was placed on the glass slide with the cell side facing down to ensure that the droplet was completely immersed in the membrane, incubated for 5 min at room temperature, covered with a cover glass, and observed under a laser confocal microscope. The number of cells and the fluorescence intensity of ZO-1, BEST-1, and PEDF were quantified using Graph Pad software. Figures 10-13 Each group showed the best expression of ZO-1, BEST-1, and PEDF on PCL-1%FBE, indicating that PCL-1%FBE is more conducive to maintaining the normal phenotype and function of RPE1 cells.

[0088] Example 3 Degradation of PCL-FBE

[0089] Each group of membranes was cut into 1 × 0.5 cm strips, three for each group, each sample was weighed and the sample weight was recorded, 10 mL of PBS containing 0.1% sodium azide was added to a sterile 15 mL centrifuge tube, one sample from each group was immersed in the PBS, the centrifuge tube was placed in a constant temperature shaker, and incubated at 37℃ with shaking, the samples were taken out at 0, 2, 4, 8, and 12 weeks, rinsed with ultrapure water, and vacuum dried to a constant weight, each sample was weighed, and the weight retention rate was calculated based on the initial weight, the macroscopic morphology of each group of samples was observed using a stereomicroscope Figure 14 , the changes in fiber morphology of the scaffold during in vitro degradation were observed by SEM Figure 15 , and the change in scaffold weight was calculated by Graph Pad software Figure 16 . The results showed that as the degradation time increased, the weight of the scaffold decreased, the SEM images showed that the internal pores of the scaffold increased and the fibers were twisted and deformed, indicating that PCL-FBE is a degradable scaffold.

[0090] Example 4 Construction of microfluidic chip

[0091] Microfluidic chip processing, comprising the following steps:

[0092] 1. Bonding of PC cover plate and PDMS film A

[0093] (1) The injection-molded PC cover plate is immersed in an activation reagent, then rinsed and dried, so that it can be better bonded to PDMS;

[0094] (2) A 25x55 mm PDMS film with a thickness of 200 µm is cut into the required hollow flow channel pattern A, i.e. PDMS film A, using a film cutting machine;

[0095] (3) The PDMS film A and the activated PC cover plate are placed in a plasma cleaning machine for about 1 min, and are lightly pressed to bond. This process should avoid the generation of air bubbles as much as possible, and a chip is obtained;

[0096] (4) A sealing ring with a thickness of 200 µm and a diameter matching the shape of the chip is cut from the PDMS film using a film cutting machine;

[0097] (5) The bonded chip and the sealing ring are placed in a plasma cleaning machine for about 1 min, and are lightly pressed to bond. Then, they are baked in an 80°C constant temperature oven for about 30 min for curing, and an upper chip is obtained;

[0098] 2. Bonding of PC bottom plate and PDMS film B

[0099] (6) The injection-molded PC bottom plate is immersed in an activation reagent, then rinsed and dried;

[0100] (7) A 25x55 mm PDMS film with a thickness of 200 µm is cut into the required hollow flow channel pattern B, i.e. PDMS film B, using a film cutting machine;

[0101] (8) The PDMS film B and the activated PC bottom plate are placed in a plasma cleaning machine for about 1 min, and are lightly pressed to bond. Then, they are baked in an 80°C constant temperature oven for about 30 min for curing, and a lower chip is obtained;

[0102] 3. Assembly of complete microfluidic chip

[0103] (9) The upper chip and the lower chip are aligned, and are sealed by a clamp, and an integrated chip is obtained;

[0104] 4. Addition of electrospun fiber membrane

[0105] (10) The electrospun fiber membrane (PCL-FBE) prepared in Example 1 is cut into small circular pieces with a diameter of 3 mm;

[0106] (11) Place the cut round into the small chamber in the middle of the chip, making sure it is between the PDMS layers of the upper and lower chips;

[0107] (13) Gently press the chip so that the electrospun membrane is tightly attached to the chamber wall, forming a stable support or functional layer.

[0108] Cells were cultured in the chamber loaded with electrospun membrane. The schematic diagram of the microfluidic system is shown in Figure 17 , and the microfluidic chip and system are shown in Figure 18 .

[0109] Example 5. Construction of vascular organoids

[0110] Human iPSC cells were treated with 0.5 mM EDTA for 2 minutes. Then treated with 0.1% StemPro Accutase for 3 minutes. 2 x 10 5 cells were resuspended in differentiation medium, which contains DMEM:F12, 20% KOSR, 10% Glutamax and 2% NEAA. 50 μΜ Y-27632 was added to promote cell aggregation. The treated cells were seeded into V-bottomed 96-well low attachment plates at a density of 1.25 x 10 4 cells per well to form cell aggregates Figures 19-20 ). On day 3, the cell aggregates were treated with 12 μΜ CHIR99021 to induce mesoderm formation. On days 5, 7 and 9, BMP4 (30 ng / mL), VEGF-A (30 ng / mL) and FGF-2 (30 ng / mL) were added to promote vascular lineage formation. On day 11, the medium was changed to one containing VEGF-A (30 ng / mL), FGF-2 (30 ng / mL) and 10 μΜ SB431542 to increase endothelial cell production and suppress excessive pericyte differentiation. On day 13, the formed cell aggregates were embedded in a gel of Matrigel and collagen I (1:1 mixture). The differentiation medium was overlaid on top of the gel, which contained 15% FBS, 100 ng / mL VEGF-A and 100 ng / mL FGF-2. The differentiation medium was changed every 2-3 days. Around day 18, vascular networks were formed, which can be further analyzed or extracted into 96-well low attachment plates for culture. These vascular networks can self-assemble into vascular organoids Figure 21 , which can be cultured in 96-well low attachment plates for up to 3 months.

[0111] Example 6. Immunofluorescence staining of vascular organoids.

[0112] Vascular organoids were fixed with 4% paraformaldehyde for 20 minutes at room temperature. For suspended vascular organoids, the fixation time was 1 hour. Fixed organoids were washed with PBS to remove excess fixative. Blocking buffer containing 3% FBS, 1% bovine serum albumin (BSA), 0.5% Triton X-100, and 0.5% Tween was used to block for 2 hours at room temperature to reduce non-specific staining. Primary antibodies BEST-1 and PEDF were diluted at 1:200 in blocking buffer and incubated overnight at 4°C. Unbound antibodies were removed by washing three times with PBS containing 0.05% Tween (PBST) for 10 minutes each. Corresponding secondary antibodies were diluted at 1:250 in blocking buffer and incubated for 2 hours at room temperature. Unbound secondary antibodies were removed by washing three times with TBST for 20 minutes each. Cell nuclei were stained with DAPI. Samples were mounted with fluorescent mounting medium. Imaging was performed under a Leica laser scanning confocal microscope.

[0113] According to the results of immunofluorescence staining, Figure 22 BEST-1 and PEDF were highly expressed in the vascular organoids, indicating that the vascular organoids successfully mimicked key functional characteristics of the retinal vascular microenvironment, especially for the study of blood-retinal barrier mechanisms and anti-angiogenic therapies.

[0114] The above merely describes the preferred embodiments of the present application, and it should be noted that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for preparing a polycaprolactone-swim bladder extract biomimetic scaffold, characterized in that, The method comprises the following steps: The polycaprolactone is dissolved in hexafluoroisopropanol, and then the swim bladder extract is added and stirred to obtain a polymer solution; the polymer solution is used to prepare an electrospun fiber membrane by electrospinning to obtain a polycaprolactone-swim bladder extract biomimetic scaffold.

2. The production method according to claim 1, characterized by, The mass ratio of the polycaprolactone to the swim bladder extract is 10:

1.

3. The preparation method according to claim 1, characterized in that, The swim bladder extract is prepared by the following method: the swim bladder is taken out from fresh silver carp, and the fat, mucous membrane and blood vessels attached to the swim bladder are removed and washed thoroughly with normal saline; after the swim bladder tissue is washed clean in a sterile PBS solution, the tissue is treated with a sodium dodecyl sulfate solution at room temperature by gently shaking; then the swim bladder is washed with pre-cooled sterile PBS solution to wash away the residual reagent to obtain a decellularized tissue; the decellularized tissue is dissolved in acetic acid, and the viscous liquid is filtered, dialyzed and freeze-dried to obtain the swim bladder extract.

4. The production method according to claim 3, characterized by, The concentration of the sodium dodecyl sulfate solution is 0.5% w / v.

5. The polycaprolactone-swim bladder extract biomimetic scaffold obtained by the preparation method according to any one of claims 1-4.

6. Application of the polycaprolactone-swim bladder extract biomimetic scaffold according to claim 5 in the preparation of a vascular organ chip.

7. A method for preparing a vascular organoid chip based on a degradable biomimetic scaffold, characterized in that, The method comprises the following steps: S1: Bonding of PC cover plate and PDMS film A 1) The injection-molded PC cover plate is immersed in an activation reagent, and then washed and dried; 2) A 25*55 mm PDMS film with a thickness of 200 µm is cut into a required hollow flow channel pattern A, i.e. PDMS film A, using a film cutting machine; 3) After the PDMS film A and the activated PC cover plate are placed in a plasma cleaning machine, they are bonded by gentle pressing to obtain a chip; 4) A sealing ring with a thickness of 200 µm and a diameter matching the shape of the chip is cut from a PDMS film using a film cutting machine; 5) After the bonded chip and the sealing ring are placed in a plasma cleaning machine, they are bonded by gentle pressing, and then baked in an 80°C constant temperature oven for 30 min for curing to obtain an upper chip; S2: Bonding of PC bottom plate and PDMS film B 6) The injection-molded PC bottom plate is immersed in an activation reagent, and then washed and dried; 7) A 25*55 mm PDMS film with a thickness of 200 µm is cut into a required hollow flow channel pattern B, i.e. PDMS film B, using a film cutting machine; 8) After the PDMS film B and the activated PC bottom plate are placed in a plasma cleaning machine, they are bonded by gentle pressing, and then baked in an 80°C constant temperature oven for 30 min for curing to obtain a lower chip; S3: Assembly of complete microfluidic chip 9) The upper chip and the lower chip are aligned and sealed by a clamp; S4: Addition of electrospun fiber membrane 10) The electrospun membrane prepared by the method of claim 1 is cut into a circular piece with a diameter of 3 mm; 11) The cut circular piece is placed in the small chamber in the middle of the chip, ensuring that it is located between the PDMS layers of the upper and lower chips; 12) The chip is gently pressed so that the electrospun membrane is tightly attached to the wall of the small chamber, forming a stable support or functional layer.

8. The vascular organ chip obtained by the preparation method of claim 7.

9. Use of the vascular organ chip of claim 8 in culturing a three-dimensional vascular organoid.

10. A method of culturing a three-dimensional vascular organoid, characterized by, The vascular organ chip of claim 8 is placed in a cell incubator, and a cell suspension is injected into the chamber, and the culturing of a vascular organoid in a microfluidic environment with an electrospun membrane is performed.

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