Methods and media for culturing lung organoids

Abstract

The application discloses a lung organoid culture method and culture medium. The culture medium comprises a basic lung organoid culture medium and an alveolar organoid culture medium. The lung organoid culture method uses the basic lung organoid culture medium and the alveolar organoid culture medium, can culture ASC in three-dimensional matrix gel to obtain a lung organoid with alveolar structure, and helps to simulate an in-vivo environment by using the lung organoid to perform various tests.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to lung organoid culture methods and culture media. Background Technology

[0002] In adult organs, most functions depend on three-dimensional structure and the synergistic effects between various cell types, which are crucial for organ function. However, most current in vitro research models utilize two-dimensional monolayer cell cultures. As early as the beginning of the 21st century, it was discovered that transferring cells from their native environment to two-dimensional cultures results in a loss of tissue specificity and function. For example, the work of Mina Bissell et al. revealed the crucial role of three-dimensional structure in development and tumorigenesis. Therefore, three-dimensional culture that mimics in vivo architecture is essential for constructing representative in vitro models.

[0003] A crucial requirement for better understanding the pathogenesis of infectious diseases is the establishment of representative model systems. However, neither two-dimensional cell models nor animal models can accurately simulate the pathogenesis of epidemic diseases. Currently, numerous research groups are using two-dimensional cell lines (such as VeroE6, Caco-2, and Calu-3 cells) for in vitro studies of the novel coronavirus (SARS-CoV-2). Although these cell lines are generally highly sensitive to SARS-CoV-2, they may fail to simulate key aspects of the viral life cycle, and most antiviral compounds that function at the cellular level are ineffective in patients. In addition to cell lines, various animal models have also been used to study novel coronavirus infection (COVID-19), but each has its limitations, demonstrating the complexity of different animal hosts' responses to viral infection. Mice are insensitive to wild-type SARS-CoV-2 infection; only overexpression of ACE-2 results in high viral loads. SARS-CoV-2 can enter using the golden Syrian hamster ACE-2 model, but it does not cause severe lung disease. Similarly, in ferrets, the disease is relatively mild, viral replication is mainly observed in the upper respiratory tract, and due to species differences, SARS-CoV-2 acquires the Spike protein, which mutates rapidly in ferrets.

[0004] Organoids are three-dimensional structures cultured from organ-specific stem cells in vitro, and they hold promise for bridging the gap between cell lines and in vivo animal models. Organoids can be derived from two different stem cell populations: adult stem cells (ASCs) and pluripotent stem cells (PSCs, including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0005] Currently, the technology for inducing lung organoids using iPSCs is relatively mature, and it is possible to induce the formation of structures composed of various airway cell types, including basal cells, rod cells, and ciliated cells, as well as alveolar cell types: alveolar type I and type II (ATI and ATII) cells. This allows for the separate induction of airway and alveolar organoids from iPSCs, but it is not yet possible to achieve a combination of airway and alveolar structures within a single organoid.

[0006] iPSC-induced lung organoids are suitable for studying viral infection mechanisms, while personalized studies for COVID-19 patients require organoids derived from ASCs. However, while airway cells are relatively easy to obtain from adult tissue cultures, alveolar cells are difficult to obtain. Furthermore, human ATII cells rapidly differentiate into ATI-like cells in 2D cell cultures, limiting the possibilities for studying ATII biology. In the past two years, to advance research on the novel coronavirus, several laboratories have attempted to obtain ASC-derived alveolar organoids and have achieved some preliminary results. In June 2022, Cell Discovery first reported alveolar organoids cultured from ASCs. The authors used low-absorption plates and a dedicated differentiation medium for suspension culture, rather than a three-dimensional culture method (using Matrigel as support). This was because, in their study, if Matrigel were used, the organoids would exhibit a thick-walled state rather than a sac-like state. Therefore, currently, there is still a lack of feasible routine three-dimensional culture methods for culturing alveolar organoids from ASCs. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for culturing lung organoids and a culture medium.

[0008] In a first aspect of the present invention, a basic lung organoid culture medium is provided. The formulation of the basic lung organoid culture medium is shown in Table 1.

[0009] Table 1. Formulation of basic lung organoid culture medium

[0010] The basic lung organoid culture medium can rapidly (approximately 15 days for passage) culture normal tissue-derived ASCs into lung organoids with transparent sac-like structures.

[0011] In a second aspect of the invention, a culture medium for alveolar organoids is provided. The formulation of the alveolar organoid culture medium is shown in Table 2.

[0012] Table 2 Formulation of alveolar organoid culture medium

[0013] ASCs cultured in the aforementioned alveolar organoid culture medium will have a slow production rate (it takes about 30 days to pass them through passage), but lung organoids cultured in this medium can differentiate and grow into alveolar-like structures.

[0014] In a third aspect, this invention provides a method for culturing lung organoids derived from alveolar scleroderma (ASC). The culturing method includes the following steps: 1) culturing ASCs in the basal lung organoid culture medium, passaged every 15 days for a total of two passages to obtain intermediate tissue; 2) culturing the intermediate tissue in the alveolar organoid culture medium, passaged every 30 days.

[0015] The culture method provided by this invention can obtain alveolar organoids (ASCs) from lung tissue entirely within a three-dimensional matrix gel environment for alveolar organoid culture. This method utilizes fewer exogenous inducing reagents, instead employing a two-stage nutrient environment adjustment to promote the differentiation of lung organoids without alveolar structures into lung organoids with alveoli.

[0016] In some embodiments of the present invention, in step 1) of the culture method, the ASC is obtained by digesting lung tissue into cell clusters using collagenase, resuspending the cells in matrix gel, adding the basic lung organoid culture medium, and changing the basic lung organoid culture medium every 3 days.

[0017] In some embodiments of the present invention, the freshly prepared basic lung organoid culture medium is preheated to 37.0±0.5 °C before each replacement of the basic lung organoid culture medium.

[0018] In some embodiments of the present invention, in step 2) of the culture method, if alveolar structures do not appear before the first passage, the intermediate tissue is moderately digested with a recombinant trypsin analog (TrypLE) before passage. To ensure an appropriate degree of digestion, microscopic observation should be performed every 15 minutes during digestion. Moderate digestion is indicated when a large number of free cell clumps are observed, while over-digestion is indicated when a large number of single cells are observed. Early comparative experiments were conducted using trypsin-EDTA and TrypLE. TrypLE digestion is gentler, allowing for better control of the degree of digestion, and the cell viability after digestion is higher than that of trypsin-EDTA. Furthermore, TrypLE can be stored at room temperature, avoiding enzyme activity errors caused by freeze-thaw cycles.

[0019] In some embodiments of the present invention, step 2) of the cultivation method specifically includes the following steps: 2-1) Remove the basic lung organoid culture medium and add cell collection buffer to the intermediate tissue; 2-2) Add matrix gel to resuspend, incubate with shaking at 4 ℃, then add Hank's balanced salt solution (HBSS), centrifuge at 4 ℃, and remove the supernatant; 2-3) The precipitate was dissociated by mechanical crushing, then HBSS was added, and the mixture was centrifuged at 4 °C to remove the supernatant; 2-4) Add matrix gel to the precipitate for resuspending, and then add the alveolar organoid culture medium; 2-5) Replace the culture medium for the alveolar organoids every 3.5 days.

[0020] In some embodiments of the present invention, the parameters for the centrifugation process in steps 2-2) and 2-3) are both set to 400 x g for 5 min.

[0021] In some embodiments of the present invention, the HBSS contains 1% fetal bovine serum (FBS).

[0022] In some embodiments of the present invention, after the supernatant is removed in step 2-2), the lung organoids obtained by precipitation are relatively large. To further decompose them, TrypLE can be added, and the mixture can be incubated at 37 °C, accompanied by mechanical methods such as pipetting and aspiration to dissociate them.

[0023] The culture medium and culture method provided by this invention can continuously culture ASCs into lung organoids with alveoli in a three-dimensional matrix gel. By simply changing the composition of the culture medium and adjusting the culture conditions, basic lung organoids can be induced into lung organoids with alveoli, resulting in better three-dimensional structures and helping to simulate the real in vivo environment. Attached Figure Description

[0024] Figure 1 This is a microscopic image of the intermediate tissue described in Example 2; Figure 2 This is a microscopic image of the lung organoid described in Example 2; Figure 3 This is a microscopic image of the tissue obtained after the culture in Control Example 1; Figure 4 This is a microscopic image of the tissue obtained after the culture in Control Example 2; Figure 5 This is an immunofluorescence image of the intermediate tissue from the Normal group described in Example 3; Figure 6 The image shows the immunofluorescence of the intermediate tissue from the Tumor group described in Example 3. Figure 7 This is an immunofluorescence image of the intermediate tissue from the Normal group described in Example 3; Figure 8This is an immunofluorescence image of lung organoids with alveolar structures from the Normal group described in Example 3. Detailed Implementation

[0025] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0026] Unless otherwise specified, the techniques or conditions described in the literature in this field or the product instructions shall be followed in the following examples. All reagents or instruments without a specified manufacturer are commercially available conventional products. Quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0027] Example 1: Preparation of Culture Medium Prepare the basic lung organoid culture medium and alveolar organoid culture medium according to the proportions in Tables 3 and 4.

[0028] Table 3. Basic Lung Organoid Culture Medium Formulation

[0029] Table 4. Culture medium ratio for alveolar organoids

[0030] Example 2: Lung organoid culture from human ASCs and lung tumor tissues Human ASCs and tumor tissues from lung cancer patients were used to cultivate organoids with alveoli.

[0031] The specific steps include: 1-1) After obtaining normal tissue and lung tumor tissue from the distal lung, immediately store them in a tissue preservation solution in an ice bath. They can be used within 24 hours after being removed from the body; ideally, the tissue should be processed immediately.

[0032] 1-2) Cut the solid tissue into pieces approximately 1 mm in size. 3 Collect small pieces and store them in 10 ml of ice-cold HBSS (containing 1% FBS). While waiting for the fragments to settle, pre-coat the pipette tips by immersing them in the ice-cold FBS 2-3 times.

[0033] 1-3) Remove the supernatant using a pipette tip pre-coated with FBS. Wash the tissue block again with 10 ml of ice-cold HBSS (containing 1% FBS).

[0034] 1-4) Add 10 ml of tissue digestion buffer (containing collagenase and cathepsin) to the tissue block, and shake it on a constant temperature shaker at 37 ℃ for 1-2 hours. Check every 30 minutes during the digestion process to assess the degree of digestion.

[0035] 1-5) When the tissue block has been digested to a large number of free cell clumps, transfer the suspension using a pre-coated pipette and filter through a 100 μm filter. For the remaining tissue block, resuspend in 10 ml of HBSS containing 1% FBS and filter again.

[0036] 1-6) Add 2% FBS to the filtered suspension to terminate digestion.

[0037] 1-7) Place the suspension in a 50 ml test tube and centrifuge at 400 xg at 4 ℃ for 5 minutes.

[0038] 1-8) If a visible red precipitate is present, resuspend the precipitate in 2-4 ml of erythrocyte lysis buffer (also known as ACK buffer) and incubate at room temperature (RT) for 5 minutes. Then add 10 ml of HBSS containing 1% FBS and centrifuge again at 400 xg. If no visible red precipitate is present, resuspend the precipitate directly in 10 ml of HBSS containing 1% FBS and centrifuge again at 400 xg.

[0039] 1-9) Resuspend the cell pellet in ice-cold matrix gel, avoiding the formation of air bubbles. The final concentration should be 1-1.5 × 10⁻⁶. 5 Cells / mL.

[0040] 1-10) Quickly add 30-40 µL of ice-cold Matrigel mixture containing ASC cells to the center of each well of a 24-well cell culture plate, approximately 2000-4000 ASC cells per well.

[0041] 1-11) Incubate the 24-well plate in an incubator at 37 °C for 10-20 minutes to allow the Matrigel to solidify.

[0042] 1-12) After gelation, add 450-500 μL of basal lung organoid culture medium to each well and culture the cells in an incubator at 37 ℃ (normally 5% CO2). Change the basal lung organoid culture medium every 3-4 days, ensuring that the fresh basal lung organoid culture medium is preheated before changing the medium.

[0043] 1-13) After culturing in basal lung organoid culture medium for 15 days, the tissue was passaged. Intermediate tissues from the second passage, such as… Figure 1As shown, the intermediate tissue of the Normal group exhibits a transparent cystic structure, consistent with existing organoid identification results. The intermediate tissue of the Tumor group exhibits a less smooth, solid structure, consistent with existing organoid identification results.

[0044] 2-1) Remove the basic lung organoid culture medium and then add 0.5-1 mL of ice-cold cell collection buffer.

[0045] 2-2) Mechanically break up the Matrigel using a pipette tip. Then incubate the 24-well plate on a shaker at 4 °C for 20-30 minutes to further dissolve the Matrigel.

[0046] 2-3) Transfer the matrix gel to a 15 mL conical tube and add 5 mL of ice-cold HBSS containing 1% FBS.

[0047] 2-4) Centrifuge at 400 xg for 5 minutes at 4 ℃ and remove the supernatant.

[0048] 2-5) If the intermediate tissue is large, add 0.5-1 mL TrypLE and incubate in a 37 ℃ water bath or incubator for 1-2 minutes.

[0049] 2-6) Use a pipette to blow and aspirate the intermediate tissue 20 times to further dissociate it.

[0050] 2-7) Add 5-10 mL of ice-cold HBSS containing 1% FBS to the test tube and centrifuge at 400 xg for 5 minutes at 4 ℃. Discard the supernatant, add 30-40 μl of frozen matrix gel (about 100 organoids) to each well, resuspend, aliquot into each well, and add 450-500 μl of alveolar organoid culture medium.

[0051] 2-8) Strictly adhere to changing the alveolar organoid culture medium every 3.5 days and continue culturing for one month. After one month of stable growth, the intermediate tissue can differentiate into lung cell-like structures, such as... Figure 2 As shown, organoids from the Normal group can differentiate and grow into lung-cell-like structures. Organoids from the Tumor group grow into relatively irregular structures.

[0052] Comparative Example 1: Basic Lung Organoid Culture Medium Control Experiment The lung organoid culture medium was cultured according to the same steps as in Example 1, but the alveolar organoid culture medium in steps 2-7) was replaced with the basal lung organoid culture medium. The basal lung organoid culture medium was changed every 3.5 days as required in steps 2-8). After one month of culture, with increasing passage number, the intermediate tissue of the Normal group maintained a hollow, transparent sac-like structure without significant change; the intermediate tissue of the Tumor group, due to nutrient supply issues, had a solid structure that limited its growth upper limit, allowing some normal cells to gain a growth advantage, thus gradually developing hollow sac-like structures similar to those of the Normal group (e.g., ...). Figure 3 (As shown). Even if the passage interval is deliberately extended to more than one month, the intermediate tissue will not develop a structure similar to alveoli; instead, it will only lack space in the matrix gel due to rapid growth.

[0053] Comparative Example 2: Controlled Experiment of Alveolar Organoid Culture Media Following the same steps as 1-1) to 1-11) in Example 1, but replacing the basic lung organoid culture medium in step 1-12) with the alveolar organoid culture medium, culture was performed. After one month of culture according to step 1-13), as follows... Figure 4 As shown, it can be observed that the intermediate tissues of both the Normal and Tumor groups develop slowly and are smaller in volume than the intermediate tissues obtained in steps 1-13 of Example 1.

[0054] Example 3 Immunofluorescence observation of lung organoids To maintain the organoid structure while ensuring its observability, we transparentized the lung organoids cultivated in Example 1. Different markers (e.g., TTF1, Ecad, Ki67, F-actin, etc.) can be labeled using immunofluorescence technology as needed. Specific steps: 3-1) Rinse each well with 1 ml of 4 °C PBS solution, taking care not to damage the matrix gel.

[0055] 3-2) Place the 24-well plate on an ice plate, add 1 ml of 4 ℃ cell recovery solution to each well, break the matrix gel and gently blow it, and incubate on a constant temperature horizontal shaker at 4 ℃ and 60 rpm for 0.5~1 h.

[0056] 3-3) Rinse the pipette tip with PBS-BSA (PBS containing 1% BSA) at 4 ℃, and then resuspend the cells with PBS-BSA.

[0057] 3-4) Transfer the intermediate tissue / lung organoids to 15 ml centrifuge tubes pre-coated with 1% PBS-BSA.

[0058] 3-5) Rinse the culture medium thoroughly with 1 ml of 1% PBS-BSA at 4℃ and collect all organic matter in the wells.

[0059] 3-6) Add PBS reagent at 4 ℃ to make the total volume in the centrifuge tube reach 10 ml, centrifuge at 4 ℃ and 100 g for 3 min, and remove the supernatant.

[0060] 3-7) Rinse the pipette tip with PBS at 4 ℃, then aspirate 1 ml of 4% paraformaldehyde and place in a 4 ℃ refrigerator for 45 min. During this period, gently shake the centrifuge tube every 10 min or so to resuspend the intermediate tissue / lung organoids.

[0061] 3-8) Add PBST (PBS containing 0.1% Tween) reagent at 4 ℃ to make the total volume of the centrifuge tube reach 10 ml, then incubate at 4 ℃ for 10 min, centrifuge at 4 ℃ and 100 g for 5 min, and remove the supernatant.

[0062] 3-9) Resuspend the organoids in 4 °C organoid washing buffer (PBS containing 0.1% Triton X-100 and 0.2% BSA) and then transfer them to a 24-well plate (add at least 200 μl of organoid washing buffer to each well).

[0063] 3-10) Incubate the 24-well plate in a refrigerator at 4 °C for 15 min.

[0064] 3-11) Add 8 μl of antibody to 600 μl of organoid washing solution, and repeatedly pipette 10-20 times to mix thoroughly to prepare the primary antibody reagent.

[0065] 3-12) Remove the organoid washing solution from each well until only 200 μl remains, and then add 200 μl of the primary antibody reagent to each well.

[0066] 3-13) After tightly wrapping the 24-well plate with tin foil, incubate it in a constant temperature shaker at 4 ℃ for 15 minutes, and then incubate it overnight in a refrigerator at 4 ℃.

[0067] 3-14) After removing the 24-well plate, add 1 ml of organoid washing solution to each well and incubate for 3 min, waiting for the cells to settle to the bottom.

[0068] 3-15) Gently aspirate 1 ml of organoid washing solution to remove it, leaving 200 μl in each well.

[0069] 3-16) After adding 1 ml of organoid washing solution to each well, incubate on a constant temperature shaker at 37 ℃ and 60 rpm for 2 h.

[0070] 3-17) Repeat cleaning steps 3-14) to 3-16) at least twice.

[0071] 3-18) Add 3 μl of fluorescent secondary antibody corresponding to the species of the primary antibody to 600 μl of organoid washing solution, and repeatedly pipette 10-20 times to mix thoroughly to prepare the secondary antibody reagent.

[0072] 3-19) Remove 1 ml of organoid washing solution until only 200 μl remains in each well.

[0073] 3-20) Add 200 μl of the secondary antibody reagent to each well, mix thoroughly, wrap tightly with tin foil, and incubate in a constant temperature shaker at 4 ℃ for 15 minutes, then incubate overnight in a refrigerator at 4 ℃.

[0074] 3-21) Repeat steps 3-14) to 3-16) for a total of 3 cleanings.

[0075] 3-22) Aliquot the organoids into 1.5 ml / 2 ml centrifuge tubes and rinse the 24-well plate with PBS (try to transfer all organoids). Centrifuge at 4 ℃ and 100 g for 2 min.

[0076] 3-23) Remove the supernatant as much as possible, and then add at least 50 μl of genomic DNA-free reverse transcription (RT) reagent to each centrifuge tube.

[0077] 3-24) Apply double-sided tape (at least 3 layers) to the four edges of the slide. To prevent liquid seepage, draw a quadrilateral inside the double-sided tape with a hydrophobic pen.

[0078] 3-25) Spread the sample evenly in the center, then slowly cover it with a coverslip, first touching the sample with one end (avoiding air bubbles as much as possible and covering the entire slide). (Note: When performing steps 23-25, use a 200μl pipette tip with the pointed end cut off).

[0079] The morphology of the intermediate tissues between the Normal and Tumor groups is as follows: Figure 5 and 6 As shown, this is consistent with the microscopic observations. Subsequently, the morphology of the intermediate tissue in the Normal group was compared with that of the lung organoids with alveoli in the Normal group, and the results are as follows... Figure 7 and 8 The lung organoids shown also exhibited multi-cystic structures in immunofluorescence experiments, while the intermediate tissue displayed a single cystic structure. This confirms that the culture method provided by this invention can utilize ASC to obtain lung organoids with alveolar structures in a three-dimensional matrix gel.

[0080] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for culturing lung organoids derived from adult stem cells, characterized in that, Including the following steps: 1) Adult stem cells derived from lung tissue were cultured in a basic lung organoid culture medium and passaged every 15 days for a total of two passages to obtain intermediate tissue; 2) The intermediate tissue was cultured using alveolar organoid culture medium and passaged every 30 days; The basic lung organoid culture medium comprises the following components: Advanced DMEM / F12, GlutaMax, 4-hydroxyethylpiperazine ethanesulfonic acid, penicillin / streptomycin bispecific antibody, B-27 Supplement, N-acetylcysteine, nicotinamide, SB202190, A-83-01, fibroblast growth factor-7, recombinant mouse head protein, recombinant human Rspo1 protein, fibroblast growth factor-10, and Y-27632. The final concentrations of the 4-hydroxyethylpiperazine ethanesulfonic acid are 10 mM, the N-acetylcysteine ​​is 1.25 mM, the nicotinamide is 5 mM, the SB202190 is 500 nM, the A-83-01 is 500 nM, the fibroblast growth factor-7 is 25 ng / mL, the recombinant mouse head protein is 100 ng / mL, and the recombinant human Rspo1 protein is 500 ng / mL. The final concentration of the fibroblast growth factor-10 was 100 ng / mL, and the final concentration of the Y-27632 was 5 μM. The alveolar organoid culture medium comprises the following components: Advanced DMEM / F12, GlutaMax, 4-hydroxyethylpiperazine ethanesulfonic acid, penicillin / streptomycin bispecific antibody, B-27 Supplement, N-acetylcysteine, nicotinamide, SB202190, A-83-01, fibroblast growth factor-7, recombinant mouse head protein, recombinant human Rspo1 protein, fibroblast growth factor-10, and Y-27632. The final concentrations of the 4-hydroxyethylpiperazine ethanesulfonic acid are 10 mM, the N-acetylcysteine, nicotinamide, SB202190, A-83-01, fibroblast growth factor-7, recombinant mouse head protein, and recombinant human Rspo1 protein are 200 ng / mL. The final concentration of the fibroblast growth factor-10 was 50 ng / mL, and the final concentration of the Y-27632 was 5 μM.

2. The cultivation method according to claim 1, characterized in that, The adult stem cells mentioned in step 1) are obtained by digesting lung tissue into cell clusters using collagenase, resuspending them in matrix gel, adding the basic lung organoid culture medium, and changing the basic lung organoid culture medium every 3 days.

3. The cultivation method according to claim 2, characterized in that, Before each change of culture medium, the fresh basal lung organoid culture medium was preheated to 37.0±0.5 ℃.

4. The cultivation method according to claim 1, characterized in that, In step 2), if no alveolar structure appears before the first passage, the intermediate tissue is digested with the recombinant trypsin analog TrypLE and then passaged.

5. The cultivation method according to claim 1 or 4, characterized in that, Step 2) specifically includes the following steps: 2-1) Remove the basic lung organoid culture medium and add cell collection buffer to the intermediate tissue; 2-2) Add matrix gel to resuspend, incubate with shaking at 4 ℃, then add HBSS, centrifuge at 4 ℃, and remove the supernatant; 2-3) The precipitate was dissociated by mechanical crushing, then HBSS was added, and the mixture was centrifuged at 4 °C to remove the supernatant; 2-4) Add matrix gel to the precipitate, resuspend it, and then add the alveolar organoid culture medium; 2-5) Replace the culture medium for the alveolar organoids every 3.5 days.

6. The cultivation method according to claim 5, characterized in that, The centrifugation parameters were set to 400 xg for 5 min.

7. The cultivation method according to claim 5, characterized in that, The HBSS contains 1% FBS.

8. The cultivation method according to claim 5, characterized in that, After removing the supernatant in step 2-2), add the recombinant trypsin analog TrypLE and incubate at 37 °C.

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