Method for producing multi-tissue lung organoid surrounded by fibroblasts, and use thereof

Abstract

The present invention relates to a method for producing a multi-tissue lung organoid structurally surrounded by fibroblasts, and a use thereof, and provides a three-dimensional structure composed of a lung organoid and a fibroblast layer surrounding the outside thereof. The organoid according to the present invention can maintain a high level of structural stability and bio-fidelity by reproducing a cell arrangement and microenvironment similar to those of actual lung tissue. The multi-tissue organoid more precisely reproduces a microenvironment in which epithelial tissues and interstitial tissues are arranged in layers as in a living body, rather than adopting a simple cell mixing method, and thus can be used in various biomedical applications, such as regenerative medicine, research on lung disease conditions, and evaluation of responsiveness to anticancer drugs and anti-fibrotic drugs. In addition, the present invention has excellent expandability to organoid-on-chip technology or bio-artificial organ development, and thus can be expected to be used as a precision medicine and next-generation cell therapy platform.

Description

Method for producing multi-tissue lung organoids surrounded by fibroblasts and their use

[0001] The present invention relates to a method for producing a next-generation multi-tissue lung organoid capable of precisely simulating the structure and function of living tissue, and its use.

[0002] In recent decades, organoid technology has emerged as a powerful biological tool that leverages the self-organizing ability of stem cells to not only recapitulate the structure and function of various organs in vitro, but also replace animal experiments. In particular, single-lineage self-organizing organoids utilizing epithelial stem cells from various organs, such as the lung, liver, small intestine, and brain, are widely utilized in research such as disease modeling, drug screening, and regenerative medicine due to their ability to induce tissue-specific differentiation and form three-dimensional structures.

[0003] However, most organoid technologies commercialized or reported to date are structures that mimic single tissues such as epithelium or muscle, and have limitations in that they do not sufficiently reflect the interactions between epithelial cells and various non-epithelial cells surrounding them (e.g., fibroblasts, vascular endothelial cells, immune cells, etc.) in actual human tissues. In particular, the presence of fibroblasts, a major cell population in stromal tissue, which provides structural support for tissues and regulates growth and differentiation signals, has often been overlooked. Consequently, existing organoids often show a gap with the body in terms of the composition of tissue-specific microenvironments, in vivo arrangement, and drug response reproducibility.

[0004] Recently, some studies have attempted co-culture models of cells from different tissues. Numerous models have been reported that co-culture immune cells with epithelial tissue organoids, and attempts have also been made to complement the microenvironment by co-culturing epithelial tissue organoids with fibroblasts or other supporting cells. However, these co-culture models simply co-culture cells from other tissues with organoids, failing to mimic the in vivo microstructural characteristics of layered epithelial and stromal tissues. In other words, the multi-tissue organoids reported to date simply mix fibroblasts with organoids, rather than forming a structure that surrounds the organoids from the outside, as in a living organism.

[0005] These structural differences pose challenges not only for organoid cultivation but also for applications such as drug response testing and chronic disease modeling. Furthermore, existing single-cell or simple mixed organoid technologies do not provide adequate models when attempting to mimic tissue-specific fibrotic responses or abnormal microenvironmental changes.

[0006] Therefore, there is a need for a next-generation organoid technology that overcomes the limitations of existing technologies and has greater physiological accuracy and reproducibility by arranging fibroblasts, which are responsible for structural and physiological support, as well as epithelial cells, on the periphery of the organoid to reproduce a structure similar to the biological arrangement.

[0007] The purpose of the present invention is to provide a method for producing a multi-tissue lung organoid in a form in which the lung organoid is structurally wrapped from the outside by fibroblasts, and a use thereof.

[0008] In order to achieve the above object, the present invention provides a method for producing a multi-tissue lung organoid surrounded by fibroblasts, comprising the steps of 1) isolating epithelial cells from lung tissue; 2) culturing the isolated lung epithelial cells in a medium in three dimensions to culture a single lung organoid; 3) isolating fibroblasts from lung tissue and culturing them in a medium; 4) mixing a suspension of a single lung organoid cultured in step 2) and a suspension of fibroblasts cultured in step 3); 5) centrifuging the mixed suspension in step 4) to attach fibroblasts to the single lung organoid; and 6) aggregating the attached single lung organoid and fibroblasts at room temperature and culturing them in a medium.

[0009] In addition, the present invention provides a multi-tissue lung organoid surrounded by fibroblasts, prepared according to the above method.

[0010] In addition, the present invention provides a method for producing a lung fibrosis disease model, comprising the steps of culturing a multi-tissue lung organoid surrounded by the fibroblasts; and the step of treating the cultured multi-tissue lung organoid surrounded by the fibroblasts with TGF-β.

[0011] In addition, the present invention provides a lung fibrosis disease model in which TGF-β is treated in a multi-tissue lung organoid surrounded by the fibroblasts.

[0012] In addition, the present invention provides a method for evaluating drug responsiveness, comprising the steps of culturing a multi-tissue lung organoid surrounded by the fibroblasts; and the steps of treating a candidate drug to the multi-tissue lung organoid surrounded by the cultured fibroblasts, and then analyzing a lung tissue-specific response.

[0013] The present invention relates to a method for producing multi-tissue lung organoids structurally encapsulated by fibroblasts, and their use. The produced organoids exhibit cell arrangements and a microenvironment similar to actual lung tissue, demonstrating excellent physiological reproducibility. In particular, fibroblasts support the organoids and provide culture stability, thereby enhancing structural maintenance. Furthermore, these organoids, rather than simply mixing different cells and organoids, precisely reproduce the biological microenvironment through an external support cell structure, demonstrating superior biomimeticity and applicability compared to existing technologies. Therefore, the present invention can be utilized as a sophisticated research platform that can replace cell and animal experiments in disease modeling, such as pulmonary fibrosis, anticancer drug responsiveness testing, regenerative medicine research, and drug efficacy and toxicity assessment. Furthermore, the present invention can be integrated with next-generation biomimetic technologies, such as organoid-on-chip and bioartificial organs, and its highly expandable nature as a multi-organ organoid system makes it highly applicable to various bio-industries.

[0014] Figure 1 illustrates an overview of a system for manufacturing multi-tissue lung organoids surrounded by fibroblasts.

[0015] Figure 2 shows the results of culturing lung organoids surrounded by fibroblasts in a multi-tissue lung organoid culture medium.

[0016] Figure 3 shows the results of immunofluorescence staining, which confirmed that fibroblasts surrounded lung organoids by the expression patterns of epithelial cell markers and fibroblast markers.

[0017] Figure 4 shows the results of confirming the expanded collagen fiber structure of fibroblasts surrounding lung organoids.

[0018] Figure 5 shows the results of sequential photography confirming the structure covered by fibroblasts from the bottom to the upper cross-section of the lung organoid.

[0019] Figure 6 shows the results of observing an increase in the amount (Figure 6A) and thickness (Figure 6B) of fibroblast strands that accelerated fibrosis when TGF-β was treated in a multi-tissue lung organoid surrounded by fibroblasts as an experimental model for pulmonary fibrosis.

[0020] The present inventors have completed the invention of a multi-tissue lung organoid having a structure in which fibroblasts surround the exterior of a single organoid formed from lung epithelial cells in order to more precisely reproduce the physiological structure of lung tissue and develop an advanced cell culture technology that can be utilized for disease modeling and drug response analysis based on this.

[0021]

[0022] The present invention provides a method for producing a multi-tissue lung organoid surrounded by fibroblasts, comprising the steps of: 1) isolating epithelial cells from lung tissue; 2) culturing the isolated lung epithelial cells in a medium in three dimensions to culture a single lung organoid; 3) isolating fibroblasts from lung tissue and culturing them in a medium; 4) mixing a suspension of the single lung organoid cultured in step 2) and a suspension of fibroblasts cultured in step 3); 5) centrifuging the mixed suspension in step 4) to attach fibroblasts to the single lung organoid; and 6) aggregating the attached single lung organoid and fibroblasts at room temperature and culturing them in a medium.

[0023] Preferably, in step 2), the medium may be a basal medium for stem cell culture containing B27, HEPES, glutamax, N-acetylcysteine, Y-27632, A83-01, R-spondin1, noggin, FGF7, FGF10, ITS, nicotinamide, and penicillin-streptomycin, but is not limited thereto.

[0024] Preferably, the method for isolating fibroblasts from lung tissue in step 3) may include, but is not limited to, finely fragmenting the lung tissue and culturing it in a fibroblast culture medium for more than a week. More preferably, the method for fragmenting the lung tissue may include, but is not limited to, mincing it with a razor blade.

[0025] Preferably, in step 3), the medium may be a fibroblast culture medium containing serum, but is not limited thereto. More preferably, the serum may include at least one selected from fetal bovine serum (FBS), bovine calf serum, horse serum, or other serum, but is not limited thereto.

[0026] Preferably, the basal media for stem cell culture and the fibroblast culture media may be any one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM / F12, and Advanced DMEM / F12, but are not limited thereto.

[0027] Preferably, instead of penicillin-streptomycin contained in the medium, an antibiotic-antifungal agent such as streptomycin, gentamicin, nystatin or amphotericin may be included, but is not limited thereto.

[0028] Preferably, the suspension of step 4) or the medium of step 6) may be mixed with the single lung organoid culture medium of step 2) and the fibroblast culture medium of step 3) in a volume ratio of 1:0.1 to 10, but is not limited thereto.

[0029] Preferably, the step 4) may be performed by mixing the cultured single lung organoid and the cultured fibroblasts at a cell ratio of 1:0.1 to 10, but is not limited thereto.

[0030] Preferably, the centrifugation in step 5) may be performed at a value of gravitational acceleration (G) of 10 to 1,000 for 1 to 100 minutes, but is not limited thereto.

[0031] Preferably, the multi-tissue lung organoid surrounded by the fibroblasts may have an average particle diameter of 50 to 500 μm, but is not limited thereto.

[0032] Preferably, the multi-tissue lung organoid surrounded by the fibroblasts can express one or more lung epithelial markers and fibroblast markers selected from the group consisting of, but not limited to, EpCAM, Surfactant protein C (SPC), Aquaporin 5 (AQ5), Vimentin, and Alpha-smooth muscle actin (α-SMA).

[0033]

[0034] In addition, the present invention provides a multi-tissue lung organoid surrounded by fibroblasts, prepared according to the above method.

[0035] Preferably, the multi-tissue lung organoid has a structure in which fibroblasts surround the inner lung organoid in a spherical shape from the outside, so that long-term culture and maintenance of the inner lung organoid and the fibroblasts surrounding it from the outside can be possible.

[0036] As used herein, "organoid" refers to a 3D cell aggregate derived from esophageal epithelial cells, including stem cells obtained from humans and animals, and refers to a model similar to a primary organ produced through an artificial culture process. The origin of the cells constituting the organoid is not limited.

[0037] In this specification, “lung” means an organ composed of alveolar epithelium, pulmonary tissue, vascular structure, and stromal cells.

[0038] In this specification, “multi-tissue lung organoid” means an organoid formed from human and animal lung-derived epithelial cells and fibroblasts coexisting, and in the present invention, means an organoid in which fibroblasts three-dimensionally surround the lung organoid from the outside to provide structural support, and includes a multi-tissue, multicellular culture that can reproduce the arrangement structure and microenvironmental characteristics similar to living lung tissue.

[0039] In this specification, “medium” means a culture medium that contains the components necessary for cells to grow, survive, and differentiate into organoids in vitro, and enables this, and includes all common media suitable for culture and differentiation used in the field of organoids. The type of medium and culture conditions may vary depending on the type of cell or the technical level of the relevant field. The medium used for culturing lung organoids and fibroblasts may generally be a minimal culture medium containing carbon sources, nitrogen sources, and trace elements.

[0040] Within this specification, "passaging" refers to the process of continuing organoid culture by isolating single cells or organoids and then transferring them to a new culture vessel, for the purpose of maintaining healthy, long-term culture. A single cell division or culture vessel replacement is referred to as passage 1.

[0041] Within this specification, “differentiation” refers to the phenomenon in which the structure or function of a cell becomes tissue-specific as the cell divides, proliferates, and grows. In other words, it refers to the process in which cells, tissues, etc. of a living organism acquire a form and function suitable for performing the role required by a specific organ. For example, differentiation can include not only the process in which lung epithelial stem cells transform into mature alveolar cells, but also the process in which hematopoietic stem cells transform into red blood cells, white blood cells, platelets, etc., i.e., all processes in which precursor cells express specific differentiation traits.

[0042]

[0043] In addition, the present invention provides a method for producing a lung fibrosis disease model, comprising the steps of culturing a multi-tissue lung organoid surrounded by the fibroblasts; and the step of treating the cultured multi-tissue lung organoid surrounded by the fibroblasts with TGF-β.

[0044] In addition, the present invention provides a lung fibrosis disease model in which TGF-β is treated in a multi-tissue lung organoid surrounded by the fibroblasts.

[0045] In addition, the present invention provides a method for evaluating drug responsiveness, comprising the steps of culturing a multi-tissue lung organoid surrounded by the fibroblasts; and the steps of treating a candidate drug to the multi-tissue lung organoid surrounded by the cultured fibroblasts, and then analyzing a lung tissue-specific response.

[0046] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0047]

[0048] <Example 1> Preparation of culture medium for multi-tissue lung organoids

[0049] A single lung organoid culture medium comprises the following composition. DMEM / F12 or Advanced DMEM / F12 was mixed with 1X B27, 1X HEPES, 1X glutamax, 1 μM N-acetylcysteine, 1 μM Y-27632, 500 nM A83-01, 100 μg / ml R-spondin1 and 100 μg / ml noggin, 25 μg / ml FGF7, 100 μg / ml FGF10, 1X ITS, 10 mM nicotinamide and 1X penicillin-streptomycin. The concentrations of these components can be adjusted between 0.1 and 10 times the suggested concentrations, and other components can be added.

[0050] The fibroblast culture medium contains the following composition: DMEM or RPMI1640 mixed with FBS at a concentration of 20%. The concentration can be adjusted to between 0.1 and 3 times the suggested concentration, or serum from other animals can be used, and other ingredients can be added.

[0051] The multi-tissue organoid culture medium surrounded by fibroblasts was prepared by mixing the single lung organoid culture medium and the fibroblast culture medium at a ratio of 1:1, and the suggested ratio can be adjusted to about 1:0.1 to 10, and other components can be added.

[0052]

[0053] <Example 2> Production of multi-tissue lung organoids surrounded by fibroblasts

[0054] 1. Isolation of epithelial cells from lung tissue

[0055] Lung specimens from patients or lung tissues from animals were washed thoroughly with phosphate-buffered saline (PBS) and minced into pieces 0.1 to 1 mm in length. The minced tissues were enzymatically dissociated in DMEM (Dulbecco's modified eagle medium; Gibco, #12430112) containing collagenase / hyaluronidase (Stem cell, #ST07912) in a cell incubator at 37°C with 5% carbon dioxide for 0.5 to 2 hours.

[0056] To dissociate epithelial cells from enzymatically dissociated lung tissue into single cells, they were placed in a 0.25% trypsin-EDTA solution (0.25% Trypsin-EDTA; Gibco, #25200-072) and enzymatically dissociated for 5 min in a cell incubator supplied with 5% carbon dioxide at 37°C.

[0057] Enzymatically dissociated lung tissue was thoroughly lysed by pipetting, and 0.25% trypsin / 0.05% EDTA was neutralized by adding phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Lung epithelial cells detached from the lung tissue were then passed through a 40 μm cell strainer (SPL, #93040), centrifuged for 5 minutes, and the supernatant was removed. The centrifuged lung stem cells were suspended in Advanced DMEM / F12 medium, and the cell number was determined.

[0058]

[0059] 2. Production of 3D single lung organoids

[0060] Lung epithelial cells extracted from the above were cultured three-dimensionally for 10 to 20 days to produce lung organoids. Specifically, 1,000 to 10,000 lung epithelial cells were suspended in 50 μl of lung organoid culture medium, mixed with matrigel (matrigel, Coring, #356231) at a 1:1 ratio, and either i) dispensed into a 24-well plate in a dome shape and cultured for 30 to 60 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C, and when the matrigel solidified, lung organoid culture medium was added; or ii) dispensed onto a 24-well cell culture transwell (Corning, #3470) and cultured for 30 to 60 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C, and when the matrigel solidified, lung organoid culture medium was supplied to the lower part of the cell culture transwell. Single lung organoids grew to sizes of 50 to 500 μm in lung organoid culture medium.

[0061]

[0062] 3. Isolation of fibroblasts from lung tissue

[0063] Fibroblasts for attachment to the lung organoids constructed above can be extracted from lung tissue. Specifically, lung tissue is placed in DMEM (Dulbecco's modified eagle medium; Gibco, #12430112) medium containing 20% ​​fetal bovine serum, minced very finely with a razor blade, and cultured in a cell incubator supplied with 5% carbon dioxide at 37°C for 5–15 days. Fibroblast culture medium is refreshed every 2–3 days.

[0064]

[0065] 4. Production of multi-tissue lung organoids surrounded by fibroblasts

[0066] The single lung organoids produced above can be re-cultured after passage and attachment with fibroblasts to produce multi-tissue lung organoids surrounded by fibroblasts. Specifically, dispase (dispase, Stem cell, #07913) was added to DMEM (Dulbecco's modified eagle medium; Gibco, #12430112), and lung organoids were enzymatically dissociated from matrigel in a cell incubator supplied with 5% carbon dioxide at 37°C for 20 to 60 minutes. The dissociated organoids were suspended in 500 μl of the multi-tissue lung organoid culture medium prepared in Example 1 and mixed with fibroblasts suspended in 50 μl of the same medium. At this time, fibroblasts were used at a cell number ratio of 0.1 to 10 times that of lung organoids corresponding to 10,000 to 100,000 cells. The suspension containing the lung organoids and fibroblasts was centrifuged at a gravitational acceleration (G) of 20 to 1,000 for 1 to 100 minutes to allow fibroblasts to attach to the organoids. The attached single lung organoids and fibroblasts were resuspended in 90 to 10 μl of multi-tissue lung organoid culture medium and incubated at room temperature for 2 to 20 minutes to consolidate the contacted organoid and fibroblast-bound structures. Afterwards, the suspension was mixed with 10 to 90 μl of Matrigel (Coring, #356231) (the ratio of the organoid-fibroblast-bound structure suspension to Matrigel was 1:0.1 to 10) i) After dispensing into a 24-well plate in a dome shape, cultured for 30 to 60 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C, and when the matrigel solidified, multi-tissue lung organoid culture medium was added; or ii) After dispensing onto a 24-well cell culture transwell (Corning, #3470), cultured for 30 to 60 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C, and when the matrigel solidified, multi-tissue lung organoid culture medium was supplied to the lower part of the cell culture transwell.

[0067] As a result, as shown in Fig. 2, multi-tissue lung organoids surrounded by fibroblasts grew to a size of 50 to 500 μm.

[0068]

[0069] <Experimental Example 1> Confirmation of structural and histological biomimetic properties of multi-tissue lung organoids.

[0070] The lungs are the primary respiratory organs responsible for oxygen exchange, and the alveoli are the fundamental structural units where gas exchange occurs within the lungs. Alveoli are composed of a thin, single-layer epithelial cell layer, surrounded by various types of fibroblasts that provide structural support and regulate biochemical signals. In particular, fibroblasts surrounding the alveoli secrete extracellular matrix components such as collagen and elastin, playing a key role in the formation and remodeling of alveoli. When lung damage occurs, these fibroblasts become activated and contribute to pathophysiological changes such as pulmonary fibrosis.

[0071] To confirm whether the multi-tissue lung organoids produced based on the above structural characteristics reproduce the cell arrangement and fibroblast distribution structure of actual alveolar tissue, immunofluorescence staining was performed on the multi-tissue lung organoids produced in Example 2. In particular, the level of tissue mimicry was evaluated by confirming the expression of epithelial cell (EpCAM) and fibroblast (Vimentin) markers and whether fibroblasts maintained a structurally enveloping form at the periphery of the organoid.

[0072]

[0073] 1. Immunofluorescence staining

[0074] Multi-tissue lung organoids embedded in Matrigel were fixed whole in 4% paraformaldehyde (Sigma, #47608) in a 24-well plate, briefly washed with phosphate-buffered saline (PBS), and blocked with PBS containing 0.1% Triton X-100 (Triton X-100, Bioworld, TR1020-500-00) and 3% bovine serum albumin (MP biomedicals, #9048-46-8) for 1 hour at room temperature. Subsequently, they were incubated overnight at 4°C with primary antibodies diluted in antibody diluent solution (REAL antibody diluent solution, Agilent, #S2022). The 24-well plate was washed three times with PBS containing 0.1% Triton X-100 / 1% bovine serum albumin, and then incubated with secondary antibodies diluted in PBS containing 1% fetal bovine serum for 1 hour at room temperature. After washing three times with PBS, the plate was incubated with DAPI (Sigma, #10236276001) diluted in PBS for 10 minutes at room temperature. After washing three times with PBS, the Matrigel was mounted on a slide and observed under a fluorescence microscope.

[0075] As a result, according to Fig. 3, the epithelial cell marker EpCAM was observed in the internal lung organoids, and the fibroblast marker Vimentin was observed in the fibroblasts surrounding the lung organoids, showing an overlapping color pattern of the two markers. This indicates that the lung organoids are surrounded by fibroblasts to form multi-tissue lung organoids, mimicking the actual alveolar environment.

[0076]

[0077] 2. Observation of the fibrotic support structure of fibroblasts surrounding lung organoids

[0078] To confirm the formation of a support structure by fibroblasts, which is one of the structural features of multi-tissue lung organoids, confocal laser scanning microscopy was used to observe the spatial arrangement and morphology of fibroblasts at high resolution, and the presence of a support structure (fibrotic scaffold-like structure) formed by fibroblasts at the periphery of the organoids was confirmed.

[0079] As a result, according to Fig. 4, a structure was confirmed in which fibroblasts formed branches along the bottom surface of the organoid and extended downward based on the central cross-section, and the presence of collagen fibers (yellow circle) was also observed within the structure.

[0080] Additionally, according to Fig. 5, in the sequential photographing results from the bottom section to the upper section, it was observed that fibroblasts (red fluorescence) were spherically wrapped around and supported the outer part of the lung organoid.

[0081]

[0082] <Experimental Example 2> Application of multi-tissue lung organoids to a pulmonary fibrosis experimental model.

[0083] In order to confirm whether the pathology of pulmonary fibrosis can be simulated using the multi-tissue lung organoids manufactured according to the present invention, an experiment was performed to activate fibroblasts and induce a fibrotic response through stimulation of TGF-β, a major factor inducing fibrosis.

[0084] The experimental group of multi-tissue lung organoids was treated with TGF-β1 (Peprotech, #100-21) at a final concentration of 2–10 ng / mL, while the control group was not treated with TGF-β under the same conditions. After culturing for 72 h, the fiber arrangement and fibrotic response extending from fibroblasts surrounding the multi-tissue lung organoids were analyzed.

[0085] As a result, as shown in Fig. 6A, the number of fibroblasts (red fluorescence) increased in the TGF-β-stimulated experimental group compared to the control group, and as shown in Fig. 6B, the thickness of the fiber bundles extending from the periphery of the organoids significantly increased. In particular, the collagen fibers formed a thicker and more complex structure in the experimental group, which is believed to reflect the activation of fibroblasts and increased production of matrix proteins.

[0086] These observations indicate that the multi-tissue lung organoids of the present invention can effectively reproduce pulmonary fibrosis responses in vitro, demonstrating their potential as a lung disease model and a platform for evaluating the efficacy of antifibrotic drugs.

[0087]

[0088] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. 1) Step of isolating epithelial cells from lung tissue; 2) A step of culturing the separated lung epithelial cells in a medium in three dimensions to cultivate a single lung organoid; 3) A step of isolating fibroblasts from lung tissue and culturing them in a medium; 4) A step of mixing the single lung organoid suspension cultured in step 2) and the fibroblast suspension cultured in step 3); 5) A step of centrifuging the mixed suspension in step 4) to attach fibroblasts to a single lung organoid; and 6) A method for producing multi-tissue lung organoids surrounded by fibroblasts, comprising a step of culturing the attached single lung organoid and fibroblasts in a medium by aggregating them at room temperature.

2. A method for producing a multi-tissue lung organoid surrounded by fibroblasts, characterized in that in the step 2), the medium is a basal medium for stem cell culture containing B27, HEPES, glutamax, N-acetylcysteine, Y-27632, A83-01, R-spondin1, noggin, FGF7, FGF10, ITS, nicotinamide, and penicillin / streptomycin.

3. A method for producing multi-tissue lung organoids surrounded by fibroblasts, characterized in that in step 3), the medium is a fibroblast culture medium containing serum.

4. A method for producing multi-tissue lung organoids surrounded by fibroblasts, characterized in that the suspension of step 4) or the medium of step 6) is a mixture of the single lung organoid culture medium of step 2) and the fibroblast culture medium of step 3) in a volume ratio of 1:0.1 to 10 in the first paragraph.

5. In the first paragraph, step 4) is a method for producing a multi-tissue lung organoid surrounded by fibroblasts, characterized in that the cultured single lung organoid and the cultured fibroblasts are mixed at a cell number ratio of 1:0.1 to 10.

6. A method for producing a multi-tissue lung organoid surrounded by fibroblasts, characterized in that in the first paragraph, the centrifugation in step 5) is performed at a gravitational acceleration (G) value of 10 to 1,000 for 1 to 100 minutes.

7. A method for producing a multi-tissue lung organoid surrounded by fibroblasts, characterized in that the multi-tissue lung organoid surrounded by fibroblasts in the first paragraph has an average particle diameter of 50 to 500 μm.

8. A method for producing a multi-tissue lung organoid surrounded by fibroblasts, characterized in that the multi-tissue lung organoid surrounded by fibroblasts in the first paragraph expresses at least one lung epithelial marker and fibroblast marker selected from the group consisting of EpCAM, Surfactant protein C (SPC), Aquaporin 5 (AQ5), Vimentin, and Alpha-smooth muscle actin (α-SMA).

9. A multi-tissue lung organoid surrounded by fibroblasts, prepared according to any one of the methods of claims 1 to 8.

10. In the 9th paragraph, the multi-tissue lung organoid is a multi-tissue lung organoid surrounded by fibroblasts, characterized in that the internal lung organoid is surrounded by fibroblasts in a spherical shape from the outside, and long-term culture and maintenance of the internal lung organoid and the fibroblasts surrounding it from the outside are possible.

11. A step of culturing a multi-tissue lung organoid surrounded by fibroblasts according to Article 9; and A method for producing a lung fibrosis disease model, comprising a step of treating TGF-β to a multi-tissue lung organoid surrounded by the above-mentioned cultured fibroblasts.

12. A lung fibrosis disease model in which TGF-β is treated in a multi-tissue lung organoid surrounded by fibroblasts according to Article 9.

13. A step of culturing a multi-tissue lung organoid surrounded by fibroblasts according to Article 9; and A method for evaluating drug responsiveness, comprising the step of treating a candidate drug to a multi-tissue lung organoid surrounded by the cultured fibroblasts, and then analyzing a lung tissue-specific response.

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