Method for preparing laryngeal organoid and use thereof

Abstract

The present invention relates to a medium composition for forming a laryngeal organoid and a method for preparing a laryngeal organoid by using same. The method for preparing a laryngeal organoid in which laryngeal epithelial cells are three-dimensionally cultured using the medium composition for forming a laryngeal organoid according to the present invention enables stable long-term serial passaging for two or more generations, so that a large quantity of cells can be obtained from a small amount of tissue. In addition, the laryngeal organoid prepared by the method includes a stratified squamous epithelium structure or pseudostratified ciliated columnar epithelium structure that is histologically highly similar to the larynx, and thus may be used as a graft material for laryngeal tissue or as a model capable of replacing cell and animal experiments in disease modeling and drug screening.

Description

Method for manufacturing laryngeal organoids and uses thereof

[0001] The present invention relates to a culture medium composition for forming laryngeal organoids and a method for producing laryngeal organoids using the same.

[0002] The larynx is an important organ located between the pharynx and the trachea; it serves as an air passage connecting airflow and protects the lungs by preventing food or foreign substances from entering the airway. It also plays a crucial role in vocalization due to the presence of vocal cords. Structurally, the larynx of humans and mice can be subdivided into three parts: the supraglottis, glottis, and subglottis, which are broadly composed of two types of tissues (Atlas of Ear, Nose and Throat Pathology, 1990, p. 82-86). The supraglottis includes the epiglottis, aryepiglottic folds, vestibular folds, and arytenoids. The glottis contains the vocal cords, which provide protection against the friction and vibration generated during vocalization. The true vocal cords and the epiglottis of the larynx form a stratified squamous epithelial structure composed of multiple layers of epithelial cells above the lamina propria. The basement layer contains basal cells that exhibit strong stem cell characteristics and divide actively; cells generated by division from these basal cells migrate to the upper layers and differentiate. Structural features are observed where cells become increasingly differentiated, flattened, and rich in keratin as they approach the surface. While the glottis is also composed of stratified squamous epithelial tissue, the subglottic region consists of pseudostratified ciliated columnar epithelial tissue; this tissue possesses the characteristics of respiratory epithelium and functions to secrete mucus and remove foreign substances through ciliary movement.It is a simple epithelium but has a pseudostratified structure that appears to be multiple layers, and it transports mucus and foreign substances while performing secretory and protective functions. It is a major component of the respiratory epithelium.

[0003] Laryngeal cancer is a representative disease affecting the larynx. While the 5-year survival rate is approximately 60–70% and the cure rate is relatively high with early diagnosis and appropriate treatment, surgical resection of the cancerous site can lead to severe vocal function deficits, which significantly impact the patient's quality of life. Therefore, identifying laryngeal stem cells and developing regenerative medicine treatments utilizing them is absolutely essential for laryngeal cancer patients.

[0004] Meanwhile, transplantation methods for reconstructing damaged human tissue include xenograft, allograft, and autograft. Xenograft presents problems such as immuno-incompatibility and the transmission of zoonotic pathogens, including retroviruses. Furthermore, allograft faces issues of donor immune rejection and unavailability, while autograft presents challenges such as difficulty in obtaining the required amount of appropriate tissue and an increased risk of trauma to the patient. To address these issues, technologies involving the direct transplantation of artificial substitutes or tissues formed by culturing cells are gaining attention. In the field of tissue engineering, organoids—also known as artificial organs or mini-organs—have recently garnered more attention than artificial tissues. Organoids are three-dimensional mini-structures (hundreds of micrometers to several millimeters) formed through self-regeneration and differentiation from stem cells; they are also referred to as mini-organs because they reproduce the histological characteristics and physiological functions of the parent organ. Additionally, they can be utilized as an important tool for expanding stem cells through serial subculture. Therefore, the development of laryngeal organoids that reproduce the histological structures of the larynx, namely stratified squamous epithelium and pseudostratified ciliated columnar epithelium, will make a significant contribution to the study of laryngeal disease mechanisms, disease modeling, and the evaluation of drug efficacy and toxicity.

[0005]

[0006] The following tasks of the present invention were carried out with the support of the present invention.

[0007] [Project ID] 2460001937

[0008] [Project No.] RS-2024-00439434

[0009] [Ministry Name] Ministry of Health and Welfare

[0010] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute

[0011] [Research Project Name] Training of Global Physician-Scientists

[0012] [Project Title] Establishment of Molecular Cancer Prevention Strategies Based on the Identification of Stem Cells and Microinvasive Leader Cells in Precancerous Lesions

[0013] [Name of Project Performing Organization] Daegu Gyeongbuk Institute of Science and Technology

[0014] [Research Period] July 1, 2024 ~ December 31, 2028

[0015]

[0016] [Project ID] 1465041595

[0017] [Project No.] RS-2023-00266627

[0018] [Ministry Name] Ministry of Health and Welfare

[0019] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute

[0020] [Research Project Name] Global Research Cooperation Support Project

[0021] [Research Project Title] Elucidating the Ferroptosis Effect of Heavy Ion and FLASH Therapy in Therapy-Resistant Non-Small Cell Lung Cancer and Overcoming Treatment Resistance

[0022] [Name of Project Performing Organization] Yonsei University Industry-Academic Cooperation Foundation

[0023] [Research Period] July 1, 2023 ~ December 31, 2025

[0024]

[0025] [Project ID] 2340013005

[0026] [Project No.] RS-2024-00451507

[0027] [Ministry Name] Ministry of Education

[0028] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0029] [Research Project Name] Establishment of Academic Research Infrastructure in Science and Engineering

[0030] [Project Title] Elucidation of the Mechanism of Stem Cell Hierarchy Regulation by Hierarchical Structure and Microstructure of Esophageal Stem Cells

[0031] [Name of Project Performing Organization] Daegu Gyeongbuk Institute of Science and Technology

[0032] [Research Period] 2024.09.01 ~ 2027.08.31

[0033]

[0034] The object of the present invention is to provide a culture medium composition for forming laryngeal organoids.

[0035] In addition, the present invention provides a method for manufacturing a laryngeal organoid.

[0036] In addition, the present invention provides a laryngeal organoid.

[0037] In addition, the present invention provides a method for evaluating laryngeal toxicity.

[0038] To achieve the above objective, the present invention provides a culture medium composition for forming laryngeal organoids comprising neureglin 1 and a bone morphogenetic protein inhibitor.

[0039] In addition, the present invention provides a method for manufacturing a laryngeal organoid using the above-mentioned culture medium composition.

[0040] In addition, the present invention provides a laryngeal organoid prepared by the above method.

[0041] In addition, the present invention provides a method for evaluating laryngeal toxicity using the above-mentioned laryngeal organoid.

[0042] The method for producing a laryngeal organoid by culturing laryngeal epithelial cells in three dimensions using the forming medium composition of the present invention enables stable long-term passage for two or more generations, allowing a large amount of cells to be obtained from a small amount of tissue. The laryngeal organoid produced through this method contains a stratified squamous epithelium or pseudostratified ciliated columnar epithelium structure that is histologically very similar to the larynx, so it has the effect of being used as a graft material for laryngeal tissue or as a model that can replace cell and animal experiments for disease modeling and drug screening.

[0043] Figure 1 is a schematic diagram showing the process of manufacturing a three-dimensional laryngeal organoid according to the present invention.

[0044] FIG. 2 is a figure showing human (human) laryngeal organoids and mouse laryngeal organoids prepared by the method of the present invention using the laryngeal organoid formation medium of the present invention:

[0045] P0: Laryngeal organoid prepared directly from a patient specimen without passage.

[0046] Figure 3 shows the growth of solid laryngeal organoids and cystic laryngeal organoids according to the number of passages:

[0047] Solid type: Solid laryngeal organoid of the present invention;

[0048] Cystic type: Cystic laryngeal organoid of the present invention;

[0049] P1: Laryngeal organoid subcultured once;

[0050] D1: Laryngeal organoid on day 1 after passage;

[0051] D2: Laryngeal organoid on day 2 after passage;

[0052] D3: Laryngeal organoid on day 3 after subculture;

[0053] D4: Laryngeal organoid on day 4 after passage;

[0054] D5: Laryngeal organoid on day 5 after passage; and

[0055] D6: Laryngeal organoid on day 6 after subculture.

[0056] Figure 4 shows the results of H&E staining of solid laryngeal organoids and cystic laryngeal organoids:

[0057] Solid type: Solid laryngeal organoid of the present invention; and

[0058] Cystic type: The cystic laryngeal organoid of the present invention.

[0059] Figure 5 shows the expression patterns of stratified squamous epithelium-specific markers and pseudostratified squamous ciliated columnar epithelium-specific markers in solid laryngeal organoids and cystic laryngeal organoids, confirmed by immunofluorescence staining analysis:

[0060] Laryngeal stratified squamous epithelial structure: Stratified squamous epithelial markers of control laryngeal tissue;

[0061] Solid organoid: Solid laryngeal organoid of the present invention;

[0062] Laryngeal pseudostratified ciliated columnar epithelial structure: pseudostratified ciliated columnar epithelial markers of control laryngeal tissue; and

[0063] Cystic organoid: Cystic laryngeal organoid of the present invention.

[0064] FIG. 6 is a figure confirming the degree of organoid formation by treating solid laryngeal organoids and cystic laryngeal organoids of the present invention with an anticancer agent:

[0065] Solid type: Solid laryngeal organoid of the present invention;

[0066] Cystic type: Cystic laryngeal organoid of the present invention;

[0067] cisplatin: cisplatin-treated organoid group; and

[0068] paclitaxel: paclitaxel-treated organoid group.

[0069] FIG. 7 is a figure quantifying the degree of organoid formation by treating solid laryngeal organoids and cystic laryngeal organoids of the present invention with an anticancer agent:

[0070] Solid type: Solid laryngeal organoid of the present invention;

[0071] Cystic type: Cystic laryngeal organoid of the present invention;

[0072] cisplatin: cisplatin-treated organoid group; and

[0073] paclitaxel: paclitaxel-treated organoid group.

[0074] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0075] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0076] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0077] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0078]

[0079] In one aspect, the present invention relates to a culture medium composition for forming a larynx organoid comprising Neureglin 1 (Nrg1) and a bone morphogenetic protein (BMP) inhibitor.

[0080] In one embodiment, the culture medium composition may be for the growth or expansion of a laryngeal organoid.

[0081] In one embodiment, the laryngeal organoid may be a solid type laryngeal organoid or a cystic type laryngeal organoid, and the solid type laryngeal organoid may include stratified squamous epithelium tissue, and the cystic type laryngeal organoid may include pseudostratified ciliated columnar epithelium tissue.

[0082] In one embodiment, the bone morphogenetic protein inhibitor may be Noggin, a cordin-like protein including cordin and a cordin domain, a polystatin-related protein including polystatin and a polystatin domain, a DAN-like protein including DAN and a DAN cysteine-note domain, sclerostin / SOST, decorin, alpha-2 macroglobulin, CER1 (Cerberus 1), or Gremlin.

[0083] In one embodiment, the medium composition may further comprise N-acetylcysteine ​​(NAC), a cAMP signaling activator, L-alanyl-L-glutamine dipeptide, a TGF-β inhibitor, a Wnt agonist, a ROCK inhibitor, a mitogenic growth factor, a zionic buffer, basal media for stem cell culture, and an antimicrobial-antifungal agent.

[0084] In one embodiment, the cAMP signaling activator may be forskolin, isoproterenol, NKH 477, isoprotereno (chemical based), PACAP 1-27, or PACAP 1-38 (peptide based).

[0085] In one embodiment, the TGF-β inhibitor may be A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, SJN-2511 or LY2157299 (galunisertib).

[0086] In one embodiment, the Wnt agonist may be Wnt3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, R-spondin-1, R-spondin-2, R-spondin-3, R-spondin-4, or Norrin.

[0087] In one embodiment, the ROCK inhibitor may be (R)-(+)-trans-4-(1-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077) or (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152).

[0088] In one embodiment, the division-promoting growth factor may be EGF (epidermal growth factor), FGF (fibroblast growth factor) 10, TGF (transforming growth factor)-α, BDNF (brainderived neurotrophic factor) or KGF (keratinocyte growth factor), and it is more preferable that it be FGF2 or FGF7.

[0089] In one embodiment, the zwitterionic buffer may be HEPES (Hydroxyethyl piperazine ethane sulfonic acid), MOPs, or bicarbonate buffer.

[0090] In one embodiment, the basic medium for stem cell culture may be any medium used for stem cell culture in the ordinary art, for example, 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, or Advanced DMEM / F12.

[0091] In one embodiment, the antibiotic-antifungal agent may be penicillin, streptomycin, gentamicin, nystatin, or amphotericin.

[0092] In one embodiment, the culture medium composition may include various compounds and elements, and such compounds and elements are known to those skilled in the art. For example, the components included in the culture medium composition of the present invention include, but are not limited to, sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts, vitamins, and amino acids. Additionally, carbohydrates and various salts (including, but not limited to, salts of calcium, magnesium, manganese, sodium, potassium, phosphorus, and sulfur) may be added to the culture medium composition.

[0093] The term "organoid" as used in this invention refers to an ex vivo three-dimensional cellular cluster composed of primary tissue, tissue subunits, or single cells (e.g., stem cells), characterized by its three-dimensional structure that allows for the creation of an environment similar to actual body organs even in a laboratory. In other words, an "organoid" refers to a three-dimensional ex vivo organ-like model that reproduces the structure and function of in vivo organs, consisting of a cluster of cells that self-organize in three dimensions and differentiate into functional cells. The origin of the cells constituting this is not limited. The organoid may have an environment that allows the cells to interact with the surrounding environment during the growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoid in this invention can almost perfectly mimic organs interacting within the body, serving as an excellent model for observing the development of treatments for diseases and the like.

[0094] In addition, the term "laryngeal organoid" used in the present invention refers to a cell aggregate having a 3D structure derived from laryngeal epithelial cells including stem cells obtained from humans and animals, and includes solid and / or cystic laryngeal organoids that exhibit a structure similar to stratified squamous epithelium and pseudostratified ciliated columnar epithelium, such as a larynx having stratified squamous epithelium and pseudostratified ciliated columnar epithelium structures.

[0095] The term "medium composition" as used in this invention refers to a medium intended for the formation and culture of laryngeal organoids. It refers to a culture medium that contains the components necessary for cells to grow, survive, and differentiate into organoids in vitro, and enables this process. It encompasses all conventional media suitable for culture and differentiation used in the field of organoids. Furthermore, the medium used for culturing laryngeal organoids may generally be a minimal culture medium containing carbon sources, nitrogen sources, and trace element components, and may include components commonly known in the technical field of this application to aid in the formation, growth, and expansion of laryngeal organoids. The medium composition may be in liquid or solid form, and a liquid (broth) medium may be solidified by mixing it with agar.

[0096] The term "bone morphogenetic protein inhibitor" as used in the present invention refers to a substance that neutralizes or inhibits the activity of BMP by competitively binding to a BMP molecule or a BMP receptor and thereby inhibiting the formation of a complex between BMP and the BMP receptor. The BMP inhibitors used in the present invention include various natural or synthetic molecules known in the art to form a binding with a BMP molecule or its receptor.

[0097] The term "Wnt agonist" as used in the present invention refers to a substance that activates TCF / LEF-mediated transcription in cells, and encompasses substances that bind to and activate any one of the Wnt family proteins, inhibit β-catenin degradation within cells, or activate TCF / LEF.

[0098] As used in this invention, the term "TGF-β inhibitor" refers to various natural or synthetic molecules that directly or indirectly inhibit or suppress the TGF-β signaling pathway.

[0099] The term "cAMP pathway activator" as used in the present invention refers to various natural or synthetic molecules that directly or indirectly promote the cAMP pathway by increasing the production of cAMP or increasing the activity or expression of adenylyl cyclase.

[0100] The term "mitogenic growth factor" as used in this invention refers to a protein secreted by specific cells that promotes mitosis and differentiation of other cells.

[0101] In one aspect, the present invention relates to a method for preparing a laryngeal organoid comprising the steps of: a) dissociating epithelial tissue from isolated laryngeal tissue; b) extracting epithelial cells from the epithelial tissue; and c) mixing the epithelial cells with a hydrogel and culturing them using a culture medium composition of the present invention.

[0102] In one embodiment, the epithelial tissue may be the epithelial tissue of the glottis and subglottis.

[0103] In one embodiment, in step c), epithelial cells can be dispensed into the upper chamber of a transwell and cultured by dispensing the medium composition of the present invention into the lower chamber.

[0104] In one embodiment, after step c), a step of classifying solid and cystic laryngeal organoids may be additionally included.

[0105] In one embodiment, the solid laryngeal organoid may include stratified squamous epithelial tissue, and the cystic laryngeal organoid may include pseudostratified ciliated columnar epithelial tissue.

[0106] In one embodiment, the solid laryngeal organoid may express one or more markers selected from the group consisting of cytokeratin 14, cytokeratin 5, cytokeratin 1, and loricrin.

[0107] In one embodiment, the cystic laryngeal organoid may express a marker of SCGB1A1 or Ace-Tub (Acetylated tubulin).

[0108] In one embodiment, the hydrogel may comprise at least one polymer selected from alginate, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), methylcellulose, carboxymethylcellulose (CMC), gelatin, collagen, fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, poly(ethylene glycol), polypropylene glycol, hyaluronan, and poly(vinylpyrrolidone), and it is more preferable that it be matrigel.

[0109] In one embodiment, the laryngeal organoid may express one or more basal cell markers or differentiated cell markers selected from the group consisting of cytokeratin 14, cytokeratin 5, cytokeratin 1, loricrin, SCGB1A1, and Ace-Tub (Acetylated tubulin).

[0110] In one embodiment, after step c), a passage culture step of dissociating into single cells and culturing them every 1 to 28 days may be additionally included, and the passage culture may be performed two or more times (2 passages), and the organoid may grow through the passage culture.

[0111] In one embodiment, to subscale the organoid, the organoid can be enzymatically dissociated (disassembled) into single cells, and for this purpose, a reagent containing trypsin can be used.

[0112] At this time, when using Trypsin / EDTA as the enzyme, it is preferable that the reaction time be 1 to 15 minutes. If the reaction time is less than the above range, there is a problem that the organoid with a three-dimensional structure is not separated into a single cell, and if it exceeds the above range, there is a problem that unexpected cell damage may occur.

[0113] The term "passage" as used in the present invention refers to the process of continuing organoid culture by separating the organoid into single cells and then transferring them to a new culture vessel in order to continuously culture the organoid in a healthy state for a long period. One instance of separating the cell group or changing the culture vessel is called one passage.

[0114] The term "Matrigel" as used in the present invention is a protein complex extracted from sarcoma cells of EHS (Engelbreth-Holm-Swarm) mice (product name of BD Bioscience) and contains an extracellular matrix (ECM) such as laminin, collagen, and heparan sulfate proteoglycan, and growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EFG), insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), and platelet-derived growth factor (PDGF). The complex constituting Matrigel is used as a substrate for cell culture by providing a complex extracellular environment found in many tissues.

[0115] The term "differentiation" as used in the present invention refers to a phenomenon in which the structure or function of a cell becomes tissue-specific while the cell divides, proliferates, and grows. That is, it refers to the process in which the cells, tissues, etc. of an organism acquire a suitable form and function to perform the role required by a specific organ. For example, differentiation can include not only the process in which laryngeal stem cells transform into other epithelial cells of the larynx, but also the process in which hematopoietic stem cells transform into red blood cells, white blood cells, platelets, etc., that is, everything in which progenitor cells come to express specific differentiation traits.

[0116] In one aspect, the present invention relates to a laryngeal organoid prepared according to the method of the present invention.

[0117] In one embodiment, the laryngeal organoid may be solid or cystic.

[0118] In one embodiment, the solid laryngeal organoid may include stratified squamous epithelial tissue (structure), and the cystic laryngeal organoid may include pseudostratified ciliated columnar epithelial tissue (structure).

[0119] In one embodiment, the solid laryngeal organoid may express one or more markers selected from the group consisting of cytokeratin 14, cytokeratin 5, cytokeratin 1, and loricrin.

[0120] In one embodiment, the cystic laryngeal organoid may express a marker of SCGB1A1 or Ace-Tub.

[0121] In one embodiment, the laryngeal organoid may have an average diameter of 30 to 2000 μm.

[0122] In one aspect, the present invention relates to a method for evaluating laryngeal toxicity caused by a drug after treating a laryngeal organoid of the present invention with a drug.

[0123] In one embodiment, the drug may be an anticancer agent, and the anticancer agent may be a pro-apoptotic peptide, an immunogenic apoptosis inducer, or an anticancer agent, SN-38 (7-ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, picantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, Ifosfamide, Carmustine (BCNU), Lomustine (CCNU), Streptozotocin, Busulfan, Thiotepa, Cisplatin, Carboplatin, Dactinomycin (Actinomycin D), Plicamycin, Mitomycin C, Vincristine, Vinblastine, Teniposide, Topotecan, Iridotecan, Uramustine, Melphalan, Bendamustine, Dacarbazine, Temozolomide, Altretamine, Duocarmycin, nedaplatin, oxaliplatin, satraplatin,Triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, It may be selected from the group consisting of maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

[0124] In one aspect, the present invention relates to an implantable composition comprising a laryngeal organoid or a support for tissue regeneration according to the present invention.

[0125] In one embodiment, the composition may be a composition for laryngeal transplantation.

[0126] In one embodiment, the implantable composition may be a cell therapy agent comprising an organoid.

[0127] The term "cell therapy agent" as used in this invention refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention, consisting of cells and tissues produced by isolation, culture, and special processing from a human being. It refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise altering the biological characteristics of living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues.

[0128] The cell therapy agent according to the present invention can be injected into the body of an individual, for example, by using the clinical method published by Lindvall et al. (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka (Pittsburgh, 1998). The formulation may include a pharmaceutically acceptable conventional carrier in addition to the laryngeal organoid which is the active ingredient, and in the case of an injectable formulation, may include a preservative, an analgesic, a solubilizing agent, or a stabilizer, and in the case of a formulation for local administration, may include a base, an excipient, a lubricant, or a preservative.

[0129] The cell therapy product according to the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person with ordinary knowledge in the ordinary art. The pharmaceutically acceptable carrier included in the cell therapy product of the present invention is one that is commonly used in formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. The cell therapeutic agent composition of the present invention may additionally include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0130] The cell therapy product according to the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally, or topically. Suitable dosages of the cell therapy product composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response responsiveness.

[0131] In the present invention, the terms “administering,” “introducing,” and “implanting” are used interchangeably and may refer to the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. At least a portion of the cells or cellular components of the composition according to one embodiment may be administered by any suitable route to deliver them to a desired location within a living individual.

[0132] In the above method, administration can be performed on the lesion site requiring the transplantation of a laryngeal organoid. Endoscopic equipment may be used for administration, but is not limited thereto. For example, administration via endoscopy is typical, but it can also be performed on the larynx through surgical procedures. For instance, if a portion of laryngeal tissue is lost, a laryngeal organoid can be transplanted to replace the damaged tissue area.

[0133]

[0134] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0135]

[0136] Example 1. Preparation of medium for laryngeal organoid formation

[0137] A medium composition was prepared for forming a larynx organoid, comprising basal media for stem cell culture, neuregulin 1, noggin, glutamax, N-acetylcysteine, forskolin, HEPES, B27, FGF2, FGF7, Wnt3a, A83-01, Y-27632, and an antibiotic-antifungal agent. Specifically, 0.1 μg / ml Neureglin 1, 0.1 μg / ml Nogin, 1X Glutamax, 1.25 mM N-acetylcysteine, 10 μM Folskolin, 1X HEPES, 1X B27, 50 ng / ml FGF2, 25 ng / ml FGF7, 50 ng / ml Wnt3a, 0.5 μM A83-01, 10 μM Y-27632, and 1X penicillin-streptomycin were mixed in the basic medium DMEM / F12 or Advanced DMEM / F12. At this time, the concentration of each of the above components can be adjusted to about 1 / 10 to 10 times the stated concentration, and other components other than those stated above may be additionally included.

[0138]

[0139] Example 2. Preparation of a 3D Laryngeal Organoid

[0140] 2-1. Separation of Laryngeal Epithelial Cells

[0141] Human laryngeal specimens or the glottis and subglottic regions of mouse larynges were thoroughly washed with phosphate buffered saline (PBS) and then cut into pieces 2 to 10 mm in length. The cut tissues were placed in DMEM (Dulbecco's modified eagle medium) (Gibco, #12430112) supplemented with dispase (Stem cell, #07913) and enzymatically dissociated for 0.5 to 2 hours in a cell culture incubator supplied with 5% carbon dioxide at 37°C. Using forceps, only the laryngeal epithelial tissue was exfoliated from the dissociated laryngeal tissue. The exfoliated epithelial tissue was placed in a 0.25% Trypsin-EDTA (0.25% Trypsin-EDTA) (Gibco, # 25200-072) solution and enzymatically dissociated for 5 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C. Epithelial cells were extracted by pipetting the dissociated laryngeal epithelial tissue, and the Trypsin / EDTA was neutralized by adding PBS containing 2% fetal bovine serum (FBS). The cells were then passed through a 40 μm cell strainer (SPL, #93040). After centrifugation for 5 minutes to remove the supernatant, the laryngeal single cells were suspended in Advanced DMEM / F12 medium, and the cell count was measured.

[0142]

[0143] 2-2. Fabrication of 3D Laryngeal Organoids

[0144] A laryngeal organoid was produced by 3D culturing epithelial cells isolated from the epithelial cells of the laryngeal tissue in Example 2-1 for 7 to 14 days (Fig. 1). Specifically, 100 to 10,000 laryngeal epithelial cells from the glottis and subglottis, respectively, were suspended in 50 μl of the medium prepared in Example 1, mixed with Matrigel (Coring, #356231) in a 1:1 ratio, dispensed into the upper chambers of 24-well cell culture transwells (Corning, #3470), and cultured for 30 to 60 minutes in a cell culture incubator supplied with 5% carbon dioxide at 37°C. Once the Matrigel solidified, the medium prepared in Example 1 was supplied to the lower chambers of the cell culture transwells to produce solid-type laryngeal organoids reproducing stratified squamous epithelium and cystic-type laryngeal organoids reproducing pseudostratified ciliated columnar epithelium. Mixed laryngeal organoids were prepared. Subsequently, solid laryngeal organoids and cystic organoids were separated through single organoid cloning, which is morphologically separated using a microscope.

[0145] As a result, both human laryngeal organoids and mouse laryngeal organoids formed laryngeal organoids in the laryngeal organoid formation medium of the present invention, and their size was found to be about 100 to 250 μm (Fig. 2).

[0146]

[0147] 2-3. Subculture of Laryngeal Organoids

[0148] The solid mouse laryngeal organoid reproducing stratified squamous epithelium and the cystic mouse laryngeal organoid reproducing pseudostratified ciliated columnar epithelium prepared in Example 2-2 were each treated with DMEM containing dispase every 14 days and dissociated into single cells for 1 to 2 hours in a cell culture incubator supplied with 5% carbon dioxide at 37°C, then placed in a 0.25% trypsin-EDTA solution and enzymatically dissociated for 5 minutes in a cell culture incubator supplied with 5% carbon dioxide at 37°C. The dissociated laryngeal epithelial tissue was pipetted to extract epithelial cells, PBS containing 2% fetal bovine serum was added to neutralize the trypsin / EDTA, and the cells were passed through a 40 μm cell strainer. Laryngeal single cells, after removing the supernatant by centrifugation for 5 minutes, were suspended in Advanced DMEM / F12 medium, and the process of forming the next generation of organoids was repeated according to the method of Example 2-2 above, thereby growing solid and cystic laryngeal organoids by subculturing multiple times over a long period (Fig. 3).

[0149]

[0150] Example 3. Characterization of a 3D laryngeal organoid

[0151] 3-1. Histological Structural Analysis

[0152] H&E staining was performed to histologically confirm whether the solid mouse laryngeal organoid composed of stratified squamous epithelium and the cystic mouse laryngeal organoid composed of pseudostratified ciliated columnar epithelium prepared in Example 2 above reproduce the histological structure of the actual larynx. Specifically, each organoid was fixed with 4% paraformaldehyde (fromaldehyde solution, Sigma, #47608), mixed with an OCT compound (Scigen, #4583) in a cryo mold (15 x 15 x 5 mm, Tissue-tek, #4566), and solidified using dry ice. Each solidified laryngeal organoid was cut into 5 μm thick slides using a frozen sectioner (Leica, #CM3050S), and H&E staining was performed using a hematoxylin and eosin stain kit (Vector laboratories, #H-3502), after which the slides were observed under a microscope.

[0153] As a result, a stratified squamous epithelial structure resembling layers of tree rings was observed in the solid mouse laryngeal organoid, and a single layer of pseudostratified ciliated columnar epithelial structure was observed in the cystic mouse laryngeal organoid (Fig. 3).

[0154]

[0155] 3-2. Marker Analysis

[0156] To confirm whether the solid mouse laryngeal organoid and cystic mouse laryngeal organoid prepared in Example 2 above reproduce the histological structure of the actual larynx, the expression of markers for stratified squamous epithelium and markers for pseudostratified ciliated columnar epithelium in the control laryngeal tissue, the solid mouse laryngeal organoid prepared in Example 2 above, and the cystic mouse laryngeal organoid was confirmed by immunofluorescence staining analysis. Specifically, slides of each laryngeal organoid were prepared by cutting them to a thickness of 10 μm in the same manner as in Example 3-1 above, and were blocked at room temperature for 1 hour with PBS containing 0.1% Triton X-100 (Biosesang, TR1020-500-00) and 3% bovine serum albumin (MP biomedicals, #9048-46-8). Each primary antibody against Keratin 14 (KRT 14), a marker of the basal layer of the stratified squamous epithelium of the larynx; Loricrin, a marker of the suprabasal layer of differentiated cells; SCGB1A1, a marker of the pseudostratified ciliated columnar epithelium of the larynx; and Acetylated tubulin (Ace-Tub) was diluted with a REAL antibody diluent solution (Agilent, #S2022) and applied to slides, which were then incubated overnight at 4°C. After washing the slides three times with PBS containing 0.1% Triton x-100 / 1% bovine serum albumin, they were each incubated with each secondary antibody diluted in PBS containing 1% fetal bovine serum for 1 hour at room temperature. After washing the slides three times with PBS, they were incubated at room temperature for 10 minutes in a DAPI (Sigma, #10236276001) solution diluted in PBS. After washing the slides three times with PBS, they were observed under a fluorescence microscope.

[0157] As a result, keratin 14 (KRT 14) and loricrin were stained at different locations in the solid laryngeal organoids, similar to the control laryngeal tissue (Fig. 4), confirming that the structure of the solid laryngeal organoids exhibits a basal layer and an upper basal layer structure similar to the stratified squamous epithelium of the larynx. In addition, immunofluorescence staining results for SCGB1A1 and Ace-Tub also showed that the stained cystic laryngeal organoids had a structure similar to the pseudostratified ciliated columnar epithelium of the control laryngeal tissue (Fig. 4).

[0158]

[0159] Example 4. Confirmation of Use for Drug Toxicity Evaluation of 3D Laryngeal Organoids

[0160] To determine whether the solid laryngeal organoids and cystic laryngeal organoids of the present invention can be utilized as a platform for evaluating drug toxicity, each laryngeal organoid formed by culturing in an organoid formation medium as in Example 2 was subcultured, and then the laryngeal organoids were treated with the anticancer drugs cisplatin and paclitaxel at concentrations of 1 to 10 μM and 0.005 to 0.01 μM, respectively. At this time, the drugs were continuously applied throughout the organoid formation period and were re-treated when the medium was replaced every 2 to 3 days.

[0161] As a result, it was found that the formation of laryngeal organoids decreased in both solid and cystic laryngeal organoids in a manner dependent on the concentration of the anticancer drug (Figures 6 and 7), confirming that both types of laryngeal organoids can be used as models to evaluate the efficacy and toxicity of the drug.

Claims

1. A culture medium composition for forming a larynx organoid comprising Neureglin 1 (Nrg1) and a bone morphogenetic protein (BMP) inhibitor.

2. A culture medium composition for forming a laryngeal organoid according to claim 1, wherein the laryngeal organoid is a solid type laryngeal organoid containing stratified squamous epithelium tissue or a cystic type laryngeal organoid containing pseudostratified ciliated columnar epithelium tissue.

3. A medium composition for forming a laryngeal organoid according to claim 1, wherein the bone morphogenetic protein (BMP) inhibitor is Noggin, a cordin-like protein comprising cordin and a cordin domain, a polystatin and a polystatin-related protein comprising a polystatin domain, a DAN and a DAN-like protein comprising a DAN cysteine-note domain, sclerostin / SOST, decorin, alpha-2 macroglobulin, CER1 (Cerberus 1), or Gremlin.

4. A medium composition for forming a laryngeal organoid according to claim 1, further comprising N-acetylcysteine ​​(NAC), a cAMP signaling activator, L-alanyl-L-glutamine dipeptide, a TGF-β inhibitor, a Wnt agonist, a ROCK inhibitor, a mitogenic growth factor, an amphoteric buffer, basal media for stem cell culture, and an antimicrobial-antifungal agent.

5. A medium composition for forming laryngeal organoids according to claim 4, wherein the cAMP signaling activator is forskolin, isoproterenol, NKH 477, isoprotereno (chemical based), PACAP 1-27, or PACAP 1-38 (peptide based).

6. A medium composition for forming laryngeal organoids according to claim 4, wherein the TGF-β inhibitor is A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, SJN-2511 or LY2157299 (galunisertib).

7. A medium composition for forming a laryngeal organoid according to claim 4, wherein the Wnt agonist is Wnt3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, R-spondin-1, R-spondin-2, R-spondin-3, R-spondin-4, or Norrin.

8. A medium composition for forming a laryngeal organoid according to claim 4, wherein the ROCK inhibitor is (R)-(+)-trans-4-(1-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077) or (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152).

9. A culture medium composition for forming laryngeal organoids, wherein, in claim 4, FGF is FGF2 or FGF7. 10.a) Step of dissociating epithelial tissue from isolated laryngeal tissue; b) a step of extracting epithelial cells from epithelial tissue; and c) A method for preparing a laryngeal organoid comprising the step of mixing epithelial cells with a hydrogel and culturing them using the culture medium composition of claim 1.

11. A method for preparing a laryngeal organoid according to claim 10, wherein in step c) above, epithelial cells are dispensed into the upper chamber of a transwell and cultured.

12. A method for manufacturing a laryngeal organoid according to claim 10, further comprising the step of classifying solid and cystic laryngeal organoids after step c).

13. A method for preparing a laryngeal organoid according to claim 10, further comprising a subculture step of dissociating into single cells and culturing every 1 to 28 days after step c).

14. Laryngeal organoid prepared according to the method of claim 10.

15. In paragraph 14, a laryngeal organoid that is solid or cystic.

16. A method for evaluating drug-induced laryngeal toxicity after treating the laryngeal organoid of Paragraph 14 with the drug.

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