Method for inducing differentiation and maturation of alveolar organoid and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for inducing differentiation and maturation of alveolar organs and application of the method. The method for inducing differentiation and maturation of the alveolar organoid comprises the following steps: performing differentiation induction on the alveolar organoid in an early alveolar stage in a suspension culture manner; the suspension culture is carried out in an alveolar organ culture medium, and polydopamine nanoparticles are additionally added into the alveolar organ culture medium. In ALO in-vitro long-term culture, through combination of suspension culture and PDA NPs, AT2 differentiation efficiency in ALO is improved, AT2 function maturation is promoted, and the application effect of ALO in the fields of lung injury repair and transplantation regeneration can be improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for inducing the differentiation and maturation of alveolar organoids and its application. Background Technology

[0002] Alveoli are hemispherical vesicles composed of a single layer of epithelial cells, located at the ends of the bronchi in the lungs, and are the core site of gas exchange. The alveolar epithelium is mainly composed of two types of cells working together: Type I alveolar cells (AT1) are flattened and cover most of the alveolar surface, enabling efficient gas exchange. Type II alveolar cells (AT2) are located between Type I alveolar cells, are more numerous, and although their coverage area is limited, they play a crucial role in maintaining alveolar homeostasis. AT2 cells not only synthesize, store, and secrete pulmonary surfactant through lamellar bodies, significantly reducing the surface tension of the air-fluid interface within the alveoli and stabilizing alveolar structure, but also possess the ability to self-proliferate and differentiate into AT1 cells. They are important adult stem cells in the alveolar region and play a role in repairing damaged alveolar epithelium. Because highly differentiated AT1 cells are almost unable to self-renew and repair, AT2 cells are activated and rapidly proliferate and differentiate into new AT1 cells when the alveoli are damaged, which is fundamental to maintaining the structural integrity and long-term functional stability of the alveoli.

[0003] In the field of lung injury repair and transplant regeneration, the viability and functional integrity of AT2 cells in donor lungs are crucial cellular bases influencing the risk of primary lung function loss, ischemia-reperfusion injury, and long-term rejection after transplantation. However, the source of functional primary human AT2 cells for clinical applications is extremely limited, their in vitro expansion capacity is poor, and they are prone to dedifferentiation, making it difficult to meet the scale required for clinical-grade cell therapy. Human pluripotent stem cells (hPSCs), with their unlimited self-renewal potential, provide a new avenue for large-scale in vitro preparation of AT2 cells. Through signal pathway-directed differentiation technology that mimics in vivo lung development, stem cells can be induced to express AT2 cell markers (such as surfactant protein B, SFTPB; surfactant protein C, SFTPC). However, AT2 cells differentiated from stem cells generally suffer from functional immaturity, such as insufficient surfactant secretion capacity and incomplete lamellar body ultrastructural development, showing a significant difference from primary human AT2 cells. The current limitations of in vitro culture systems further exacerbate this challenge.

[0004] Polydopamine nanoparticles (PDA NPs) are black, insoluble biopolymers formed by the self-oxidative polymerization of dopamine (DA). The catechol structure of PDA endows it with excellent antioxidant properties; it can quench free radicals by donating hydrogen atoms through its phenolic hydroxyl groups or reduce compounds through electron transfer, thereby exerting antioxidant activity. Currently, there are methods that first modify cell culture plates with a PDA coating, and then add Matrigel for cell differentiation and adherent culture. This method uses the PDA coating as an antioxidant coating, increasing the yield of adherent cells by improving the levels of reactive oxygen species (ROS) in the environment that do not enter the cells to participate in the cell differentiation process. In addition, there are methods that use PDA NPs to improve cell adhesion during culture, but none of these address how to solve the problem of immature AT2 function obtained from stem cell differentiation. Therefore, further research is needed on methods for differentiating and culturing alveolar organoids. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for promoting the differentiation efficiency and functional maturation of AT2 in alveolar organoids in vitro.

[0006] This invention provides a method for inducing the differentiation and maturation of alveolar organoids, which induces the differentiation of alveolar organoids in the early alveolar stage by suspension culture; the suspension culture is carried out in an alveolar organoid culture medium, and polydopamine nanoparticles are additionally added to the alveolar organoid culture medium.

[0007] This invention has found that current methods for differentiating and culturing alveolar organoids (ALOs) from stem cells result in low differentiation efficiency and immature function of AT2 cells, which are significantly different from human primary AT2 cells. Consequently, when applied to the fields of lung injury repair and transplant regeneration, there is a high risk of primary lung function loss, ischemia-reperfusion injury, and long-term rejection after transplantation.

[0008] Therefore, this invention has conducted extensive research on methods for inducing alveolar organoid formation. Ultimately, it was discovered that in early-stage alveolar organoids (derived from lung progenitor cell differentiation or early-stage epithelial cell organoids from fetal lung alveolar formation), culture in suspension combined with the addition of polydopamine nanoparticles (PDA NPs) for alveolar epithelial differentiation and maturation can allow PDA NPs to enter the cell interior through endocytosis, eliminate ROS enriched in ALO, improve the differentiation efficiency of AT2 cells in ALO, and interact with lysosomes to promote lamellar body formation, thereby improving the functional maturity of alveolar organoids and solving the current problem of immature AT2 cell function and low differentiation efficiency.

[0009] Specifically, this invention has found that PDA NPs are small in size and have a large specific surface area, enabling them to diffuse in biological systems and be taken up by ALO cells via endocytosis. They then accumulate in ROS-rich lysosomal regions, effectively exerting antioxidant effects while participating in the development and maturation of AT2, thereby continuously promoting the differentiation and maturation of ALO into AT2.

[0010] Compared to the conventional use of PDA coatings as an intermediate layer between cell culture plates and cells to optimize the state of 2D adherent cells by promoting cell adhesion or improving ROS enrichment in the culture environment (without involving interactions and influences with the cell interior), this invention innovatively disperses PDA NPs in the culture medium and allows them to enter 3D organoids (mimicking the 3D structure of in vivo organs and the three-dimensional interactions between cells) through endocytosis, enabling them to directly participate in the cell development and maturation process. This breaks with the conventional use of PDAs and reveals a new efficacy of PDAs in alveolar organoid culture.

[0011] Specifically, lysosomes are the precursor structures of AT2 lamellar bodies (LB). In the ALO suspension stage, the PDA NPs of this invention are specifically taken up by AT2 into lysosomes (adding them during the suspension culture stage can disperse the PDA NPs in the culture medium, which is conducive to organoid uptake). By exerting antioxidant properties (effectively reducing the intracellular ROS content during ALO culture) and interacting with organelles in the cell, they promote the maturation of lamellar body function (promote the maturation of AT2 lamellar bodies and enhance the secretion capacity of AT2), thereby improving the differentiation efficiency of AT2 in ALO.

[0012] In this invention, the early alveolar stage corresponds to the luminal stage of human embryonic development, which has AT2 precursor cells, expresses SFTPB, and has not yet produced mature AT2 and AT1. Early alveolar organoids (LPCs) show SFTPB expression (qPCR detection relative to GAPDH expression level of 50-100) after 7-10 days of culture in alveolar organoid culture medium, but have not yet shown large-scale expression of SFTPC, AGER, and HOPX (qPCR detection relative to GAPDH expression level, SFTPC is less than 30, and AGER and HOPX are both less than 10).

[0013] In the method of the present invention, the concentration of polydopamine nanoparticles added to the alveolar organoid culture medium is 10-100 μg / mL, preferably 25 μg / mL; And / or, the particle size of the polydopamine nanoparticles is 100-200 nm; And / or, each 500 μL of the alveolar organoid culture medium contains 50-200 alveolar organoids in the early alveolar stage.

[0014] The culture conditions of this invention can achieve better culture results.

[0015] In this invention, polydopamine nanoparticles can be prepared using methods known in the art.

[0016] The polydopamine nanoparticles of the present invention have small particle size, good dispersibility, antioxidant properties, and can be endocytosed by cells.

[0017] As a specific implementation method, the preparation method of polydopamine nanoparticles includes: Dopamine hydrochloride undergoes oxidative self-polymerization in an alkaline solution system and under the action of oxygen to generate polydopamine nanoparticles (PDA NPs).

[0018] Preferably, the alkaline solution system includes anhydrous ethanol, deionized water and ammonia, with a volume ratio of (10-12):(26-28):(0.15-0.3), wherein the mass concentration of ammonia is 28-30%; the concentration of dopamine hydrochloride in the alkaline solution system is 1-5 mg / mL, more preferably, the concentration of dopamine hydrochloride is 2 mg / mL.

[0019] Preferably, after the oxidative self-polymerization reaction of dopamine hydrochloride is completed, purified PDA NPs are obtained by centrifugation, resuspension, washing, freeze drying, etc.

[0020] In this invention, culture media known in the art can be used for cell culture at each stage.

[0021] Early alveolar organoids obtained by methods known in the art are all applicable to the method of the present invention for improving the differentiation and maturation of alveolar organoids.

[0022] In the method of the present invention, the alveolar organoid culture medium comprises one or more of the following: basal culture medium, protein supplement, nutritional supplement, antibiotic, glutamine supplement, insulin-transferrin-selenium supplement, glucocorticoid, component one, fibroblast growth factor, TGFβ signaling pathway inhibitor, and component two; wherein component one is a cAMP signaling pathway agonist and / or phosphodiesterase inhibitor; and component two is a Wnt signaling pathway agonist and / or GSK3β signaling pathway inhibitor. Preferably, the basal culture medium in the alveolar organoid culture medium is Advanced DMEM / F-12; Preferably, the protein supplement in the alveolar organoid culture medium includes albumin at a concentration of 0.1%-1%; more preferably, 0.25%. Preferably, the nutrient supplement in the alveolar organoid culture medium includes B-27 supplement at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the antibiotic in the alveolar organoid culture medium is penicillin / streptomycin (P / S) at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the glutamine supplement in the alveolar organoid culture medium is GlutaMAX, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the insulin-transferrin-selenium supplement in the alveolar organoid culture medium is ITS-X, with a concentration of 0.05%-1%; more preferably, 0.1%. Preferably, the glucocorticoid in the alveolar organoid culture medium is dexamethasone, with a concentration of 10-200 nM, more preferably 50 nM; Preferably, the cAMP signaling pathway agonist in the alveolar organoid culture medium is 8-Br-cAMP at a concentration of 10-000 nM, more preferably 100 nM; Preferably, the fibroblast growth factor in the alveolar organoid culture medium is FGF7, with a concentration of 1-50 ng / mL, more preferably 10 ng / mL; Preferably, the phosphodiesterase inhibitor in the alveolar organoid culture medium is IBMX, with a concentration of 10-1000 nM, more preferably 100 nM; Preferably, the TGFβ signaling pathway inhibitor in the alveolar organoid culture medium is SB431542, with a concentration of 1-20 μM, more preferably 10 μM; Preferably, the Wnt signaling pathway agonist and / or GSK3β signaling pathway inhibitor in the alveolar organoid culture medium is CHIR99021, with a concentration of 0.5-10 μM, more preferably 3 μM.

[0023] In the method of the present invention, the alveolar organoids of the early alveolar stage are obtained as follows: on the seventh to fourteenth day of lung progenitor cell differentiation, after qPCR detection of NKX2-1 relative GAPDH expression level ≥100, the lung progenitor cell culture medium is replaced with alveolar organoid culture medium to induce the lung progenitor cells into alveolar organoids of the early alveolar stage.

[0024] In the method of the present invention, the alveolar organoids in the early alveolar stage are obtained by inducing lung progenitor cells in 3D culture hydrogel using alveolar organoid culture medium. The lung progenitor cells are obtained by inducing single cells of the foregut endoderm in a 3D culture hydrogel using a lung progenitor cell culture medium (preferably, each 25 μL of 3D culture hydrogel before solidification contains 8,000-10,000 single cells of the foregut endoderm during induction culture). The anterior foregut endoderm single cell is obtained by dissociating the anterior foregut endoderm cell mass; The anterior foregut endoderm cell mass is obtained by induction of shaped endoderm cells; The defined endoderm cells are induced from human pluripotent stem cells.

[0025] The 3D culture hydrogel includes one or more of Matrigel, Cultrex BME, dECM hydrogel, collagen hydrogel, fibrin gel, gelatin, hyaluronic acid, and polyethylene glycol hydrogel, preferably Matrigel; more preferably GFR-Matrigel.

[0026] In this invention, the gelatin may be methacrylamide gelatin.

[0027] The Matrigel mainly includes standard Matrigel, Growth Factor Reduced-Matrigel (GFR-Matrigel), Phenol Red-Free Matrigel, and LDEV-Free Matrigel, with GFR-Matrigel being the preferred choice.

[0028] Matrigel is a natural extracellular matrix (ECM) extract derived from mouse Engelbreth-Holm-Swarm (EHS) tumors. The Matrigel used in this embodiment was purchased from Corning. The main components of Matrigel include structural proteins: Laminin-111, collagen IV, Entactin, and Heparan Sulfate Proteoglycans complex; it also includes growth factors: EGF, TGF-β, FGF, IGF, PDGF, etc.

[0029] In this invention, alveolar organoids in the early alveolar stage of culture can be collected from the solidified 3D culture hydrogel using methods known in the art.

[0030] For example, after the alveolar organoids in the early alveolar stage have been cultured, alveolar organoids in the early alveolar stage can be collected using DPBS, treated with Cell Recovery Solution until the Matrigel in the 3D culture hydrogel is completely lysed, DPBS can be added to stop the reaction, the supernatant can be removed by centrifugation, and then fresh alveolar organoid culture medium can be added for resuspension.

[0031] ALO can then be transferred to a low-adsorption culture plate, changing the culture method from 3D culture to suspension culture.

[0032] In the method of the present invention, the lung progenitor cell culture medium includes one or more of the following: basal culture medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, Notch signaling pathway inhibitor, BMP4, fibroblast growth factor 7, fibroblast growth factor 10, GSK3β signaling pathway inhibitor, and retinoic acid. Preferably, in the lung progenitor cell culture medium, the basal culture medium is DMEM / F-12 medium and / or modified DMEM / F-12 medium, more preferably Advanced DMEM / F-12; Preferably, in the lung progenitor cell culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the nutritional supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, the antibiotic in the lung progenitor cell culture medium is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the Notch signaling pathway inhibitor is selected from N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) or dibenzozazepine; more preferably, the Notch signaling pathway inhibitor is N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) at a concentration of 15-25 μM, and more preferably 20 μM; Preferably, the concentration of BMP4 in the lung progenitor cell culture medium is 15-25 ng / mL, more preferably 20 ng / mL; Preferably, in the lung progenitor cell culture medium, the concentrations of fibroblast growth factor 7 and / or fibroblast growth factor 10 are 5-15 ng / mL, more preferably 10 ng / mL. Preferably, in the lung progenitor cell culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 1-5 μM, and even more preferably, a concentration of 3 μM; Preferably, the concentration of retinoic acid in the lung progenitor cell culture medium is 40-60 nM, more preferably 50 nM.

[0033] In the method of the present invention, the anterior foregut endoderm culture medium used to differentiate and form the anterior foregut endoderm cell mass includes one or more of the following: basal culture medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist and GSK3β signaling pathway inhibitor. Preferably, in the pre-endoderm culture medium, the basal culture medium is Advanced DMEM / F-12; Preferably, in the pre-endoderm culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the nutrient supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, in the pre-endoderm culture medium, the antibiotic is penicillin / streptomycin, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the TGF-β signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542; more preferably, the TGF-β signaling pathway inhibitor is SB431542, with a concentration of 5-15 μM; even more preferably, it is 10 μM; Preferably, in the pre-endoderm culture medium, the BMP signaling pathway inhibitor is selected from Noggin, Dorsomorphin, DMH1, or LDN-193189; more preferably, the BMP signaling pathway inhibitor is Noggin, with a concentration of 150 ng / mL to 250 ng / mL; even more preferably, it is 200 ng / mL. Preferably, the concentration of FGF4 in the pre-endoderm culture medium is 400-600 ng / mL; Preferably, in the pre-endoderm culture medium, the SHH agonist is selected from SHH or SAG; more preferably, the SHH agonist is SAG with a concentration of 0.5-1.5 μM; even more preferably, it is 1 μM. Preferably, in the pre-endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021 at a concentration of 1-3 μM; even more preferably, it is 2 μM. Preferably, the time for differentiation to form the anterior foregut endoderm cell mass is 2-7 days, more preferably 4 days.

[0034] In the method of the present invention, the culture medium used to differentiate and form the fixed endoderm cells is a fixed endoderm complete culture medium, which includes a fixed endoderm basal culture medium, a TGF-β signaling pathway activator and a GSK3β signaling pathway inhibitor. The shaped endoderm basal culture medium includes one or more of the following: core basal culture medium, protein supplement, nutrient supplement, antibiotic, pH stabilizer, and glutamine supplement; Preferably, in the shaped endoderm basal culture medium, the core basal culture medium is MCDB131; Preferably, in the basal culture medium for shaping endoderm, the protein supplement is bovine serum albumin with a concentration of 0.4%-0.6%, more preferably 0.5%; Preferably, in the basal culture medium for shaping endoderm, the nutrient supplement is glucose with a concentration of 8-12 mM, more preferably 10 mM; Preferably, in the basal culture medium for shaping endoderm, the antibiotic is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the basal culture medium for shaping endoderm, the pH stabilizer is sodium bicarbonate with a concentration of 1.5-2 mg / mL; more preferably, it is 1.7 mg / mL. Preferably, in the basal culture medium for shaping endoderm, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the complete endoderm culture medium, the TGF-β signaling pathway activator is selected from Activin A, Activin B, TGF-β1, TGF-β2 or TGF-β3; more preferably, the TGF-β signaling pathway activator is Activin A, with a concentration of 80-120 ng / mL, and even more preferably 100 ng / mL; Preferably, in the complete endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 0.1-3 µM. Preferably, the time for differentiation to form the defined endoderm cells is 2.5-4 days, more preferably 3 days; More preferably, the complete culture medium for the typed endoderm used on the first day of differentiation to form the typed endoderm cells includes: the typed endoderm basal culture medium, 100 ng / mL Activin A and 3 µM CHIR99021; More preferably, the complete culture medium for the finalized endoderm cells used on the second day after differentiation comprises: the basal culture medium for the finalized endoderm cells, 100 ng / mL Activin A and 0.1 µM CHIR99021; More preferably, the complete culture medium for the finalized endoderm cells used on the third day of differentiation comprises: the basal culture medium for the finalized endoderm cells and 100 ng / mL Activin A.

[0035] If the time for differentiation to form the defined endoderm cells is 4 days, then the culture medium used on day 3 should still be used on day 4.

[0036] The present invention also provides the application of the above-mentioned method for inducing alveolar organoid differentiation and maturation in promoting the differentiation efficiency of type II alveolar cells in alveolar organoids.

[0037] The present invention also provides the application of the above-mentioned method for inducing alveolar organoid differentiation and maturation in promoting the functional maturation of type II alveolar cells in alveolar organoids.

[0038] The methods and applications of this invention are not for disease diagnosis or treatment purposes, but are mainly used for the differentiation and culture of alveolar organoids in vitro.

[0039] The methods of this invention do not include applications involving the destruction of human embryos for industrial or commercial purposes.

[0040] In this invention, "foregut endoderm" refers to the endoderm that forms the anterior portion of the liver endoderm. "Foregut endoderm" includes, for example, the pharyngeal endoderm and other more highly differentiated endoderm cell populations, and contains multiple cell types exhibiting different expression patterns of molecular markers. "Foregut endoderm" will develop into various tissues, such as the tonsils, tympanic membrane, thyroid gland, parathyroid glands, thymus, trachea, esophagus, stomach, lungs, larynx, and / or pharynx.

[0041] "Definitive endoderm cells (DE)" refer to cells that express one or more markers of a defined endoderm lineage. These markers include, but are not limited to, CXCR4, SOX17, GATA-4, FOXA2, AFP, CER1, C-KIT, EPCAM, SNAI1, GSC, E-Cad, and / or N-Cad. The defined endoderm is functionally defined by one or more cells that further differentiate from endoderm tissues. This can include the lung, thyroid, liver, pancreas, or intestine.

[0042] Important markers of "lung progenitor cells" are NKX2-1, SOX9, and / or SOX2.

[0043] "Organoids" are three-dimensional, multicellular aggregates derived from stem cells that differentiate and self-organize, encapsulating the structural features and cell-cell interactions of mature tissues. These organoids are three-dimensional aggregates of one or more cell types that mimic the surface appearance, structure, or function of tissues or organs.

[0044] "Induction" or "induce" relates to processes or behaviors that cause a specific effect on a cell's phenotype. Such effects can take the form of causing a phenotypic change, such as differentiating to another cell phenotype; or they can take the form of maintaining a cell in a specific cellular state, such as preventing dedifferentiation or promoting cell survival.

[0045] Pluripotent stem cells are multipotent cells with the ability to self-renew and self-replicate, and can differentiate into various cell types under certain conditions.

[0046] The terms "precursor cell," "progenitor cell," and "stem cell" are used interchangeably in the art, and in this invention refer to pluripotent or lineage-determined progenitor cells that have the potential to undergo an unlimited number of mitotic divisions to renew themselves or differentiate into daughter cells of a desired cell type. Compared to pluripotent stem cells, lineage-determined progenitor cells are generally considered incapable of generating numerous cell types that are phenotypically different from each other. Instead, progenitor cells will be able to generate one or possibly two lineage-determined cell types.

[0047] In this invention, "differentiation" refers to the process by which less specialized cells, such as stem cells or induced pluripotent stem cells, become more specialized cell types so that they become specific lineages, including but not limited to certain progenitor cells and more specialized somatic cells. The conditions for stem cell differentiation are well known in the art.

[0048] In this invention, "differentiation medium" refers to a cell growth medium containing or lacking certain additives, so that when cultured in the medium, stem cells, induced pluripotent cells, or incompletely differentiated cells develop into differentiated cells or cells that exhibit some or all of the characteristics of cells that are more differentiated than stem cells, induced pluripotent cells, or other similar cells.

[0049] In this application, "dissociation" refers to the breaking down of cell aggregates or clusters into smaller aggregates or single-cell suspensions. Dissociation of cell aggregates can be achieved by conventional methods, including but not limited to enzymatic, chemical, or mechanical methods. Enzymatic dissociation can be performed using, for example, accutase, dispersase, or trypsin.

[0050] The DMEM / F-12 can also be replaced by one or more of the following: William's E cell culture medium, Neurobasal Medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F-12 cell culture medium.

[0051] The DMEM / F-12 culture medium contains a 1:1 mixture of DMEM and Ham's F-12.

[0052] The DMEM / F-12 comprises a modified DMEM / F-12 culture medium with its composition adjusted according to the actual application.

[0053] The DMEM / F-12 modified culture medium includes, but is not limited to, DMEM-low-glucose-pyruvate-glutamine-free-phenol red-free, and DMEM / F-12-GlutaMAX. TMDMEM / F-12-HEPES (DMEM / F-12 with HEPES), DMEM-lowglucose-pyruvate-HEPES, Advanced DMEM / F-12 (containing glucose, non-essential amino acids, sodium pyruvate and phenol red, but without L-glutamine and HEPES).

[0054] The beneficial effects of this invention are at least as follows: This invention improves the differentiation efficiency of AT2 in ALO (alveolar organoid lamellae) during a specific stage of long-term in vitro culture by using suspension culture combined with PDANPs, thereby promoting the maturation of AT2 function (promoting the maturation of AT2 lamellar bodies in alveolar organoids and enhancing their secretory function). This reduces the risk of primary lung function loss, ischemia-reperfusion injury, and long-term rejection when applying ALO to lung injury repair and transplant regeneration. Attached Figure Description

[0055] Figure 1 The following are the characterization results of PDA NPs. A shows the Fourier transform infrared spectroscopy analysis of PDA NPs; B shows the scanning electron microscopy of PDA NPs (scale bar is 100 nm); C shows the transmission electron microscopy of PDA NPs (scale bar is 100 nm); D shows the dynamic light scattering particle size distribution of PDA NPs; E shows the Zeta potential of PDA NPs; and F shows the DPPH radical scavenging test results of PDA NPs.

[0056] Figure 2 To validate the results of lung progenitor cells and early alveolar organoid differentiation indicators. In this study, A represents the relative expression level of NKX2-1 to GAPDH in LPCs on day 7; B represents the relative expression level of alveolar marker genes to GAPDH in differentiated LPCs after 7 days of culture in alveolar organoid culture medium (early alveolar stage, ALO day 7).

[0057] Figure 3 This image shows the results validating the maturation of alveolar organoid cultures using polydopamine nanoparticles. In the image, A is a bright-field diagram of culture with PDA NPs added during the ALO stage (scale bar: 100 μm); B shows the qPCR results; C and D show the staining (scale bar: 50 μm) and quantification results, respectively. This means P < 0.05.

[0058] Figure 4 The results of ROS and Superoxide staining in the ALO phase.

[0059] Figure 5The staining results of lysosomes were obtained by co-incubating FITC-labeled PDA NPs (green) with ALO for 24 h, followed by labeling with Lysotracker (red). The scale bar in the figure is 20 μm.

[0060] Figure 6 The results show the maturation of AT2 function in alveolar organoids cultured using polydopamine nanoparticles. In Figure A, AT2 secretion function is validated; the area circled in yellow indicates co-localization of lysosomes and pulmonary surfactant. The scale bar in the figure is 50 μm. In Figure B, the staining results show the marker gene for AT2 functional maturation. The scale bar in the figure is 50 μm.

[0061] Figure 7 The image shows an ALO transmission electron microscope image. The red arrows indicate lamellar bodies; the yellow arrows indicate lipid droplets; and the blue arrows indicate PDA NPs. The scale bar in the image is 100 nm. Detailed Implementation

[0062] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0064] Example 1 This embodiment provides a method for preparing polydopamine nanoparticles and characterizing their properties, as detailed below: 1. Synthesis of polydopamine nanoparticles 12 mL of anhydrous ethanol and 28 mL of deionized water were added to a flask and placed on a magnetic stirrer. 200 μL of ammonia (28%) was added to adjust the pH to alkaline, and the mixture was stirred for 30 minutes to ensure homogeneity. Dopamine hydrochloride was then added to achieve a final concentration of 2 mg / mL, and the mixture was allowed to react for 18 hours with stirring. The reaction mixture was then transferred to a centrifuge tube and centrifuged at 12,000 rpm. The mixture was washed three to five times with deionized water until the supernatant was clear, and the precipitate at the bottom was collected. This precipitate was then lyophilized to obtain polydopamine nanoparticles (PDA NPs).

[0065] 2. Performance characterization of polydopamine nanoparticles The microstructure and morphology of the prepared PDA NPs were characterized using scanning electron microscopy and transmission electron microscopy. Fourier transform infrared spectroscopy was used to determine the functional groups of the PDA NPs, and their chemical structure was qualitatively analyzed to verify the successful synthesis of the PDA NPs. The Zeta potential of the PDA NPs was measured using a Zeta potential analyzer, and the hydrodynamic diameter of the nanoparticles was measured using a nanoparticle size analyzer. The free radical scavenging rate of the nanoparticles against 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH·) at different concentrations was measured to evaluate the antioxidant activity of the PDA NPs. Results are shown below. Figure 1 In this embodiment, PDA NPs with a particle size of 100-200 nm were successfully obtained and they have antioxidant properties.

[0066] Example 2 This embodiment provides a method for culturing alveolar organoids differentiated from human pluripotent stem cells using polydopamine nanoparticles prepared in Example 1, and verifies the maturation of the cultured alveolar organoids.

[0067] 1. Human pluripotent stem cells are directed to differentiate into definitive endoderm (DE) cells using a culture medium supplemented with specific small molecules, and then differentiate into foregut endoderm cells (AFE cells). AFE cells form spherical bodies (foregut spheroids) that separate from adherent cells and float in the culture medium, as shown below: (1) Matrigel coating construction Thaw Matrigel (previously stored at -20°C) on ice. Add 100 µL of the thawed Matrigel (Corning, membrane matrix, 354277) to 10 mL of pre-chilled DMEM / F-12 medium (Gibco). TM Resuspend the cells in DMEM / F-12 medium (11320033), mix thoroughly, and place on ice for later use. Add 300 µL of DMEM / F-12 medium containing Matrigel to each well of a 24-well plate and incubate at 37°C for 2 hours. After 2 hours, discard the remaining DMEM / F-12 medium containing Matrigel and immediately seed the cells.

[0068] (2) Stem cell inoculation and differentiation With a density of 1.6 × 10 5 Human pluripotent stem cells per cell / mL were seeded into 24-well culture plates with a Matrigel coating, 500 μL per well.

[0069] The medium was cultured at a constant temperature of 37 degrees Celsius and 5% CO2, with the medium changed every 24 hours. The medium used was mTeSR (Stemcell).

[0070] After 1-2 days, the stem cell proliferation was monitored under an optical microscope. When the spread area reached approximately 70-80% of the well area, DE medium was added to induce the stem cells to differentiate into DE cells. After 3 days, AFE medium was added to induce the DE cells into AFE cell spheroids, and the cells were cultured for a total of 7 days.

[0071] The cell culture medium should be changed every 24 hours.

[0072] The DE medium formula is as follows: Basic culture medium formula: MCDB131 + glucose (10 mM) + NaHCO3 (1.7 mg / mL) + BSA (0.5%) + GlutaMAX (1:100) + penicillin / streptomycin (1:100).

[0073] On day 1, replace the DE basal medium with Activin A (100 ng / mL) and CHIR99021 (3 µM). On the second day, the DE basal medium was replaced with Activin A (100 ng / mL) and CHIR99021 (0.1 µM). On day 3, replace the DE basal medium with Activin A (100 ng / mL).

[0074] The AFE medium formula is as follows: Basic culture medium formulation: Advanced DMEM / F-12 (Gibco) TM Advanced DMEM / F-12,12634010) + HEPES (1:100) + B-27 (1:50) + N-2 (1:100) + GlutaMAX (1:100) + Penicillin / Streptomycin (1:100).

[0075] Complete culture medium formula: basal medium + SB431542 (10µM) + CHIR99021 (2µM) + Noggin (200ng / mL) + SAG (1µM) + FGF4 (500ng / mL).

[0076] 2. Without damaging the underlying cells, transfer the floating foregut spheres in the liquid to a centrifuge tube and allow them to settle for 10-15 minutes until they settle at the bottom of the tube. Digest the aspirated spheres into single cells using Accutase and count the number of cells using a hemocytometer.

[0077] Thaw GFR-Matrigel (Corning, Growth Factor Reduced Basement Membrane Matrix, 354230) stored at -20°C on ice beforehand. Then, seed 8000 cells per 25 μL GFR-Matrigel, gently pipette to mix, and drop into the center of a well plate. Invert the plate at 37°C for 10-15 minutes. After the GFR-Matrigel solidifies, add lung progenitor cell culture medium to differentiate foregut spheroids into lung progenitor cells (LPCs). Culture LPCs at a constant temperature of 37°C and 5% CO2 for seven days, changing the medium every 48 hours. The lung progenitor cell culture medium formula is: Advanced DMEM / F-12 (Gibco... TM Advanced DMEM / F-12, 12634010), HEPES (1:100), N-2 (1:100), B-27 (1:50), penicillin / streptomycin (1:100), GlutaMAX (1:100), N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) (20 μM), BMP4 (20 ng / mL), fibroblast growth factor 7 (10 ng / mL), fibroblast growth factor 10 (10 ng / mL), CHIR99021 (3 μM) and retinoic acid (50 nM).

[0078] 3. On day 7 of LPC culture, qPCR was used to detect NKX2-1 expression relative to GAPDH levels ≥100 (see results). Figure 2 (A) Then the lung progenitor cell culture medium was changed to alveolar organoid culture medium.

[0079] The alveolar organoid culture medium formula is: Advanced DMEM / F-12 (Gibco) TM Advanced DMEM / F-12,12634010), BSA (0.25%), B-27 (1:100), penicillin / streptomycin (1:100), GlutaMAX (1:100), insulin-transferrin-selenium supplement ITS-X (0.1%), dexamethasone (50 nM), 8-Br-cAMP (100 nM), IBMX (100 nM), FGF7 (10 ng / mL), SB431542 (10 μM), CHIR99021 (3 μM).

[0080] Seven days after changing to alveolar organoid culture medium, qPCR analysis showed that SFTPB expression relative to GAPDH was 50-100, SFTPC was below 30, and AGER and HOPX were both below 10, confirming alveolar organoids in the early alveolar stage (ALO). Results are shown below. Figure 2 B in the middle.

[0081] ALOs were collected into centrifuge tubes using pre-cooled DPBS (4°C). Cell Recovery Solution was added, and the tubes were incubated on ice for 45-60 minutes to lyse GFR-Matrigel and release early alveolar organoids. After lysis, DPBS was added to stop the reaction. After centrifugation to remove the supernatant, fresh alveolar organoid culture medium was added, and the ALOs were resuspended and transferred to a low-adsorption culture plate. The culture mode was changed from 3D culture to suspension culture. Each 500 μL of the alveolar organoid culture medium contained 100 early alveolar organoids. PDA NPs prepared in Example 1 were added to the culture medium at a final concentration of 25 μg / mL. The tubes were co-cultured for one week and then characterized.

[0082] Validation of alveolar organoid morphology, marker gene and protein expression levels: To assess the impact of PDA NPs on ALO development, bright-field imaging of the organoids was performed using a desktop binocular stereomicroscope, and the morphology of the ALOs was recorded. A control group (Ctrl) was established where PDA NPs were not added during the suspension culture stage. Results are shown in [link to results]. Figure 3 A in the middle.

[0083] In addition, to further explore the effects of different groups on ALO differentiation process, mRNA was extracted from the two ALO ​​groups using an RNA purification kit, and the mRNA was reverse transcribed into cDNA using a Maxima H Minus kit. Subsequently, the expression levels of marker genes for ALO development were detected by qPCR, including: (1) lung cell marker genes NKX2-1 and SOX9; (2) AT1 markers HOPX and AGER; (3) AT2 markers SFTPB and SFTPC. The role of PDA NPs in ALO development was analyzed by comparing the differences in expression of key genes among the groups. Each sample was prepared in 3 replicate wells, with GAPDH as the internal reference gene. The relative expression level was calculated using ΔCt relative to GAPDH to ensure the comparability and reliability of the results. The results are shown in […]. Figure 3 B in the middle.

[0084] To further validate the qPCR results, immunofluorescence staining was used. ALO cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with DPBS, and then treated with immunofluorescence permeation buffer containing 0.3% Triton X-100 at room temperature for 1 hour, followed by immunofluorescence blocking buffer for 30 minutes. After blocking, primary antibodies (SFTPB, AGER) were added and incubated overnight at 4°C. The next day, secondary antibodies of the corresponding species and Hoechst 33342 were added and incubated at room temperature in the dark for 1 hour. Finally, microscopic imaging was performed using a 980 laser confocal microscope to quantify the nuclear percentage of ALO cells and determine the proportion of AGER and SFTPB-positive cells. Results are shown below. Figure 3 C and D in the example.

[0085] pass Figure 3 The results showed that the levels of SFTPB gene and protein in ALO were significantly increased after PDA NPs treatment, indicating that the differentiation efficiency of AT2 in ALO was significantly improved.

[0086] Example 3 This embodiment verifies the intracellular antioxidant effect of polydopamine nanoparticles in alveolar organoids as described in Example 2. A culture group without the addition of PDA NPs during the suspension culture stage was used as a control (Ctrl).

[0087] To investigate the antioxidant effects of PDA NPs in organoids, a Fluorimetric Hydrogen Peroxide Assay Kit was used to stain reactive oxygen species (ROS) and superoxide ions, and their content in the final ALO obtained in Example 2 was measured. Results are shown below. Figure 4 This indicates that PDA NPs can effectively remove intracellular ROS during ALO culture.

[0088] Example 4 This embodiment verifies the ability of alveolar organoids to take up polydopamine nanoparticles.

[0089] Method for labeling PDA NPs with FITC: The PDA NPs prepared in Example 1 were labeled with FITC. Specifically, 0.5 mg of FITC powder was completely dissolved in 5 mL of a 2 mg / mL PDA NPs solution. The pH of the system was adjusted to 8.5 using Tris buffer, and the reaction was carried out overnight at room temperature in the dark with stirring. After the reaction was completed, the mixture was transferred to a centrifuge tube and centrifuged at 18,000 rpm for 10 minutes at 4°C. The supernatant was discarded. The precipitate was washed three times with deionized water to remove free FITC. Finally, the labeled nanoparticles were redispersed in PBS for subsequent use.

[0090] To assess the uptake capacity of ALOs of PDA NPs, FITC-labeled PDA NPs and early alveolar organoids (ALOs) obtained in Example 2 were co-incubated in alveolar organoid culture medium (see Example 2) at 37°C and 5% CO2 for 24 hours. After washing with alveolar organoid culture medium, 100 nM Lysotracker Red was added and incubated at room temperature in the dark for 30 minutes to label lysosomes. To clearly visualize the cell nucleus and cell membrane outlines, the cell membrane was stained with Hoechst 33342 and CellMask deep red plasma membrane staining agent for 10 minutes.

[0091] See results Figure 5 Lysosomes in ALO were labeled with Lysotracker Red (red fluorescence); PDA NPs were labeled with FITC (green fluorescence). Co-staining was observed, indicating that PDA NPs were taken up by lysosomes of AT2 in ALO alveolar organoids in the early alveolar stage.

[0092] Example 5 This example verifies the maturation of AT2 function in alveolar organoids cultured using polydopamine nanoparticles in Example 2. A control group (Ctrl) was used, in which PDA NPs were not added during the suspension culture stage. Details are as follows: 1. Verification of surfactant secretion function: After incubating the ALO cells obtained in Example 2 with 1 μM β-BODIPY for 24 hours, they were washed with alveolar organoid culture medium (see Example 2). Then, the ALO cells were incubated with 100 nM Lysotracker Red at room temperature in the dark for 30 minutes. Subsequently, the nuclei and cell membranes of the ALO cells were stained with Hoechst 33342 and CellMask deep red plasma membrane staining agent. Finally, the co-localization of β-BODIPY and Lysotracker was observed and photographed using a 980 laser confocal microscope.

[0093] See results Figure 6 In the A group, β-BODIPY was used to label AT2-secreted surfactant, and LysotrackerRed was used to label lysosomes in ALO. Lysosomes are important precursor organelles for AT2 lamellar body formation. The observation of co-localization between lysosomes and pulmonary surfactant indicates that AT2 can effectively synthesize and store pulmonary surfactant, marking the maturity of AT2 function. Staining results showed co-localization between lysosomes and pulmonary surfactant, and AT2 secretion capacity was enhanced after PDA NPs treatment.

[0094] 2. Immunofluorescence staining was used to stain the marker gene for AT2 functional maturity in the final ALO obtained in Example 2. The specific procedure is described in Example 2, with SFTPB and ABCA3 added as primary antibodies. Results are shown below. Figure 6 B in the middle.

[0095] Immunofluorescence staining results from ABCA3 showed that AT2 cells treated with PDA NPs were more mature. PDA can promote the functional maturation of AT2 cells in ALO.

[0096] 3. Ultrastructural imaging: The ALO obtained in Example 2 was first fixed with 2.5% glutaraldehyde at room temperature, rinsed, and then fixed with osmium tetroxide. Subsequently, it underwent gradient dehydration with alcohol, and after dehydration, it was embedded and ultrathinly sectioned. The sections were stained and observed using transmission electron microscopy (TEM), and the morphology of lamellar bodies, lipid droplets, and nanoparticles within the alveolar organoids was photographed and recorded.

[0097] See results Figure 7 Transmission electron microscopy results showed that PDA NPs treatment promoted the maturation of AT2 lamellar bodies and the secretion of lipid droplets.

[0098] In summary, this invention applies polydopamine nanoparticles to ALO ​​suspension culture. PDA NPs enter organoids through endocytosis (and exert antioxidant effects intracellularly via ALO uptake), participating in organoid development and promoting the formation of LB bodies (lamellar bodies) and the maturation of AT2 function.

[0099] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for inducing differentiation and maturation of alveolar organoids, characterized in that, Alveolar organoids in the early alveolar stage were induced to differentiate by suspension culture; the suspension culture was carried out in an alveolar organoid culture medium, which was additionally supplemented with polydopamine nanoparticles.

2. The method for inducing alveolar organoid differentiation and maturation according to claim 1, characterized in that, The concentration of polydopamine nanoparticles added to the alveolar organoid culture medium is 10-100 μg / mL, preferably 25 μg / mL; And / or, the particle size of the polydopamine nanoparticles is 100-200 nm; And / or, each 500 μL of the alveolar organoid culture medium contains 50-200 alveolar organoids in the early alveolar stage.

3. The method for inducing alveolar organoid differentiation and maturation according to claim 1 or 2, characterized in that, The alveolar organoid culture medium includes one or more of the following: basal culture medium, protein supplement, nutritional supplement, antibiotic, glutamine supplement, insulin-transferrin-selenium supplement, glucocorticoid, component one, fibroblast growth factor, TGFβ signaling pathway inhibitor, and component two. Component one is a cAMP signaling pathway agonist and / or a phosphodiesterase inhibitor; component two is a Wnt signaling pathway agonist and / or a GSK3β signaling pathway inhibitor. Preferably, the basal culture medium in the alveolar organoid culture medium is Advanced DMEM / F-12; Preferably, the protein supplement in the alveolar organoid culture medium includes albumin at a concentration of 0.1%-1%; more preferably, 0.25%. Preferably, the nutrient supplement in the alveolar organoid culture medium includes a B-27 supplement at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the antibiotic in the alveolar organoid culture medium is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the glutamine supplement in the alveolar organoid culture medium is GlutaMAX, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, the insulin-transferrin-selenium supplement in the alveolar organoid culture medium is ITS-X, with a concentration of 0.05%-1%; more preferably, 0.1%. Preferably, the glucocorticoid in the alveolar organoid culture medium is dexamethasone at a concentration of 10-200 nM, more preferably 50 nM; Preferably, the cAMP signaling pathway agonist in the alveolar organoid culture medium is 8-Br-cAMP at a concentration of 10-000 nM, more preferably 100 nM; Preferably, the fibroblast growth factor in the alveolar organoid culture medium is FGF7, with a concentration of 1-50 ng / mL, more preferably 10 ng / mL; Preferably, the phosphodiesterase inhibitor in the alveolar organoid culture medium is IBMX, with a concentration of 10-1000 nM, more preferably 100 nM; Preferably, the TGFβ signaling pathway inhibitor in the alveolar organoid culture medium is SB431542, with a concentration of 1-20 μM, more preferably 10 μM; Preferably, the Wnt signaling pathway agonist and / or GSK3β signaling pathway inhibitor in the alveolar organoid culture medium is CHIR99021, with a concentration of 0.5-10 μM, more preferably 3 μM.

4. The method for inducing alveolar organoid differentiation and maturation according to any one of claims 1-3, characterized in that, The method for obtaining alveolar organoids in the early alveolar stage is as follows: on the seventh to fourteenth day of lung progenitor cell differentiation, after qPCR detection shows that the relative expression level of NKX2-1 with GAPDH is ≥100, the lung progenitor cell culture medium is replaced with alveolar organoid culture medium to induce the lung progenitor cells into alveolar organoids in the early alveolar stage.

5. The method for inducing alveolar organoid differentiation and maturation according to claim 4, characterized in that, The alveolar organoids in the early alveolar stage were obtained by inducing lung progenitor cells in 3D culture hydrogels using alveolar organoid culture medium. The lung progenitor cells were obtained by inducing single cells of the foregut endoderm in a 3D culture hydrogel using a lung progenitor cell culture medium. The 3D culture hydrogel includes one or more of Matrigel, Cultrex BME, dECM hydrogel, collagen hydrogel, fibrin hydrogel, gelatin, hyaluronic acid, and polyethylene glycol hydrogel, preferably Matrigel; more preferably GFR-Matrigel. The anterior foregut endoderm single cell is obtained by dissociating the anterior foregut endoderm cell mass; The anterior foregut endoderm cell mass is obtained by induction of shaped endoderm cells; The defined endoderm cells are induced from human pluripotent stem cells.

6. The method for inducing alveolar organoid differentiation and maturation according to claim 5, characterized in that, The lung progenitor cell culture medium includes one or more of the following: basal culture medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, Notch signaling pathway inhibitor, BMP4, fibroblast growth factor 7, fibroblast growth factor 10, GSK3β signaling pathway inhibitor, and retinoic acid. Preferably, in the lung progenitor cell culture medium, the basal culture medium is DMEM / F-12 medium and / or modified DMEM / F-12 medium, more preferably Advanced DMEM / F-12; Preferably, in the lung progenitor cell culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the nutritional supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, the antibiotic in the lung progenitor cell culture medium is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the lung progenitor cell culture medium, the Notch signaling pathway inhibitor is selected from N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) or dibenzozazepine; more preferably, the Notch signaling pathway inhibitor is N-(N-(3,5-difluorophenylacetyl)-L-alanyl)-S-phenylglycine t-butyl ester) at a concentration of 15-25 μM, and more preferably 20 μM; Preferably, the concentration of BMP4 in the lung progenitor cell culture medium is 15-25 ng / mL, more preferably 20 ng / mL; Preferably, in the lung progenitor cell culture medium, the concentrations of fibroblast growth factor 7 and / or fibroblast growth factor 10 are 5-15 ng / mL, more preferably 10 ng / mL. Preferably, in the lung progenitor cell culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 1-5 μM, and even more preferably, a concentration of 3 μM; Preferably, the concentration of retinoic acid in the lung progenitor cell culture medium is 40-60 nM, more preferably 50 nM.

7. The method for inducing alveolar organoid differentiation and maturation according to claim 5 or 6, characterized in that, The anterior foregut endoderm culture medium used for differentiating and forming the anterior foregut endoderm cell mass includes: basal medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist and GSK3β signaling pathway inhibitor, or one or more of these. Preferably, in the pre-endoderm culture medium, the basal culture medium is Advanced DMEM / F-12; Preferably, in the pre-endoderm culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the nutrient supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, in the pre-endoderm culture medium, the antibiotic is penicillin / streptomycin, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the TGF-β signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542; more preferably, the TGF-β signaling pathway inhibitor is SB431542, with a concentration of 5-15 μM; even more preferably, it is 10 μM; Preferably, in the pre-endoderm culture medium, the BMP signaling pathway inhibitor is selected from Noggin, Dorsomorphin, DMH1, or LDN-193189; more preferably, the BMP signaling pathway inhibitor is Noggin, with a concentration of 150 ng / mL to 250 ng / mL; even more preferably, it is 200 ng / mL. Preferably, the concentration of FGF4 in the pre-endoderm culture medium is 400-600 ng / mL; Preferably, in the pre-endoderm culture medium, the SHH agonist is selected from SHH or SAG; more preferably, the SHH agonist is SAG with a concentration of 0.5-1.5 μM; even more preferably, it is 1 μM. Preferably, in the pre-endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021 at a concentration of 1-3 μM; even more preferably, it is 2 μM. Preferably, the time for differentiation to form the anterior foregut endoderm cell mass is 2-7 days, more preferably 4 days.

8. The method for inducing alveolar organoid differentiation and maturation according to any one of claims 5-7, characterized in that, The culture medium used for differentiating and forming the defined endoderm cells is a defined endoderm complete culture medium, which includes a defined endoderm basal culture medium, a TGF-β signaling pathway activator and a GSK3β signaling pathway inhibitor. The shaped endoderm basal culture medium includes one or more of the following: core basal culture medium, protein supplement, nutrient supplement, antibiotic, pH stabilizer, and glutamine supplement; Preferably, in the shaped endoderm basal culture medium, the core basal culture medium is MCDB131; Preferably, in the basal culture medium for shaping endoderm, the protein supplement is bovine serum albumin with a concentration of 0.4%-0.6%, more preferably 0.5%; Preferably, in the basal culture medium for shaping endoderm, the nutrient supplement is glucose with a concentration of 8-12 mM, more preferably 10 mM; Preferably, in the basal culture medium for shaping endoderm, the antibiotic is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the basal culture medium for shaping endoderm, the pH stabilizer is sodium bicarbonate with a concentration of 1.5-2 mg / mL; more preferably, it is 1.7 mg / mL. Preferably, in the basal culture medium for shaping endoderm, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the complete endoderm culture medium, the TGF-β signaling pathway activator is selected from Activin A, Activin B, TGF-β1, TGF-β2 or TGF-β3; more preferably, the TGF-β signaling pathway activator is Activin A, with a concentration of 80-120 ng / mL, and even more preferably 100 ng / mL; Preferably, in the complete endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 0.1-3 µM. Preferably, the time for differentiation to form the defined endoderm cells is 2.5-4 days, more preferably 3 days; More preferably, the complete culture medium for the typed endoderm used on the first day of differentiation to form the typed endoderm cells includes: the typed endoderm basal culture medium, 100 ng / mL Activin A and 3 µM CHIR99021; More preferably, the complete culture medium for the finalized endoderm cells used on the second day after differentiation comprises: the basal culture medium for the finalized endoderm cells, 100 ng / mL Activin A and 0.1 µM CHIR99021; More preferably, the complete culture medium for the finalized endoderm cells used on the third day of differentiation comprises: the basal culture medium for the finalized endoderm cells and 100 ng / mL Activin A.

9. The method for inducing differentiation and maturation of alveolar organoids according to any one of claims 1-8 is used to improve the differentiation efficiency of type II alveolar cells in alveolar organoids.

10. The method for inducing differentiation and maturation of alveolar organoids according to any one of claims 1-8 is used to promote the functional maturation of type II alveolar cells in alveolar organoids.

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