Nucleocapsid microcapsules loaded with nasal mucosa epithelial cells, and preparation method and application thereof

Abstract

The application discloses a nucleus-shell microcapsule loaded with nasal mucosa epithelial cells and a preparation method and application thereof, and belongs to the technical field of biological medicine. A coaxial microfluidic electrospray technology is used to pass a sodium carboxymethyl cellulose inner phase solution containing nasal mucosa epithelial cells and a sodium alginate outer phase solution into an outer tube and an inner tube of a coaxial microfluidic device, respectively, and under the action of an applied electric field, the nucleus-shell microcapsule loaded with the nasal mucosa epithelial cells is formed in a calcium ion-containing collection solution. The application constructs a three-dimensional microenvironment with clear components and controllable physical properties, completely replaces an animal source matrix glue used in traditional culture, significantly reduces the cost, and improves the repeatability and standardization degree of experiments. After being induced by an organoid culture medium, the nasal mucosa organoids can be derived and formed in the microcapsule.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nuclear-shell microcapsule loaded with nasal mucosal epithelial cells, its preparation method, and its application. Background Technology

[0002] The nasal mucosa, as the first line of defense in the respiratory tract, plays a crucial role in olfactory formation, air warming and humidification, and immune defense. Damage or dysfunction of nasal mucosal epithelial cells is closely related to various diseases such as chronic sinusitis, allergic rhinitis, olfactory dysfunction, and cystic fibrosis. In recent years, organoid technology has become an important tool for studying the physiological and pathological mechanisms of the nasal mucosa, drug screening, and regeneration and repair due to its ability to mimic the structure and function of in vivo tissues. However, traditional nasal mucosal organoids are usually cultured in three dimensions using matrix gel, which suffers from problems such as large batch-to-batch variability, uneven size, limited nutrient diffusion, and difficulty in achieving high-throughput preparation and standardized control, thus limiting their clinical and industrial applications.

[0003] To address the aforementioned issues, microcapsule technology has been introduced into the field of organoid culture. Core-shell microcapsules provide mechanical support and a selective permeability barrier through their outer shell, while the inner shell provides a growth microenvironment for cells, thereby achieving confined growth and functional regulation of organoids. Currently, commonly used microcapsule preparation methods include coaxial electrospraying, microfluidics, and emulsification. Among these, microfluidic electrospraying technology can precisely control droplet size and structure, offering advantages such as high throughput, good monodispersity, and mild conditions, making it suitable for live cell encapsulation. However, systematic reports on core-shell microcapsule preparation systems for nasal mucosa organoid loading have not been found, particularly regarding the correlation between material composition, process parameters, and cell viability and organoid formation efficiency. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a core-shell microcapsule loaded with nasal mucosal epithelial cells, its preparation method, and its application. Sodium alginate is used as the outer shell material, and sodium carboxymethyl cellulose mixed with nasal mucosal epithelial cells is used as the inner phase to construct core-shell microcapsules with uniform size, controllable structure, and good permeability. By optimizing key parameters such as flow rate, voltage, and height, the cell encapsulation efficiency and organoid formation quality are improved, overcoming the shortcomings of traditional culture methods such as large batch-to-batch variability, poor reproducibility, and difficulty in large-scale application. This provides a new technical solution for basic research and translational applications of nasal mucosal organoids.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing core-shell microcapsules loaded with nasal mucosal epithelial cells. The method employs coaxial microfluidic electrospray technology, in which a sodium carboxymethyl cellulose inner phase solution containing nasal mucosal epithelial cells and a sodium alginate outer phase solution are respectively introduced into the outer tube and inner tube of a coaxial microfluidic device. Under the action of voltage, core-shell microcapsules loaded with nasal mucosal epithelial cells are formed in a collection solution containing calcium ions.

[0006] Furthermore, the sodium carboxymethyl cellulose inner phase solution containing nasal mucosal epithelial cells is obtained by mixing nasal mucosal epithelial cells and sodium carboxymethyl cellulose solution, with each milliliter of sodium carboxymethyl cellulose solution containing 3 × 10⁻⁶ cells. 6 -6×10 6 One nasal mucosal epithelial cell.

[0007] Furthermore, the sodium carboxymethyl cellulose solution is a 0.8-1.2 wt% solution; the nasal mucosal epithelial cells are obtained by the nasal brush method.

[0008] Method for obtaining nasal mucosal epithelial cells using a nasal brush: 1) Prepare a sterile nasal brush, sterile saline, 15mL sterile centrifuge tubes, DMEM / F-12 culture medium containing 10% fetal bovine serum, crushed ice, etc.

[0009] 2) The operator wears sterile gloves and a mask, rinses both nasal cavities with physiological saline 2-3 times each, and the subject blows their nose, tilting their head back to fully expose the anterior part of the nasal cavity; after moistening the nasal brush with PBS, the operator holds the sterile nasal brush and gently inserts it into the subject's nostril and inferior turbinate, slowly rotating it 3-5 times along the nasal mucosa surface to ensure the brush head fully contacts the mucosal epithelium; the operator removes the nasal brush and immerses the brush head in a 15mL centrifuge tube containing 10mL of DMEM / F-12 culture medium containing 1% penicillin and streptomycin, and uses a new brush to clean the contralateral nasal cavity. The procedure was repeated in the nasal cavity. The brush tips were collected in the same 15 mL centrifuge tube, labeled, and stored on ice. The tip of a sterile 1250 µL pipette tip was cut off with sterile scissors, allowing the nasal brush to pass through. The brush was then inserted into the pipette tip, and the tip was repeatedly scraped up and down to detach cells into the culture medium. The nasal brush was then discarded, and the process was repeated with a second brush. The centrifuge tube was placed in a centrifuge and centrifuged at 1200 rpm for 5 min at 4 °C. The supernatant was discarded, and the cell pellet at the bottom was retained. The cell pellet was resuspended in 2 mL of sterile saline, and centrifuged again at 1200 rpm for 5 min. The supernatant was discarded, and this step was repeated twice to remove residual mucus and impurities. After discarding the supernatant, add approximately 5-8 times the volume of the precipitate with 0.25% TrypLE express enzyme, resuspend the precipitate, and incubate at 37°C for 5 min. Remove the resuspended cells and continue incubation for 5 min. Add 2 times the volume of DMEM / F-12 medium containing 10% FBS to terminate digestion. After resuspending, filter through a 70 μm cell sieve, collect the filtrate, centrifuge at 1200 r / min at 4°C for 5 min, discard the supernatant, and retain the cell pellet at the bottom, which is the nasal mucosal epithelial cells.

[0010] Furthermore, the sodium alginate external phase solution is a 1.8-2.2 wt% solution.

[0011] Furthermore, the outer tube of the coaxial microfluidic device is a capillary tube with a diameter of 300-350 μm, and the inner tube is a capillary tube with a diameter of 80-100 μm; the mass concentration of calcium chloride in the calcium ion-containing collection solution is 1.5-2.5%.

[0012] Furthermore, the flow rate of the sodium carboxymethyl cellulose inner phase solution containing nasal mucosal epithelial cells is 5-20 μL / min, and the flow rate of the sodium alginate outer phase solution is 60-100 μL / min.

[0013] Furthermore, the voltage is 3.5-7.5kV, and the distance between the spray tip of the coaxial microfluidic device and the surface of the collected liquid is 2-6cm.

[0014] In a second aspect, the present invention provides a core-shell microcapsule loaded with nasal mucosal epithelial cells prepared by the above preparation method, wherein the core-shell microcapsule loaded with nasal mucosal epithelial cells has calcium alginate as the outer shell and sodium carboxymethyl cellulose containing nasal mucosal epithelial cells as the core.

[0015] Furthermore, the outer diameter of the nuclear-shell microcapsule loaded with nasal mucosal epithelial cells is 375-435 μm, and the inner diameter is 210-270 μm.

[0016] In a third aspect, the present invention provides the application of the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells in the culture of nasal mucosal organoids, specifically: placing the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells in a nasal mucosal organoid culture medium for culture to obtain nasal mucosal organoids; Furthermore, the aforementioned nasal mucosa-loaded organoids can be used in drug screening, disease model construction, toxicity testing, and as reagents or tools for regenerative medicine.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) This invention is the first to apply core-shell microcapsule technology to the culture of nasal mucosa organoids. Sodium alginate is used as the outer shell and sodium carboxymethyl cellulose containing nasal mucosa epithelial cells is used as the core to construct a three-dimensional microenvironment with well-defined composition and controllable physical properties. This completely replaces the animal-derived matrix gel used in traditional culture, significantly reducing costs and improving the reproducibility and standardization of experiments. After induction by organoid culture medium, nasal mucosa organoids can be derived and formed in the microcapsule.

[0018] (2) The present invention uses microfluidic technology to prepare core-shell microcapsules loaded with nasal mucosal epithelial cells. The operation is simple and can generate core-shell microcapsules with uniform size and stable structure in one step, realizing precise control of the organoid formation microenvironment. The method can produce microcapsules continuously and in high throughput, and is easy to automate, providing a reliable tool for large-scale drug screening and preclinical research.

[0019] (3) The substrate-free culture method provided by the present invention enables nasal mucosal epithelial cells to rapidly proliferate in microcapsules and self-assemble into three-dimensional organoids with regular morphology and good activity. This microenvironment not only supports cell proliferation and differentiation, but also avoids the heterogeneity caused by organoid fusion in traditional culture, and the resulting organoids have higher consistency in morphology and function. Attached Figure Description

[0020] Figure 1 This is a front view of a coaxial microfluidic device; Figure 2 This is a 3D view of a coaxial microfluidic device; Figure 3It 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 physical pictures of a coaxial microfluidic device and a coaxial microfluidic electrospray device, where (a) is an overall structural diagram of the coaxial microfluidic device, (b) is a detailed enlarged view of the outlet of the coaxial microfluidic device, (c) is an overall schematic diagram of the coaxial microfluidic electrospray device, and (d) is a partial enlarged view of the coaxial microfluidic electrospray device. Figure 8 A schematic diagram of the preparation process for core-shell microcapsules loaded with nasal mucosal epithelial cells; Figure 9 Generate images of the droplets in real time; Figure 10 An optical microscope image of a nuclear-shell microcapsule loaded with nasal mucosal epithelial cells; Figure 11 The diagram shows the particle size analysis of the inner and outer diameters and shell thickness of the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells, where a is the outer diameter of the shell, b is the inner diameter of the shell, and c is the shell thickness. Figure 12 The curves show the average particle size relationship of the nucleoshell microcapsules loaded with nasal mucosal epithelial cells, where a is the relationship between different internal phase flow rates and particle size, b is the relationship between different external phase flow rates and particle size, c is the relationship between different voltages and particle size, and d is the relationship between collection distance and particle size. Figure 13 To illustrate the growth morphology and live / dead staining of nasal mucosal organoids cultured at different time points (days 1, 3, 7, and 14); Explanation of reference numerals in the attached figures: 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 present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0023] Example 1 (1) The front view and perspective view of the coaxial microfluidic device are shown below. Figure 1 and Figure 2 As shown, the construction method is as follows: 1) Prepare two capillary glass tubes with inner diameters of 300 μm and 100 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. Small-diameter capillary glass tube 2 is coaxially inserted into large-diameter capillary glass tube 1, with one end of small-diameter capillary glass tube 2 flush with one end of large-diameter capillary glass tube 1. The other end of small-diameter capillary glass tube 2 extends from the other end of 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 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. The three-dimensional and sectional views of the two coaxially inserted capillary glass tubes are shown below. Figure 3 and Figure 4 As shown.

[0024] 2) Take a clean glass slide 3 as the substrate, and place the coaxially fitted capillary glass tube on top of the glass slide 3; to establish a sealed fluid passage, use two flat-tipped needles (first flat-tipped needle 4 and second flat-tipped needle 5). The three-dimensional views of the first flat-tipped needle 4 and the second flat-tipped needle 5 are shown below. Figure 5 and Figure 6 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 is provided with a large and a small first groove, namely the first large groove 411 and the first small groove 412. The second flat-mouth needle 5 is composed of a second needle seat 51 and a second needle 52. The bottom surface of the second needle seat 51 is provided with a second groove 511.

[0025] 3) 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 extension 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; adjust the position to ensure that all components are coaxial, fill all gaps with epoxy resin glue and cure, so that each needle holder forms a stable and sealed connection with 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. A physical diagram of the coaxial microfluidic device is shown below. Figure 7 As shown in (a) and (b), (a) is the overall structural diagram and (b) is a detailed enlarged view of the exit.

[0026] (2) Construction of coaxial microfluidic electrospray device The prepared coaxial microfluidic device 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 outlet end of the coaxial microfluidic device (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 electrospray device is shown below. Figure 7 As shown in (c) and (d), (c) is an overall physical diagram of the coaxial microfluidic electrospray device, and (d) is a partial enlarged view. This device can generate 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 collection liquid and solidify into microcapsules.

[0027] Example 2 Isolation of nasal mucosal epithelial cells: (1) Prepare sterile nasal brush, sterile physiological saline, 15mL sterile centrifuge tube, DMEM / F-12 medium containing 10% fetal bovine serum, 0.25% TrypLE express enzyme, crushed ice, etc. (2) The operator wore sterile gloves and a mask, rinsed both nasal cavities with physiological saline three times each, and the subject blew his nose and tilted his head back to fully expose the anterior part of the nasal cavity. After moistening the nasal brush with PBS, the operator held the sterile nasal brush and gently inserted it into the subject's nostril and inferior turbinate, slowly rotating it 5 times along the surface of the nasal mucosa to ensure that the brush head fully contacts the mucosal epithelium. The nasal brush was removed and the brush head was immersed in a 15 mL centrifuge tube containing 10 mL of DMEM / F-12 medium containing 1% penicillin and streptomycin. The operation was repeated on the lateral nasal cavity with a new brush. The brush head was collected in the same 15 mL centrifuge tube, labeled and stored on ice. The brush head was cut off with sterile scissors. Using a sterile 1250µL pipette tip, insert a nasal brush through the tip and repeatedly scrape the brush up and down to detach cells into the culture medium. Discard the nasal brush and repeat the process with a second brush. Centrifuge the tube at 1200 rpm for 5 minutes at 4°C, discarding the supernatant and retaining the cell pellet at the bottom. Resuspend the cell pellet in 2 mL of sterile saline and centrifuge again at 1200 rpm for 5 minutes, discarding the supernatant. Repeat this step twice to remove residual mucus and impurities. After discarding the supernatant, add approximately 0.25% of the pellet volume (about 6 times its volume). TrypLE express enzyme, resuspend the pellet and incubate at 37°C for 5 min; remove the resuspended cells and continue incubation for 5 min, add 2 volumes of DMEM / F-12 medium containing 10% FBS to stop digestion; after resuspending, filter through a 70 μm cell sieve, collect the filtrate, centrifuge at 1200 r / min at 4°C for 5 min, discard the supernatant and retain the bottom cell pellet, which is the nasal mucosal epithelial cells.

[0028] Example 3 The preparation process of a nuclear-shell microcapsule loaded with nasal mucosal epithelial cells is shown in the schematic diagram below. Figure 8 As shown: (1) Internal phase solution: Weigh sterile sodium carboxymethyl cellulose powder, add sterile deionized water to prepare a 1% carboxymethyl cellulose solution, thoroughly mix the nasal mucosal epithelial cell precipitate prepared in Example 1 with the carboxymethyl cellulose solution, and blow evenly (each milliliter of sodium carboxymethyl cellulose solution contains 5 × 10⁻⁶ ppm). 6 (each cell), so that the nasal mucosal epithelial cells are evenly dispersed in sodium carboxymethyl cellulose solution as the inner phase solution; (2) External phase solution: Weigh the sterile sodium alginate powder, add sterile deionized water, and prepare a sodium alginate solution with a mass concentration of 2% as the external phase solution; (3) Collection liquid: Weigh the sterile anhydrous calcium chloride granules, add sterile deionized water, and prepare a calcium chloride solution with a mass concentration of 2%, which is the collection liquid containing calcium ions. (4) Construct a coaxial microfluidic electrospray device according to the method of Example 1; inject the inner phase solution in step (1) and the outer phase solution in step (2) into the small-diameter capillary glass tube 2 and the large-diameter capillary glass tube 1 of the coaxial microfluidic electrospray device, respectively; set the inner phase flow rate to 15 μL / min, the outer phase flow rate to 80 μL / min, the voltage range to 5.5 kV, and the distance between the spray tip of the coaxial microfluidic device and the surface of the collected liquid (collection distance) to 4 cm (the core-shell structure droplets formed at the device outlet (such as...) Figure 9 The cells fall into the collection liquid and stand for 10 minutes (sodium alginate and calcium ions crosslink and solidify), forming a nucleoshell microcapsule loaded with nasal mucosal epithelial cells in the collection liquid. The outer shell of the nucleoshell microcapsule loaded with nasal mucosal epithelial cells is sodium alginate hydrogel, and the inner core is an aqueous solution of sodium carboxymethyl cellulose containing nasal mucosal epithelial cells.

[0029] A light micrograph of the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells prepared in Example 3 is shown. Figure 10 As shown; the microcapsule has a core-shell structure.

[0030] The particle size analysis diagram of the core-shell microcapsules loaded with nasal mucosal epithelial cells prepared in Example 3 is shown below. Figure 11 As shown in the figure, a is the outer diameter of the shell, b is the inner diameter, and c is the shell thickness. It can be seen from the figure that the outer diameter of the microcapsule shell is 375-435μm, the inner diameter of the microcapsule is 210-270μm, the thickness distribution is 155-175μm, and the particle size distribution is relatively uniform.

[0031] Example 4 The procedure was the same as in Example 3, except that the flow rate of the inner phase solution in step (4) was changed to 5, 10, 15, 20, and 25 μL / min, while other conditions remained unchanged. The changes in the outer diameter of the shell and the inner diameter of the prepared nuclear-shell microcapsules loaded with nasal mucosal epithelial cells are shown in the figure. Figure 12 As shown in Figure a, it can be seen that when the external phase flow rate, voltage, and collection distance are kept constant, the particle size of the nucleoshell microcapsules loaded with nasal mucosal epithelial cells increases with increasing the internal phase flow rate.

[0032] Example 5 The procedure is the same as in Example 3, except that the flow rate of the external phase solution in step (4) is changed to 60, 70, 80, 90, and 100 μL / min, while other conditions remain unchanged. The changes in the outer shell diameter and inner core diameter of the prepared nuclear-shell microcapsules loaded with nasal mucosal epithelial cells are shown in the figure. Figure 12 As shown in Figure b, it can be seen that when the internal phase flow rate, voltage, and collection distance are kept constant, increasing the external phase flow rate will increase the particle size of the nucleoshell microcapsules loaded with nasal mucosal epithelial cells.

[0033] Example 6 The operation is the same as in Example 3, except that the voltage in step (4) is changed to 3.5, 4.5, 5.5, 6.5, and 7.5 kV respectively, while the other conditions remain unchanged. The changes in the outer diameter of the shell and the inner diameter of the prepared nuclear-shell microcapsules loaded with nasal mucosal epithelial cells are shown in the figure. Figure 12 As shown in Figure c, it can be seen that when the flow rate of the inner and outer phases and the collection distance are kept constant, by increasing the voltage, the particle size of the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells decreases due to the increase in the electric field force.

[0034] Example 7 The operation is the same as in Example 3, except that the distance between the spray tip of the coaxial microfluidic device and the surface of the collection liquid in step (4) is changed to 2, 3, 4, 5, and 6 cm respectively, while other conditions remain unchanged. The changes in the outer diameter of the shell and the inner diameter of the prepared nuclear-shell microcapsules loaded with nasal mucosal epithelial cells are shown in the figure. Figure 12 As shown in Figure d, when the internal and external phase flow rates and voltage are kept constant, increasing the collection distance results in a relative decrease in electric field strength, leading to an increase in the particle size of the generated nuclear-shell microcapsules loaded with nasal mucosal epithelial cells.

[0035] Application Examples The nuclear-shell microcapsules loaded with nasal mucosal epithelial cells prepared in Example 3 were washed three times with DPBS, transferred to 6-well culture plates, and cultured in nasal mucosal organoid culture medium at 37°C in a 5% CO2 incubator. The culture medium was changed every 48 h. The outer shell of the microcapsule provided a stable three-dimensional growth space for the cells, while sodium carboxymethyl cellulose in the inner phase provided initial growth support. The cells proliferated and self-assembled within the microcapsules to form morphologically uniform nasal mucosal organoids.

[0036] A method for qualitative and quantitative detection of cell viability in nasal mucosal organoids within microcapsules was employed using a live / dead cell staining kit. This method was used to evaluate the ability of microcapsule carriers to support the in vitro survival, proliferation, and structural maintenance of nasal mucosal organoids. The specific steps are as follows: (1) Preparation of experimental materials and reagents: Purchase commercially available Calcein-AM / PI staining kits. Calcein-AM is a live cell-specific fluorescent probe that can penetrate intact cell membranes and emit green fluorescence under the action of intracellular esterases. PI is a dead cell-specific dye that can penetrate damaged cell membranes, embed into nucleic acid double strands, and emit red fluorescence. Sterile calcium- and magnesium-free Duchenne phosphate buffer solution (DPBS, pH 7.2~7.4) is used to remove residual culture medium and unbound staining reagents to avoid background fluorescence interference.

[0037] (2) Specific testing operation steps: 1) On days 1, 3, 7 and 14 of the culture of nasal mucosa organoid microcapsules, the culture system at the corresponding time points was collected under aseptic conditions in a clean bench; the complete culture medium was discarded by a Pasteur pipette, and an appropriate amount of sterile DPBS was added to resuspend the microcapsules, and the capsules were gently inverted and washed 3 times. 2) Take out the Calcein-AM / PI staining kit and working solution stored at -20℃ and equilibrate at room temperature for 30 minutes; prepare the staining working solution in a sterile EP tube according to the proportions in the kit instructions: take an appropriate amount of Calcein-AM stock solution and PI stock solution, add them to the working solution and mix well. Prepare and use immediately to avoid dye degradation or hydrolysis.

[0038] 3) Add an appropriate amount of the above staining working solution to the washed microcapsule precipitate, and gently blow to suspend the microcapsules completely; seal and place in a dark environment, and incubate in a 37°C incubator for 30 min, gently inverting and mixing once every 10 min to ensure that the staining reagent fully penetrates into the microcapsule and fully contacts the organoid cells. 4) After staining, wash the microcapsules twice with DPBS to thoroughly remove unbound free dye, reduce non-specific fluorescence background, and ensure that the fluorescence signal comes only from the cells themselves. 5) Place the washed and stained microcapsules in a well plate and observe them under an inverted fluorescence microscope, switching between the green fluorescence channel (excitation wavelength 488 nm, emission wavelength 530 nm) and the red fluorescence channel (excitation wavelength 535 nm, emission wavelength 617 nm). Simultaneously acquire bright-field images to display the morphology and structure of the microcapsules and organoids. Record the distribution of live cells (green fluorescence) and dead cells (red fluorescence) within the nasal mucosa organoids at different time points, as well as the fluorescence intensity and cell morphology. The results are as follows: Figure 13 As shown; Nasal mucosal organoids within the microcapsules exhibited uniform green fluorescence, indicating intact cell membranes, normal intracellular esterase activity, and thus, viable cells. The appearance of red fluorescence suggested damaged cell membranes and exposed intracellular nucleic acids, indicating dead cells. Simultaneously, the morphological integrity of the organoids was observed to assess the microcapsules' ability to maintain their structure. Figure 13On day 1 of culture, the nasal mucosal organoids within the microcapsules exhibited a loose distribution, high-intensity green fluorescence, and minimal red fluorescence from dead cells, with a high proportion of live cells. This indicates that the microcapsule encapsulation process and the initial culture environment did not significantly damage the cells, and the organoids showed good initial activity. On days 3 and 7, the organoids within the microcapsules gradually aggregated, with uniform green fluorescence distribution and only a small amount of weak red fluorescence. The proportion of live cells remained above 90%, suggesting that the organoids continued to survive and slowly proliferate within the microcapsules, and the microcapsule carrier provided a stable microenvironment for cell survival. On day 14 of culture, the organoids within the microcapsules maintained their morphology intact, with green fluorescence continuing to dominate, and the proportion of live cells did not decrease significantly. This indicates that the nasal mucosal organoids can survive stably within the microcapsules for a long time and maintain good proliferative activity, demonstrating that the microcapsule carrier has the ability to support long-term in vitro culture of nasal mucosal organoids.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a nuclear-shell microcapsule loaded with nasal mucosal epithelial cells in the culture of nasal mucosal organoids, characterized in that, Nuclear-shell microcapsules loaded with nasal mucosal epithelial cells were placed in nasal mucosal organoid culture medium and cultured to obtain nasal mucosal organoids. The method for preparing the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells is as follows: using coaxial microfluidic electrospray technology, the inner phase solution of sodium carboxymethyl cellulose containing nasal mucosal epithelial cells and the outer phase solution of sodium alginate are respectively passed into the inner tube and outer tube of the coaxial microfluidic device. Under the action of voltage, the nuclear-shell microcapsules loaded with nasal mucosal epithelial cells are formed in the collection liquid containing calcium ions. The sodium carboxymethyl cellulose inner phase solution containing nasal mucosal epithelial cells is obtained by mixing nasal mucosal epithelial cells and sodium carboxymethyl cellulose solution, with each milliliter of sodium carboxymethyl cellulose solution containing 3 × 10⁻⁶ cells. 6 -6×10 6 One nasal mucosal epithelial cell, a sodium carboxymethyl cellulose solution of 0.8-1.2 wt%; The sodium alginate external phase solution is a 1.8-2.2 wt% solution; The outer tube of the coaxial microfluidic device is a capillary tube with a diameter of 300-350 μm, and the inner tube is a capillary tube with a diameter of 80-100 μm; the mass concentration of calcium chloride in the calcium ion-containing collection solution is 1.5-2.5%; The flow rate of the sodium carboxymethyl cellulose inner phase solution containing nasal mucosal epithelial cells is 5-20 μL / min, and the flow rate of the sodium alginate outer phase solution is 60-100 μL / min.

2. The application according to claim 1, characterized in that, The nasal mucosal epithelial cells were obtained using a nasal brush method.

3. The application according to claim 1, characterized in that, The voltage is 3.5-7.5kV, and the distance between the spray tip of the coaxial microfluidic device and the surface of the collected liquid is 2-6cm.

4. The application according to claim 1, wherein the core-shell microcapsule loaded with nasal mucosal epithelial cells has a sodium alginate gel as the outer shell and a sodium carboxymethyl cellulose solution containing nasal mucosal epithelial cells as the core.

5. The application according to claim 1, characterized in that, The nuclear-shell microcapsules loaded with nasal mucosal epithelial cells have an outer shell diameter of 375-435 μm and an inner core diameter of 210-270 μm.

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