Lung tissue organoid culture method
By transplanting alveolar organoids into the spleen, the vascular network and immune microenvironment of the spleen are utilized to solve the problems of vascularization and mechanical stress simulation in lung organoid technology, enabling rapid maturation and functional improvement of alveolar organoids, which are suitable for disease models and drug screening.
Patent Information
- Application Number
- CN202511960823.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-02-10
AI Technical Summary
Existing lung organoid technologies lack a functional vascular network, cannot simulate the mechanical stress generated by respiratory movements, and lack a complex immune microenvironment in the body. This leads to hypoxia inside the organoid, insufficient nutrient delivery, and low gas exchange efficiency. Furthermore, traditional methods are complex, costly, or invasive.
Basal cells were isolated from lung tissue and cultured in three dimensions using enzymatic digestion and specific inducing factors. Subsequently, alveolar organoids were transplanted into mouse spleens along with hyaluronic acid gel during ectopic spleen surgery. The rich vascular network and immune microenvironment of the spleen were utilized to promote the vascularization and functional maturation of the organoids.
It has achieved rapid vascularization and functional maturation of alveolar organoids, forming hollow alveolar structures with barrier properties similar to those of physiology, making them suitable for disease model construction and drug screening, and providing an efficient in vivo biomimetic model.
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Figure CN121495832A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for culturing lung tissue organoids, belonging to the fields of biotechnology and regenerative medicine. Background Technology
[0002] Respiratory diseases, such as acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD), are leading causes of death and disability worldwide. The common pathological basis of these diseases is the structural destruction and functional loss of the alveolar-vascular unit. The alveolar-vascular unit is the core functional module for gas exchange in the lungs, intricately composed of alveolar epithelial cells (including type I alveolar cells AT1 and type II alveolar cells AT2), fused basal layers, and capillary endothelial cells.
[0003] Currently, clinical treatments such as medication and mechanical ventilation are insufficient to reverse substantial damage to lung tissue. Lung transplantation is the only curative treatment for end-stage lung disease, but it faces challenges such as donor scarcity and immune rejection. Therefore, developing new strategies to promote lung tissue regeneration and repair is urgently needed. Organoid technology offers hope for this field. Lung organoids can simulate the structure and function of lung tissue in three dimensions outside the body, providing a novel platform for disease mechanism research and drug screening.
[0004] However, existing lung organoid technologies, especially alveolar organoids, have significant limitations: (1) lack of functional vascular networks, resulting in hypoxia and insufficient nutrient delivery inside the organoids, and gas exchange efficiency far below physiological levels; (2) traditional static culture systems cannot simulate the periodic mechanical stress generated by respiratory movements, affecting the differentiation of alveolar wall cells and the maturation of barrier function; (3) lack of complex in vivo immune microenvironment and intercellular interactions, limiting their realism in pathological simulation.
[0005] While lung-on-a-chip technology attempts to simulate fluid shear forces and mechanical stretching through microfluidics, its fabrication is complex and costly, and it still struggles to fully replicate the complex microenvironment in vivo. In vivo transplantation (such as kidney cyst transplantation) is another way to promote organoid maturation, but orthotopic lung transplantation is a difficult and invasive procedure, and the pathological microenvironment of the lung itself (such as high expression of TGF-β) may inhibit organoid survival and differentiation.
[0006] Therefore, there is an urgent need in this field for a new method that can effectively integrate the advantages of vascularization, mechanical stress simulation and in vivo microenvironment to construct structurally complete and functionally mature alveolar organoids. Summary of the Invention
[0007] The purpose of this invention is to provide a method for culturing lung tissue organoids, which can efficiently construct vascularized and functionally mature lung tissue organoids.
[0008] The technical solution adopted in this invention is as follows: A method for culturing lung tissue organoids, characterized by the following steps: (1) Cell isolation and culture: Basal cells were isolated from lung tissue by enzymatic digestion and expanded and cultured on feeder cells; (2) Three-dimensional differentiation culture: The expanded basal cells were seeded on the surface of solid matrix gel and cultured in proliferation medium to form cell clusters. Then, the culture medium was replaced with alveolar-directed differentiation medium containing specific inducing factors and cultured for 10-14 days to form alveolar organoids. (3) Functional maturation: The recipient mice underwent ectopic spleen surgery to transfer the spleen to the subcutaneous tissue to form a "subcutaneous spleen" transplantation site. The alveolar organoids cultured in step (2) were digested and collected from the matrix gel, premixed with hyaluronic acid gel containing specific factors, and transplanted into the parenchyma of the above-mentioned subcutaneous spleen. The recipient mice were fed until the alveolar organoids grew in the spleen. The amount of hyaluronic acid gel added was 0.1-5 wt% of the alveolar organoids.
[0009] Mice were fed using standard methods, and after transplantation, the spleen's highly abundant vascular network (capillary density of 300-500 / mm²) was utilized. 2 With its pulsatile blood flow (shear force 0.8-1.2 Pa, highly consistent with the physiological range of 0.6-1.5 Pa for pulmonary capillaries), organoids can rapidly acquire blood supply, achieving vascularization and functional maturation. The loose lymphoid tissue within the spleen provides a low-stress, highly inclusive growth space for the organoids, and factors such as HGF and FGF2 secreted by its stromal cells contribute to the maturation of the air-blood barrier. Typically, after transplantation, they integrate with the host and form hollow alveolar organoids approximately 4 weeks later. These alveolar organoids can be retained in mice for subsequent animal experiments or extracted from the mouse spleen via micromanipulation for further experiments.
[0010] Preferably, the enzymatic digestion method in step (1) refers to cutting the lung tissue into small pieces and then treating it with a digestive solution containing DNase and protease.
[0011] Preferably, the trophoblast cells in step (1) refer to 3T3-J2 cells treated with mitomycin C.
[0012] Preferably, the proliferation medium in step (2) is PriMed-iCELL-037 medium containing 10% fetal bovine serum, with an additional 5 μM of Y-27632, and the proliferation culture time is 24-48 h.
[0013] Preferably, the alveolar-directed differentiation medium in step (2) is PriMed-iCELL-037 medium without fetal bovine serum, supplemented with the following specific inducing factors: FGF10 50ng / ml, HGF 30ng / ml Insulin 10ug / ml Transferrin 5.5 μg / ml Sodium selenite 6.7 ng / ml 1 mg / ml bovine serum albumin, 5uM Y-27632.
[0014] Preferably, the recipient mouse in step (3) is an immunodeficient mouse.
[0015] Preferably, in step (3), a microinjector is used for transplantation, with a transplantation density of 180-250 organoids / spleen.
[0016] Preferably, the specific inducing factors added in step (3) include: FGF10, HGF, VEGF and Apelin.
[0017] Preferably, the concentration of specific factors in the hyaluronic acid in step (3) is: 50 ng / ml FGF10, 30 ng / ml HGF, 20 ng / ml VEGF, and 20 ng / ml Apelin.
[0018] This invention also discloses a method for establishing a respiratory disease model. Transplanted mice obtained using the aforementioned lung tissue organoid culture method are given LPS to construct an acute lung injury model. Specifically, the acute injury model can be constructed by intraperitoneal injection of LPS (5 mg / kg). Forty-eight hours after injection, the mice are sacrificed, and their spleens are harvested. Pathological staining is used to detect indicators of immune cell infiltration and inflammatory damage in the transplanted lung organoids, such as q-PCR detection of inflammatory factors TNF-α, IL-1, and IL-6.
[0019] The beneficial effects of this invention are as follows: Temporal control: This invention first uses differentiation medium to culture alveolar organoids containing both lung progenitor cells and alveolar epithelial cells in matrix gel. Then, during transplantation, this invention encapsulates alveolar organoids with hyaluronic acid gel along with FGF10, HGF, VEGF, and Apelin and transplants them into the spleen after ectopic surgery. FGF10 and HGF can maintain the alveolar morphology of alveolar organoids in the spleen microenvironment. VEGF can recruit splenic endothelial progenitor cells to the vicinity of alveolar organoids, promoting rapid vascularization of alveolar organoids. Apelin can induce endothelial progenitor cells to differentiate into lung gas exchange endothelium, forming a mosaic structure of alveolar epithelial cells and gas exchange endothelium. This epithelial-endothelial mosaic structure is the structural basis of the classic lung gas exchange barrier. This continuation of temporal control allows the alveolar organoids in the spleen to further mature.
[0020] Efficient vascularization and maturation: By utilizing the spleen's natural rich vascular microenvironment, the problem of vascularization of organoids in vitro has been solved. Transplanted organoids can quickly obtain oxygenation and nutritional support, and their endothelial cells can be "recruited" by the host blood vessels and integrated to form functional blood flow, which significantly promotes the structural and functional maturation of alveolar organoids, making them closer to natural alveoli.
[0021] Unique site advantages: The "subcutaneous spleen" model not only provides an ideal biological microenvironment, but also has the advantages of relatively simple surgery, minimal trauma, and good host tolerance (splenic function can be compensated). Compared with the spleen in vivo, the subcutaneous location is more conducive to in vivo observation and intervention, such as integrating mechanical stimulation devices to simulate respiratory stress, or using in vivo imaging technology (such as two-photon microscopy) to monitor dynamic parameters such as air-blood barrier thickness and oxygen diffusion rate in real time.
[0022] Good functional integrity: The alveolar organoids produced by the method of this invention contain both AT1 and AT2 cells, express function-related proteins, and have barrier properties similar to those of physiology, which surpasses the functional limitations of traditional in vitro models.
[0023] Broad application prospects: This method provides a highly biomimetic in vivo model for studying alveolar development and disease mechanisms (such as ARDS, COPD, pulmonary fibrosis, and viral infection), and also provides a powerful technical platform for drug toxicology evaluation, personalized medicine, and the development of future organoid-based regenerative therapy strategies. Attached Figure Description
[0024] Figure 1 : Schematic diagram of the technical route of this invention. (Illustration: The entire process from basal cell extraction, in vitro alveolar organoid construction, spleen heterotopic surgery to intrasplenic transplantation and in vivo maturation) Figure 2Immunofluorescence staining image of in vitro constructed alveolar organoids, showing co-expression of AQP5 (AT1 marker, green) and pro-SPC (AT2 marker, red).
[0025] Figure 3 Tissue sections (HE staining and immunofluorescence staining) four weeks after alveolar organoid transplantation into the spleen show that the organoids have successfully colonized, maintained a hollow alveolar-like structure, and continued to express alveolar cell markers. Detailed Implementation
[0026] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.
[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, while this document may provide examples of parameters containing specific values, it should be understood that the parameters need not be exactly equal to the corresponding values, but may approximate the corresponding values within acceptable error tolerances or design constraints.
[0028] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.
[0029] Example 1: Lung tissue organoid culture method, the process is as follows: Figure 1 As shown Extraction of mouse lung basal cells: C57BL / 6 mice (or immunodeficient mice) were euthanized and their lungs were aseptically harvested. The lung tissue was minced and digested overnight at 4°C with a digestive solution containing DNase (10 μg / mL) and protease (1 mg / mL). The next day, the cells were pipetted into a single-cell suspension, passed through a 70 μm cell sieve, centrifuged and resuspended, and then seeded onto mitomycin C-treated 3T3-J2 feeder cells (brand: BDBIO, catalog number: C6204) for expansion culture. The proliferation medium was prepared as a complete medium using PriMed-iCELL-037-500ml, with an additional 5 μM of Y-27632 added.
[0030] In vitro differentiation of alveolar organoids: Expanded basal cells were collected by digestion with Accutase and counted. Cell suspension (density 1×10^5 cells / mL, 500 μL per well) resuspended in proliferation medium was added to 8-well slides pre-coated with 50 μL of matrix gel and cured. After 48 hours of culture, the medium was replaced with alveolar-directed differentiation medium, PriMed-iCELL-037, supplemented with the following specific inducing factors: FGF10 50 ng / ml, HGF 30 ng / ml, insulin 10 μg / ml, transferrin 5.5 μg / ml, sodium selenite 6.7 ng / ml, 1 mg / ml bovine serum albumin, and 5 μM Y-27632.
[0031] Change the medium every 2 days and culture for 14 days. Figure 2 As shown, immunofluorescence assay revealed that 3D organoids were formed in vitro, expressing AQP5, PDPN, pro-SPC, E-cadherin, and Collagen I.
[0032] Splenic heterotopia and transplantation: Standard C57BL / 6 mice were used (if the alveolar organoids were derived from humans, immunodeficient mice were required, such as NOD / ShiLtJGpt-Prkdc mice purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). em26Cd52 Il2rg em26Cd22 After anesthesia, a ectopic spleen transplant was performed. While maintaining the integrity of the arteriovenous pedicle, the spleen was relocated and fixed to the subcutaneous tissue of the mouse's lateral abdomen, forming a "subcutaneous spleen" transplantation site. The patient recovered for one week post-surgery. Alveolar organoids cultured in vitro for 14 days were digested and collected, and transplanted into the subcutaneous spleen using a microinjector (approximately 200 organoids / spleen). Post-operative hemostasis was carefully achieved, and the skin was sutured.
[0033] Results: Spleens were harvested 2 and 4 weeks post-transplantation. Gross examination revealed the presence of grafts within the spleen. Paraffin sections were stained with H&E and immunofluorescence staining, as shown... Figure 3 As shown in the figure. The results showed that, unlike transplanted lung tissue blocks which could only preserve airway structures, the alveolar organoids constructed by this method successfully colonized in the spleen, forming typical, hollow alveolar-like structures, and stably expressed the AT1 and AT2 cell markers PDPN and pro-SPC, demonstrating that they achieved better maturity in the in vivo microenvironment.
[0034] Example 2: Co-culturing alveolar organoids with spleen endothelial progenitor cells to verify the plasticity and differentiation potential of spleen endothelial progenitor cells. Method for extracting spleen endothelium: C57BL / 6 mice (or immunodeficient mice) were used. Before dissecting the spleen, the spleen was perfused through the left ventricle with ice-cold PBS at a rate of 2 mL / min for 5 min. The spleen was then aseptically harvested. Spleens were washed with pre-cooled HBSS to remove residual blood. The spleen was then placed in digestion buffer, minced, and further ground. The mixture was then incubated at 37°C for 20 minutes with shaking every 5 minutes. After digestion, digestion was stopped with HBSS solution containing 10% serum. Cells were passed through a 70μm cell sieve, centrifuged, and washed again with HBSS solution containing 10% serum. The cells were then resuspended in 0.5% BSA solution and loaded into Ficoll buffer. After density gradient centrifugation, the Ficoll layer was collected and seeded into cell plates containing rat tail collagen. One week later, splenic endothelial progenitor cells were obtained through negative selection with CD45 magnetic beads and positive selection with CD34 magnetic beads. The sorted cells were further expanded and cultured in splenic endothelial progenitor cell culture medium (catalog number: iCell-i011-002m-500ml) and used for downstream vascularized lung organoids.
[0035] In vitro differentiation of vascularized lung organoids: Expanded basal cells were collected by digestion with Accutase and counted. Cell suspension (density 1×10^5 cells / mL, 500 μL per well) was added to 8-well slides pre-coated with 50 μL of cured matrix gel. After 48 hours of culture, expanded splenic endothelial progenitor cells were resuspended in co-culture differentiation medium and loaded onto matrix gel containing alveolar organoids. The medium was changed every 2 days for 14 days. Bright-field, hematoxylin and eosin (HE) and immunofluorescence assays showed that the co-cultured vascularized lung organoids formed a mosaic structure of alveolar epithelial and endothelial cells. Immunofluorescence confirmed that PDPN is a type I alveolar cell marker and Car4 is a marker for gas-exchange endothelial cells, indicating that splenic endothelial progenitor cells can further differentiate into lung-specific endothelial markers. This indicates that alveolar organoids and splenic endothelial progenitor cells can work synergistically to form more functionally mature and morphologically more biomimetic organoids, demonstrating that the spleen has significant advantages as a transplantation site for lung organoids, and that alveolar organoids have the potential to form more functionally mature lung organoids in the spleen.
[0036] The co-culture differentiation medium consisted of spleen endothelial progenitor cells (catalog number: iCell-i011-002m-500ml) and basal cell expansion medium (PriMed-iCELL-037-500ml). First, the basal media from both kits were mixed 1:1. Then, spleen endothelial progenitor cell additives and basal cell additives were added separately, along with 1% penicillin-streptomycin solution, 50 ng / ml FGF10, 30 ng / ml HGF, 1 mg / ml BSA, 1X transferrin-insulin solution (catalog number: 41400045), and 5 μm Y-27632.
[0037] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for culturing lung tissue organoids, characterized in that... The steps include: (1) Cell isolation and culture: Basal cells were isolated from lung tissue by enzymatic digestion and expanded and cultured on trophoblast cells; (2) Three-dimensional differentiation culture: The expanded basal cells were seeded on the surface of solid matrix gel and cultured in proliferation medium to form cell clusters. Then, the culture medium was replaced with alveolar-directed differentiation medium containing specific inducing factors and cultured for 10-14 days to form alveolar organoids. (3) Functional maturation: The recipient mice underwent ectopic spleen surgery to transfer the spleen to the subcutaneous tissue to form a "subcutaneous spleen" transplantation site. The alveolar organoids cultured in step (2) were digested and collected from the matrix gel, premixed with hyaluronic acid gel containing specific factors, and transplanted into the parenchyma of the above-mentioned subcutaneous spleen. The recipient mice were fed until the alveolar organoids grew in the spleen. The amount of hyaluronic acid gel added was 0.1-5 wt% of the alveolar organoids.
2. The lung tissue organoid culture method according to claim 1, characterized in that, The enzymatic digestion method in step (1) refers to cutting the lung tissue into small pieces and then treating it with a digestive solution containing DNase and protease.
3. The lung tissue organoid culture method according to claim 1, characterized in that, The trophoblast cells in step (1) refer to 3T3-J2 cells treated with mitomycin C.
4. The lung tissue organoid culture method according to claim 1, characterized in that, The proliferation medium in step (2) is PriMed-iCELL-037 medium containing 10% fetal bovine serum, with an additional 5 μM of Y-27632, and the proliferation culture time is 24-48 h.
5. The lung tissue organoid culture method according to claim 4, characterized in that, The alveolar-directed differentiation medium in step (2) is PriMed-iCELL-037 medium, with the following specific inducing factors added: FGF10 50ng / ml, HGF 30ng / ml, Insulin 10ug / ml Transferrin 5.5 μg / ml Sodium selenite 6.7 ng / ml 1 mg / ml bovine serum albumin, 5uM Y-27632.
6. The lung tissue organoid culture method according to claim 5, characterized in that, The recipient mice in step (3) are immunodeficient mice.
7. The lung tissue organoid culture method according to claim 6, characterized in that, In step (3), transplantation is performed using a microinjector at a density of 180-250 organoids / spleen.
8. The method for culturing lung tissue organoids according to any one of claims 1-7, characterized in that, The specific factors added in step (3) include: FGF10, HGF, VEGF and Apelin.
9. The lung tissue organoid culture method according to claim 6, characterized in that, In step (3), the concentrations of specific factors in the hyaluronic acid are: 50 ng / ml FGF10, 30 ng / ml HGF, 20 ng / ml VEGF, and 20 ng / ml Apelin.
10. A method for establishing a respiratory system disease model, characterized in that, Transplanted mice obtained using the lung tissue organoid culture method described in any one of claims 1-9 were given LPS to construct an acute injury model by intraperitoneal injection of LPS.