Organ-like culture solution for directional differentiation of Club cells and culture method

Abstract

The invention provides an organ culture solution for directional differentiation of Club cells. The organ culture solution is prepared from the following components in concentration: 1 * Advanced DMEM (dulbecco's modified eagle medium) / F12, 1 * GlutaMax, 1 * Hepes, 1 * penicillin / streptomycin, 500ng / ml R-spondin1, 100ng / ml FGF10 (fibroblast growth factor 10), 25ng / ml FGF7 (fibroblast growth factor 7), 5mu M Y27632, 500nM SB202190, 1.25 mM N-Acetylcysteine, 5mM Nicotinamide, 1x trophic factor B27 and 1-3mu g / ml Jag1. The invention further provides a Club cell directional differentiation organoid culture kit and a Club cell directional differentiation organoid culture method. The invention discloses a culture method of tracheal organoids for directional differentiation of in-vitro Club cells and a corresponding kit. The directional differentiation of the Club cells of the individual source trachea organs of the patient is realized, and the Club cells are stably cultured, passaged and stored in vitro for a long time; a research tool is provided for research of Club cell related disease mechanisms, and a novel organoid research model is provided for drug research and development.

Description

Technical Field

[0001] The present invention relates to a culture medium and a culture method for the directed differentiation of club cells into organoids. Background Art

[0002] Since it is difficult to stably culture human lung epithelial cells in vitro and it is also difficult to specifically sort them by flow cytometry, there is currently no human club cell line for basic research. Organoid models have shown great potential in basic biological research and medical drug R & D. Compared with the limitations of cell culture technology for human cells, the construction of an organoid culture system not only requires a very small amount of tissue samples, but also can be passaged, cultured, cryopreserved and resuscitated infinitely. More importantly, organoid models can accurately reproduce the phenotypic characteristics of in situ tissues at the gene, transcriptional and protein levels in vitro. [1] In recent years, tracheal organoids have been used in the study of virus infection mechanisms, drug screening and evaluation of curative effects, which has promoted the research progress and clinical transformation in related fields. [2] However, the proportion of club cells in the traditional human tracheal organoid culture system is often low, which is difficult to support in-depth mechanism research on the related functions of club cells. Therefore, we tried to individually or associatively dynamically regulate Notch signaling [3] , TGF-β / BMP signaling [4] , FGF7 and FGF10, WNT / β-catenin signaling [5] , Hippo-Yap signaling [6] , protein methyltransferase Ezh2 [7] , IL-33-ST2 axis [8] , and immune factors IL-4 and IL-13 signaling [9] and other signaling pathways and molecules to promote the proliferation and differentiation of club cells, so as to construct a human club cell directed differentiation system. A culture system for realizing the in vitro directed differentiation of club cells will provide a research tool for the study of the disease mechanism related to club cells and a new organoid research model for drug R & D. Summary of the Invention

[0003] The present invention provides a culture medium and a culture method for the directed differentiation of club cells into organoids.

[0004] The present invention provides a culture medium for the directed differentiation of club cells into organoids, which contains components with the following concentrations:

[0005] Advanced DMEM / F12 1×, GlutaMax 1×, Hepes 1×, penicillin / streptomycin 1×, 500 ng / ml R-spondin1, 100 ng / ml FGF10, 25 ng / ml FGF7, 5 μM Y27632, 500 nM SB202190, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1x B-27 supplement, 1 - 3 μg / ml Jag1.

[0006] Preferably, it contains components at the following concentrations:

[0007] Advanced DMEM / F12 1×, GlutaMax 1×, Hepes 1×, penicillin / streptomycin 1×, 500 ng / ml R-spondin1, 100 ng / ml FGF10, 25 ng / ml FGF7, 5 μM Y27632, 500 nM SB202190, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1x B-27 supplement, 1 μg / ml Jag1.

[0008] The present invention also provides a Club cell-directed differentiation organoid culture kit, which includes the Club cell-directed differentiation organoid culture medium described above.

[0009] The present invention provides a method for culturing Club cell-directed differentiation organoids, which includes the following steps:

[0010] a. Select a sample: Bronchial epithelial tissue of patients who have indications for bronchoscopy and mucosal biopsy clinically and have no significant PCD-related gene mutations after combining electron microscopy biopsy and whole-exome sequencing results.

[0011] b. Primary culture of tracheal organoids; The time for primary culture is 20 days.

[0012] c. Differentiation induction culture of tracheal organoids: Culturing with the Club cell-directed differentiation organoid culture medium described in claim 1 or 2 for 8 days, and replacing the Club cell-directed differentiation organoid culture medium every 4 days.

[0013] Among them, the primary culture method in step b includes the following steps:

[0014] a. Sample washing: Repeatedly pipetting and washing the biopsy sample with DPBS.

[0015] b. Tissue digestion: Transfer to an EP tube containing 1 ml of tracheal organoid digestion solution, place on a shaker at 37°C (100 rpm, for 40 - 60 min), pipette and mix every 10 min to observe the digestion status; after the tissue is dissociated, add 100 μL of FBS to terminate the reaction;

[0016] c. Preparation of single cells: Filter through a 40-μm filter, centrifuge the filtrate (200 g, 3 min), and retain the cell pellet;

[0017] d. Washing and purification: Add 1 ml of Wash solution containing DMEM / F12, GlutaMax, Hepes, and P / S, pipette to mix, and centrifuge to remove the supernatant;

[0018] e. Matrigel coating: Resuspend the cells on ice at 30 μL / well, spread them on a 24-well plate, and let the gel solidify at 37°C for 15 min;

[0019] f. Culture and maintenance: Add 500 μL of tracheal organoid medium containing growth factors, small molecules, antioxidants, and B27 to each well, culture at 37°C and 5% CO2, and change the medium every four days.

[0020] Among them, the tracheal organoid digestion solution in step b is: 500 mL of 1x Advanced DMEM / F12 medium, 5 mL of GlutaMax, 5 mL of Hepes, 5 mL of P / S, 400 U / ml of collagenase I, 0.25 mg / ml of protease E, 10 U / ml of DNase, and 10 μM of Y-27632.

[0021] Among them, the Wash solution described in step d consists of: 500 mL of 1x Advanced DMEM / F12 medium, 5 mL of GlutaMax, 5 mL of Hepes, and 5 mL of P / S.

[0022] Among them, the tracheal organoid medium described in step f is: Add growth factors to the Wash solution: 500 ng / ml of R-spondin1, 100 ng / ml of Noggin, 100 ng / ml of FGF10, and 25 ng / ml of FGF7, small molecules: 500 nM of A8301, 5 μM of Y-27632, 500 nM of SB202190, antioxidant factors: 1.25 mM of N-Acetylcysteine, 5 mM of Nicotinamide, and 1x Nutrient B27 additive.

[0023] The present invention constructs a method for culturing tracheal organoids with directional differentiation of Club cells in vitro and a corresponding kit, realizes the long-term stable culture, passage and preservation of tracheal organoids with directional differentiation of Club cells of individual patients in vitro, provides a research tool for the study of the mechanism of Club cell-related diseases, and provides a new organoid research model for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Schematic diagram of the operation (constructing primary tracheal organoids and maintaining the culture with tracheal organoid medium (AO) until about the 20th day. At this time, the diameter of most organoids is about 100 μm - 150 μm, and then introducing a differentiation induction culture system to maintain the culture for about 8 days);

[0025] Figure 2 : qPCR experiment preliminarily determines the induction culture protocol (Figure A - C: The results of the qPCR experiment suggest that the AO - Noggin - A8301 protocol, AO - Noggin - A8301 + RA protocol, and AO - Noggin - A8301 + Jag1 (1 μg / ml) protocol significantly increased the transcriptional level expression of CC10 compared with the AO protocol. Figure D: The results of the qPCR experiment suggest that compared with the AO protocol, the AO - Noggin - A8301 + Jag1 (1 μg / ml) protocol increased the transcriptional level of CC10 by nearly 3 times. Figures E - F: qPCR verifies the changes in the expression levels of the Goblet cell marker gene MUC5AC, the ciliated cell marker gene FOXJ1, and the basal cell marker gene P63);

[0026] Figure 3 : Identifying the induction culture protocol for Club cells (Figure A: Bright - field morphology of tracheal organoids in the induction group and the control group. The vehicle group is: human tracheal organoid culture system + corresponding vehicle. Figure B: Statistical analysis of the diameters of organoids in the induction group and the control group grown for the same number of days. Figure C: Representative HE staining of each group. Figure D: Immunofluorescence staining of the induction group and the control group. Figure E: Proportion of Club cells in the induction group and the control group in immunofluorescence staining). DETAILED DESCRIPTION OF THE INVENTION

[0027] Example 1 Method for culturing organoids with directional differentiation of Club cells of the present invention

[0028] 1. Ethical and sample selection

[0029] According to the Declaration of Helsinki, this research protocol (project approval number KL118) has been approved by the Ethics Committee of West China Second University Hospital, Sichuan University. All participants have signed written informed consent forms.

[0030] The samples were from pediatric patients who had indications for bronchoscopy and mucosal biopsy clinically, that is, those with manifestations such as recurrent respiratory tract infections, and were highly suspected of having primary ciliary dyskinesia (PCD) clinically. All the pediatric clinical samples involved in this study were from the Pediatric Bronchoscopy Diagnosis and Treatment Center of West China Second University Hospital, Sichuan University. During the clinical diagnosis and treatment process, bronchoscopy and tracheal mucosal biopsy were performed on the children who met the indications, and the tissue of the bronchial epithelial area with normal mucosal morphology at the 3-4th level bronchi was precisely obtained using a tracheal clamp. Some specimens were used for transmission electron microscopy to evaluate the ciliary ultrastructure; the remaining tissue samples were used as the source of primary cells for the construction of pediatric tracheal organoids. Patients without significant PCD-related gene mutations in combination with electron microscopy biopsy and whole exome sequencing results after the operation could be included in this study.

[0031] 2. Primary culture of tracheal organoids

[0032] The pediatric clinical samples collected in this study were from children who underwent bronchoscopic interventional diagnosis and treatment in the Pediatric Bronchoscopy Diagnosis and Treatment Center of West China Second University Hospital, Sichuan University. A tracheal clamp was used to precisely sample the bronchial epithelial tissue with normal mucosal morphology at the 3-4th level bronchial branches. Some specimens were subjected to transmission electron microscopy ultrastructure analysis to systematically evaluate the ciliary structure; the remaining tissue was treated with pre-cooled biological sample preservation solution (1x DMEM medium), placed in a constant temperature specimen transport box (4°C) to maintain cell viability, and strictly transported to the laboratory for primary culture treatment within 6 hours.

[0033] Primary treatment was carried out in the laboratory, and the specific experimental steps were as follows:

[0034] 1) Use a pipette to gently aspirate the biopsy sample and place it in a 3 cm culture dish containing DPBS solution for repeated pipetting and washing until there is no visible red color in the biopsy tissue specimen.

[0035] 2) Transfer the biopsy tissue sample after washing to a 1.5 ml EP tube containing 1 ml of tracheal organoid digestion solution. Seal the tube with parafilm and place the EP tube containing the biopsy sample and digestion solution on a pre-warmed shaker at 37°C, 100 rpm, for 40 - 60 min. The EP tube can be taken out every 10 min, pipetted up and down repeatedly to mix well, and observe the digestion of the biopsy sample. Preparation of Wash solution: Take 500 mL of 1x Advanced DMEM / F12 medium, add 5 mL of GlutaMax, 5 mL of Hepes, and 5 mL of P / S, and mix well. Store at 4°C in the refrigerator for no more than one week. Preparation method of tracheal organoid digestion solution: Add collagenase I (400 U / ml), protease E (0.25 mg / ml), DNase (10 U / ml), and Y-27632 (10 μM) to the Wash solution. The tracheal organoid digestion solution for children is best prepared and used immediately.

[0036] 3) Until most of the biopsy samples are dissociated, add 100 μl of 100% FBS and pipette up and down to mix well to terminate digestion.

[0037] 4) Filter all the liquid through a 40 μm filter. The filtrate is the single-cell suspension of tracheal epithelium. After centrifuging the filtrate (200G, 3 min), discard the supernatant and retain the precipitate. At this time, pay attention to observing whether there are blood cells in the precipitate, that is, whether there is visible red color to the naked eye. If there are blood cells, 1 ml of red blood cell lysate needs to be added and placed at room temperature for 3 - 5 min for lysis. After lysis is completed, centrifuge (200G, 3 min) and discard the supernatant to retain the precipitate.

[0038] 5) Add 1 ml of Wash solution and pipette up and down to mix well. Centrifuge (200G, 3 min) and discard the supernatant to retain the precipitate.

[0039] 6) Add 1 ml of Wash solution and pipette up and down to mix well. Take 10 μl and use a cell counter to count the cells. Centrifuge the remaining liquid at 200G for 3 min, discard the supernatant, and retain the cell pellet.

[0040] 7) Resuspend the cell pellet with 30 μl / well of Matrigel and quickly spread the gel in a 24-well plate. Place it in a 37°C incubator for 15 min to allow the Matrigel to completely solidify. Usually, 8000 cells / well are seeded in a 24-well culture plate, the volume of Matrigel is 30 μl / well, and the volume of the medium is 500 μl / well. Note: Matrigel needs to be pre-melted on ice.

[0041] 8) Add 500 μl / well of tracheal organoid medium and place it in an incubator at 37 °C with 5% CO2 for culture. Subsequently, change the medium every 4 days or when the medium turns yellow. Preparation method of tracheal organoid medium (AO): Add growth factors (500 ng / ml R-spondin1, 100 ng / ml Noggin, 100 ng / ml FGF10, and 25 ng / ml FGF7), small molecules (500 nM A8301, 5 μM Y27632, 500 nM SB202190), antioxidant factors (1.25 mM N-Acetylcysteine, 5 mM Nicotinamide), and 1x nutrient factor B27 supplement to the Wash solution and mix evenly. The tracheal organoid medium is preferably prepared and used immediately. If it is prepared in advance, it can be stored in a 4 °C refrigerator for no more than one week to prevent the inactivation of active ingredients such as growth factors in the medium and affect the culture effect.

[0042] 3. Maintain the culture with tracheal organoid medium (AO) until about day 20. At this time, the diameter of most organoids is about 100 μM - 150 μM, and then introduce a differentiation induction culture system to maintain the culture for about 8 days.

[0043] The experimental groups are as follows:

[0044] 1) AO (i.e., add an equal amount of solvent to the tracheal organoid medium)

[0045] 2) AO + IL-4 (10 ng / mL)

[0046] 3) AO + IL-13 (5 ng / mL)

[0047] 4) AO + RA (50 nM)

[0048] 5) AO-Noggin-A8301

[0049] 6) AO-Noggin-A8301 + IL-4 (10 ng / mL)

[0050] 7) AO-Noggin-A8301 + IL-13 (5 ng / mL)

[0051] 8) AO-Noggin-A8301 + RA (50 nM)

[0052] 9) AO-Noggin-A8301 + TGF-β (100 ng / ml)

[0053] 10) AO-Noggin-A8301 + BMP4 (100 ng / ml)

[0054] 11) AO-Noggin-A8301 + TGF-β (100 ng / ml) + BMP4 (100 ng / ml)

[0055] 12) AO-Noggin-A8301 + TGF-β (100 ng / ml) + BMP4 (100 ng / ml) + RA (50 nM)

[0056] 13) AO-Noggin-A8301 + TGF-β (100 ng / ml) + BMP4 (100 ng / ml) + IL-4 (10 ng / mL)

[0057] 14) AO-Noggin-A8301 + TGF-β (100 ng / ml) + BMP4 (100 ng / ml) + IL-13 (5 ng / mL)

[0058] 15) AO-Noggin-A8301 + Jag1 (1 μg / ml)

[0059] 16) AO-Noggin-A8301 + DLL1 (1 μg / ml)

[0060] 17) AO-Noggin-A8301 + VAP (1 mM)

[0061] 18) AO-Noggin-A8301 + DLL4 (500 ng / ml)

[0062] 19) AO-Noggin-A8301 + Jga1 (3 μg / ml)

[0063] 20) AO-Noggin-A8301 + Jag1 (1 μg / ml) + DLL1 (1 μg / ml)

[0064] Above - indicates removal, + indicates addition.

[0065] 4. Collect experimental samples for qPCR experiments to preliminarily determine the transcriptional level expression of the Club cell marker gene CC10. The experimental steps of the qPCR experiment are as follows:

[0066] 1) Operate according to the RNA extraction kit instructions as follows:

[0067] a) Collect the organoid sample precipitate according to the experimental needs. Add 350 μl / tube of RL lysis buffer (containing 1% β-mercaptoethanol) and pipette up and down repeatedly to lyse it. It can be temporarily stored in a -80 °C refrigerator for a short time.

[0068] b) Take out the sample to be operated and place it on ice at 4°C. Add 350 μl of 70% ethanol and pipette to mix well. The 70% ethanol is prepared by mixing 100% ethanol and enzyme-free water at a ratio of 7:3.

[0069] c) Immediately transfer the well-mixed liquid to adsorption column CR1 and cover the tube cap. Set the conditions of the high-speed low-temperature centrifuge in advance as: 24°C, 12000 rpm, 60 s. After centrifugation, discard the waste liquid in the collection tube.

[0070] d) Add 350 μl of protein removal solution RW1 to adsorption column CR1 and centrifuge at 24°C and 12000 rpm for 1 min. After centrifugation, discard the waste liquid in the collection tube.

[0071] e) Add 500 μl of wash solution RW to adsorption column CR1, let it stand at room temperature for 2 min, and centrifuge at 24°C and 12000 rpm for 60 s. After centrifugation, discard the waste liquid in the collection tube.

[0072] f) Repeat step 5, without standing, directly centrifuge, and discard the waste liquid in the collection tube after centrifugation.

[0073] g) Transfer adsorption column CR1 to a new enzyme-free 1.5 ml EP tube. Place it at room temperature, open the lid of the adsorption column, and let it stand for 5 min to dry the residual liquid.

[0074] h) Add 14 μl of enzyme-free water to the center of the adsorption column membrane, cover the tube cap, and let it stand at room temperature for 2 min. Centrifuge at 24°C and 12000 rpm for 1 min. After centrifugation, remove adsorption column CR1 and retain the RNA solution in the EP tube.

[0075] i) Use Nanodrop to measure the concentration of the RNA solution.

[0076] j) The RNA solution can be temporarily stored in a -20°C refrigerator for a short time.

[0077] 2) Operate according to the reverse transcription kit instructions as follows:

[0078] a) In this project, 200 ng - 500 ng of RNA is used for reverse transcription for quantitative PCR experiments, and 100 ng of RNA is used for reverse transcription for digital PCR. Calculate the required volume of RNA according to the experimental conditions and needs, and make up to a 10 μl system with enzyme-free water. Add 1 μl of Oligo(dT)18 primer and 2 μl of Random primer to configure a 13 μl first-step reaction system mixture and place it in a 200 μl enzyme-free EP tube.

[0079] b) Centrifuge instantaneously for 30 s to ensure that all the reaction solutions converge at the bottom of the tube.

[0080] c) Place a 200 μl enzyme-free EP tube in a PCR instrument, and set the program to 65 °C for 5 min; 4 °C for 5 min.

[0081] d) Take out the EP tube and add the pre-prepared reaction system at 7 μl per portion. The pre-prepared reaction system of 7 μl per portion in the second step is as follows:

[0082] e) Centrifuge instantaneously for 30 s to ensure that all the reaction solutions gather at the bottom of the tube.

[0083] f) Place a 200 μl enzyme-free EP tube in a PCR instrument, and set the program to 25 °C for 10 min; 50 °C for 60 min; 85 °C for 5 min; 4 °C forever.

[0084] g) Centrifuge instantaneously for 30 s to ensure that all the liquid gathers at the bottom of the tube.

[0085] h) Add 180 μl of enzyme-free water, which is a 10-fold dilution, and store it briefly in a -20 °C refrigerator.

[0086] 3) Operate according to the GoTaq qPCR Master Mix instruction manual as follows:

[0087] a) Prepare a reaction system of 10 μl per well, including: 5 μl of GoTaq qPCR Master Mix, 1 μl of primer (F), 1 μl of primer (R), 1 μl of enzyme-free water, and 2 μl of sample cDNA. Set up 3 replicate wells for each sample.

[0088] b) Add the 10 μl per well reaction system into a dedicated 96-well plate or 384-well plate for the qPCR instrument as per the planned protocol. Seal the plate to ensure the overall seal of the well plate. Centrifuge using a plate centrifuge for 1 min.

[0089] c) Set the qPCR instrument program as follows:

[0090] d) The qPCR instrument records the Ct value. A difference in Ct values between three replicate wells of less than 0.5 is determined to be within the allowable range of technical error. Using GAPDH as an internal reference gene, the relative quantification method is used to compare the mRNA expression differences of related genes. According to the formula, the relative expression level of the target gene is 2−ΔΔCt. ΔΔCt = (average Ct value of the gene in the sample group - average Ct value of GAPDH in the sample group) - (average Ct value of the gene in the control group - average Ct value of GAPDH in the control group).

[0091] 5. Collect the experimental samples with positive results in step 3 for qPCR experiments to further confirm the transcriptional level expressions of the goblet cell marker gene MUC5AC, the ciliated cell marker gene FOXJ1, and the basal cell marker gene P63.

[0092] 6. Collect experimental samples for HE experiment to finally determine the changes in the cell tissue morphology of the organoids. The HE experiment is operated as follows:

[0093] a) In the fume hood, soak the basket containing the sections in sequence to complete dewaxing and hydration. The sequence and soaking time are: xylene 1 (10 min), xylene 2 (10 min), xylene 3 (10 min), 100% alcohol (5 min), 100% alcohol (5 min), 95% alcohol (5 min), 80% alcohol (5 min), 75% alcohol (5 min), double-distilled water (3 min).

[0094] b) Immerse the whole basket in hematoxylin solution for 3 min.

[0095] c) Take out the basket and place it in a jar filled with tap water, and rinse it with a large amount of tap water until the water in the jar is clear and colorless.

[0096] d) Place the basket in double-distilled water and let it stand for 3 min.

[0097] e) Immerse the whole basket in eosin solution and let it stand for 3 min.

[0098] f) Take out the basket and place it in a jar filled with tap water, and rinse it with a large amount of tap water until the water in the jar is clear and colorless.

[0099] g) In the fume hood, soak the basket containing the sections in sequence to complete permeabilization. The soaking sequence and time are: 75% alcohol (2 min), 80% alcohol (2 min), 95% alcohol (2 min), 100% alcohol (2 min), 100% alcohol (2 min), xylene 1 (10 min), xylene 2 (10 min), xylene 3 (10 min).

[0100] h) After air-drying the remaining xylene in the fume hood, take an appropriate amount of neutral gum, cover with a coverslip for mounting, and collect pictures with an Olympus microscope.

[0101] 7. Collect experimental samples for immunofluorescence staining experiment to finally determine the protein level expression of the Club cell marker gene CC10.

[0102] The operation steps of the immunofluorescence staining experiment are as follows:

[0103] a) Collect tracheal organoid precipitates according to experimental requirements.

[0104] b) Add 1 ml of Wash for washing, centrifuge horizontally at 70G for 3 min, discard the supernatant and retain the organoid precipitate.

[0105] c) Add 1 ml of 4% PFA, gently pipette to mix well, and place in a 4°C refrigerator overnight to complete fixation.

[0106] d) Add 1 ml of DPBS solution for washing, centrifuge horizontally (70G, 3 min), discard the supernatant, and retain the organoid pellet.

[0107] e) Preheat the metal bath to 70°C in advance. Prepare the mold in advance, that is, cut off the tip part of the bottom of a 200 μl PCR tube and retain the cylindrical part, close the tube cap, place it upside down, and place it horizontally. Prepare the solidified 3% agarose solution in advance, that is, dissolve agarose powder in PBS solution to prepare a 3% agarose solution, melt it in a water bath, dispense 1 ml into an EP tube, store it in a 4°C refrigerator, and place it in a water bath to melt before use and then place it in a 70°C metal bath to keep it in a liquid state.

[0108] f) Add 70 μL of 3% agarose liquid to the EP tube containing the organoid pellet, and gently mix to resuspend the organoids.

[0109] g) Quickly transfer the resuspended organoids and agarose liquid into the cylindrical mold. Place it in a 4°C refrigerator for more than 30 min to solidify.

[0110] h) Open the tube cap of the cylindrical mold, gently take out the solidified organoid and agarose mixture. Transfer it to a tissue embedding cassette and soak it in 70% ethanol for more than 10 minutes.

[0111] i) Transfer to a dehydrator, and set the program as follows: 75% alcohol, 1 h; 85% alcohol, 1 h; 90% ethanol 1, 30 mins; 90% ethanol 2, 30 min; 100% ethanol 1, 30 minutes; 100% ethanol 2, 30 minutes; xylene 15 min; 65°C wax 1 h; wax 2, 1 h.

[0112] j) Start the embedding machine in advance to ensure that the wax in the wax pool melts. Take out the tissue embedding cassette from the dehydrator and place it in the wax pool at the lower layer of the embedding machine.

[0113] k) Clean the metal embedding cassette in advance. Embed the organoid and agarose mixture with paraffin liquid in the metal embedding cassette, cool for more than 1 h, and transfer it to a 4°C refrigerator and refrigerate overnight to ensure solidification.

[0114] l) Separate the wax block and the metal embedding cassette the next day. The wax block can be stored in a 4°C refrigerator for a long time.

[0115] m) Use a microtome to cut the wax block into 5-μm-thick sections. Observe the morphology and quantity of organoids on the sections under bright field microscopy during sectioning to meet the requirements of subsequent experiments.

[0116] n) Dewax and hydrate the basket containing the sections in sequence in the fume hood. The sequence and soaking time are as follows: Xylene 1 (10 min), Xylene 2 (10 min), Xylene 3 (10 min), 100% alcohol (5 min), 100% alcohol (5 min), 95% alcohol (5 min), 80% alcohol (5 min), 75% alcohol (5 min), double-distilled water (3 min).

[0117] o) Use an immunohistochemistry pen to draw a circle closely around the position of the organoids on each section.

[0118] p) Prepare 250 ml of antigen retrieval solution: Add 5 ml of 50x EDTA to 245 ml of double-distilled water. Place the sections and the antigen retrieval solution in an antigen retrieval box, and place the antigen retrieval box in a box containing an appropriate amount of tap water. Transfer the whole to a microwave oven. Heat at high power in the microwave oven for 10 min until the temperature of the antigen solution reaches about 98°C, then change to medium power and maintain for 20 min. Finally, let it cool to room temperature.

[0119] q) Wash once with PSBS buffer.

[0120] r) Prepare 5% BSA and add it to the circled position on the glass slide. Incubate at room temperature in a humidified box for 1 h.

[0121] s) Prepare the primary antibody solution incubation solution (20 μl / well) in advance, add it and add it to the corresponding position on the glass slide. Incubate overnight in a 4°C refrigerator in a humidified box. The dilution ratio of the primary antibody is as follows:

[0122] t) After taking it out of the 4°C refrigerator and restoring to room temperature the next day, wash 3 times with PBST, 10 min each time.

[0123] u) Prepare the secondary antibody solution incubation solution (20 μl / well) in advance, add it to the corresponding position on the glass slide. Incubate at room temperature in the dark for 1 h. The dilution ratios of the nuclear staining antibody and the secondary antibody are as follows:

[0124] v) Wash 3 times with PBST in the dark box, 10 min each time.

[0125] w) Wipe off most of the liquid on the glass slide, add an appropriate amount of fluorescent anti-quenching agent, and cover with a coverslip for mounting.

[0126] x) After placing it overnight at room temperature under light-proof conditions, collect images with an Olympus FV3000 confocal microscope.

[0127] 8. Experimental results:

[0128] The schematic diagram of the operation process for inducing the directional differentiation of Club cells is as Figure 1As shown. The preliminary identification results using qPCR experiments indicated that the AO-Noggin-A8301 protocol, the AO-Noggin-A8301+RA protocol, and the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol significantly increased the transcriptional level expression of CC10 compared to the AO protocol ( Figure 2 A-C). Further comparing the degree of transcriptional expression elevation of the CC10 gene among the AO-Noggin-A8301+Jag1 (1 μg / ml), AO-Noggin-A8301+Jag1 (3 μg / ml), and AO-Noggin-A8301+Jag1 (1 μg / ml)+DLL1 protocols, the qPCR experimental results indicated that compared to the AO protocol, the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol increased the transcriptional level of CC10 by nearly 3-fold ( Figure 2 D). To compare the specificity of the AO-Noggin-A8301 protocol, the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol, and the AO-Noggin-A8301+RA protocol in inducing Club cell differentiation, the expression changes of the Goblet cell marker gene MUC5AC, the ciliated cell marker gene FOXJ1, and the basal cell marker gene P63 were verified using qPCR. The results are shown ( Figure 2 E-G): Compared to the AO protocol group, in the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol group, the transcriptional level expressions of MUC5AC and FOXJ1 were significantly reduced. Therefore, the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol group was determined as the optimal induction protocol group, abbreviated as the Club group (the components of the AO-Noggin-A8301+Jag1 (1 μg / ml) protocol group are: Advanced DMEM / F12 1×, GlutaMax 1×, Hepes 1×, penicillin / streptomycin 1×, 500 ng / ml R-spondin1, 100 ng / ml FGF10, 25 ng / ml FGF7, 5 μM Y27632, 500 nM SB202190, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1x nutrient factor B27, Jag1 1 μg / ml).

[0129] The bright-field morphological changes of tracheal organoids were recorded using a microscope. As shown in the figure, the organoids in all groups showed 3D spherical cavity-like growth. Figure 3A). Among them, the Vehicle group was: human tracheal organoid culture system + corresponding vehicle. The diameters of the organoids in each group on the same growth day were recorded and statistically analyzed. The results showed that the diameters of the organoids in the Club group were slightly smaller than those in the Vehicle group, but there was no significant statistical difference ( Figure 3 B). HE staining showed no obvious difference in the structure of the organoids between the two groups ( Figure 3 C).

[0130] By using immunofluorescence staining technology to localize the expression of Club cells and CC10 protein, it was found that in the Vehicle group, Club cells and CC10 in tracheal organoids were located on the inner side of the organoid lumen. In the Club group, Club cells and CC10 were not only visible on the inner side of the organoid lumen but also occasionally visible on the outer side of the lumen (Figure L). Through statistical analysis of the immunofluorescence staining of CC10 protein, the results showed that the proportion of Club cells in the Club group was higher than that in the Vehicle group ( Figure 3 D - E).

[0131] [1] Kim M, Mun H, Sung C O, et al. Patient - derived lung cancer organoids as in vitro cancer models for therapeutic screening[J]. Nat Commun, 2019, 10(1):3991.

[0132] [2] Zhu L, Yang W, Luo J, et al. Comparison of characteristics and immune responses between paired human nasal and bronchial epithelial organoids[J]. Cell & Bioscience, 2025, 15(1):18.

[0133] [3] Choi J, Jang Y J, Dabrowska C, et al. Release of Notch activity coordinated by IL - 1beta signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration[J]. Nat Cell Biol, 2021, 23(9):953 - 966.

[0134] [4] Tian J, Ouyang H, Wu J, et al. Inactivation of the TGF-β1 / ALK5 axis enhances club cell function and alleviates lung tissue damage to ameliorate COPD progression through the MEK / ERK signaling pathway[J]. Gen Physiol Biophys, 2024, 43(1): 37-48.

[0135] [5] Mou H, Vinarsky V, Tata Purushothamar, et al. Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells[J]. Cell Stem Cell, 2016, 19(2): 217-231.

[0136] [6] Zhao R, Fallon T R, Saladi S V, et al. Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells[J]. Dev Cell, 2014, 30(2): 151-65.

[0137] [7] Snitow M E, Li S, Morley M P, et al. Ezh2 represses the basal cell lineage during lung endoderm development[J]. Development, 2015, 142(1): 108-17.

[0138] [8] Dagher R, Copenhaver AM, Besnard V, et al. IL-33-ST2 axis regulates myeloid cell differentiation and activation enabling effective club cell regeneration[J]. Nat Commun, 2020, 11(1): 4786.

[0139] [9] Zhu L, An L, Ran D, et al. The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma[J]. Am J Respir Cell Mol Biol, 2019, 60(6): 695 - 704。

Claims

1. A culture medium for Club cell-directed differentiation organoids, characterized in that: It contains components with the following concentrations: Advanced DMEM / F12 1×, GlutaMax 1×, Hepes 1×, penicillin / streptomycin 1×, 500 ng / ml R-spondin1, 100 ng / ml FGF10, 25 ng / ml FGF7, 5 μM Y27632, 500 nM SB202190, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1x Neurobasal B27, Jag1 1 - 3 μg / ml.

2. The Club cell-directed differentiation organoid culture medium according to claim 1, characterized in that: It contains components with the following concentrations: Advanced DMEM / F12 1×, GlutaMax 1×, Hepes 1×, penicillin / streptomycin 1×, 500 ng / ml R-spondin1, 100 ng / ml FGF10, 25 ng / ml FGF7, 5 μM Y27632, 500 nM SB202190, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1x Neurobasal B27, Jag1 1 μg / ml.

3. A Club cell-directed differentiation organoid culture kit, characterized in that: It includes the Club cell-directed differentiation organoid culture medium described in claim 1 or 2.

4. A method for culturing Club cell-directed differentiated organoids, characterized in that: It includes the following steps: a. Select a sample: Bronchial epithelial tissue of patients who have indications for bronchoscopy and mucosal biopsy clinically and have no significant PCD-related gene mutations after combining electron microscopy biopsy and whole-exome sequencing results. b. Primary culture of tracheal organoids; The primary culture time is 20 days. c. Differentiation induction culture of tracheal organoids: Culture with the Club cell-directed differentiation organoid culture medium described in claim 1 or 2 for 8 days, and replace the Club cell-directed differentiation organoid culture medium every 4 days.

5. The method for culturing club cell-derived organoids according to claim 4, wherein: The primary culture method described in step b includes the following steps: a. Sample washing: Repeatedly pipette and wash the biopsy sample with DPBS. b. Tissue digestion: Transfer to an EP tube containing 1 ml of tracheal organoid digestive solution, place on a 37°C shaker (100 rpm, 40 - 60 min), pipette and mix well every 10 min to observe the digestion situation; After the tissue is dissociated, add 100 μL of FBS to terminate the reaction. c. Preparation of single cells: Filter through a 40 μm filter, centrifuge the filtrate (200 g, 3 min), and retain the cell pellet. d. Washing and purification: Add 1 ml of Wash solution containing DMEM / F12, GlutaMax, Hepes, P / S, pipette, and centrifuge to remove the supernatant. e. Matrigel coating: Resuspend the cells at 30 μL / well on ice, spread them on a 24-well plate, and let the gel solidify at 37°C for 15 min. f. Culture and maintenance: Add 500 μL of tracheal organoid medium containing growth factors, small molecules, antioxidants, and B27 to each well, culture at 37°C and 5% CO2, and replace the medium every four days.

6. The method for culturing Club cell-derived organoids according to claim 5, characterized in that: The digestive fluid for tracheal organoids in step b is: 500 mL of 1x Advanced DMEM / F12 medium, 5 mL of GlutaMax, 5 mL of Hepes, 5 mL of P / S, 400 U / ml of collagenase I, 0.25 mg / ml of protease E, 10 U / ml of DNase, and 10 μM of Y-27632.

7. The method for culturing Club cell-directed differentiated organoids according to claim 5, wherein: The composition of the Wash solution in step d is: 500 mL of 1x Advanced DMEM / F12 medium, 5 mL of GlutaMax, 5 mL of Hepes, and 5 mL of P / S.

8. The method for culturing Club cell-directed differentiated organoids according to claim 5, characterized in that: The tracheal organoid medium in step f is: adding growth factors to the Wash solution: 500 ng / ml of R-spondin1, 100 ng / ml of Noggin, 100 ng / ml of FGF10, and 25 ng / ml of FGF7, small molecules: 500 nM of A8301, 5 μM of Y27632, 500 nM of SB202190, antioxidant factors: 1.25 mM of N-Acetylcysteine, 5 mM of Nicotinamide, and 1x nutritional factor B27 additive.

Images