Respiratory organoids with ciliated epithelial cells attached to the outside and method for producing 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 having 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 body that reproduces all the morphological and functional aspects of a tissue or organ created by three-dimensional culturing, aggregating, or recombining 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 because it has a morphology and structural organization similar to that of an actual tissue or organ, 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 a very useful technology 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, which plays a crucial role in supporting cells to attach and grow, is not only vital from a biomedical engineering perspective but also plays a vital role in the growth of cells seeded within a porous structure or cells that have migrated from the surrounding tissue. Most cells in the human body are adherent cells that attach and grow; if there is no place to attach, the cells cannot grow and will die. Therefore, the support must provide a suitable environment for cell attachment, differentiation, growth, and migration. Such supports can be made from various materials, but research is actively being conducted on developing supports using natural materials, synthetic polymers, bioceramics, and polymer-ceramic composite materials. As a result, organoids that mimic various organs have been developed to date, and these are mainly cultured in Matrigel, a three-dimensional culture environment.

[0004] However, although Matrigel is widely used in organoid culture, it is derived from mouse sarcoma, and there are no viable alternatives. This presents a limitation, as we are forced to rely on Matrigel despite its high cost. Furthermore, it is dominated by specific components, limiting its ability to reflect tissue-specific characteristics. To complement and replace these limitations, decellularization of tissues and organs is being studied as a promising method for creating functional supports or scaffolds for cell culture and transplantation, and its need is increasing. However, currently, organoids produced using organoid culture technology differ significantly from actual human tissue in terms of differentiation and function, and there is a need for further technological development for culturing more mature organoids.

[0005] As described above, various studies are being conducted to establish organoids, and lung organoids have also been developed. Conventional techniques for manufacturing lung organoids include Non-Patent Documents 1 and 2, which disclose a method for manufacturing lung organoids from hPSCs (human pluripotent stem cells).

[0006] However, conventional respiratory organoids manufactured using conventional methods have the drawback that, because the epithelial cells are located inside the organoid, when evaluating tissue damage caused by toxic substances or harmful bacteria entering through respiration using such organoids, an additional step of injecting the harmful substances into the organoid is required. To solve this, respiratory organoids in an apical-out form, where ciliated cells are located on the outside, have been manufactured. However, these organoids lack goblet cells, which limits their ability to accurately elucidate the cellular infection response to pathogen exposure (Non-Patent Literature 3).

[0007] Therefore, the inventors attempted to adjust the polarity of respiratory-derived stem cell basal cells by adjusting the extracellular matrix components. By differentiating these cells in three-dimensional culture using laminin instead of Matrigel, they confirmed that they could produce apical-out organoids, i.e., respiratory organoids that better realize the structure of actual respiratory tissue, with ciliated epithelial cells located on the outside, stem cell basal cells located on the inside, and goblet cells (secretory cells) located inside. This led to the completion of the present invention. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Development, 144: 986-997, 2017 [Non-Patent Document 2] Nature cell biology, 19(5): 542-549, 2017 [Non-Patent Document 3] Scientific Reports, 12:7673, 2022 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a method for producing respiratory organoids in which ciliated epithelial cells are attached to the outside, and respiratory organoids obtained by the production method. [Means for solving the problem]

[0010] To achieve the above objective, the present invention provides a method for producing respiratory organoids in which ciliated epithelial cells are exposed externally, comprising the steps of (a) culturing respiratory cells on a plate coated with an extracellular matrix material, (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 a certain number of cells onto a plate and culturing them to induce differentiation.

[0011] Furthermore, the present invention provides a respiratory organoid manufactured by the method described above.

[0012] Furthermore, the present invention provides a method for evaluating the toxicity of a hazardous substance, comprising the steps of (a) treating respiratory organoids produced by the above method with a hazardous substance, (b) measuring the survival rate of respiratory organoids treated with the hazardous substance in step (a) and respiratory organoids not treated with the hazardous substance, and (c) determining the toxicity of the hazardous substance by comparing the survival rates of cells measured in step (b). [Effects of the Invention]

[0013] This invention allows for the production of respiratory organoids by proliferating and differentiating normal human bronchial epithelial cells (NHBE cells). The respiratory organoids produced by this method have an apical-out form in which the ciliary structure is visible on the outside of the organoid, with goblet cells (secretory cells) located inside and basal stem cells (stem cells) present internally. This form is even more similar to that of actual organs in the body, and considering the intended use of organoids to mimic actual organs, it is expected that they can replicate / realize reactions that are close to those that occur in actual organs. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the results of observing organoids at each differentiation stage after dispensing 500 or 1000 cells cultured in growth medium into 96-well plates. [Figure 2a] This figure shows the results of observing respiratory organoids on day 21 of differentiation. [Figure 2b] This is an enlarged view of Figure 2a. [Figure 3] This figure shows the results of a Live / dead staining assay performed using respiratory organoids in the differentiation process. [Figure 4] This figure shows respiratory organoids prepared using the culture medium composition shown in Table 1, followed by confirmation of the organoid's cellular composition by immunofluorescence staining. Ciliated epithelial cells, secretory cells (goblet cells), and cell nuclei are visible. [Figure 5] This figure shows the results of preparing respiratory organoids using the culture medium composition shown in Table 1, and then confirming the distribution of stem cell-like basal cells within the organoids using immunofluorescence staining. [Figure 6]This is a diagram showing the production of respiratory organoids using the composition of the medium shown in Table 2, and then confirming the cell composition of the organoids by immunofluorescence staining. Ciliated epithelial cells, secretory cells (goblet cells), and cell nuclei are confirmed. [Figure 7] This is a diagram showing the results of producing respiratory organoids using the composition of the medium shown in Table 2, and then confirming the distribution of stem cell-like basal cells in the organoids by immunofluorescence staining.

Embodiments 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, which 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.

[0017] The organoid refers to an artificial in vitro construct that cultures or recombines cells separated from stem cells or organ cells, and mimics or resembles the functionality and / or histological structure of an organ or a part thereof.

[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 aforementioned respiratory cells may be any stem cells that differentiate into respiratory epithelial cells having respiratory cilia, and may also 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 stem cell cells changes depending on the extracellular matrix substance to which the cells are exposed. Conventional methods often use Matrigel, but Matrigel is expensive, which not only increases the cost of organoid production but also increases the time required. Therefore, in this invention, we have attempted 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 this invention, "culture medium" refers to a substance that enables the growth and survival of cells, and contains components suitable for cell culture, encompassing all conventional culture media used in this field. The culture medium and culture conditions can be selected according to the type of cultured cell. Examples of such basic cell culture media include 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, and BEGM (bronchial epithelial growth medium). Antibiotics such as penicillin and streptomycin, supplements, etc., may be added as needed. In this invention, bronchial epithelial growth medium (BEGM) may contain A8301, Y-27632, CHIR99021, and SB202190.

[0023] A8301 (A83-01) is an inhibitor of the TGFβ / SMAD signaling pathway and is known to be used as an essential compound for stem cell expansion. In this invention, A8301 is added at a concentration of 0.5 to 2 μM, preferably 0.75 to 1.5 μM, and more preferably 1 to 1.3 μM. Excessive inhibition of the TGFβ / SMAD signaling pathway can lead to stem cell hyperplasia.

[0024] Y-27632 is an inhibitor of the ROCK signaling pathway and is known to play a role in maintaining the stem cell function of primary cells. In this invention, Y-27632 is added at a concentration of 2 to 10 μM, preferably 3 to 7 μM, and more preferably 4 to 6 μM. Since the ROCK signaling pathway plays a role in regulating stem cell differentiation, excessive or insufficient inhibition may prevent normal stem cell differentiation.

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

[0026] SB202190 is a MAPK signaling pathway inhibitor compound and is known to be used in the proliferation of stem cells. In this invention, SB202190 is added at a concentration of 0.1 to 2 μM, preferably 0.3 to 1.5 μM, and more preferably 0.3 to 1 μM. Excessive inhibition of the MAPK signaling pathway can suppress mucus production and prevent normal differentiation into secretory cells (goblet cells).

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

[0028] The differentiation medium may be PneumaCult-ALI medium containing hydrocortisone and heparin.

[0029] The aforementioned hydrocortisone is an analog of the steroid hormone cortisol and plays a role in promoting differentiation. In this invention, hydrocortisone is included in the culture medium at a concentration of 0.5 to 1.0 ug / ml, preferably 0.75 to 1.0 ug / ml. While hydrocortisone plays a role in promoting the growth and differentiation of respiratory cells, using an excessive amount may alter gene expression and the physiological characteristics of cells, preventing normal differentiation.

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

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

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

[0033] The respiratory organoid may contain ciliated epithelial cells in the outer layer (external layer), goblet cells (secretory cells) below them (intermediate layer), and basal stem cells (stem cells) in the inner layer (internal layer).

[0034] The ciliated epithelial cells may also be motile.

[0035] Furthermore, the present invention provides a respiratory organoid manufactured by the respiratory organoid manufacturing method described above.

[0036] Furthermore, the present invention provides a method for evaluating the toxicity of a hazardous substance, comprising the steps of (a) treating respiratory organoids according to the present invention with a hazardous substance, (b) measuring the viability of cells in the treated group respiratory organoids treated with the hazardous substance in step (a) and the viability of cells in the control group respiratory organoids not treated with the hazardous substance, and (c) determining the toxicity of the hazardous substance by comparing the viability measured in step (b).

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

[0038] The aforementioned chemical substances refer to, but are not limited to, substances that irritate and damage the respiratory system upon exposure, such as chlorine, sulfur dioxide, hydrogen sulfide, cyanide, methane, carbon monoxide, nitrogen dioxide, and / or ammonia. When organisms are exposed to these chemicals, they typically experience a tingling sensation in the respiratory system, coughing and hemoptysis, nausea, and shortness of breath. In the long term, respiratory function is impaired, leading to chronic cough and shortness of breath.

[0039] The aforementioned pathogens are microorganisms or substances that cause disease, and include, but are not limited to, viruses, viroids, mycoplasmas, bacteria, fungi, algae, parasitic plants, nematodes and / or mites.

[0040] Conventional lung or respiratory organoids were manufactured in a form where epithelial cells were located inside the organoid. Therefore, when evaluating tissue damage caused by harmful substances, the harmful substances had to be injected inside the organoid. This could introduce experimental variables, such as organoid damage during the injection process, which could reduce accuracy. In the organoid of the present invention, the epithelial cells are exposed to the outside, so the toxicity of harmful substances can be evaluated simply by exposing the organoid to the harmful substance.

[0041] If the survival rate of cells constituting the organoids in the treatment group exposed to the hazardous substance is very low, and there is a statistically significant difference between the survival rate of cells constituting the control group organoids (not exposed to the hazardous substance) and the survival rate of cells constituting the treatment group organoids exposed to the hazardous substance, the hazardous substance is judged to be harmful. Furthermore, the greater the difference in survival rates, the higher the degree of harmfulness is judged, and this 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 were produced by proliferating bronchial epithelial cells, inoculating 500 or 1000 cells in each well, and differentiating them. The size of the apical-out respiratory organoids decreased as the differentiation period lengthened, but it was confirmed that the size remained constant at around 100 μm from day 7 onwards, and the organoids produced by inoculating 500 cells maintained a good spherical shape (Figure 1).

[0043] Furthermore, it was confirmed that ciliated cells are formed and maintained from day 14 of differentiation (Figure 1), and observation of respiratory organoids on day 21 of differentiation revealed that ciliated structures were formed on the outside of the organoids, the cilia were motile (beating), and the ciliated epithelial cells were functioning normally (Figures 2a and 2b).

[0044] When organoids become excessively large, it becomes difficult for internal cells to interact with the outside, which can lead to necrosis of internal cells. To confirm this, a live / dead staining assay was performed. As a result, in the respiratory organoids of the present invention, some cells were observed to have died inside the organoid on day 21, but no necrotic core was observed (Figure 3).

[0045] Immunofluorescence staining was performed to confirm the types of cells constituting the organoid of the present invention. It was confirmed that ciliated epithelial cells were located in the outer layer of the organoid, with goblet cells present beneath them (Figure 4), and that stem cell-like basal cells were located inside the organoid (Figure 5). Therefore, it can be seen that the organoid of the present invention is similar in structure to actual respiratory tissue.

[0046] To confirm the importance of the additive composition for organoid production according to the present invention, organoids were produced by changing the composition of some additives, and immunofluorescence staining was performed to confirm the cellular composition. As a result, it was found that in organoids with altered additive compositions, the ciliated structure was located in the outer layer of the organoid, and goblet cells were located further inside, similar to the original composition, but stem cell-like basal cells were not included (Figures 6 and 7). In other words, it can be seen that changing the composition alters the cellular composition of the organoid.

[0047] Therefore, it has been found that the manufacturing method of the present invention can be used to produce respiratory organoids having a structure similar to actual respiratory tissue. It is predicted that these respiratory organoids will be able to accurately mimic actual organs, and that they will allow for more accurate verification of respiratory injury models and the like.

[0048] The present invention will be described in more detail below with reference to specific examples.

[0049] However, these embodiments are merely illustrative of the present invention, and the present invention is not limited to these embodiments. [Examples]

[0050] Manufacturing of respiratory organoids Cell culture plates were 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 into the plates, and the cells were cultured in expansion medium for 3 days to promote growth. The composition of the expansion medium is shown in Table 1.

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

[0052] The number of cells that were resuspended was measured and 5 × 10 3 The cell / ml concentration was adjusted, and 100 µl was dispensed into each well of a 96-well ultra-low attachment plate. The plates containing the cells were centrifuged at 1000 rpm for 2 minutes to collect the cells in the center of each well. After confirming that the cells had collected in the center using a microscope, the cells were differentiated for 21 days at 37°C and 5% CO2, with the differentiation medium being changed every two days. The differentiation medium was changed by pipetting out 50 µl of the existing differentiation medium per well and adding 50 µl of new differentiation medium in each well.

[0053] [Table 1] [Examples]

[0054] Morphological observation of respiratory organoids The respiratory organoids produced by the method of Example 1 were observed under a microscope to confirm their properties.

[0055] First, to determine the optimal cell density for organoid formation and maintenance, we inoculated 500 or 1000 cells per well and cultured them. In both groups (500 cells / well and 1000 cells / well), cell debris adhered to the bottom of the well, while the organoids were observed to be floating in the culture medium. Observation of the organoid morphology of the two groups revealed that the group inoculated with 500 cells maintained a better spherical shape compared to the group inoculated with 1000 cells. Therefore, in subsequent experiments, we inoculated 500 cells per well (Figure 1).

[0056] Furthermore, compared to day 1 of differentiation, the size of the organoid decreased over time, reaching approximately 100 μm by day 7 of differentiation, and was maintained thereafter (Figure 1).

[0057] Conventional organoids, particularly those manufactured using Matrigel, are not uniform in size and can become excessively large, making it difficult to supply nutrients to the cells inside the organoid. However, the organoids of the present invention are manufactured while maintaining a consistent size.

[0058] In addition, cilia were observed to appear on the outer edge of the organoids in both groups, with cilia forming on day 14 of differentiation and the morphology being maintained thereafter (Figure 2). Furthermore, microscopic observation revealed that the ciliary structure exhibited motility (beating).

[0059] In other words, it was observed that the organoids of the present invention have a fixed spherical shape and size, and that not only are ciliated epithelial cells located outside the organoid, but the ciliated structure also functions normally. [Examples]

[0060] Survival rate analysis of respiratory organoid constituent cells If the inside of an organoid cannot interact with the external environment, nutrients will not be supplied, and the internal cells will die. Therefore, organoids of this form cannot mimic the function of a normal organ. To confirm whether or not cell death occurs in the respiratory organoids manufactured in Example 1, a live / dead staining assay was performed.

[0061] Specifically, organoids were transferred to 1.5 ml tubes and left for 1 hour to allow them to sink naturally to the bottom. The settled organoids were washed with DPBS (Dulbecco's Phosphate-Buffered Saline), and then calcein-AM and ethidium-homodimer-1 were added. After being left at room temperature for 1 hour, the organoids were stained and then observed using a fluorescence microscope. With this staining method, living cells were stained yellow-green (calcein-AM), and dead cells were stained red (ethidium-homodimer-1).

[0062] As a result, on days 1 and 14 of differentiation, most cells were observed to be alive, and no dead cells were observed. In the organoid on day 21 of differentiation, some cells in the central part were observed to have died, but most cells were observed to be alive (Figure 3).

[0063] Furthermore, if the organoids are not uniform in size or are formed to be large, a problem may arise in which they have a necrotic core, a condition in which the cells in the central part die continuously and only the cells in the outer layer survive. However, 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 possess stability that prevents cell death during the differentiation process, maintain an appropriate size that prevents the formation of a necrotic core, and allow for appropriate interaction between the internal cells of the organoid and the external environment. [Examples]

[0065] Confirmation of whether respiratory organoids are apical-out. Immunofluorescence staining was performed to confirm whether lung epithelial cells were produced in respiratory organoids located externally, similar to the structure of the actual respiratory system.

[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 then treated with 3% BSA solution for 30 minutes to block unspecified binding, the primary antibody was diluted with 3% BSA and allowed to stand overnight at 4°C, and then washed three times. The secondary antibody was diluted with DPBS and allowed to stand at room temperature for 1 hour, and then washed three times. For primary antibodies, antibodies against Acetylated-tubulin (Sigma, #T7451), a marker for ciliated epithelial cells; Muc5ac (Abcam, #198294), a marker for goblet cells; or KRT5 (Thermofisher, #MA5-17057), a marker for stem cell basal cells, were used. For secondary antibodies, Alexafluor488 (Thermofisher, #A11001) and Alexafluor 594 (Thermofisher, #A11012) were used. For the cell nuclei, DAPI (4',6-Diamidino-2-phenylindole dihydrochloride) was diluted in tertiary distilled water to 5 ug / ml, and then treated with this solution for 3 minutes after secondary antibody staining was complete. The organoids were then washed and stained again.

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

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

[0069] Considering that in the actual respiratory system, ciliated epithelial cells are located at the apical region exposed to air, mucus-secreting cells such as goblet cells are located below them, and stem cell-like basal cells exist at the innermost layer as reserve cells for epithelial cell replacement, it has been confirmed that the organoid structure of the present invention has a structure similar to that of actual respiratory tissue. [Examples]

[0070] Culture medium composition test To confirm the organoid properties of the culture medium additive of the present invention, respiratory organoids were prepared by similarly performing the method of Example 1 with the culture medium compositions shown in Table 2, excluding CHIR99021 and varying the concentration of Y-27632. Immunofluorescence staining was performed in the same manner as in Example 4 to observe the constituent cells of the organoids.

[0071] [Table 2]

[0072] As a result, it was observed that ciliated epithelial cells were distributed on the outside of the organoid and goblet cells were distributed on the inside (Figure 6), but basal stem cells were not maintained inside the organoid (Figure 7).

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

[0074] According to this embodiment, apical-out respiratory organoids can be produced by growing and differentiating cells in a laminin-coated environment instead of Matrigel using the culture medium composition of the present invention. These respiratory organoids mimic actual respiratory tissue, with ciliary structures on the outside of the organoid, secretory cells located inside, and stem cell-like basal cells present internally.

Claims

1. A method for producing respiratory organoids in which ciliated epithelial cells are present on the outside, (a) A step of culturing respiratory cells in a growth medium on a laminin-coated plate, (b) The step of obtaining cells cultured in step (a), removing the growth medium by centrifugation to obtain a cell pellet, (c) The step of resuspending the cell pellet in a differentiation medium, and then inoculating a certain number of cells onto a plate and culturing them to induce differentiation, The growth medium comprises bronchial epithelial growth medium (BEGM), 0.5 to 2 μM of A8301, 2 to 10 μM of Y-27632, 0.5 to 3 μM of CHIR99021, and 0.1 to 2 μM of SB202190, and A method for producing respiratory organoids, wherein the number of cells inoculated in step (c) is 400 to 700.

2. The method for manufacturing a respiratory organoid according to claim 1, wherein the respiratory organoid is a lung organoid.

3. The method for producing respiratory organoids according to claim 1, wherein the respiratory cells are normal human bronchial basal epithelial cells (NHBE cells).

4. The method for manufacturing a respiratory organoid according to claim 1, wherein step (c) is performed for 15 to 30 days.

5. The method for producing a respiratory organoid according to claim 1, wherein the respiratory organoid contains stem cell-derived basal cells inside the organoid.

6. (a) The step of treating a respiratory organoid produced by the method of claim 1 with a hazardous substance, (b) A step of measuring the viability of cells in respiratory organoids treated with the hazardous substance of step (a) and cells in respiratory organoids not treated with the hazardous substance of the control group, (c) A step of determining the toxicity of a harmful substance by comparing it with the cell viability measured in step (b), Methods for evaluating the toxicity of hazardous substances.

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