Lung airway organoid culture system with tracheal branch morphological structure and application and culture method thereof

Abstract

The invention discloses a lung airway organoid culture system with a trachea branch morphological structure as well as application and a culture method of the lung airway organoid culture system, and relates to the technical field of lung airway organoid culture. The hydrogel culture system is prepared from I-type rat tail collagen, gelatin, hyaluronic acid, laminin, low-melting agarose, chondroitin sulfate, fibronectin, elastin, epidermal growth factors, fibronectin L and transglutaminase; the induction culture medium is prepared from a 1XB27 supplement, a fiber growth factor, glutamine, glutathione, A8301, SB202190, IBMX, 8-Br-cAMP, FGF7, FGF10, Shh and ascorbic acid. The culture system is clear in component, low in price and cost and simple and convenient in preparation method, more importantly, the lung airway organoid which is closer to the physiological state can be efficiently induced to be formed within a short time, and the generated organoid has a branch form similar to an in-vivo trachea and contains various types of functional airway epithelial cells at the same time; and a more preferable scheme is provided for constructing an airway organ with an airway branch form based on the adult lung stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of lung airway organoid culture, and more specifically to a lung airway organoid culture system with a tracheal branching morphological structure, and its application, culture method and organoid. Background Art

[0002] In the respiratory system, the lung is an important organ for gas exchange and immune defense in animals. It is extremely vulnerable to infection or damage caused by pathogens or foreign objects in the external environment. Any disorder or abnormality in its function may cause irreversible damage to the animal's lung tissue, induce severe respiratory diseases and even cause death. At present, the pathogenic mechanisms of various lung diseases remain to be explored.

[0003] As physiological organs artificially constructed in vitro, organoids are called "organs in a dish". They are 3D "human organoids" obtained by inducing differentiation of pluripotent stem cells through different culture media and culture conditions. They are currently the closest system model to human physiological organs. The construction of organoids has promoted scholars' research on the development process of human organs, the mechanism of developmental diseases, and clinical drug testing. Given that organoids represented by brain organoids have gradually become physiological models for studying organ development, tissue homeostasis, and pathological conditions, and are used to study disease mechanisms and clinical drug screening, lung airway organoids have also gradually become a good research model for the physiological development of respiratory organs, the mechanism of respiratory diseases, and drug screening. Lung airway organoids have gradually become an important carrier that can replace animals as a targeted drug screening and efficacy evaluation for respiratory diseases.

[0004] According to current literature reports, the in vitro induction and culture of pulmonary airway organoids must rely on the 3D Matrigel gel system. Matrigel gel is derived from mouse tumor tissue extracts and contains a variety of protein molecules and growth factors. The specific composition is unclear, the batch product differences are relatively large, and the price is expensive. Considering the cell composition of pulmonary airway organoids and the stability of the culture system, using this system for large-scale induction and culture of pulmonary airway organs in vitro not only requires high costs, but also has the important problem of batch instability.

[0005] Therefore, establishing a culture system with clear components and capable of efficiently and stably culturing lung airway organoids is an urgent problem that needs to be solved by technical personnel in this field. Summary of the invention

[0006] In view of this, the present invention aims to provide a lung airway organoid culture system and culture method with a tracheal branching morphological structure.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A lung airway organoid culture system with a tracheal branching morphological structure, comprising a hydrogel culture system and an induction culture medium;

[0009] The hydrogel culture system includes: type I rat tail collagen 1.0-1.5 mg / mL, gelatin 100-200 mg / mL, hyaluronic acid 25-200 μg / mL, laminin 50-300 μg / mL, low melting agarose 5-20 mg / mL, chondroitin sulfate 50-300 μg / mL, fibronectin 20-200 μg / mL, elastin 20-200 μg / mL, epidermal growth factor 2.5-100 ng / mL, fibroblast growth factor 5-150 ng / mL, and transglutaminase 15 U / mL.

[0010] The induction medium includes: 1×B27 supplement, fibroblast growth factor 200 ng / mL, glutamine 2 mM, glutathione 100 μg / mL, A830 11 μM, SB202190 1 μM, IBMX 100 μM, 8-Br-cAMP 100 μM, FGF 75 ng / mL, FGF 105 ng / mL, Shh 100 ng / mL, and ascorbic acid 1 mM.

[0011] Preferably, the hydrogel culture system uses DMEM / F12 as solvent.

[0012] Preferably, the preparation process of the hydrogel culture system comprises the following steps:

[0013] (1) Dissolve type I rat tail collagen, gelatin, hyaluronic acid, laminin, chondroitin sulfate, epidermal growth factor, and fibroblast growth factor in DMEM / F12 to prepare a stock solution;

[0014] (2) Add the mother solutions of step (1) to DMEM / F12 according to the final concentration of the hydrogel system, and finally add 0.023 times the volume of type I rat tail collagen 1M NaOH as a cross-linking agent for cross-linking, so that the pH value of the 3D gel system is 7.2.

[0015] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the culture system in inducing lung airway stem cells to differentiate into lung airway organoids having a tracheal branching morphological structure.

[0016] As an inventive concept identical to the above technical solution, the present invention also seeks protection for a method for culturing lung airway organoids, comprising the following steps:

[0017] (1) Digesting tracheal basal stem cells with a cell confluence of 80%-90% with 0.25% trypsin to make them fall off the bottom of the cell culture dish, and then terminate the digestion with a basal medium with 10% FBS;

[0018] (2) Centrifuge and collect lung stem cells, resuspend the cells in DMEM / F12 basal medium containing 10 μM Y-27632, and dilute to 5×10 5 / mL, and a cell suspension was obtained;

[0019] (3) 100 μL of the cell suspension was mixed with 500 μL of the hydrogel system, and then 11.5 μL of 1 M NaOH was added for mixing and cross-linking;

[0020] (4) After culturing in a cell culture incubator for 30-45 minutes to form a 3D gel, add the induction medium and replace it with new induction medium every 3 days until an organoid with a significantly enlarged morphology and a significantly complex branching structure is obtained, then stop induction.

[0021] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a lung airway organoid obtained by culturing using the above culturing method.

[0022] As an inventive concept identical to the above technical solution, the present invention also claims protection for an application of the pulmonary airway organoid, which is any one of the following:

[0023] A: Study the regulatory mechanisms and physiological functions of lung airway development in vitro;

[0024] B: In vitro study of the pathological characteristics of the pulmonary airway system and the pathogenic mechanisms of respiratory diseases;

[0025] C: Drug development, screening, and pharmacological efficacy evaluation related to pulmonary airway system diseases;

[0026] D: Establish respiratory system disease models to study the corresponding pathogenic mechanisms and drug action mechanisms;

[0027] E: Testing the toxicity of inhaled substances or the effectiveness of inhaled drugs;

[0028] F: Study the infection of respiratory viruses on airway epithelial cells;

[0029] G: Further construction and development of functional bronchial organoids.

[0030] It can be seen from the above technical solutions that compared with the existing technology, the culture system has clear ingredients, low price and cost, simple preparation method, and more importantly, it can efficiently induce the formation of lung airway organoids that are closer to the physiological state in a short time. The generated organoids have a branching morphology similar to the trachea in vivo and contain multiple types of functional airway epithelial cells at the same time, which provides a more preferred solution for constructing airway organoids with airway branching morphology based on adult lung stem cells. It is of great significance to promote the research on the regulation mechanism of lung airway development, the pathogenesis of respiratory diseases, the development process of lung organs and the evaluation of clinical drug efficacy, the damage of viral infection to the respiratory system, and the construction of vascularized lung airway organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 The accompanying drawings are, from left to right, morphological diagrams of lung airway organoids at day 14 of induction culture of lung airway stem cells in Example 1, Example 2, and Example 3 of the present invention, respectively, with a scale of 400 μm.

[0033] Figure 2 The accompanying drawings are, from left to right, morphological diagrams of lung airway organoids at day 21 of induction culture of lung airway stem cells in Example 1, Example 2, and Example 3 of the present invention, with a scale of 400 μm.

[0034] Figure 3 The accompanying drawings are representative fluorescence images of basal cells (KRT5), MUC5B+ secretory cells (MUC5B), ciliated cells (ACT), Club endocrine cells (SCGB1A1), Goblet cells (MUC5AC), etc. in lung airway organoids cultured for 21 days in Example 2 of the present invention.

[0035] Figure 4 The accompanying figure is a representative diagram of cells with proliferation capacity (Ki67, green fluorescent marker) in lung airway organoids cultured for 21 days in Example 2 of the present invention.

[0036] Figure 5 The attached figure is a representative image of apoptotic cells in lung airway organoids cultured for 21 days in Example 2 of the present invention (Caspase-3, green fluorescent marker).

[0037] Figure 6The accompanying figure shows the identification results of airway epithelial cell marker genes after qPCR identification of lung airway organoids cultured for 21 days in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] Preparation of the lung airway organoid hydrogel system

[0041] Using DMEM / F12 as solvent, a culture medium containing the following final concentrations was prepared, including: type I rat tail collagen 1.0 mg / mL, gelatin 150 mg / mL, hyaluronic acid 25 μg / mL, laminin 50 μg / mL, low melting agarose 5 mg / mL, chondroitin sulfate 50 μg / mL, fibronectin 20 μg / mL, elastin 20 μg / mL, epidermal growth factor 2.5 ng / mL, fibroblast growth factor 5 ng / mL, 15 U / mL glutamyl transferase and 1 M NaOH as a cross-linking agent, the volume of 1 M NaOH being 0.023 times the volume of type I rat tail collagen 1.5 mg / mL.

[0042] Example 2

[0043] Preparation of the lung airway organoid hydrogel system

[0044] Using DMEM / F12 medium as solvent, the system includes: type I rat tail collagen 1.3 mg / mL, gelatin 100 mg / mL, hyaluronic acid 100 μg / mL, laminin 150 μg / mL, low melting agarose 10 mg / mL, chondroitin sulfate 200 μg / mL, fibronectin 150 μg / mL, elastin 150 μg / mL, epidermal growth factor 50 ng / mL, fibroblast growth factor 100 ng / mL, glutamyl transferase 15 U / mL and 1 M NaOH as cross-linking agent.

[0045] Example 3

[0046] Preparation of the lung airway organoid hydrogel system

[0047] Using DMEM / F12 as solvent, prepare a culture medium containing the following final concentrations: 1.5 mg / mL type I rat tail collagen, 200 mg / mL gelatin, 200 μg / mL hyaluronic acid, 300 μg / mL laminin, 20 mg / mL low melting agarose, 300 μg / mL chondroitin sulfate, 200 μg / mL fibronectin, 200 μg / mL elastin, 100 ng / mL epidermal growth factor, 150 ng / mL fibroblast growth factor, 15 U / mL transglutaminase, 1 M NaOH as a cross-linking agent, and the volume is 0.023 times the volume of 1.5 mg / mL type I rat tail collagen.

[0048] Example 4

[0049] Preparation of lung airway organoid induction medium

[0050] Using DMEM / F12 as solvent, a medium containing the following final concentration components was prepared, including: 1XB 27 supplement, fibroblast growth factor 200 ng / mL, glutamine 2 mM, glutathione 100 μg / mL, A830 11 μM, SB202190 1 μM, IBMX 100 μM, 8-Br-cAMP 100 μM, FGF 75 ng / mL, FGF 105 ng / mL, Shh 100 ng / mL, ascorbic acid 1 mM. The induction medium uses DMEM / F12 as solvent.

[0051] Example 5

[0052] Methods for culturing lung airway organoids

[0053] (1) Lung airway stem cells with a cell confluence of 80%-90% in a 6 cm cell culture dish were digested with 1.5 mL of 0.25% trypsin to make them fall off the bottom of the collagen-coated cell culture dish. The digestion was terminated with an equal volume of 10% FBS basal medium, and the lung basal stem cells were collected by centrifugation at 1000 rpm for 5 min.

[0054] (2) Resuspend the cells in 1 mL of DMEM / F12 basal medium containing 10 μM Y-2763 and dilute to 5 × 10 5 / mL to obtain cell suspension.

[0055] (3) Take 100 μL of the cell suspension and mix it with 500 μL of the lung airway organoid culture hydrogel system required by Example 1, Example 2, and Example 3, then mix and cross-link it with 5.7 μL of 1M NaOH, add it to a 12-well cell culture dish, and place it in a cell culture incubator for 30-45 minutes to form a solid gel.

[0056] (4) 1 mL of the lung airway organoid induction medium prepared in Example 4 was added for culture, and the cell morphology was observed every day. Fresh medium was replaced every three days. After about 14 days of culture, lung airway organs with branched morphological structures visible to the naked eye were formed in the 3D gel. By the 21st day, the morphological structure of the organoids was significantly enlarged and the branching structure was significantly complicated (see Figure 1-2 ).

[0057] Example 6

[0058] Characterization of the cellular composition of lung airway organoids

[0059] In order to verify that the pulmonary airway organoid gel system pulmonary airway organoid induction culture medium described in the present invention can be stably and effectively used for the culture of pulmonary airway organoids, the pulmonary airway organoids cultured for 21 days in Example 2 were selected to verify the types of their lineage cells. The pulmonary airway organoids cultured for 21 days in Example 2 were already visible to the naked eye. The culture medium was discarded, the gel was washed with PBS, fixed with 4% paraformaldehyde solution at room temperature for 2 hours, and dehydrated with 30% sucrose until the gel sank to the bottom of the dish. The gel was embedded in OCT and frozen sectioned with a thickness of 10um. The frozen sections were subjected to immunofluorescence staining, using markers that can mark basal cells (KRT5), ciliated cells (FOXj1), endocrine Club cells (SCGB1A1) and MUC5AC + 、MUC5B + The antibody protein of the secretory cell was used to trace the lung cells with the corresponding characteristics. The antibody and tissue sections were incubated overnight at 4°C. The tissue sections were washed 3 times with PBS, and then the corresponding immunofluorescence secondary antibody was added. The sections were incubated at room temperature for 1 hour. The tissue sections were washed 3 times with PBS, and then incubated with DAPI stain for 5 minutes. After washing 3 times with PBS, the sections were observed under a fluorescence microscope (see Figure 3 , are the fluorescent labeling results of various types of lung cells generated in cultured lung airway organs, including KRT5 for basal cells; MUC5B for MUC5B+ secretory cells; ACT for ciliated cells, SCGB1A1 for inner club secretory cells; and MUC5AC for Goblet cells. At the same time, the identification of cell proliferation and apoptosis in organoids found that there were still cells with proliferation ability at the edge of the organoids ( Figure 4 , Ki67 marks proliferating cells), while a small amount of apoptotic cells begin to appear in the center ( Figure 5 , Caspase-3 marked the proliferating cells). This showed that the 3D hydrogel system and lung organoid induction medium could induce lung stem cells to form organoids composed of multiple types of mature lung epithelial cells.

[0060] Example 7

[0061] Real-time fluorescence quantitative PCR verified that lung airway organoids expressed genes of mature pneumocytes in adult lung tissue.

[0062] The lung airway organoids cultured for 21 days in Example 2 and the cells extracted from adult lung tissue were washed with PBS, fully lysed with Trizol, and chloroform with 1 / 2 volume of Trizol was added. The mixture was centrifuged at 4°C, 12000 rpm, and 15 min. The upper aqueous phase was taken and an equal volume of isopropanol was added to mix thoroughly. The mixture was allowed to stand at room temperature for 10 min, and centrifuged at 4°C, 12000 rpm, and then the precipitate was fully washed with 75% ethanol, dried at room temperature until translucent, and RNase-free ddH2O water was added to dissolve the RNA.

[0063] RNA reverse transcription was performed using a reverse transcription kit (Promega, A5001). First, 500 ng RNA was added with 1 μL primer dT, and the volume was adjusted to 5 μL with ddH2O. The mixture was heated at 70°C for 5 min to open the helical structure in the RNA, and then immediately placed on ice for more than 5 min. Then, 4 μL 5X Reaction buffer, 4 μL MgCl2, 1 μL PCR dNTP, 0.5 μL RNase inhibitors, 1 μL Reverse Transcriptase and 4.5 μL ddH2O were added to the reaction system. After sufficient mixing, the reverse transcriptase was inactivated at 25°C for 5 min, 42°C for 1 h, and 70°C for 15 min. The expression level of the corresponding gene was detected by real-time fluorescence quantitative PCR. The reaction system of real-time fluorescence quantitative PCR is shown in Table 1, the reaction conditions are shown in Table 2, and the primer sequences are shown in Table 3.

[0064] Table 1 Reaction system of real-time fluorescence quantitative PCR

[0065] Reagents Volume (μL) 2XSYBGreenTaq 10.0 Forwardprimer 0.6 Reverseprimer 0.6 cDNA 0.6 <![CDATA[ddH2O]]> 8.2 Total volume 20

[0066] Table 2 Reaction conditions of real-time fluorescence quantitative PCR

[0067]

[0068]

[0069] Table 3 Real-time fluorescence quantitative PCR primer sequence list

[0070]

[0071] like Figure 6 The lung airway organoids cultured in vitro for 14 days expressed genes of epithelial cells in adult lung airway tissue, indicating that lung epithelial cells of the corresponding cell lineage were generated in the lung organoids.

[0072] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0073] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A lung airway organoid culture system with tracheal branching morphology, characterized in that: It includes a hydrogel culture system and an induction culture medium; The hydrogel culture system includes: type I rat tail collagen 1.0-1.5 mg / mL, gelatin 100-200 mg / mL, hyaluronic acid 25-200 μg / mL, laminin 50-300 μg / mL, low melting agarose 5-20 mg / mL, chondroitin sulfate 50-300 μg / mL, fibronectin 20-200 μg / mL, elastin 20-200 μg / mL, epidermal growth factor 2.5-100 ng / mL, fibroblast growth factor 5-150 ng / mL, and transglutaminase 15 U / mL. The induction medium includes: 1×B27 supplement, fibroblast growth factor 200 ng / mL, glutamine 2 mM, glutathione 100 μg / mL, A830 11 μM, SB202190 1 μM, IBMX 100 μM, 8-Br-cAMP 100 μM, FGF 75 ng / mL, FGF 105 ng / mL, Shh 100 ng / mL, and ascorbic acid 1 mM.

2. The lung airway organoid culture system with tracheal branching morphology according to claim 1, characterized in that: The hydrogel culture system uses DMEM / F12 as solvent.

3. The lung airway organoid culture system with tracheal branching morphology according to claim 1, characterized in that: The preparation process of the hydrogel culture system comprises the following steps: (1) Dissolve type I rat tail collagen, gelatin, hyaluronic acid, laminin, chondroitin sulfate, epidermal growth factor, and fibroblast growth factor in DMEM / F12 to prepare a stock solution; (2) Add the mother solutions of step (1) to DMEM / F12 according to the final concentration of the hydrogel system, and finally add 0.023 times the volume of type I rat tail collagen 1M NaOH as a cross-linking agent for cross-linking, so that the pH value of the 3D gel system is 7.

2.

4. Use of the culture system according to claim 1 in inducing lung airway stem cells to differentiate into lung airway organoids having a tracheal branching morphological structure.

5. A method for culturing lung airway organoids, characterized in that: The following steps are involved: (1) Digesting tracheal basal stem cells with a cell confluence of 80%-90% with 0.25% trypsin to make them fall off the bottom of the cell culture dish, and then terminate the digestion with a basal medium with 10% FBS; (2) Centrifuge and collect lung stem cells, resuspend the cells in DMEM / F12 basal medium containing 10 μM Y-27632, and dilute to 5×10 5 / mL, and a cell suspension was obtained; (3) taking 100 μL of the cell suspension and mixing it with 500 μL of the hydrogel system of claim 1, and then adding 11.5 μL of 1M NaOH for mixing and cross-linking; (4) After culturing in a cell culture incubator for 30-45 minutes to form a 3D gel, add the induction medium described in claim 1, replace the new induction medium every 3 days, and culture until an organoid with a significantly enlarged morphology and a significantly complex branching structure is obtained, then stop induction.

6. A lung airway organoid, characterized in that: Obtained by culturing using the culturing method according to claim 5.

7. A use of the lung airway organoid according to claim 6, characterized in that: The application is any of the following: A: Study the regulatory mechanisms and physiological functions of lung airway development in vitro; B: In vitro study of the pathological characteristics of the pulmonary airway system and the pathogenic mechanisms of respiratory diseases; C: Drug development, screening, and pharmacological efficacy evaluation related to pulmonary airway system diseases; D: Establish respiratory system disease models to study the corresponding pathogenic mechanisms and drug action mechanisms; E: Testing the toxicity of inhaled substances or the effectiveness of inhaled drugs; F: Study the infection of respiratory viruses on airway epithelial cells; G: Further construction and development of functional bronchial organoids.

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