Respiratory organoids with externally attached ciliated epithelial cells and method for preparing the same

Abstract

To provide a method for preparing a respiratory organoid by proliferating and differentiating respiratory epithelial cells.SOLUTION: Unlike conventional respiratory organoids in which epithelial cells existing in an apical region of a respiratory organ are generated on an interior of the organoid, the respiratory organoid of the present disclosure is shown in a form in which ciliated structures, which are epithelial cells, are located in an outer layer of the organoid, secretory cells are present on the interior, and basal stem cells existing at a base of respiratory tissue are also present on the interior of the organoid. It has a form similar to an actual respiratory tissue, and it was observed to exhibit normal functions of the epithelial cells, such as motility of ciliated cells. Therefore, when an organoid is prepared using the preparation method of the present disclosure, a respiratory organoid that can well mimic the actual tissue can be prepared.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a respiratory organoid and a method for producing the same, specifically, a respiratory organoid with ciliated epithelial cells attached to the outside and a method for producing the same.

Background Art

[0002] An organoid, also referred to as a biomimetic or mini-organ, means a small culture that reproduces all the morphological and functional aspects of a tissue or organ created by three-dimensional culture, aggregation, or recombination of cells isolated from stem cells or organ-derived cells. Such an organoid contains various specific cell populations that make up an organ or tissue, and has a morphological and structural organization similar to that of an actual tissue or organ, so it can reproduce the special functions of each organ. Organoids are formed by a common series of processes, where cells of the same function aggregate and are arranged in appropriate positions, and when cell compartments are separated, further fine differentiation occurs. Organoids have a morphology similar to that of actual organs and can realize in vitro studies that are difficult to achieve in animal models such as molecular signal control. Naturally, they are very useful for basic research, and furthermore, they are very useful technologies in various fields such as the human development process, establishment of disease models, screening for evaluation of drug efficacy, and development of cell therapy agents.

[0003] In the process of manufacturing organoids, the support or scaffold that plays a role in supporting cell adhesion and growth not only plays a very important role in tissue engineering but also plays an important role in the growth of cells seeded in the porous structure and cells that have migrated from the tissue periphery. Most cells in the human body are adherent cells that grow by adhering. Without a place to adhere, the cells cannot grow and will die. Therefore, the support must provide a suitable environment for cell adhesion, differentiation, growth, and cell migration. Such supports can be made of various materials, but generally, research on developing supports using natural materials, synthetic polymers, bioceramics, and polymer-ceramic composite materials has been actively conducted. Therefore, organoids mimicking various organs have been developed to date, and they are mainly cultured in Matrigel, which is a three-dimensional culture environment.

[0004] However, although Matrigel is widely used for organoid culture, it is derived from mouse sarcoma, there is no powerful alternative, and despite the high cost, there is a limit in that it has to rely on the use of Matrigel. Also, specific components are dominant, and there are limitations in reflecting tissue-specific characteristics. To complement and replace them, the decellularization of tissues and organs has been studied as a promising method for producing functional supports or scaffolds for cell culture and transplantation, and the need for it is increasing. However, currently, organoids produced by organoid culture technology have a large difference from actual human tissues in terms of differentiation degree and function, and there is a current situation where further technological development for more mature organoid culture is required.

[0005] Thus, various studies for the establishment of organoids have been conducted, and lung organoids have also been developed. As prior art regarding the method for manufacturing lung organoids, Non-Patent Documents 1 and 2, which disclose methods for manufacturing lung organoids from hPSCs (human pluripotent stem cells), can be cited.

[0006] However, in the respiratory organoids produced by conventional manufacturing methods, since the epithelial cells are located inside the organoids, when evaluating tissue damage caused by toxic substances or harmful bacteria that enter by respiration using such organoids, there is a drawback that the process of injecting the harmful substances into the inside of the organoids must be added. To solve this problem, apical-out type respiratory organoids in which ciliated cells are located on the outside have been produced, but since goblet cells do not exist in the organoids, there is a limit that it is difficult to accurately elucidate the infection reaction of cells due to pathogen exposure (Non-Patent Document 3).

[0007] Therefore, the present inventors attempted to regulate the polarity of respiratory-derived stem cell-like basal cells by regulating extracellular matrix components, and when differentiating by three-dimensional culture using laminin instead of Matrigel, ciliated epithelial cells were located on the outside, stem cell-like basal cells were located on the inside, and goblet cells, which are secretory cells, were present inside, that is, it was confirmed that respiratory organoids that better realize the structure of actual respiratory tissue can be produced, and the present invention was completed.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide a method for producing a respiratory organoid in which ciliated epithelial cells are attached to the outside, and a respiratory organoid obtained by the production method.

Means for Solving the Problems

[0010] To achieve the above object, the present invention includes: (a) culturing respiratory cells on a plate coated with an extracellular matrix substance; (b) obtaining the cells cultured in step (a), and removing the growth medium by centrifugation to obtain a cell pellet; and (c) resuspending the cell pellet in a differentiation medium, and then inoculating and culturing a certain number of cells on the plate to differentiate them. The present invention provides a method for producing a respiratory organoid in which ciliated epithelial cells appear on the outside.

[0011] The present invention also provides a respiratory organoid produced by the above method.

[0012] Furthermore, the present invention includes: (a) treating a respiratory organoid produced by the above method with a harmful substance; (b) measuring the survival rates of the respiratory organoid treated with the harmful substance in step (a) and the respiratory organoid not treated with the harmful substance; and (c) comparing the survival rates of the cells measured in step (b) to determine the harmfulness of the harmful substance. The present invention provides a method for evaluating the harmfulness of a harmful substance.

Advantages of the Invention

[0013] The present invention can produce respiratory organoids by proliferating and differentiating normal human bronchial epithelial cells (NHBE cells). The respiratory organoids produced by the method of the present invention are in an apical-out form in which the ciliary structure appears outside the organoid. Inside the organoid, goblet cells, which are secretory cells, are located, and basal stem cells with stem cell properties are present inside. This is a form that is more similar to the original in-vivo organs, and considering the use of organoids to mimic actual organs, it is expected to mimic / realize reactions similar to those occurring in actual organs.

Brief Description of Drawings

[0014]

Figure 1

Figure 2a

Figure 2b

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention will be described in detail.

[0016] The present invention provides a method for producing respiratory organoids, comprising: (a) culturing respiratory cells on a plate coated with an extracellular matrix substance; (b) obtaining the cells cultured in step (a), and removing the growth medium by centrifugation to obtain a cell pellet; and (c) resuspending the cell pellet in a differentiation medium, and then inoculating and culturing a certain number of cells on the plate to differentiate them.

[0017] The organoid refers to an artificial in vitro construct that mimics or is similar to the functionality and / or histological structure of an organ or a part thereof, which is obtained by culturing or recombining cells separated from stem cells or organ cells.

[0018] The respiratory organ of the respiratory organoid is a part of the upper and lower respiratory tracts including the lungs, preferably the lungs.

[0019] The respiratory cells may be any stem cells that differentiate into respiratory epithelial cells having respiratory cilia, and may be normal human bronchial basal epithelial cells (NHBE cells).

[0020] The extracellular matrix substance in step (a) may be laminin, collagen I, or collagen IV. The polarity of the stem cell-like cells is changed by the extracellular matrix substance exposed to the cells. Although many conventional methods use Matrigel, Matrigel is expensive, which not only increases the cost of manufacturing organoids but also takes a long time. Therefore, in the present invention, an attempt was made to eliminate the use of such Matrigel.

[0021] The growth medium used in step (a) may contain A8301, Y-27632, CHIR99021, and SB202190.

[0022] In the present invention, the "medium" refers to a substance capable of maintaining cell growth and survival, contains components suitable for cell culture, and includes all ordinary media used in the art. The medium and culture conditions can be selected according to the type of cultured cells. As such a basic cell culture medium, for example, DMEM (Dulbeco’s Modified Eagle’s Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12 (Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove’s Modified Dulbecco’s Medium, BEGM (bronchial epithelial growth medium), etc. can be used, and antibiotics such as penicillin and streptomycin, supplements, etc. may be further added as necessary. In the present invention, it may also be a bronchial epithelial growth medium (BEGM) containing A8301, Y-27632, CHIR99021 and SB202190.

[0023] A8301 (A83-01) is an inhibitor of the TGFβ / SMAD signaling system and is known to be used as an essential compound for stem cell expansion. In the present invention, A8301 is added at a concentration of 0.5 to 2 uM, preferably 0.75 to 1.5 uM, more preferably 1 to 1.3 uM. If the inhibition of the TGFβ / SMAD signaling system is excessive, hyperplasia of stem cells may occur.

[0024] Y-27632 is an inhibitor of the ROCK signaling system and is known to play a role in maintaining the stem cell ability of primary cells. In the present invention, Y-27632 is added at a concentration of 2 to 10 μM, preferably at a concentration of 3 to 7 μM, more preferably at a concentration of 4 to 6 μM. Since the ROCK signaling system plays a role in regulating the differentiation of stem cells, if the inhibition is excessive or insufficient, the differentiation of stem cells may not occur normally.

[0025] CHIR99021 is a compound that activates the Wnt signaling system and inhibits the GSK3β signaling system. In the present invention, CHIR99021 is added at a concentration of 0.5 to 3 μM, preferably at a concentration of 0.5 to 2 μM, more preferably at a concentration of 0.5 to 1 μM. Since the Wnt signaling system plays a role in regulating cell differentiation, if the concentration of CHIR99021 is low or it is absent, basal stem cells with stem cell properties may not be maintained.

[0026] SB202190 is an inhibitor compound of the MAPK signaling system and is known to be used when proliferating stem cells. In the present invention, SB202190 is added at a concentration of 0.1 to 2 μM, preferably at a concentration of 0.3 to 1.5 μM, more preferably at a concentration of 0.3 to 1 μM. If the MAPK signaling system is excessively inhibited, mucus production may be inhibited and the differentiation into secretory cells (goblet cells) may not occur normally.

[0027] The differentiation medium used in step (c) may be any medium known as a differentiation medium for respiratory epithelial cell proliferation, but is preferably Air-Liquid Interface Medium (Promocell, #C-21080) or PneumaCult-ALI medium (Stemcell Technologies, #05001).

[0028] The differentiation medium may contain hydrocortisone and heparin in PneumaCult-ALI medium.

[0029] The hydrocortisone is an analog of the steroid hormone cortisol and plays a role in promoting differentiation. In the present invention, hydrocortisone is contained in the medium at a concentration of 0.5 to 1.0 μg / ml, preferably 0.75 to 1.0 μg / ml. Hydrocortisone plays a role in promoting the growth and differentiation of respiratory cells, but when an excessive amount is used, gene expression and the physiological characteristics of cells may change, and differentiation may not occur normally.

[0030] The heparin is a polysaccharide having an anticoagulant effect and is currently widely used as an anticoagulant, but it plays a role in supporting the stability of growth factors such as FGF (fibroblast growth factor) and EGF (epidermal growth factor). In the present invention, it is used to induce differentiation. In the present invention, heparin is used at a concentration of 0.05 to 0.5%, preferably 0.1 to 0.3%. Excessive treatment with heparin may rather inhibit cell growth and cause cell senescence.

[0031] The number of cells inoculated in step (c) may be 400 to 700, may be 450 to 650, or may be 450 to 550.

[0032] Step (c) may be carried out for 15 to 30 days, may be carried out for 17 to 30 days, or may be carried out for 20 to 30 days.

[0033] The respiratory organoid may contain ciliated epithelial cells in the outer side (outer layer) of the organoid, goblet cells, which are secretory cells, in the lower part (intermediate layer) thereof, and stem cell-like basal cells in the inner side (inner layer) of the organoid.

[0034] The ciliated epithelial cells may exhibit motility.

[0035] The present invention also provides a respiratory organoid produced by the method for producing a respiratory organoid.

[0036] Furthermore, the present invention provides a method for evaluating the toxicity of a harmful substance, comprising: (a) treating the respiratory organoid according to the present invention with a harmful substance; (b) measuring the survival rate of cells in the treated group of respiratory organoids treated with the harmful substance in step (a) and the survival rate of cells in the control group of respiratory organoids not treated with the harmful substance; and (c) comparing the survival rates measured in step (b) to determine the toxicity of the harmful substance.

[0037] The harmful substance may be a toxic substance such as particulate matter, dust, smoke, air pollution, chemical substances, etc., or a pathogen such as a respiratory virus or infectious bacteria.

[0038] The chemical substance means a substance that stimulates and damages the respiratory tract when exposed, such as chlorine, sulfur dioxide, hydrogen sulfide, cyanide, methane, carbon monoxide, nitrogen dioxide, and / or ammonia, but is not limited thereto. When an organism is exposed to a chemical substance, it generally causes a tingling sensation in the respiratory tract, hemoptysis and cough, nausea and shortness of breath. In the long term, the function of the respiratory tract is inhibited, causing chronic cough and shortness of breath.

[0039] The pathogen is a microorganism or substance that causes disease, and includes, but is not limited to, viruses, viroids, mycoplasmas, bacteria, fungi, algae, parasitic plants, nematodes, and / or mites.

[0040] In conventional lung or respiratory organoids, since the epithelial cells were produced in a form where they were located inside the organoid, when evaluating tissue damage caused by harmful substances, the harmful substances had to be injected inside the organoid. Therefore, experimental variables such as damage to the organoid during the injection process may occur, which may reduce the accuracy. In the organoid of the present invention, since the epithelial cells are exposed to the outside, it is possible to evaluate the harmfulness of harmful substances only by exposing the organoid to the harmful substances.

[0041] If the survival rate of the cells constituting the treated group organoids exposed to harmful substances is very low, and there is a statistically significant level difference between the survival rate of the cells constituting the control group organoids not exposed to harmful substances and the survival rate of the cells constituting the treated group organoids exposed to harmful substances, the harmful substance is determined to be harmful. Also, the greater the difference in the survival rate, the higher the determined harmfulness, and the difference may be 1.2 times or more, 1.5 times or more, 1.7 times or more, 2 times or more, or 2.3 times or more.

[0042] In a specific embodiment of the present invention, organoids produced by growing bronchial epithelial cells and inoculating 500 or 1000 cells per well and differentiating them were observed. The produced apical-out respiratory organoids showed that the size of the organoids became smaller as the differentiation period became longer, but it was confirmed that the size was maintained at around 100 μm from the 7th day, and it was confirmed that the morphology of the organoids produced by inoculating 500 cells maintained a good spherical shape (Figure 1).

[0043] Also, it was confirmed that ciliated cells were formed and maintained from the 14th day of differentiation (Figure 1). As a result of observing the respiratory organoids on the 21st day of differentiation, it was found that a ciliary structure was formed outside the organoids, the cilia had motility (beating), and the ciliated epithelial cells were functioning normally (Figure 2a and Figure 2b).

[0044] When the organoid becomes excessively large, it becomes difficult for the internal cells to communicate with the outside, and necrosis of the internal cells may occur. Therefore, a live / dead staining assay was performed to confirm this. As a result, in the organoids of the present invention on the 21st day, it was observed that some cells died inside the organoids, but no necrotic core was observed (Figure 3).

[0045] To confirm the cell types constituting the organoids of the present invention, an immunofluorescence staining method was performed. As a result, it was confirmed that ciliated epithelial cells are located in the outer layer of the organoids, and goblet cells are present below them (Figure 4). Since it was confirmed that stem cell-like basal cells are located inside the organoids (Figure 5), it can be seen that the organoids of the present invention are similar to the structure of actual respiratory tissues.

[0046] To confirm the importance of the additive composition for the production of the organoids of the present invention, organoids were produced by changing the composition of some additives, and an immunofluorescence staining method was performed to confirm the cell composition. As a result, in the organoids with the changed additive composition, it was found that the ciliated structure is located in the outer layer of the organoids, and the goblet cells are located inside it, but it does not contain stem cell-like basal cells (Figures 6 and 7). That is, it can be seen that changing the composition of the composition changes the cell composition of the organoids.

[0047] Therefore, it can be seen that using the production method of the present invention, a respiratory organoid having a structure similar to that of actual respiratory tissue can be produced. The respiratory organoid is predicted to be able to well mimic an actual organ, and it is considered that a respiratory injury model and the like can be confirmed more accurately.

[0048] Hereinafter, the present invention will be described in more detail with specific examples.

[0049] However, these examples are only illustrative of the present invention, and the present invention is not limited to these examples.

Example

[0050] Production of Respiratory Organoids The cell culture plate was coated with laminin (Laminin-511, iMatrix-511) at a concentration of 0.25 μg / cm2, then normal human bronchial epithelial cells (NHBE cells) were dispensed, and then cultured in an expansion medium for 3 days to allow expansion. The composition of the expansion medium is shown in Table 1.

[0051] When the NHBE cells reached a cell confluency of approximately 80%, they were dissociated using trypsin (Trypsin-EDTA), then centrifuged to remove the supernatant to obtain a cell pellet, and then the differentiation medium was added to the cell pellet and resuspended. The composition of the differentiation medium is shown in Table 1.

[0052] The number of resuspended cells was measured and adjusted to 5×10 3 cells / ml, and 100 μl per well was dispensed into a 96-well ultra-low attachment plate. The plate containing the cells was centrifuged at 1000 rpm for 2 minutes to collect the cells at the center of the well. It was confirmed under a microscope that the cells had gathered at the center, and then they were differentiated for 21 days at 37°C and 5% CO2 while changing the differentiation medium once every 2 days. The method for changing the differentiation medium was to remove 50 μl of the existing differentiation medium per well with a pipette and add 50 μl of the new differentiation medium.

[0053] [Table 1]

Example

[0054] Observation of the Morphology of Respiratory Organoids To confirm the characteristics of the respiratory organoids produced by the method of Example 1, they were observed under a microscope.

[0055] First, to confirm the cell density suitable for the formation and maintenance of organoids, 500 or 1000 cells were inoculated per well and cultured. As a result, the cell debris in both of the two groups (500 cells / well or 1000 cells / well) adhered to the bottom of the well, and it was observed that the organoids were in a floating form in the medium. When observing the morphology of the organoids in the two groups, it was confirmed that the group inoculated with 500 cells maintained a better spherical morphology compared to the group inoculated with 1000 cells. Therefore, in subsequent experiments, 500 cells were inoculated per well (Figure 1).

[0056] Also, it was confirmed that as time passed compared to the first day of differentiation, the size of the organoids became smaller, and on the seventh day of differentiation, the size was around 100 μm, and then the size was maintained (Figure 1).

[0057] In organoids formed by conventional methods, especially those produced using Matrigel, the size was not uniform, the size became excessively large, and it was sometimes difficult to supply nutrients to the cells inside the organoids. However, it can be seen that the organoids of the present invention are produced while maintaining a certain size.

[0058] At the same time, it was confirmed that cilia appeared in the outer edge part of both groups of organoids. Cilia were formed on the 14th day of differentiation, and it was observed that the morphology was maintained thereafter (Figure 2). Also, when observed under a microscope, the cilia structure had motility.

[0059] That is, it was observed that the organoids of the present invention have a certain spherical shape and a certain size, and not only are ciliated epithelial cells located outside the organoids, but also the cilia structure has a normal function.

Example

[0060] Analysis of the survival rate of cells constituting the respiratory organoid If the inside of the organoid cannot communicate with the external environment, nutrients are not supplied and the internal cells die, so an organoid of this form cannot mimic the function of a normal organ. To confirm whether cell death occurs in the cells constituting the respiratory organoid produced in Example 1, a live / dead staining assay was performed.

[0061] Specifically, the organoid was transferred to a 1.5 ml tube and left for 1 hour so that the organoid would naturally sink to the bottom. The settled organoid was washed with DPBS (Dulbecco’s Phosphate-Buffered Saline), and then Calcein-AM and Ethidium-homodimer-1 were added. It was left at room temperature for 1 hour for staining and then observed using a fluorescence microscope. With such staining, living cells are stained yellow-green (Calcein-AM), and dead cells are stained red (Ethidium-homodimer-1).

[0062] As a result, on the 1st and 14th days of differentiation, it was observed that most cells were in a living state, and no dead cells were observed. In the organoids on the 21st day of differentiation, it was observed that some cells in the central part had died, but most cells were observed to be in a living state (Figure 3).

[0063] In addition, if the size of the organoid is not uniform or it is formed large, there may be a problem of having a necrotic core in which the cells in the central part continue to die and only the external (outer layer) cells survive, but no necrotic core was observed in the respiratory organoids produced by the method of Example 1 (Figure 3).

[0064] Therefore, it is predicted that the organoids of the present invention have stability such that cells do not die during the differentiation process, maintain an appropriate size without the occurrence of a necrotic core, and the interaction between the internal cells of the organoids and the external environment is appropriately carried out.

Example

[0065] Confirmation of whether the respiratory organoid is apical-out In order to confirm whether a respiratory organoid in which lung epithelial cells are located outside, similar to the structure of an actual respiratory organ, was produced, immunofluorescence staining was performed.

[0066] Specifically, the organoids were fixed with 4% paraformaldehyde at room temperature for 10 minutes, then washed with DPBS, and treated with 0.5% Triton X-100 for 10 minutes to increase permeability. They were blocked by treatment with a 3% BSA solution for 30 minutes to inhibit non-specific binding, the primary antibody was diluted with 3% BSA and left standing at 4°C overnight, and then washed three times. The secondary antibody was diluted with DPBS and left standing at room temperature for 1 hour, and then washed three times. As the primary antibody, an antibody against Acetylated-tubulin (Sigma, #T7451), which is a marker for ciliated epithelial cells, Muc5ac (Abcam, #198294), which is a marker for goblet cells, or KRT5 (Thermofisher, #MA5-17057), which is a marker for stem cell-like basal cells, was used. As the secondary antibody, Alexafluor488 (Thermofisher, #A11001), Alexafluor 594 (Thermofisher, #A11012) was used. For the cell nucleus, DAPI (4’,6-Diamidino-2-phenylindole dihydrochloride) was diluted to 5 μg / ml in triple-distilled water, and then the organoids after the secondary antibody staining was completed were treated for 3 minutes, and then washed for staining.

[0067] As a result, on the outer surface of the respiratory organoids produced in Example 1, ciliated epithelial cells (yellow-green) were observed, and inside the ciliated epithelial cells, goblet cells (red), which are secretory cells, were observed (Figure 4).

[0068] In addition, inside the organoids, stem cell-like basal cells (yellow-green) were observed (Figure 5).

[0069] In the structure of the actual respiratory organ, ciliated epithelial cells are located at the apical site exposed to air, mucus-secreting cells such as goblet cells are located below them, and stem cell-like basal cells exist as reserve cells for epithelial cell replacement at the innermost side. In view of this, it was confirmed that the organoid structure of the present invention has a structure similar to that of actual respiratory tissue.

Example

[0070] Composition test of the medium To confirm the organoid characteristics by the medium additive of the present invention, the method of Example 1 was carried out in the same manner using the medium compositions in Table 2 in which CHIR99021 was excluded and the concentration of Y-27632 was changed to produce respiratory organoids. To observe the constituent cells of the organoids, immunofluorescence staining was performed in the same manner as in Example 4.

[0071]

Table 2

[0072] As a result, it was observed that ciliated epithelial cells were distributed on the outside of the organoids, and secretory cells (goblet cells) were distributed on the inside in the same manner (Figure 6), but stem cell-like basal cells (basal stem cell) were not maintained inside the organoids (Figure 7).

[0073] Therefore, it was confirmed that the composition of the growth medium shown in Table 1 is suitable for the production of apical-out respiratory organoids in which stem cell-like basal cells are located inside, ciliated epithelial cells are located outside, and secretory cells are located under the ciliated epithelial cells.

[0074] According to this example, by growing and differentiating in an environment coated with laminin instead of Matrigel using the composition of the medium of the present invention, apical-out respiratory organoids can be produced, and the respiratory organoids can produce organoids that mimic the actual respiratory tissue in which the ciliary structure is outside the organoid, secretory cells are located inside, and stem cell-like basal cells are present inside.

Claims

**Claim 1** A method for producing a respiratory organoid in which ciliated epithelial cells are present on the outside, comprising: (a) culturing respiratory cells using a growth medium in a plate coated with an extracellular matrix substance; (b) obtaining the cells cultured in step (a), removing the growth medium by centrifugation to obtain a cell pellet; (c) resuspending the cell pellet in a differentiation medium, and then inoculating and culturing a certain number of cells on a plate to differentiate them. A method for producing a respiratory organoid. **Claim 2** The method for producing a respiratory organoid according to claim 1, wherein the respiratory organoid is a lung organoid. **Claim 3** The method for producing a respiratory organoid according to claim 1, wherein the respiratory cells are normal human bronchial basal epithelial cells (NHBE cells). **Claim 4** The method for producing a respiratory organoid according to claim 1, wherein the extracellular matrix substance in step (a) is laminin, collagen I or collagen IV. **Claim 5** The method for producing a respiratory organoid according to claim 4, wherein the extracellular matrix substance in step (a) is laminin. **Claim 6** The method for producing a respiratory organoid according to claim 1, wherein the growth medium used in step (a) contains A8301, Y-27632, CHIR99021 and SB202190. **Claim 7** The method for producing a respiratory organoid according to claim 6, wherein the growth medium contains bronchial epithelial growth medium (BEGM), 0.5 - 2 μM of A8301, 2 - 10 μM of Y-27632, 0.5 - 3 μM of CHIR99021, and 0.1 - 2 μM of SB202190. **Claim 8** The method for producing a respiratory organoid according to claim 1, wherein the number of cells inoculated in step (c) is 400 - 700. **Claim 9** The method for producing a respiratory organoid according to claim 1, wherein step (c) is carried out for 15 - 30 days. **Claim 10** The method for manufacturing a respiratory organoid according to claim 1, wherein the respiratory organoid contains stem cell-derived basal cells inside the organoid.

11. A respiratory organoid produced by the method according to claim 1.

12. The respiratory organoid according to claim 11, wherein ciliated cells are located outside the organoid, goblet cells, which are secretory cells, are located inside the ciliated cells, and stem cell-derived basal cells are located inside the organoid.

13. (a) Treating the respiratory organoid produced by the method according to claim 1 with a harmful substance; (b) Measuring the survival rate of cells in the treated group of respiratory organoids treated with the harmful substance in step (a) and cells in the control group of respiratory organoids not treated with the harmful substance; (c) Comparing the survival rate of the cells measured in step (b) to determine the harmfulness of the harmful substance. A method for evaluating the harmfulness of a harmful substance.

Images