Preparation method of a load blood-vesselized nasal mucosa organoid microcapsule and application thereof

Abstract

The present application belongs to the field of biomedical materials, and particularly relates to a preparation method of a microcapsule loaded with a vascularized nasal mucosa organoid and application thereof. The present application utilizes a coaxial microfluidic electrospinning technology, takes sodium carboxymethyl cellulose containing a nasal mucosa organoid and vascular endothelial cells as an inner phase, takes sodium alginate as an outer phase, and is solidified in a calcium chloride collecting liquid under the action of an electric field force to form a core-shell structure hydrogel microcapsule. The microcapsule has a uniform structure and a controllable size, and the core provides a three-dimensional protective microenvironment for the organoid and promotes vascularization. Animal experiments show that the microcapsule can significantly improve the survival rate and retention capacity of the organoid, accelerate the regeneration of the epithelium and the formation of new blood vessels on the nasal mucosa wound, promote wound healing, and has good biological safety. The present application provides an efficient and safe active microcarrier for nasal mucosa injury repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a method for preparing organoid microcapsules loaded with vascularized nasal mucosa and their application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nasal mucosal injury is a common clinical problem in otolaryngology, frequently resulting from trauma, surgical procedures, infections, and chronic inflammatory diseases. As a vital barrier of the respiratory tract, the nasal mucosa plays a crucial role in warming, humidifying, and providing immune defense; its structural integrity is essential for maintaining normal nasal physiological function. Damage to the nasal mucosa can easily trigger local inflammatory responses, ciliary dysfunction, and abnormal mucus secretion. In severe cases, it can lead to tissue adhesions, scarring, and even permanent loss of function.

[0004] Currently, clinical treatments for nasal mucosal damage mainly include drug therapy (such as antibiotics, glucocorticoids, and growth factors), physical barrier material coverage, and surgical repair. Although these methods can alleviate symptoms and promote healing to some extent, they still have many limitations: local administration is difficult to maintain effective drug concentrations, and frequent use leads to poor adherence; systemic administration may cause systemic side effects; traditional biomaterials mostly only provide physical support and lack the ability to actively regulate tissue regeneration.

[0005] In recent years, advancements in tissue engineering technology have provided new avenues for nasal mucosal repair. Organoids, as three-dimensional structures derived from stem cells or tissue cells, can mimic the microstructure and some functions of natural tissues in vitro and have been widely applied in regenerative medicine research. Nasal mucosal organoids, in particular, have shown promising potential in rebuilding the epithelial barrier and restoring local function. However, organoid transplantation in vivo still faces challenges such as low survival rates, insufficient nutrient supply, and limited vascularization, hindering its therapeutic efficacy. Vascularization is a key factor in achieving long-term survival and functional integration of tissue-engineered constructs. Although studies have attempted to construct vascularized organoids, their stability during delivery and effective retention at injury sites remain challenging. Furthermore, direct transplantation of organoids is susceptible to mechanical damage and the immune microenvironment, leading to reduced cell activity or even cell death.

[0006] Microencapsulation technology, as a cell delivery and protection strategy, can provide physical protection for cells or organoids and allow the exchange of nutrients and metabolites, thereby improving their survival rate and functional stability in vivo. Furthermore, microfluidics, due to its ability to precisely control the size, structure, and composition of microcapsules, has become an important means of constructing homogeneous and highly controllable microcarriers. However, existing microcapsule systems still have limitations in loading complex three-dimensional organoid structures, promoting vascularization, and achieving efficient tissue repair; their structural design and functional integration still need further optimization. Therefore, developing a microcapsule capable of efficiently loading vascularized nasal mucosal organoids, exhibiting good stability and excellent biocompatibility, and significantly promoting nasal mucosal wound repair, along with its preparation method, has significant clinical implications and application value. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing vascularized nasal mucosa organoid microcapsules and their application in nasal mucosal wounds.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing organoid microcapsules loaded with vascularized nasal mucosa, comprising the following steps: (1) Construction of coaxial microfluidic device: Two capillary glass tubes of different diameters are coaxially installed and fixed. The annular gap formed between the large-diameter and small-diameter capillary glass tubes serves as the outer phase channel, while the small-diameter capillary glass tube serves as the inner phase channel. Epoxy resin adhesive is used to fix the two capillary glass tubes to the glass slide. Flat-tipped needles are used to fix the inlet of both capillary glass tubes to ensure the coaxiality and sealing of the channels, forming a sealed connection and preventing solution leakage.

[0009] (2) Preparation of inner and outer phase solutions: The dissociated nasal mucosal organoids and vascular endothelial cells were resuspended in sodium carboxymethyl cellulose dissolved in physiological saline as the inner phase solution, and sodium alginate dissolved in physiological saline as the outer phase solution. The concentration of sodium carboxymethyl cellulose (CMC-Na) in the inner phase solution was 1 wt%, and the concentration of sodium alginate (ALG) in the outer phase solution was 1.5 wt%. This concentration ratio can take into account both the fluidity of the solution and the stability of the microcapsule formation, while providing a suitable growth environment for the organoids.

[0010] (3) Preparation of microcapsules: The inner phase solution and outer phase solution prepared in step (2) are simultaneously introduced into the inner phase channel and outer phase channel of step (1), respectively, and an electric field is applied at the channel outlet. After being electrosprayed, the microcapsules fall into the calcium chloride collection solution to obtain organoid microcapsules loaded with vascularized nasal mucosa. Then, the obtained microcapsules are placed in cell culture medium and cultured in an incubator at 37 ℃ to further enhance organoid activity. Specifically, in step (1), the outer phase channel is a glass capillary with an inner diameter of 500 μm and the inner phase channel is a glass capillary with an inner diameter of 200 μm. This size ratio can ensure that the inner phase solution is uniformly wrapped by the outer phase solution to form a regular core-shell structure.

[0011] Furthermore, in step (3), the flow rates of the inner phase solution and the outer phase solution affect the diameter and core-shell ratio of the microcapsules. When the flow rate of the sodium alginate solution into the outer phase channel is 120 μL / min and the flow rate of the inner phase solution into the inner phase channel is 40 μL / min, the obtained microcapsules have good monodispersity and optimal size uniformity.

[0012] Furthermore, in step (3), the electric field force applied at the channel outlet is 8-10 kV. This range of electric field force can ensure the stability of the electro-spraying process, avoid droplet aggregation or rupture, and at the same time ensure the integrity of the core-shell structure of the microcapsule.

[0013] Furthermore, in step (3), the collecting liquid is a calcium chloride solution with a concentration of 2 wt%. Calcium ions can undergo a displacement reaction with sodium ions in sodium alginate, causing sodium alginate to crosslink rapidly to form a hydrogel shell, thereby improving the structural stability of the microcapsules. The distance between the channel outlet and the collecting liquid is 5-7 cm. This distance can prevent the microcapsules from breaking due to excessive impact when they fall into the collecting liquid, while ensuring that the microcapsules are formed uniformly.

[0014] Furthermore, the prepared microcapsules have a diameter of 420-450 μm, a core diameter of 340-360 μm, and a shell thickness of 65-85 μm. This size range is suitable for the local environment of nasal mucosal wounds, which facilitates the retention of microcapsules on the wound surface and is also conducive to nutrient exchange and organoid proliferation.

[0015] Furthermore, the vascularized nasal mucosa organoid microcapsules prepared by the above-described method are also within the scope of protection of this invention. These microcapsules have a core-shell structure, with the core layer being a sodium carboxymethyl cellulose hydrogel containing nasal mucosa organoids and vascular endothelial cells, and the shell layer being a sodium alginate hydrogel. They exhibit uniform structure, controllable size, and good biocompatibility, stability, and permeability.

[0016] This invention provides the application of the above-mentioned vascularized nasal mucosa organoid microcapsules in the preparation of drugs for treating nasal mucosal wounds. These microcapsules can be applied directly to nasal mucosal wounds, or they can be combined with other drugs to form compound preparations for treating various nasal mucosal injuries, including nasal mucosal wounds caused by trauma, surgical trauma, infection, and chronic inflammation.

[0017] The beneficial effects of the technical solution provided by this invention are: (1) The core-shell structured hydrogel microcapsules prepared by the present invention using coaxial microfluidic electrospray technology have uniform structure and controllable size, which can provide a three-dimensional protective culture microenvironment for nasal mucosal organoids, effectively promote the proliferation and vascularization of organoids, and solve the problem of reduced activity during in vitro culture and in vivo transplantation of organoids.

[0018] (2) The coaxial microfluidic control process parameters used in this invention are adjustable. By precisely controlling the flow rate, voltage and collection distance, the particle size and core-shell ratio of microcapsules can be stably controlled. The preparation process is highly standardized and has the potential for large-scale production and clinical translation.

[0019] (3) The vascularized nasal mucosa organoid microcapsules prepared in this invention have good biocompatibility and blood compatibility. The hemolysis rate was only 2.25% ± 0.13% (n = 3) as verified by experiments, and there was no obvious biological toxicity. In vivo application can effectively improve the survival rate of organoids on nasal mucosal wounds, promote epithelial regeneration and angiogenesis, accelerate wound healing, improve the local inflammatory microenvironment, realize functional repair of nasal mucosal tissue, have high biosafety and broad clinical application prospects. Attached Figure Description

[0020] Figure 1 This is a front view of a coaxial microfluidic chip; Figure 2 This is a 3D view of a coaxial microfluidic chip; Figure 3 It is a three-dimensional view of two coaxially fitted capillary glass tubes; Figure 4 It is a cross-sectional view of two coaxially fitted capillary glass tubes; Figure 5 It is a three-dimensional diagram of the first flat-mouthed needle; Figure 6 It is a 3D diagram of the second flat-mouthed needle. Figure 7 The images show the actual structure of the capillary glass tube coaxial microfluidic chip and the device prepared by the present invention. (a) is the overall structure of the coaxial microfluidic chip, (b) is a detailed magnified view of the chip outlet, (c) is a schematic diagram of the device for preparing microcapsules using microfluidic electrospray technology, and (d) is a partial magnified view of the device. Figure 8The following diagrams illustrate the process and characterization of microcapsules prepared using microfluidic electrospray technology in this invention: (a) is an optical image of microdroplet formation during electrospraying; (b) is a bright-field microscope image of the obtained core-shell structured microcapsules; (c) and (d) are scanning electron microscope images of the microcapsules; (e) is a statistical diagram of the overall particle size distribution of the microcapsules; (f) is a statistical diagram of the outer shell thickness distribution of the microcapsules; and (g) is a statistical diagram of the core size distribution of the microcapsules. Figure 9 The diagram shows the effects of different parameters on microcapsule size and core-shell ratio. (a) shows the effect under different voltages, (b) shows the effect under different receiving distances, (c) shows the effect under different external phase flow velocities, and (d) shows the effect under different internal phase flow velocities. Figure 10 The results are for the blood compatibility test of the hydrogel microcapsules; Figure 11 The proliferation of vascularized nasal mucosal organoids inside core-shell microcapsules; (a) bright-field micrographs of vascularized nasal mucosal organoids inside microcapsules from day 1 to day 5 of culture; (b) fluorescence staining results of live / dead cells from day 1 to day 5 of culture. Figure 12 The images show the effect of applying vascularized nasal mucosa organoid microcapsules to nasal mucosal wounds in New Zealand white rabbits. (a) is a schematic diagram of the animal experiment, (b) is a photograph of the wound healing process in each group on day 7, and (c) is a hematoxylin-eosin staining image of the nasal septum and nasal mucosa wounds in each group on day 7 and day 14. Figure 13 Immunofluorescence staining and statistical images after treatment with vascularized nasal mucosa organoid microcapsules: (a) Immunofluorescence staining images of α-SMA, Ki67, and CD31 on the nasal septum and nasal mucosa wounds of New Zealand white rabbits on day 7; (b) Quantitative statistical results of α-SMA immunofluorescence; (c) Quantitative statistical results of Ki67 immunofluorescence; (d) Quantitative statistical results of CD31 immunofluorescence. Figure 14 Images of the kidneys, lungs, heart, liver, and spleen of New Zealand white rabbits treated with the vascularized nasal mucosa organoid microcapsules used in this invention, stained with hematoxylin and eosin. (Figure reference numerals:) 1. Large-diameter capillary glass tube; 2. Small-diameter capillary glass tube; 3. Glass slide; 4. First flat-mouth needle; 41. First needle seat; 411. First large groove; 412. First small groove; 42. First needle; 5. Second flat-mouth needle; 51. Second needle seat; 52. Second needle; 511. Second groove. Detailed Implementation

[0021] The specific embodiments of the present invention are described in detail below. These embodiments are intended to more fully demonstrate the technical content of the present invention and help to understand the specific implementation process of the present invention, but their content should not be construed as limiting the scope of the claims of the present invention in any way. For those skilled in the art, various adjustments, modifications, and substitutions made to the embodiments without departing from the spirit and scope of the present invention are all within the scope of protection sought by the present invention.

[0022] Example 1 Prepare two capillary glass tubes with inner diameters of 500 μm and 200 μm, respectively. The larger inner diameter tube is designated as large-diameter capillary glass tube 1, and the smaller inner diameter tube as small-diameter capillary glass tube 2. Insert the small-diameter capillary glass tube 2 coaxially into the large-diameter capillary glass tube 1, ensuring one end of the small-diameter capillary glass tube 2 is flush with one end of the large-diameter capillary glass tube 1. The other end of the small-diameter capillary glass tube 2 extends from the other end of the large-diameter capillary glass tube 1, forming an extension section. The inlet of this extension section serves as the inlet of the inner phase channel; the end of the large-diameter capillary glass tube 1 closest to this extension section serves as the inlet of the outer phase channel. The annular gap formed between the two is the outer phase channel, as shown below. Figure 3-4 As shown.

[0023] Take a clean glass slide 3 as the substrate. Place the coaxially fitted capillary glass tube on top of the glass slide 3. To establish a sealed fluid passage, use two specially designed flat-tipped needles—a first flat-tipped needle 4 and a second flat-tipped needle 5. Figure 5 As shown, the first flat-mouth needle 4 is composed of a first needle seat 41 and a first needle 42. The bottom surface of the first needle seat 41 has a large and a small first groove, namely a large first groove 411 and a small first groove 412; as shown Figure 6 As shown, the second flat needle 5 is composed of a second needle seat 51 and a second needle 52, and a second groove 511 is formed on the bottom surface of the second needle seat 51.

[0024] During fixation, the first large groove 411 of the first flat-mouth needle 4 is engaged on the outer wall of the large-diameter capillary glass tube 1, and the first small groove 412 is engaged on the outer wall of the small-diameter capillary glass tube 2, so that the bottom surface of the first needle holder 41 is in contact with the glass slide 3; the second groove 511 of the second flat-mouth needle 5 is engaged on the extended section of the small-diameter capillary glass tube 2, and the inlet of the inner phase channel extends into the interior of the second needle holder 51, and the bottom surface of the second needle holder 51 is also in contact with the glass slide 3. The positions are adjusted to ensure that all components are coaxial, and epoxy resin is used to fill all gaps and cure, so that a stable and sealed connection is formed between each needle holder and the capillary glass tube and the glass slide 3. Thus, the first needle 42 communicates with the outer phase channel through the internal cavity of the needle holder, and the second needle 52 communicates with the inner phase channel through the internal cavity of the needle holder, forming a coaxial microfluidic chip with an independent injection port. Its main view and perspective view are shown below. Figure 1 and Figure 2 As shown, the actual object is as follows Figure 7 As shown in (a) and 7(b).

[0025] (2) Assembly of coaxial micro-controlled flow electro-injection device: The prepared microfluidic chip is fixed on a support. A dual-channel injection pump is used, connected to the first flat-mouth needle 4 and the second flat-mouth needle 5 respectively, to drive the external and internal phase solutions. A conductive metal needle is fixed as an electrode at the chip's outlet end (i.e., the outlet of the internal and external phase fluids), and connected to the positive terminal of a high-voltage electrostatic generator. A receiving container filled with calcium chloride collection solution is placed directly below the chip outlet, and the negative terminal or ground terminal of the high-voltage electrostatic generator is connected to the collection solution to create a stable high-voltage electric field between the outlet and the liquid surface. By adjusting the support, the distance between the channel outlet and the receiving liquid surface can be precisely controlled. The final coaxial microfluidic electro-injection device is shown below. Figure 7 As shown in (c) and 7(d), this device generates highly uniform core-shell microdroplets at the outlet under the combined action of electric field force, fluid shear force, and surface tension, which then fall into the collecting liquid and solidify into microcapsules. By adjusting the internal and external phase flow rates, voltage, and receiving distance, the particle size, core diameter, and shell thickness of the microcapsules can be controlled.

[0026] Example 2 Description of vascular endothelial cells: Vascular endothelial cells are human umbilical vein endothelial cells (HUVECs), which are cultured to the 3rd-5th generation using conventional cell passage methods before use.

[0027] Pre-culture of nasal mucosal organoids: (1) Tissue collection and pretreatment: Healthy New Zealand white rabbits were anesthetized and euthanized. The nasal mucosa tissue on both sides of the nasal septum and the bilateral turbinates was quickly dissected and dissected using sterile instruments such as sterile scissors and forceps. The collected tissue was immediately placed in pre-cooled PBS buffer containing 2% penicillin-streptomycin and rinsed repeatedly to remove surface mucus, blood and impurities.

[0028] (2) Tissue digestion and cell dissociation: Nasal mucosa tissue was cut into 1 mm pieces using sterile surgical scissors. 3 Tissue fragments were transferred to centrifuge tubes, and 1.5-2.0 ml of 0.25% Trypsin-EDTA digestive enzyme was added. The mixture was incubated in a 37°C shaker for 30-60 minutes, with the tubes being pipetted every 10 minutes to promote tissue dissociation.

[0029] (3) Cell filtration and collection: After digestion, add an equal volume of DMEM / F12 medium containing 10% fetal bovine serum to terminate digestion. Filter the digestion solution through a 70 μm cell sieve to remove undigested tissue residue. Collect the filtrate, centrifuge at 4℃ and 1000 rpm for 5 minutes, discard the supernatant, and collect the nasal mucosal epithelial cell pellet at the bottom.

[0030] (4) Organoid seeding culture: After washing the cell pellet 1-2 times with cold PBS, resuspend the cells with an appropriate amount of matrix gel and adjust the cell density to 1×10⁻⁶. 5 Cells / mL. Inoculate 40-50 μL of the matrix gel-cell mixture into preheated 24-well plates, forming hemispherical droplets. Incubate the plates at 37 ℃, 5% CO2 for 20-30 minutes until the matrix gel is completely cured. Add 500 μL of nasal mucosa organoid culture medium (DMEM + B27 (1×) + EGF (50 ng / mL) + 1% vol penicillin-streptomycin) to each well. Change the medium every 2-3 days and observe the growth of the organoids. When the organoid diameter reaches 200-500 μm or a distinct cavitary structure appears, recover the organoids using cell recovery buffer and mechanically dissociate them for subsequent experiments.

[0031] Example 3 Preparation and characterization of organoid microcapsules loaded with vascularized nasal mucosa: (1) Prepare an aqueous solution containing 1.5 wt% high-viscosity sodium alginate and 1 wt% sodium carboxymethyl cellulose as the raw material solution; using the microfluidic chip prepared in Example 1, use organoid dissociation reagent to dissociate nasal mucosal organoids into single cells and resuspend them in culture medium to form a single-cell solution. Mix the organoid single-cell solution and vascular endothelial cell single-cell solution with the sodium carboxymethyl cellulose solution and introduce them into the inner phase of the device. Introduce 1.5% sodium alginate into the outer phase. Collect the liquid containing 2% calcium chloride. Use two syringe pumps to push the two-phase liquid at a constant speed, adjust the flow rate and voltage, and form a core-shell microcapsule encapsulating the vascularized nasal mucosal organoid, such as Figure 8 As shown in (a).

[0032] (2) Figure 8 (b) It can be seen that in the calcium chloride collection solution, calcium ions replace sodium ions in sodium alginate to form a calcium alginate hydrogel shell, and finally obtains a core-shell structured microcapsule with stable morphology. Figure 9 (a)- Figure 9(d) It can be seen that by adjusting the flow rates of the inner and outer phases and the voltage, we found that as the outer phase flow rate increases, the core diameter becomes smaller; as the inner phase flow rate increases, the core diameter becomes larger; as the voltage increases, the microcapsule size becomes smaller; and as the receiving distance increases, the microcapsule size becomes larger. Experiments showed that the most stable microcapsules were formed when the outer phase microchannel flow rate was 120 μL / min, the inner phase microchannel flow rate was 40 μL / min, and the electric field at the outlet was 8-10 kV. These microcapsules had a particle size of 420-450 μm, a core diameter of 340-360 μm, and a shell thickness of 65-85 μm. The results are as follows: Figure 8 (e)- Figure 8 As shown in (g). The obtained microcapsules were freeze-dried and then photographed using a scanning electron microscope, as shown. Figure 8 (c)- Figure 8 As shown in (d), its complete outer shell and clearly defined core-shell structure are visible.

[0033] Example 4 Cell compatibility properties of microcapsule hydrogels: Fresh rabbit blood was collected, centrifuged multiple times, and red blood cells were purified. The collected red blood cell pellet was resuspended in physiological saline. Three groups were set up: a positive group (1 mL red blood cell suspension + 1 mL water), a negative group (1 mL red blood cell suspension + 1 mL PBS), and a material group (1 mL red blood cell suspension + 1 mL microcapsule extract). The mixture was incubated at 37°C for 4 hours, photographed, and the absorbance at 545 nm was measured using a multi-mode microplate reader. Figure 10 It can be seen that the purified red blood cells naturally precipitated without rupture, and the hemolysis rate was 2.25% ± 0.13% (n = 3). According to ISO 10993-4 "Biological evaluation of medical devices - Part 4: Tests for interaction with blood" and ASTM F756 hemolysis test standard, a hemolysis rate of less than 5% can be considered as the material having good blood compatibility. This proves that the prepared microcapsules have good blood compatibility.

[0034] Example 5 Analysis of vascularized nasal mucosa organoid culture within microcapsules: The microcapsules containing vascularized nasal mucosa organoids prepared in Example 2 were washed three times with physiological saline and then placed in 24-well cell culture dishes containing cell culture medium (DMEM + B27 (1×) + EGF (50 ng / mL) + 1% vol penicillin-streptomycin) for 9-10 days, with the culture medium changed every two days. The cell proliferation process was observed and recorded. The cell viability of the vascularized nasal mucosa organoids in the hydrogel microcapsules was detected using a live / dead cell staining kit. On days 1, 2, 3, 4, and 5 of culture, the microcapsules were recovered from the culture medium. After washing three times with 2 mL PBS, the live / dead cell staining reagent was added and incubated in the dark for 30 minutes. Figure 11 (a) Bright-field micrographs of vascularized nasal mucosa organoids in microcapsules from day 1 to day 5 of culture, showing the gradual proliferation and increase in volume of the organoids over time; Figure 11 (b) The results of fluorescence staining of live / dead cells from day 1 to day 5 of culture, where: the first row is Calcein-AM staining (green) representing live cells; the second row is PI staining (red) representing dead cells; the third row is a merged image, showing that the organoids in the microcapsules have good survival rate and maintain stable proliferation over time.

[0035] Example 6 Application of microcapsules loaded with vascularized nasal mucosa organoids in the treatment of nasal septum mucosal wounds: (1) After anesthetizing New Zealand white rabbits, a 15 mm × 10 mm nasal mucosal wound was created in the right nasal septum mucosa to construct a wound model. The New Zealand white rabbits were randomly divided into five groups: normal group, control group, MC group, MC@MO group, and MC@VMO group. Wound changes were recorded by photograph during different treatment processes. The control group did not receive any treatment intervention. In the MC group, empty microcapsules were applied to the nasal mucosal wound. In the MC@MO group, microcapsules loaded with nasal mucosal organoids were applied to the nasal mucosal wound. In the MC@VMO group, microcapsules loaded with vascularized nasal mucosal organoids were applied to the nasal mucosal wound.

[0036] We found that compared to other groups, the MC@VMO group showed faster wound healing and more angiogenesis after microcapsule treatment with vascularized nasal mucosa organoids, as shown in the results. Figure 12 As shown in (a) and 12(b). Further hematoxylin-eosin staining of the nasal septum and mucosa in each group revealed that the wounds treated with microcapsules loaded with vascularized nasal mucosal organoids showed more complete mucosal repair and fewer inflammatory cells.

[0037] (2) Further immunostaining with α-SMA, Ki67, and CD31 was performed on the nasal septum and mucosal wound tissue, and quantitative analysis was conducted. Figure 13It can be seen that the nasal mucosal wound tissue treated with microcapsules loaded with vascularized nasal mucosal organoids showed greater positivity for α-SMA, Ki67, and CD31, indicating better wound repair, reduced inflammation, and increased angiogenesis after treatment. Simultaneously, hematoxylin-eosin staining was performed on the major organs of New Zealand white rabbits (kidney, lung, heart, liver, and spleen) to... Figure 14 The results showed no significant differences between the groups, confirming that the microcapsule therapy with vascularized nasal mucosa organoids is biosafety.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing organoid microcapsules loaded with vascularized nasal mucosa, characterized in that, The following steps are adopted: (1) Two capillary glass tubes of different diameters are fixed coaxially, wherein the annular gap formed between the large-diameter capillary glass tube and the small-diameter capillary glass tube is used as the outer phase channel and the small-diameter capillary glass tube is used as the inner phase channel. (2) The dissociated nasal mucosal organoids and vascular endothelial cells were resuspended in sodium carboxymethyl cellulose dissolved in physiological saline as the inner phase solution, and sodium alginate was dissolved in physiological saline as the outer phase solution. (3) The inner phase solution and the outer phase solution described in step (2) are respectively introduced into the inner phase channel and the outer phase channel of step (1). The inner phase solution is wrapped by the outer phase solution to form a core-shell structure droplet. An electric field force is applied at the channel outlet, and after being electro-sprayed, it falls into the calcium chloride collection liquid to obtain the loaded vascularized nasal mucosa organoid microcapsule.

2. The method for preparing vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (1), the method for coaxially fixing the two capillary glass tubes is as follows: two capillary glass tubes of different diameters are coaxially fitted together, with one end of the smaller diameter capillary glass tube flush with one end of the larger diameter capillary glass tube, and the other end of the smaller diameter capillary glass tube passing through the other end of the larger diameter capillary glass tube to form an extension section. The end of the extension section serves as the inlet of the inner phase channel, and the end of the larger diameter capillary glass tube near the inlet of the inner channel serves as the inlet of the outer channel. Take a glass slide, a first flat-mouth needle, and a second flat-mouth needle. Place the coaxially fitted capillary glass tubes above the glass slide, and place the first flat-mouth needle at the inlet of the outer channel to establish a sealed connection with the outer channel. Place the second flat-mouth needle at the inlet of the inner channel to establish a sealed connection with the inner channel.

3. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (3), the flow rate of the external phase solution into the external phase channel is 120 μL / min; the flow rate of the internal phase solution into the internal phase channel is 40 μL / min.

4. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (2), the concentration of sodium carboxymethyl cellulose in the inner phase solution is 0.8-1 wt%, and the concentration of sodium alginate in the outer phase solution is 1.5-2 wt%.

5. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (3), the electric field force applied at the channel outlet is 8-10 kV; the concentration of the calcium chloride collection solution is 1.5-2 wt%.

6. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (3), the distance between the channel outlet and the collected liquid is 5-7 cm.

7. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, In step (1), the outer phase channel has an inner diameter of 450-500 μm; the inner phase channel has an inner diameter of 150-200 μm.

8. The method for preparing the vascularized nasal mucosa organoid microcapsules according to claim 1, characterized in that, The prepared microcapsules have a diameter of 420-450 μm, a core diameter of 340-360 μm, and a shell thickness of 65-85 μm.

9. The vascularized nasal mucosa organoid microcapsules prepared by the preparation method according to any one of claims 1-8.

10. The use of the vascularized nasal mucosa organoid microcapsule according to claim 9 in the preparation of a medicament for repairing nasal mucosal wounds.

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