A lacrimal passage epithelial organoid and a construction method and application thereof

Abstract

The application discloses a lacrimal passage epithelial organoid and a construction method and application thereof, and the construction method comprises the following steps: S1, preparation of a three-dimensional biomimetic support; S2, acquisition of lacrimal passage epithelial stem cells; and S3, construction and culture of the organoid. The PCL / GEL nanofiber support constructed by the electrospinning technology is combined with Matrigel coating, the extracellular matrix (ECM) microenvironment of the in-vivo lacrimal passage mucosa is successfully simulated, and the technical difficulties that stem cells are prone to aging and differentiation direction disorder in traditional two-dimensional culture are fundamentally solved. This breakthrough result promotes the lacrimal passage tissue engineering from simple 'cell adhesion growth' to a new height of'structure-function synchronous reconstruction', and provides a double technical guarantee of seed cells and tissue engineering supports for solving the clinical core pain point of postoperative scar adhesion and re-occlusion of the lacrimal passage obstruction disease.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, specifically to a lacrimal duct epithelial organoid, its construction method, and its application. Background Technology

[0002] Lacrimal duct obstruction is one of the most common blinding eye diseases in ophthalmology. It is often caused by congenital developmental abnormalities, inflammation, trauma, or other factors that obstruct the tear drainage channel, leading to tear overflow. In severe cases, it can lead to secondary chronic dacryocystitis, keratitis, and even endophthalmitis, significantly impairing the patient's visual health and quality of life. Currently, the mainstream clinical treatment strategy is to surgically restore or reconstruct the lacrimal drainage channel. However, long-term efficacy remains limited by the critical technical bottleneck of impaired lacrimal duct epithelial repair. Postoperatively formed lacrimal ducts or anastomoses often lack timely coverage by functional epithelium, leading to excessive proliferation of fibroblasts and scar adhesions, ultimately causing re-obstruction of the lumen. Existing auxiliary methods such as anti-inflammatory irrigation or silicone prostheses only provide physical support or short-term anti-inflammatory effects, failing to fundamentally promote the directed proliferation and differentiation of epithelial cells and thus failing to meet the physiological repair needs of the lacrimal duct mucosa.

[0003] To address the challenge of lacrimal duct epithelial repair, isolating and culturing lacrimal duct epithelial stem cells (Lacrimal duct stem cells) as "seed cells" for tissue engineering has become a research hotspot. Studies have confirmed that a population of stem cells with self-renewal and multi-directional differentiation potential exists in lacrimal duct epithelial tissue, which is core for maintaining lacrimal duct patency and repairing damage. However, current lacrimal duct epithelial stem cell culture techniques are mainly limited to the traditional two-dimensional (2D) culture stage. Due to the lack of extracellular matrix (ECM) network and three-dimensional spatial structure support, the two-dimensional culture system cannot simulate the complex biomimetic microenvironment in vivo. As a result, the isolated stem cells easily lose their stemness characteristics during in vitro expansion, exhibiting low proliferation efficiency and disordered differentiation direction. They cannot reproduce the physiological function and structural integrity of the lacrimal duct epithelium in vivo, thus limiting their application in clinical regenerative medicine.

[0004] Organoid technology, as a three-dimensional culture model capable of highly mimicking the structure and function of in vivo tissues, has achieved breakthroughs in the construction of luminal tissues such as the intestine and urethra, as well as epithelial tissues such as the conjunctiva and lacrimal ducts of the eye. Although there have been research reports on organoids of the ocular surface and glands, the construction of organoids for the specific anatomical site of the lacrimal duct epithelium remains a technological gap. Because lacrimal duct epithelial cells differ significantly from conjunctival or lacrimal gland cells in physiological characteristics, gene expression, and microenvironmental requirements, existing ocular organoid culture systems (including culture medium components and scaffold materials) cannot meet the specific growth and directed differentiation requirements of lacrimal duct epithelial stem cells. Therefore, developing a three-dimensional organoid culture system specifically for the expansion and differentiation of lacrimal duct epithelial stem cells has significant clinical significance and application value for overcoming the treatment bottleneck of lacrimal duct obstructive diseases. Summary of the Invention

[0005] The main objective of this invention is to propose a lacrimal duct epithelial organoid, its construction method, and its application, aiming to solve the problems of existing lacrimal duct obstruction treatments, which lack functional epithelial repair methods, leading to postoperative scar adhesion and re-obstruction, as well as the loss of stemness and poor functional adaptability of traditional two-dimensional cultured lacrimal duct epithelial stem cells, thus failing to meet clinical needs.

[0006] To achieve the above objectives, this invention proposes a method for constructing a lacrimal duct epithelial organoid, comprising the following steps: S1. Preparation of three-dimensional biomimetic scaffold: Polycaprolactone and gelatin were mixed, and hexafluoroisopropanol was added as a solvent. The mixture was heated and stirred to obtain a spinning solution. Hollow nanofiber tubes were prepared by electrospinning technology. After cross-linking treatment, the surface of the matrix adhesive was coated to obtain a three-dimensional biomimetic scaffold. S2. Acquisition of lacrimal duct epithelial stem cells: Lacrimal duct epithelial stem cells are isolated and purified from lacrimal duct mucosal epithelial tissue and expanded and cultured in vitro; S3. Organoid construction and culture: The expanded lacrimal duct epithelial stem cells are seeded onto the three-dimensional biomimetic scaffold and induced to undergo three-dimensional induction culture in organoid culture medium. The cells are induced to differentiate on the three-dimensional biomimetic scaffold to form organoids with lacrimal duct epithelial structures, namely lacrimal duct epithelial organoids.

[0007] Preferably, in step S1, the mass ratio of polycaprolactone to gelatin in the spinning solution is 5:(4~6); preferably, the mass ratio of polycaprolactone to gelatin in the spinning solution is 5:5. The electrospinning parameters are: positive high voltage 13 kV, negative high voltage 1 kV, spinning solution flow rate 18~22 μL / min, receiving distance 10~20 cm, and receiving device rotation speed 45 rpm. The hollow nanofiber tube has a fiber diameter of 1~3 μm, a porosity of 60%~80%, and the longest diameter of the pores between fibers is 3-10 μm.

[0008] Preferably, in step S1, the specific steps of the crosslinking treatment are as follows: the hollow nanofiber tube is immersed in a MES buffer containing 20 mM EDC and 10 mM NHS, and reacted at 4 °C for 12 h.

[0009] Preferably, in step S1, the specific steps for coating the surface of the matrix gel are as follows: the Matrigel stock solution and DMEM / F12 culture medium are mixed at a volume ratio of 5:(90~100) to prepare a dilution solution, the sterilized three-dimensional biomimetic scaffold is completely immersed in the dilution solution, and incubated at 36~38 ℃ for 0.8~1.2 h.

[0010] Preferably, in step S2, the acquisition of lacrimal duct epithelial stem cells specifically includes the following steps: first, using collagenase to digest the lacrimal duct mucosal epithelial tissue, then using trypsin-EDTA digestion to obtain a single-cell suspension, and then inoculating the cell suspension for 1.5~2.5 h and then aspirating the supernatant for transfer culture to remove adherent fibroblast impurities.

[0011] Preferably, in step S3, the organoid culture medium is a basal culture medium supplemented with the following components: 1×B27, 100 ng / mL Rspondin-1, 1.25 mM N-acetylcysteine, 100 ng / mL Noggin, 50 ng / mL epidermal growth factor, 10 ng / mL basic fibroblast growth factor, 5 μM transforming growth factor β inhibitor, 10 μM ROCK inhibitor, 15 ng / mL interleukin-10, and 10 μM PDGF antagonist.

[0012] Preferably, in step S3, the cell density for seeding is 5 × 10⁻⁶. 5 The cells / mL, the three-dimensional induction culture period is 14 days, and the organoid culture medium is changed every 3 days during the period.

[0013] The present invention also proposes a transplantable lacrimal duct epithelial organoid, which is prepared by the construction method described above. The epithelial organoid has a tubular structure with a superficial columnar epithelium and a basal squamous epithelium. The epithelial thickness of the epithelial organoid is 65±8 μm. The epithelial organoid is double positive for the stem cell markers P63 and KRT17.

[0014] The present invention also proposes the application of a transplantable lacrimal duct epithelial organoid as described above in the preparation of lacrimal duct repair implants.

[0015] The present invention also proposes the application of the transplantable lacrimal duct epithelial organoid as described above in the preparation of a drug for treating lacrimal duct obstructive diseases, including lacrimal duct obstruction, lacrimal duct malformation, or chronic dacryocystitis complicated with infection.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention utilizes a PCL / GEL nanofiber scaffold constructed through electrospinning technology, combined with Matrigel coating, to successfully simulate the extracellular matrix (ECM) microenvironment of the lacrimal duct mucosa in vivo, fundamentally solving the technical problems of easy aging and disordered differentiation of stem cells in traditional two-dimensional culture. Experimental results show that the stemness marker (P63) of lacrimal duct epithelial stem cells in this three-dimensional culture system... + / KRT17 +The double-positive expression was significantly superior to that in the two-dimensional culture group, confirming that the three-dimensional scaffold effectively maintained the self-renewal capacity of stem cells through the synergistic effect of spatial structural constraint and gradient release of growth factors. Furthermore, the induction phase of this invention utilized organoid culture medium, in which key factors such as EGF, FGF-basic, and the TGF-β inhibitor (SB431542) synergistically induced stem cells to differentiate into functional epithelial cells under the constraint of the three-dimensional scaffold. This dual-regulation system of "scaffold-culture medium" successfully induced stem cells to form a natural tubular structure with "surface columnar epithelium + basal squamous epithelium," indicating that it possesses functions similar to those of the lacrimal duct epithelium in vivo. This breakthrough elevates lacrimal duct tissue engineering from simple "cell adhesion growth" to a new level of "simultaneous reconstruction of structure and function," providing dual technical support of seed cells and tissue engineering scaffolds for addressing the core clinical challenge of postoperative scar adhesion and re-occlusion.

[0017] (2) The optimized PCL / GEL composite scaffold of this invention cleverly balances the contradiction between mechanical strength and biocompatibility: the PCL component provides sufficient mechanical support to resist the pressure of lacrimal duct tissue (appropriate compressive strength) and prevents luminal collapse; while the Gelatin component, combined with a nanoporous structure (porosity 60%-80%), significantly improves cell adhesion and promotes nutrient exchange, overcoming the repair failure problem caused by the "epithelial creep barrier" on the surface of pure synthetic materials (such as silicone). More importantly, the composite material has good biodegradability and can be gradually absorbed and replaced by autologous tissue regeneration after implantation, avoiding the pain and risk of re-blockage caused by the need for a second surgery to remove traditional silicone prostheses. At the same time, the electrospinning process can flexibly adjust the length and diameter of the scaffold, enabling personalized customization for different clinical scenarios such as lacrimal duct obstruction and malformation, significantly reducing postoperative scar tissue formation, and providing a new treatment option for lacrimal duct obstructive diseases that combines anatomical reconstruction and functional repair. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the hollow nanofiber tubular three-dimensional biomimetic scaffold constructed by electrospinning according to the present invention. Figure 2 Electron micrographs at different magnifications of the three-dimensional nanofiber structure formed by electrospinning in this invention; Figure 3 This is a diagram of the lacrimal duct support of the present invention; Figure 4 This is a flowchart illustrating the isolation, digestion, and in vitro culture of lacrimal duct epithelial stem cells according to the present invention. Figure 5 This is a fluorescence micrograph of the lacrimal duct epithelial organoid stained with Calcein AM / PI according to the present invention; Figure 6 The images show an optical microscope image (A) of lacrimal duct epithelial organoids stained with hematoxylin and eosin (HE) and an immunofluorescence staining image (B) of epithelial organoids cultured on a scaffold. Figure 7 This is an immunofluorescence staining image of 2D cultured epithelial organoids from this invention; Figure 8 These are diagrams illustrating the anatomical structure, modeling verification, and pathological changes of the rabbit lacrimal duct obstruction model of this invention; A1: Anatomical location of the rabbit lacrimal punctum; A2: Anatomical location of the rabbit lacrimal sac; A3: Anatomical location of the rabbit nasolacrimal duct; A4: HE-stained optical microscope image of normal rabbit lacrimal canaliculi; A5: HE-stained optical microscope image of normal rabbit lacrimal sac tissue; A6: HE-stained optical microscope image of normal rabbit nasolacrimal duct tissue; B1: Gross appearance of a normal rabbit; B2: Gross appearance of a rabbit with dacryocystitis; B3: CT angiography image of a normal lacrimal duct; B4: CT angiography image of dacryocystitis; B5: HE-stained optical microscope image of a normal nasolacrimal duct; B6: HE-stained optical microscope image of the nasolacrimal duct after 2 weeks of obstruction; B7: HE-stained optical microscope image of the nasolacrimal duct after 4 weeks of obstruction; B8: HE-stained optical microscope image of the nasolacrimal duct after 8 weeks of obstruction. Figure 9 This is a grouping diagram after constructing the rabbit lacrimal duct obstruction model according to the present invention; A: Blank control group; B: Control group; C: Experimental group; Figure 10 The following images are for evaluating the repair effect of this invention: A: Normal lacrimal duct mucosa; B: Lacrimal duct obstruction; C: Endoscopic lacrimal duct mucosa of a rabbit model with implanted electrospinning scaffold; D: Endoscopic lacrimal duct mucosa of a rabbit model with implanted pure PLLA scaffold; E: Electron micrograph of rabbit lacrimal duct epithelium with normal mucosa; F: Electron micrograph of rabbit lacrimal duct epithelium with implanted electrospinning scaffold; G: Electron micrograph of rabbit lacrimal duct epithelium with implanted pure PLLA scaffold. Figure 11 This is a flowchart illustrating the overall research process of this invention.

[0020] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0023] The English abbreviations and their explanations involved in this invention are as follows: PCL: Polycaprolactone, a synthetic polymer material with good biocompatibility and biodegradability; GEL: Gelatin, a natural collagen derivative with excellent biocompatibility and cell adhesion. Matrigel: A matrix gel that mimics the natural matrix material of the extracellular matrix (ECM); R-spondin 1: provides key signaling support for the sustained growth and phenotypic maintenance of organoids; Noggin: Head protein, inhibits bone morphogenetic protein signaling; EGF: Epidermal Growth Factor, a growth factor that promotes the proliferation of epithelial cells; FGF-basic: Basic Fibroblast Growth Factor, a growth factor that promotes stem cell proliferation and differentiation; SB431542: A transforming growth factor-β (TGF-β) inhibitor that inhibits fibrosis and abnormal differentiation; Y-27632: A selective Rho-associated coiled-coil-containing kinase (ROCK) inhibitor that regulates cytoskeleton remodeling, inhibits apoptosis, and promotes cell survival by blocking the Rho / ROCK signaling pathway. IL-10: Interleukin-10, an anti-inflammatory factor that inhibits the expression of pro-inflammatory factors; AG1296: A platelet-derived growth factor (PDGF) antagonist that inhibits fibroblast proliferation; FBS: Fetal Bovine Serum, a serum product obtained by separating, purifying and aseptically processing the blood of unborn fetuses; EDTA: Ethylenediaminetetraacetic acid, a broad-spectrum metal ion chelating agent; PBS: Phosphate Buffered Saline, an isotonic buffer solution.

[0024] IL-6: Interleukin-6, a pro-inflammatory factor that initiates and amplifies inflammatory responses; TNF-α: Tumor necrosis factor-α, which induces the expression of other pro-inflammatory factors (such as IL-6 and IL-1β) and amplifies inflammatory signals. MMA: Methyl methacrylate is an organic monomer with strong free radical polymerization ability; PMMA: Poly(methyl methacrylate) is a thermoplastic, amorphous acrylic resin. HFIP: Hexafluoroisopropanol is an organic solvent used to dissolve PCL and GEL; MES: 2-(N-Morpholino)ethanesulfonic acid, a water-soluble weak acid buffer; P63: A stem cell marker, a protein specifically expressed by lacrimal duct epithelial stem cells; KRT17: Cytokeratin 17, a specific marker of epithelial basal cells; KI67: A cell proliferation marker, expressed in cells with proliferative capacity; The raw materials and culture media used in the following examples: (1) Raw material components Cell source: Lacrimal duct epithelial stem cells (isolated from the lacrimal sac and upper segment of the nasolacrimal duct mucosa epithelium of cadaveric eyes or New Zealand white rabbits).

[0025] (2) Culture medium system Basic culture medium: Modified embryonic stem cell culture medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antibiotics, 2mM glutamine, and 1% non-essential amino acids; Organoid culture medium: Add specific additives to the basal culture medium to the following final concentrations: 1×B27, 100 ng / mL Rspondin-1, 1.25 mM N-acetylcysteine, 100 ng / mL Noggin, 50 ng / mL epithelial growth factor (EGF), 10 ng / mL fibroblast growth factor (FGF-basic), 5 μM transforming growth factor β inhibitor (SB431542), 10 μM ROCK inhibitor (Y-27632), 15 ng / mL anti-inflammatory factor (IL-10), and 10 μM PDGF antagonist (AG1296). Primary complete culture medium: basal medium + 10% FBS, 2ng / ml EGF, 0.1 nM cholera toxin, 0.5μg / ml hydrocortisone, 1×ITS, 10μM Y-276232 for stem cell isolation and expansion.

[0026] Auxiliary reagents: 0.25% trypsin-EDTA digestion solution, PBS buffer, 4% paraformaldehyde fixative, immunofluorescence staining kit (containing antibodies such as P63), and blood agar medium; Animal model construction reagents: methyl methacrylate (MMA) and polymethyl methacrylate (PMMA) were used to prepare a rabbit lacrimal duct obstruction model.

[0027] Example 1: Preparation of lacrimal duct epithelial organoids 1. Fabrication of a three-dimensional biomimetic scaffold (1) Preparation of spinning solution: Weigh 0.6 g of polycaprolactone (PCL, Mn=80,000) and 0.6 g of gelatin (GEL, Type A) accurately at a mass ratio of 5:5, and add them together to 10 mL of hexafluoroisopropanol (HFIP) solvent. Place the mixture on a constant temperature magnetic stirrer and stir continuously at 40°C for 12 h until the solute is completely dissolved to obtain a uniform spinning solution with a mass concentration of 12%, which is ready for use.

[0028] (2) Electrospinning and Fiber Tube Preparation: An electrospinning apparatus was constructed, using a thin metal rod wrapped in a silicone tube as the receiving device. The spinning parameters were set as follows: receiving device rotation speed 45 rpm, high voltage power supply output positive voltage 13 kV, negative voltage 1 kV, receiving distance 15 cm, and micro-injection pump flow rate 20 μL / min. Spinning was carried out continuously for 20 minutes at room temperature (25℃) and relative humidity 40%, and hollow nanofiber tubes with a thickness of approximately 200 μm were collected. The fiber tubes, along with the mold, were frozen at -20℃ for 4 h before demolding, and then dried in a vacuum freeze dryer for 48 h.

[0029] (3) Crosslinking modification: Prepare a crosslinking solution (containing 20 mM EDC and 10 mM NHS, in 0.05 M MES buffer with 50% (v / v) ethanol as the solvent). Completely immerse the dried fiber tube in the crosslinking solution and react at 4 °C for 12 h. After the reaction, soak and rinse with PBS buffer for 2 h, then rinse continuously with deionized water for 24 h to completely remove residual crosslinking agent. Finally, freeze-dry for 48 h to obtain the crosslinked fiber tube.

[0030] (4) Cutting and sterilization: Cut the dried cross-linked fiber tube into tubular segments with a length of 5 mm, sterilize with ethylene oxide for 3 h, and then place in a sterile environment for later use.

[0031] (5) Matrigel coating (construction of biomimetic microenvironment): Under aseptic conditions, 50 μL of Matrigel stock solution was added to 950 μL of pre-cooled DMEM / F12 basal medium and gently mixed to prepare a 5% (v / v) Matrigel dilution. The sterilized scaffold was placed in a 24-well plate, moistened with a small amount of serum-free medium, and then 500 μL of Matrigel dilution was added to ensure that the scaffold was completely submerged. The plate was incubated at 37 ℃ for 1 h, and excess liquid was removed to obtain the three-dimensional biomimetic scaffold. A schematic diagram of the structure of the hollow nanofiber tubular three-dimensional biomimetic scaffold constructed by electrospinning is shown below. Figure 1 As shown; electron microscope images of the three-dimensional nanofiber structure formed by electrospinning at different magnifications are as follows. Figure 2 As shown, the lacrimal duct stent is as follows Figure 3 As shown.

[0032] 2. Isolation and expansion of lacrimal duct epithelial stem cells (1) Tissue processing: Under sterile conditions, the lacrimal sac and upper segment of the nasolacrimal duct of New Zealand white rabbits (or human cadaver eyes) were obtained (approximately 0.5 cm³ in volume). The tissue was repeatedly rinsed three times with PBS containing double antibodies. The submucosal connective tissue was dissected under a microscope, and the translucent mucosal epithelial tissue was preserved.

[0033] (2) Double enzyme digestion: The retained tissue was cut into small pieces of 1 mm³, transferred to centrifuge tubes, and 2 mL of collagenase (2 mg / mL) digestion solution was added for the first digestion. The tubes were then incubated at 37 ℃ for 4 h. After centrifugation and resuspending the cells, 0.25% trypsin-0.1% EDTA digestion solution was added, and the tubes were incubated at 37 ℃ for 15 minutes, with gentle shaking every 5 minutes to promote digestion. After digestion, an equal volume of primary complete culture medium was added to terminate the digestion. The solution was then filtered through a 100 μm cell sieve, and the filtrate was collected.

[0034] (3) Cell purification: Place the filtered cell suspension in a centrifuge and centrifuge at 1000 rpm for 5 minutes, then discard the supernatant; resuspend the cell pellet in primary complete culture medium and purify using differential adhesion method: inoculate the cell suspension into a culture flask and incubate at 37 ℃ for 2 hours, then gently aspirate the supernatant (containing non-adherent epithelial stem cells) and transfer it to a new culture flask to remove the fibroblast impurities that have adhered to the culture flask.

[0035] (4) Stem cell expansion culture: The purified lacrimal duct epithelial stem cells were cultured at a ratio of 5×10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in culture flasks, primary complete culture medium was added, and the flasks were incubated at 37 ℃ in a 5% CO2 incubator. The culture medium was replaced for the first time after 24 hours to remove viable cells. Thereafter, the culture medium was replaced every 2 days for 5-7 days until cell confluence reached 80%. Cells were then passaged at a 1:3 ratio for expansion, and second-generation cells were used for organoid construction (ensuring cell stemness and proliferative activity). The isolation, digestion, and in vitro culture process of lacrimal duct epithelial stem cells is as follows: Figure 4 As shown.

[0036] 3. Construction and induction of organoids (1) Cell seeding: Second-generation lacrimal duct epithelial stem cells were prepared into 5×10 cells. 5 A high-density suspension of cells / mL was added evenly to the surface of the three-dimensional biomimetic scaffold prepared above at a rate of 0.5 mL per scaffold. The scaffold was then placed in a clean bench and allowed to stand for 2 hours to allow the cells to fully adhere to the biomimetic scaffold material.

[0037] (2) Functional induction culture: Add organoid culture medium (containing inducing factors such as EGF, FGF-basic, SB431542, etc.) to 24-well plates after cell seeding, and place them in a 37 ℃, 5% CO2 incubator for 14 days. During this period, replace the fresh organoid culture medium every 3 days. The cells proliferate and differentiate on the scaffold and secrete extracellular matrix to form lacrimal duct organoids with complete epithelial structure and function.

[0038] Example 2: Testing of the cultured lacrimal duct epithelial organoids prepared in Example 1.

[0039] (1) When the organoids were cultured for 72 hours, the cell-scaffold complex was stained with Calcein AM / PI and then observed using an inverted fluorescence microscope to obtain... Figure 5 .

[0040] (2) Cell viability assay: Figure 5 The results showed that cells migrated and spread along the fiber network to form a multi-layered cell structure with high cell survival rate, demonstrating the scaffold's excellent cell affinity and ability to support three-dimensional growth.

[0041] (3) Morphological and proliferative capacity detection Organoids were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope to examine the multilayered cell structure and distribution. The results are as follows: Figure 6 As shown in Figure A, the results showed that the organoids formed a typical epithelial structure with an average thickness of 65 ± 8 μm, highly replicating the histological characteristics of the natural lacrimal duct mucosa.

[0042] By comparing with traditional 2D culture, P63, KI67, and KRT17 staining was performed for verification, and the results are as follows: Figure 6 B. Figure 7 As shown, the three-dimensional microenvironment effectively maintains the stemness and proliferative potential of stem cells.

[0043] Example 3: Animal in vivo transplant repair experiment 1. Construction of a rabbit lacrimal duct obstruction model Healthy New Zealand white rabbits (weighing 2-2.5 kg) were selected. After anesthesia, a nasolacrimal duct obstruction model was constructed by injecting a mixture of MMA / PMMA into the lacrimal duct. The success rate of obstruction was verified by lacrimal duct irrigation to ensure complete patency of the lacrimal duct. The construction process of the rabbit lacrimal duct obstruction model is as follows: Figure 8 As shown, Figure 8 The results confirmed the successful establishment of the lacrimal duct obstruction model.

[0044] 2. Grouping and Transplantation Experimental group: The stent prepared in Example 1 was implanted via lacrimal duct intubation.

[0045] Control group: A conventional biodegradable stent was implanted via lacrimal duct intubation.

[0046] Blank control group: No materials were implanted; observation was conducted to allow for natural repair. Each group is as follows: Figure 9 As shown.

[0047] 3. Evaluation of Repair Results The wound coverage was observed by lacrimal duct endoscopy at 1 month and 3 months, and the epithelial coverage area ratio was calculated. Lacrimal duct irrigation was performed 3 months post-transplantation, and the lacrimal duct patency rate was calculated. After euthanasia, lacrimal duct tissue was obtained, paraffin-embedded, and sectioned to observe epithelial lesions and repair. Results are as follows: Figure 10 . Figure 11 This is a flowchart of the overall research process.

[0048] Depend on Figure 10 The results show that the lacrimal duct epithelial organoids prepared in this invention can quickly integrate and cover the wound after implantation. Through the dual effects of structural support and functional epithelial repair, they significantly inhibit scar hyperplasia and effectively prevent lacrimal duct re-obstruction.

[0049] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for constructing a lacrimal duct epithelial organoid, characterized in that, Includes the following steps: S1. Preparation of three-dimensional biomimetic scaffold: Polycaprolactone and gelatin were mixed, and hexafluoroisopropanol was added as a solvent. The mixture was heated and stirred to obtain a spinning solution. Hollow nanofiber tubes were prepared by electrospinning technology. After cross-linking treatment, the surface of the matrix adhesive was coated to obtain a three-dimensional biomimetic scaffold. S2. Acquisition of lacrimal duct epithelial stem cells: Lacrimal duct epithelial stem cells are isolated and purified from lacrimal duct mucosal epithelial tissue and expanded and cultured in vitro; S3. Organoid construction and culture: The expanded lacrimal duct epithelial stem cells are seeded onto the three-dimensional biomimetic scaffold and induced to undergo three-dimensional induction culture in organoid culture medium. The cells are induced to differentiate on the three-dimensional biomimetic scaffold to form organoids with lacrimal duct epithelial structures, namely lacrimal duct epithelial organoids.

2. The construction method according to claim 1, characterized in that, In step S1, the mass ratio of polycaprolactone to gelatin in the spinning solution is 5:(4~6). The electrospinning parameters are: positive high voltage 13 kV, negative high voltage 1 kV, spinning solution flow rate 18~22 μL / min, receiving distance 10~20 cm, and receiving device rotation speed 45 rpm. The hollow nanofiber tube has a fiber diameter of 1~3 μm, a porosity of 60%~80%, and the longest diameter of the pores between fibers is 3-10 μm.

3. The construction method according to claim 1, characterized in that, In step S1, the specific steps of the crosslinking treatment are as follows: the hollow nanofiber tube is immersed in a MES buffer containing 20 mM EDC and 10 mM NHS and reacted at 4 °C for 12 h.

4. The construction method according to claim 1, characterized in that, In step S1, the specific steps for coating the surface of the matrix gel are as follows: the Matrigel stock solution and DMEM / F12 culture medium are mixed at a volume ratio of 5:(90~100) to prepare a dilution solution, the sterilized three-dimensional biomimetic scaffold is completely immersed in the dilution solution, and incubated at 36~38 ℃ for 0.8~1.2 h.

5. The construction method according to claim 1, characterized in that, In step S2, the acquisition of lacrimal duct epithelial stem cells specifically includes the following steps: first, the lacrimal duct mucosal epithelial tissue is digested with collagenase, then a single-cell suspension is obtained by digestion with trypsin-EDTA, and then the cell suspension is seeded and cultured for 1.5~2.5 h, and the supernatant is aspirated for transfer culture to remove adherent fibroblast impurities.

6. The construction method according to claim 1, characterized in that, In step S3, the organoid culture medium is a basal culture medium supplemented with the following components: 1×B27, 100 ng / mL Rspondin-1, 1.25 mM N-acetylcysteine, 100 ng / mL Noggin, 50 ng / mL epidermal growth factor, 10 ng / mL basic fibroblast growth factor, 5 μM transforming growth factor β inhibitor, 10 μM ROCK inhibitor, 15 ng / mL interleukin-10, and 10 μM PDGF antagonist.

7. The construction method according to claim 1, characterized in that, In step S3, the seeded cell density is 5 × 10⁻⁶. 5 The cells / mL, the three-dimensional induction culture period is 14 days, and the organoid culture medium is changed every 3 days during the period.

8. A transplantable lacrimal duct epithelial organoid, characterized in that, Prepared by the construction method according to any one of claims 1 to 7, the epithelial organoid has a tubular structure of superficial columnar epithelium and basal squamous epithelium; the epithelial thickness of the epithelial organoid is 65±8 μm; and the epithelial organoid expresses double positive expression of stem cell markers P63 and KRT17.

9. The use of a transplantable lacrimal duct epithelial organoid as described in claim 8 in the preparation of lacrimal duct repair implants.

10. The use of the transplantable lacrimal duct epithelial organoid as described in claim 8 in the preparation of a drug for treating lacrimal duct obstructive diseases, characterized in that, The lacrimal duct obstruction diseases include lacrimal duct obstruction, lacrimal duct malformation, or chronic dacryocystitis complicated by infection.

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