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KR20240176372A - Method for preparing sheeted oral mucosa organoids - Google Patents

  • ️Tue Dec 24 2024

KR20240176372A - Method for preparing sheeted oral mucosa organoids - Google Patents

Method for preparing sheeted oral mucosa organoids Download PDF

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Publication number
KR20240176372A
KR20240176372A KR1020230077056A KR20230077056A KR20240176372A KR 20240176372 A KR20240176372 A KR 20240176372A KR 1020230077056 A KR1020230077056 A KR 1020230077056A KR 20230077056 A KR20230077056 A KR 20230077056A KR 20240176372 A KR20240176372 A KR 20240176372A Authority
KR
South Korea
Prior art keywords
oral mucosal
manufacturing
organoid
oral
paragraph
Prior art date
2023-06-15
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
KR1020230077056A
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Korean (ko)
Inventor
정용택
오수민
김진우
Original Assignee
고려대학교 산학협력단
이화여자대학교 산학협력단
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2023-06-15
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2023-06-15
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2024-12-24
2023-06-15 Application filed by 고려대학교 산학협력단, 이화여자대학교 산학협력단 filed Critical 고려대학교 산학협력단
2023-06-15 Priority to KR1020230077056A priority Critical patent/KR20240176372A/en
2024-12-24 Publication of KR20240176372A publication Critical patent/KR20240176372A/en
Status Pending legal-status Critical Current

Links

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0632—Cells of the oral mucosa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007—Drug-containing films, membranes or sheets
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Abstract

본 발명은 평면화 구강점막 오가노이드 제조방법, 상기 제조방법으로 제조된 평면화 구강점막 오가노이드, 상기 평면화 구강점막 오가노이드를 포함하는 세포 시트제 및 상기 평면화 구강점막 오가노이드를 유효성분으로 포함하는, 구강점막 질환 예방 또는 치료용 의약외품 조성물에 관한 것이다.
본 발명의 제조방법으로 제조된 평면화 구강점막 오가노이드는 세포 시트제로서 구강병변 부위에 생착하여 구강점막 질환을 예방 또는 치료하는데 유용하게 사용될 수 있다.The present invention relates to a method for producing a planarized oral mucosal organoid, a planarized oral mucosal organoid produced by the method, a cell sheet comprising the planarized oral mucosal organoid, and an over-the-counter pharmaceutical composition for preventing or treating oral mucosal diseases, comprising the planarized oral mucosal organoid as an effective ingredient.
The planarized oral mucosal organoids manufactured by the manufacturing method of the present invention can be usefully used to prevent or treat oral mucosal diseases by engrafting on oral lesion sites as a cell sheet.

Description

평면화 구강점막 오가노이드 제조방법 {Method for preparing sheeted oral mucosa organoids}Method for preparing sheeted oral mucosa organoids {Method for preparing sheeted oral mucosa organoids}

본 발명은 평면화 구강점막 오가노이드 제조방법, 상기 제조방법으로 제조된 평면화 구강점막 오가노이드, 상기 평면화 구강점막 오가노이드를 포함하는 세포 시트제 및 상기 평면화 구강점막 오가노이드를 유효성분으로 포함하는, 구강점막 질환 예방 또는 치료용 의약외품 조성물에 관한 것이다.The present invention relates to a method for producing a planarized oral mucosal organoid, a planarized oral mucosal organoid produced by the method, a cell sheet comprising the planarized oral mucosal organoid, and an over-the-counter pharmaceutical composition for preventing or treating oral mucosal diseases, comprising the planarized oral mucosal organoid as an effective ingredient.

구강은 인후와 식도로 이어지는 소화관의 첫 부분으로서 구강의 표면은 피부와 비슷하지만 보다 무르고 습한 점막으로 덮여 있다. 이러한 구강점막은 심부 조직을 구강의 환경으로부터 보호하고 기회감염을 일으킬 수 있는 미생물로부터 방어막 역할을 한다. The oral cavity is the first part of the digestive tract that leads to the pharynx and esophagus. The oral cavity's surface is covered with a mucous membrane that is similar to the skin but softer and more moist. This oral mucosa protects the deep tissues from the oral environment and acts as a barrier against microorganisms that can cause opportunistic infections.

구강점막은 신체의 외부와 내부를 연결하는 위치에 있기 때문에 신체의 내부적 변화와 외부적 자극으로부터 모두 영향을 받게 되어 매우 다양한 질병 상태를 나타낸다. 구강점막의 백색 변화나 어두운 색조로의 변화, 조직 결손 또는 부피 증가와 같은 양상은 관련된 병적 과정을 반영하므로 구강점막질환의 진단에 중요한 단서를 제공한다.Since the oral mucosa is located at a position that connects the outside and inside of the body, it is affected by both internal changes in the body and external stimuli, and thus exhibits a wide variety of disease states. Changes in the oral mucosa, such as white or dark color changes, tissue loss, or volume increase, reflect related pathological processes, and thus provide important clues for the diagnosis of oral mucosal diseases.

현재까지 구강점막 질환을 치료하는 방법은 병변 부위를 보호하여 통증을 줄이고 지연치유를 기다리는 것에 그치며, 종래 구강점막 궤양 치료제 또한 구강패치나 스프레이 등을 이용한 처치제로 증상을 완화하는 것에 불과하다. 따라서, 구강병변을 직접적으로 치료하여 치유시간을 줄이고 통증 및 부작용을 최소화하기 위한 연구가 시급한 실정이다.Up to now, the treatment methods for oral mucosal diseases are limited to protecting the lesion site to reduce pain and waiting for delayed healing, and existing oral mucosal ulcer treatments only alleviate symptoms with oral patches or sprays. Therefore, research is urgently needed to directly treat oral lesions to reduce healing time and minimize pain and side effects.

한편, 종래 2D 배양 기법의 줄기세포 연구는 세포 본연의 성질과 조직 단위의 특성을 잃어버리기 쉽고, 동물모델은 인체의 장기와 생물학적 차이가 있어 조직 재생의 일반화에 부적합하다. 이러한 문제점을 극복하기 위해 3D 배양기법을 통한 오가노이드 제조가 대두되고 있다.Meanwhile, stem cell research using conventional 2D culture techniques tends to lose the original properties of cells and the characteristics of tissue units, and animal models are not suitable for generalizing tissue regeneration due to biological differences from human organs. To overcome these problems, organoid production using 3D culture techniques is emerging.

다능성줄기세포 및 성체줄기세포를 세포외기질 유사 매트릭스에 포매하여 3차원으로 배양함으로써 생성할 수 있는 오가노이드는 줄기세포의 분화능을 인공적으로 조작함으로써 모사하고자하는 장기의 세포, 장기의 특이 구조 및 기능 등을 갖는 세포의 집합체이다. 오가노이드 배양법을 활용해 구강점막 주변에 존재하는 구강점막 줄기세포를 배양함으로써 구강점막 오가노이드를 형성할 수 있다. 다만, 오가노이드를 통한 조직 재현에 있어 침샘, 눈물샘 등 내강이 존재하는 기관은 오가노이드화되었을 때 본래의 극성을 잘 형성할 수 있으나, 구강점막, 미뢰와 같은 이장 상피는 내강이 존재하지 않고 정단부(apical) 부위가 생체 외부로 노출되어 있는 구조이므로, 3D 배양을 통한 오가노이드 형성 시 본래 조직과 정반대의 극성을 가지게 된다. Organoids, which can be created by embedding pluripotent stem cells and adult stem cells in an extracellular matrix-like matrix and culturing them three-dimensionally, are aggregates of cells that have the cells of the organ, specific structures, and functions of the organ to be simulated by artificially manipulating the differentiation potential of stem cells. Oral mucosal organoids can be formed by culturing oral mucosal stem cells existing around the oral mucosa using the organoid culture method. However, when reproducing tissues using organoids, organs with lumens such as salivary glands and lacrimal glands can form their original polarity well when organoidized, but intestinal epithelium such as the oral mucosa and taste buds do not have lumens and their apical part is exposed to the outside of the body, so when organoids are formed through 3D culture, they have a polarity that is exactly the opposite of the original tissue.

이러한 배경 하에, 본 발명자들은 구강점막 질환을 치료하기 위한 세포 유래 소재를 제조하는 방법을 예의 연구 노력한 결과, 평면화된 구강점막 오가노이드 제조방법을 확립하고, 이러한 방법으로부터 형성한 세포 시트가 구강점막의 병변 부위에 생착될 수 있음을 확인하여, 본 발명을 완성하였다.Against this backdrop, the inventors of the present invention have conducted extensive research efforts to develop a method for producing a cell-derived material for treating oral mucosal diseases, and as a result, have established a method for producing a flat oral mucosal organoid, and confirmed that a cell sheet formed from this method can be engrafted on a lesion site of the oral mucosa, thereby completing the present invention.

대한민국 공개특허공보 제10-2021-0078704호Republic of Korea Patent Publication No. 10-2021-0078704

본 발명은 전술한 문제 및 이와 연관된 다른 문제를 해결하는 것을 목적으로 한다.The present invention aims to solve the above-mentioned problems and other problems related thereto.

본 발명의 일 예시적 목적은 다음의 단계를 포함하는, 평면화 구강점막 오가노이드 제조방법을 제공하는 것이다:One exemplary object of the present invention is to provide a method for producing planarized oral mucosal organoids, comprising the following steps:

(a) 세포외기질에 포매되어 있는 구강점막 상피세포를 배양하여 구강점막 오가노이드를 함유한 배양물을 수득하는 단계;(a) a step of culturing oral mucosal epithelial cells embedded in an extracellular matrix to obtain a culture containing oral mucosal organoids;

(b) 상기 배양물에서 세포외기질을 제거하는 단계;(b) a step of removing the extracellular matrix from the culture;

(c) 상기 세포외기질이 제거된 배양물에 효소를 첨가하고 물리적 압력을 가하여 단일세포를 수득하는 단계;(c) a step of adding an enzyme to a culture from which the extracellular matrix has been removed and applying physical pressure to obtain single cells;

(d) 상기 단일세포를 함유한 배양물을 원심분리하여 얻은 단일세포펠렛을 배지에 현탁하여 현탁액을 수득하는 단계;(d) a step of obtaining a suspension by suspending a single cell pellet obtained by centrifuging a culture containing the single cell in a medium;

(e) 상기 현탁액을 배양 용기에 시딩(seeding)하는 단계; 및(e) a step of seeding the suspension into a culture vessel; and

(f) 상기 배양 용기에 성장인자를 포함한 배지를 첨가한 후 배양하여 평면화 구강점막 오가노이드를 수득하는 단계.(f) A step of adding a medium containing growth factors to the culture vessel and culturing it to obtain a flat oral mucosal organoid.

본 발명의 다른 예시적 목적은 상기 제조방법으로 제조된 평면화 구강점막 오가노이드를 제공하는 것이다.Another exemplary object of the present invention is to provide a planarized oral mucosal organoid manufactured by the above manufacturing method.

본 발명의 또 다른 예시적 목적은 상기 평면화 구강점막 오가노이드를 포함하는 세포 시트제를 제공하는 것이다.Another exemplary object of the present invention is to provide a cell sheet comprising the flattened oral mucosal organoid.

본 발명의 또 다른 예시적 목적은 상기 평면화 구강점막 오가노이드를 유효성분으로 포함하는, 구강점막 질환 예방 또는 치료용 의약외품 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating oral mucosal diseases, comprising the flat oral mucosal organoid as an effective ingredient.

본 명세서에 개시된 발명의 기술적 사상에 따라 이루고자 하는 기술적 과제는 이상에서 언급한 문제점을 해결하기 위한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제는 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The technical problems to be achieved according to the technical idea of the invention disclosed in this specification are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

상기 목적을 달성하기 위한 일 양태로서, 본 발명은 다음의 단계를 포함하는, 평면화 구강점막 오가노이드 제조방법을 제공한다.As one aspect for achieving the above object, the present invention provides a method for producing a planarized oral mucosal organoid, comprising the following steps.

(a) 세포외기질에 포매되어 있는 구강점막 상피세포를 배양하여 구강점막 오가노이드를 함유한 배양물을 수득하는 단계;(a) a step of culturing oral mucosal epithelial cells embedded in an extracellular matrix to obtain a culture containing oral mucosal organoids;

(b) 상기 배양물에서 세포외기질을 제거하는 단계;(b) a step of removing the extracellular matrix from the culture;

(c) 상기 세포외기질이 제거된 배양물에 효소를 첨가하고 물리적 압력을 가하여 단일세포를 수득하는 단계;(c) a step of adding an enzyme to a culture from which the extracellular matrix has been removed and applying physical pressure to obtain single cells;

(d) 상기 단일세포를 함유한 배양물을 원심분리하여 얻은 단일세포펠렛을 배지에 현탁하여 현탁액을 수득하는 단계;(d) a step of obtaining a suspension by suspending a single cell pellet obtained by centrifuging a culture containing the single cell in a medium;

(e) 상기 현탁액을 배양 용기에 시딩(seeding)하는 단계; 및(e) a step of seeding the suspension into a culture vessel; and

(f) 상기 배양 용기에 성장인자를 포함한 배지를 첨가한 후 배양하여 평면화 구강점막 오가노이드를 수득하는 단계.(f) A step of adding a medium containing growth factors to the culture vessel and culturing it to obtain a flat oral mucosal organoid.

본 발명의 용어 "구강점막(oral mucosa)"은 입안 전체를 덮고 있는 점막으로, 치은, 경구개, 구순, 협부(볼), 구강전정, 구강저, 혀, 연구개 및 치조 점막을 포함한다.The term "oral mucosa" of the present invention refers to a mucosa covering the entire inside of the mouth, including the gingiva, hard palate, lips, buccal region (cheeks), oral vestibule, floor of the mouth, tongue, soft palate, and alveolar mucosa.

본 발명의 용어 "오가노이드(organoid)"는 1차 조직(primary tissue), 조직 하위단위 또는 단일세포(예를 들어 줄기세포)로 구성된 생체 외 3차원 세포 집합체(3D cellular cluster)를 의미한다. 오가노이드는 자가재생(self-renewal) 및 자가조직화(self-organization)가 가능한 특징을 가지며, 본래 조직과 유사한 표현형 및 기능을 구현할 수 있으므로, '소형 유사 장기' 또는 '장기 유사체'로도 명명될 수 있다.The term "organoid" of the present invention refers to an in vitro 3D cellular cluster composed of primary tissue, tissue subunit, or single cell (e.g., stem cell). Organoids have the characteristics of self-renewal and self-organization, and can implement a phenotype and function similar to that of the original tissue, and therefore can also be called 'small-sized organ-like' or 'organ-like'.

본 발명의 용어 "평면화 구강점막 오가노이드"는 3D 배양을 통해 형성된 오가노이드가 평면화된 형태로, 평면화를 통해 구강점막 자극부위가 노출된 형태를 확보할 수 있으며, 세포 시트제로서 생체 구강병변에 생착시켜 구강점막 질환의 예방 및 치료에 이용될 수 있다.The term "flattened oral mucosal organoid" of the present invention refers to an organoid formed through 3D culture in a flattened form, and can secure a form in which an oral mucosal stimulation site is exposed through flattening, and can be used to prevent and treat oral mucosal diseases by engrafting it on a living oral lesion as a cell sheet.

본 발명의 용어 "배양"은 생체로부터 분리된 세포, 이들의 2차원 또는 3차원적 집합체, 조직 또는 조직의 일부를 체외에서 증식, 성장, 유지 및 분화시키는 것을 의미한다. 이에, 용어“배양”은 출발 물질(세포, 조직 또는 조직 유사체)을 이용하여 인공적인 환경 하에서 목적 물질을 수득하는 전 과정을 포괄하는 의미이며, 이에“배양용 조성물”은“증식용 조성물”, “성장용 조성물”, “유지용 조성물” 및 “분화 유도용 조성물”을 모두 포함하는 개념이다.The term "culture" of the present invention means the proliferation, growth, maintenance and differentiation of cells separated from a living organism, a two-dimensional or three-dimensional aggregate thereof, a tissue or a part of a tissue in vitro. Accordingly, the term "culture" encompasses the entire process of obtaining a target substance under an artificial environment using a starting material (cell, tissue or tissue analog), and thus the "culture composition" is a concept that includes all of the "proliferation composition", the "growth composition", the "maintenance composition" and the "differentiation-inducing composition".

본 발명에 있어서, 상기 (a)단계의 배양은 EGF, Y27632 및 A8301을 포함하는 배지에서 배양되는 것일 수 있다.In the present invention, the culture in step (a) may be cultured in a medium containing EGF, Y27632 and A8301.

본 발명에 있어서, 상기 세포외기질은 콜라겐(collagen)일 수 있으나 이에 제한되지 않는다.In the present invention, the extracellular matrix may be collagen, but is not limited thereto.

본 발명에 있어서, 상기 (b)단계의 세포외기질이 콜라겐(collagen)인 경우 상기 배양물에 콜라게나제(collagenase)를 처리하여 세포외기질을 제거할 수 있다.In the present invention, if the extracellular matrix in step (b) is collagen, the extracellular matrix can be removed by treating the culture with collagenase.

본 발명의 일 구현예로, 상기 콜라게나제를 처리한 후 10분 내지 30분, 구체적으로 15분 내지 25분, 더욱 구체적으로 15분 내지 20분간 37℃에서 인큐베이션하는 단계를 더 포함할 수 있다.In one embodiment of the present invention, the method may further include a step of incubating at 37° C. for 10 to 30 minutes, specifically 15 to 25 minutes, and more specifically 15 to 20 minutes after treating the collagenase.

본 발명에 있어서, 상기 (e) 단계의 현탁액 내 1 x 104 개 내지 1 x 108 개일 수 있으며, 구체적으로 1 x 105 개 내지 1 x 107 개일 수 있으며, 더욱 구체적으로 1 x 105 개 내지 1 x 106 개 일 수 있으나 이에 제한되지 않는다.In the present invention, the number of the particles in the suspension of step (e) may be 1 x 10 4 to 1 x 10 8 , specifically 1 x 10 5 to 1 x 10 7 , and more specifically 1 x 10 5 to 1 x 10 6 , but is not limited thereto.

본 발명에 있어서, 상기 성장인자는 EGF 및 Y27632 중 선택되는 하나 이상일 수 있으나 이에 제한되지 않는다.In the present invention, the growth factor may be at least one selected from EGF and Y27632, but is not limited thereto.

상기 EGF는 상피 성장인자로 세포 내 신호경로를 조절하여 증식, 분화 및 자가 복제에 영향을 미치며, 배지 조성물 내 EGF의 농도는 구강점막 구성 세포를 효율적으로 증식, 분화시키기 위해 적절히 선택될 수 있다.The above EGF is an epithelial growth factor that regulates intracellular signaling pathways to affect proliferation, differentiation, and self-renewal, and the concentration of EGF in the medium composition can be appropriately selected to efficiently proliferate and differentiate oral mucosal cells.

상기 Y-27632(디하이드로클로라이드)는 Rho/Rock 신호 경로를 억제하는 화합물로서 하기 화학식 1의 구조를 가지며, 배지 조성물 내 Y-27632의 농도는 구강점막 구성 세포를 효율적으로 증식, 분화시키기 위해 적절히 선택될 수 있다.The above Y-27632 (dihydrochloride) is a compound that inhibits the Rho/Rock signal pathway and has a structure represented by the following chemical formula 1. The concentration of Y-27632 in the medium composition can be appropriately selected to efficiently proliferate and differentiate oral mucosal cells.

[화학식 1][Chemical Formula 1]

본 발명에 있어서, 상기 효소는 세포외기질이 제거된 후 획득한 오가노이드 세포군을 물리적, 화학적으로 단일 세포화 하기위해 사용되는 것으로, 일 예로, 트리플(Tryple), 트립신(Trypsin), EDTA 또는 아큐테이즈(Accutase)일 수 있고, 구체적으로 트리플일 수 있으나 이에 제한되지 않는다.In the present invention, the enzyme is used to physically and chemically single-cell the organoid cell group obtained after the extracellular matrix is removed, and may be, for example, Tryple, Trypsin, EDTA or Accutase, and may be specifically, Tryple, but is not limited thereto.

본 발명에 있어서, 상기 (f)단계의 배양은 5일 내지 15일간 수행될 수 있고, 구체적으로 5일 내지 12일간 또는 5일 내지 10일간 수행될 수 있으며, 더욱 구체적으로 7일 내지 10일간 수행될 수 있으나 이에 제한되지 않는다.In the present invention, the culturing in step (f) may be performed for 5 to 15 days, specifically for 5 to 12 days or 5 to 10 days, and more specifically for 7 to 10 days, but is not limited thereto.

본 발명에 있어서, 상기 (f)단계의 배양은 35℃ 내지 45℃에서 수행될 수 있고, 구체적으로 35℃ 내지 38℃에서 수행될 수 있으나 이에 제한되지 않는다.In the present invention, the culturing in step (f) can be performed at 35°C to 45°C, and specifically, can be performed at 35°C to 38°C, but is not limited thereto.

본 발명에 있어서, 상기 (f)단계의 배양 이후 20℃ 내지 30℃에서 배양하는 단계를 더 포함할 수 있으며, 구체적으로 20℃ 내지 25℃에서 배양하는 단계를 더 포함할 수 있으나 이에 제한되지 않는다.In the present invention, a step of culturing at 20°C to 30°C may be further included after the culturing in step (f), and specifically, a step of culturing at 20°C to 25°C may be further included, but is not limited thereto.

본 발명에 있어서, 상기 배양 용기의 표면은 N-isopropylacrylamide(PIPAAm)으로 코팅된 것일 수 있다. 37℃ 이상의 온도에서는 상기 코팅 층이 소수성이므로 세포의 부착 및 성장이 가능하나, 32℃ 이하의 온도에서는 코팅 층이 친수성으로 바뀌면서 물과 결합하여 부풀어올라 세포가 용기로부터 탈착될 수 있다.In the present invention, the surface of the culture vessel may be coated with N-isopropylacrylamide (PIPAAm). At a temperature of 37°C or higher, the coating layer is hydrophobic, allowing attachment and growth of cells. However, at a temperature of 32°C or lower, the coating layer becomes hydrophilic, combines with water, swells, and allows cells to detach from the vessel.

상기 목적을 달성하기 위한 다른 양태로서, 본 발명은 상기 제조방법으로 제조된 평면화 구강점막 오가노이드를 제공한다.As another aspect for achieving the above object, the present invention provides a planarized oral mucosal organoid manufactured by the above manufacturing method.

본 발명의 용어 구강점막, 오가노이드, 평면화 구강점막 오가노이드는 전술한 바와 같다.The terms oral mucosa, organoid, and planarized oral mucosa organoid of the present invention are as described above.

구체적으로, 본 발명의 실시예에서는, 상기 제조방법으로 평면화 구강점막 오가노이드를 제조한 후 이를 이용한 세포 시트제를 제조하였으며, 면역형광염색을 통해 제조된 세포 시트제가 구강병변 부위에 잘 생착하는 것을 확인하였다.Specifically, in an embodiment of the present invention, a planarized oral mucosal organoid was manufactured using the above-described manufacturing method, and then a cell sheet was manufactured using the same. Through immunofluorescence staining, it was confirmed that the manufactured cell sheet was well established in the oral lesion area.

상기 목적을 달성하기 위한 또 다른 양태로서, 본 발명은 상기 평면화 구강점막 오가노이드를 포함하는 세포 시트제를 제공한다.As another aspect for achieving the above object, the present invention provides a cell sheet comprising the flat oral mucosal organoid.

본 발명에 있어서, 상기 세포 시트제는 평면화 구강점막 오가노이드를 포함하는 시트 형태로 형성되는 것일 수 있다.In the present invention, the cell sheet agent may be formed in a sheet shape including a flat oral mucosal organoid.

본 발명에 있어서, 상기 세포 시트제는 구강점막 병변 부위에 부착시켜 구강점막 질환을 예방 또는 치료하는 용도로 사용될 수 있다.In the present invention, the cell sheet agent can be used to prevent or treat oral mucosal diseases by attaching it to an oral mucosal lesion site.

상기 목적을 달성하기 위한 또 다른 양태로서, 본 발명은 상기 평면화 구강점막 오가노이드를 유효성분으로 포함하는, 구강점막 질환 예방 또는 치료용 의약외품 조성물을 제공한다.As another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating oral mucosal disease, comprising the flat oral mucosal organoid as an effective ingredient.

본 발명의 용어 구강점막, 오가노이드, 평면화 구강점막 오가노이드는 전술한 바와 같다.The terms oral mucosa, organoid, and planarized oral mucosa organoid of the present invention are as described above.

본 발명에 있어서, 상기 구강점막 질환은 구내염, 아프타성 구내염, 편평태선, 약물에 의한 접촉성 병소, 천포창 및 점막 유천포창으로 이루어진 군에서 선택되는 1종 이상일 수 있다.In the present invention, the oral mucosal disease may be at least one selected from the group consisting of stomatitis, aphthous stomatitis, lichen planus, drug-induced contact lesions, pemphigoid, and mucosal pemphigoid.

구체적으로, 본 발명의 의약외품 조성물은 구강용　의약외품 또는 동물용 의약외품을 포함하는 것일 수 있다. 본 발명의　의약외품　조성물은 상기 평면화 구강점막 오가노이드 이외에　의약외품　조성물에 통상적으로 사용되는 성분들을 포함할 수 있으며, 예컨대 연마제, 습윤제, 결합제, 기포제, 감미제, 방부제, 약효성분, 향미제, 색소, 용제, 증백제, 가용화제 또는 pH 조정제를 포함할 수 있다.Specifically, the quasi-drug composition of the present invention may include an oral quasi-drug or an veterinary quasi-drug. The quasi-drug composition of the present invention may include, in addition to the planarized oral mucosal organoid, components commonly used in quasi-drug compositions, and may include, for example, an abrasive, a wetting agent, a binder, a foaming agent, a sweetener, a preservative, a medicinal ingredient, a flavoring agent, a pigment, a solvent, a whitening agent, a solubilizer, or a pH adjuster.

본 발명의 의약외품 조성물은 패치, 외용제, 산제, 살균소독제, 치약제, 연고제, 로션제, 물티슈, 스프레이 파스 또는 밴드 등을 예시할 수 있으나 이에 특별히 제한되는 것은 아니며, 의약외품의 제제화 방법, 용량, 이용방법, 구성성분 등은 당해 기술분야에 공지된 통상의 기술로부터 당업자에 의해 적절히 선택될 수 있다.The pharmaceutical composition of the present invention may include, but is not limited to, patches, external preparations, powders, sterilizing disinfectants, toothpaste, ointments, lotions, wet tissues, spray patches, or bandages, and the like, and the formulation method, dosage, usage method, components, etc. of the pharmaceutical composition may be appropriately selected by those skilled in the art from common techniques known in the art.

또한, 본 발명의　의약외품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 구강용 의약외품으로서 구체적으로는 구강필름, 구강스프레이, 구강용 연고제, 치약, 구강청결제, 껌, 캔디류, 구강용 바니쉬(varnish) 및 잇몸 마사지 크림으로 구성된 군으로부터 선택되는 하나 이상의 제형일 수 있으나 이에 특별히 제한되는 것은 아니다. In addition, the quasi-drug composition of the present invention can be manufactured in any formulation commonly manufactured in the art, and specifically, as an oral quasi-drug, it can be at least one formulation selected from the group consisting of an oral film, an oral spray, an oral ointment, a toothpaste, a mouthwash, a gum, a candy, an oral varnish, and a gum massage cream, but is not particularly limited thereto.

상기 목적을 달성하기 위한 다른 양태로서, 본 발명은 상기 평면화 구강점막 오가노이드를 구강 내 생착시켜 구강점막 질환을 예방 또는 치료하는 방법을 제공한다.As another aspect for achieving the above purpose, the present invention provides a method for preventing or treating oral mucosal disease by engrafting the flat oral mucosal organoid into the oral cavity.

상기 목적을 달성하기 위한 다른 양태로서, 본 발명은 상기 상기 평면화 구강점막 오가노이드의 구강점막 질환 예방 또는 치료 용도를 제공한다. As another aspect for achieving the above purpose, the present invention provides a use of the flat oral mucosal organoid for preventing or treating oral mucosal diseases.

본 발명의 제조방법으로 제조된 평면화 구강점막 오가노이드는 세포 시트제로서 구강병변 부위에 생착하여 구강점막 질환을 예방 또는 치료하는데 유용하게 사용될 수 있다.The planarized oral mucosal organoids manufactured by the manufacturing method of the present invention can be usefully used to prevent or treat oral mucosal diseases by engrafting on oral lesion sites as a cell sheet.

다만, 본 명세서에 개시된 기술의 일 실시예에 따른 효과는 이상에서 언급한 것들로 제한되지 않으며, 언급하지 않은 또 다른 효과들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the effects according to one embodiment of the technology disclosed in this specification are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

본 명세서에서 인용되는 도면을 보다 충분히 이해하기 위하여 각 도면의 간단한 설명이 제공된다.
도 1은 구강점막 오가노이드 제작 과정을 도시한 것이다.
도 2는 본 발명의 제조방법으로 제조된 평면화 구강점막 오가노이드(세포 시트제)를 확인한 결과이다(a : 업셀 디쉬에 시딩한 직후 세포 모습, b : 7일간 배양 후 형성된 평면화 구강점막 오가노이드, c : 평면화 구강점막 오가노이드를 디쉬에서 탈착하여 실리콘패드에 위치시킨 모습).
도 3은 면역형광염색을 통해 본 발명의 제조방법으로 제조된 평면화 구강점막 오가노이드의 병변부위에의 생착을 확인한 결과이다(a: 10 배율, b: 25 배율).To facilitate a more thorough understanding of the drawings cited herein, a brief description of each drawing is provided.
Figure 1 illustrates the process of producing oral mucosal organoids.
Figure 2 shows the results of confirming the flat oral mucosal organoid (cell sheet) manufactured by the manufacturing method of the present invention (a: appearance of cells immediately after seeding on an upsell dish, b: flat oral mucosal organoid formed after culturing for 7 days, c: appearance of the flat oral mucosal organoid removed from the dish and placed on a silicone pad).
Figure 3 shows the results of confirming the engraftment of planarized oral mucosal organoids manufactured by the manufacturing method of the present invention into a lesion site through immunofluorescence staining (a: 10x magnification, b: 25x magnification).

이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples.

실시예 1: 구강점막 오가노이드 제조Example 1: Preparation of oral mucosal organoids

환자의 동의하에 구강점막(혀 및 볼점막)을 채취하여, 생리식염수에 희석한 dispase II 효소(2mg/mL)(Roche, cat #4942078001)를 점막 상피와 상피 하 결합조직의 사이 공간에 주입하여 팽창시키고, 이렇게 팽창된 구강점막 조직을 dispase II 효소 희석액에서 37℃에서 30분 배양하였다. 상피 하 결합조직으로부터 상피층을 박리해 상피층판을 분리하고, 이를 trypsin/EDTA (Gibco, cat #25200072)용액 내부에서 멸균된 수술용 가위, 칼 등의 도구를 이용하여 최대한 작은 면적으로 잘게 잘랐다. 잘게 자른 조직을 37℃에서 30분 배양하였다. 이후, 필터에 연속적인 피페팅으로 잘게 자른 조직에 압력을 가하여 조직으로부터 세포를 추출하여 단일세포 현탁액을 형성하였다. 이 현탁액을 원심분리를 하여 얻은 세포 펠렛을 적절한 비율로 콜라겐(collagen) 등의 세포 외 기질에 포매하였으며, 기본 배양액(DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 보충액 + 1x B27 보충액 + 1x penicillin/streptomycin)을 기초로 배지 조성물(20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50), A8301(Tocris, cat #2939))을 함유한 배지에서 수일간 배양하였다. 총 7일간 배양하여 오가노이드 세포집합체를 함유한 배양물을 수득하였다.With the patient's consent, oral mucosa (tongue and buccal mucosa) was collected, dispase II enzyme (2 mg/mL) (Roche, cat #4942078001) diluted in saline was injected into the space between the mucosal epithelium and the subepithelial connective tissue to cause expansion, and the expanded oral mucosal tissue was incubated in the dispase II enzyme dilution solution at 37°C for 30 minutes. The epithelial layer was peeled from the subepithelial connective tissue to separate the epithelial lamina, and this was minced into the smallest possible area using sterilized surgical scissors, a knife, etc. in a trypsin/EDTA (Gibco, cat #25200072) solution. The minced tissue was incubated at 37°C for 30 minutes. Thereafter, cells were extracted from the tissue by applying pressure to the minced tissue by continuous pipetting through a filter to form a single-cell suspension. This suspension was centrifuged to obtain a cell pellet, which was embedded in an extracellular matrix such as collagen at an appropriate ratio, and cultured for several days in a medium containing a medium composition (20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50), A8301 (Tocris, cat #2939)) based on a basic culture medium (DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 supplement + 1x B27 supplement + 1x penicillin/streptomycin). The cells were cultured for a total of 7 days to obtain a culture containing organoid cell aggregates.

도 1은 인체유래 구강점막 오가노이드 제작 과정을 도시한 것이다.Figure 1 illustrates the process of producing human-derived oral mucosal organoids.

실시예 2: 평면화 구강점막 오가노이드 제조Example 2: Preparation of planarized oral mucosal organoids

실시예 1에서 수득한 배양물에 콜라게나제(gollagenase)를 첨가하고 37℃에서 15 내지 20분 처리하여 세포외기질인 콜라겐을 제거하였다. 이후 배양물에 Tryple(Gibco, cat #12604013)을 첨가하고 5분동안 37℃에 위치시킨 뒤 유리 파이펫을 이용해 연속적인 압력을 5분 이상 가하여 오가노이드를 깨서 단일세포형태로 만들었다. 이후 RCF 45 g, 4℃ 조건에서 4분 30초간 원심분리를 통해 단일세포 펠렛을 수득한 후 37℃로 가열한 배지(기본 배양액(DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 보충액 + 1x B27 보충액 + 1x penicillin/streptomycin)을 기초로 배지 조성물(20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50) 추가)에 첨가된 펠렛을 풀고 1 x 106 개의 세포를 PIPAAm으로 표면이 코팅된 업셀 디쉬(upcell dish)에 시딩(seeding)하였다. 기본 배양액(DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 보충액 + 1x B27 보충액 + 1x penicillin/streptomycin)을 기초로 배지 조성물(20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50))이 첨가된 배지를 3 mL 첨가한 후 3~4일에 한번씩 교체하며 37℃에서 7 내지 10일간 배양하여, 세포 confluency(부착세포로 덮인 배양 접시 표면 백분율)가 90% 이상인 평면화 구강점막 오가노이드(세포 시트제)를 제조하였다(도 2, a : 업셀 디쉬에 시딩한 직후 세포 모습, b : 7 내지 10일간 배양 후 형성된 평면화 구강점막 오가노이드, c : 평면화 구강점막 오가노이드를 디쉬에서 탈착하여 실리콘패드에 위치시킨 모습).Collagenase was added to the culture obtained in Example 1 and treated at 37°C for 15 to 20 minutes to remove collagen, which is an extracellular matrix. Thereafter, Tryple (Gibco, cat #12604013) was added to the culture and placed at 37°C for 5 minutes. Then, continuous pressure was applied using a glass pipette for more than 5 minutes to break the organoids into single-cell forms. After centrifugation at 45 g RCF for 4 minutes and 30 seconds at 4℃, a single cell pellet was obtained. The pellet was dissolved in a medium (basal medium (DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 supplement + 1x B27 supplement + 1x penicillin/streptomycin) heated to 37℃ with medium composition (20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50) added) and 1 x 106 cells were seeded on an upcell dish whose surface was coated with PIPAAm. Basic medium (DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x After adding 3 mL of medium containing medium composition (20% R-spondin 1, 10% Noggin, 50 ng/mL EGF2, 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50)) based on N2 supplement + 1x B27 supplement + 1x penicillin/streptomycin, the medium was replaced once every 3-4 days and cultured at 37°C for 7-10 days to produce planarized oral mucosal organoids (cell sheets) with a cell confluency (the percentage of the culture dish surface covered with adherent cells) of 90% or higher (Fig. 2, a: appearance of cells immediately after seeding on an upsell dish, b: flattened oral mucosal organoids formed after culturing for 7-10 days, c: appearance of flattened oral mucosal organoids detached from the dish and placed on a silicone pad).

이후 배양물이 담긴 디쉬를 20℃ 내지 25℃의 환경으로 옮긴 후 배지를 제거하고 운반용 멤브레인(Thermo, cat #174904)을 올리고, 평면화 오가노이드의 건조를 막기 위해 0.1 mL 배지((DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 보충액 + 1x B27 보충액 + 1x penicillin/streptomycin)를 멤브레인에 뿌려주었다. 10분간 방치하여 세포 시트제가 디쉬에서 탈착되어 멤브레인에 부착된 상태가 되었다. 마이크로포셉을 이용해 멤브레인의 가장자리를 잡고 천천히 시트를 들어올렸다. 멸균된 실리콘 패드 위에 멤브레인을 놓고 배지를 넣어 멤브레인에서 시트를 분리한 뒤, 구강병변에 맞게 세포 시트제 크기를 조절하여 절단하였다. 제작된 세포 시트제를 새 멤브레인에 다시 부착하여 구강병변 부위로 옮긴 후 수분을 처리하여 세포 시트제가 멤브레인에서 탈착되면서 병변에 생착하도록 하였다. 이후 물기를 제거하고 종래 구강패치제품(큐라틱, TBM㈜)을 생착 부위에 부착시켜 세포 시트제가 병변 부위에 생착하는 동안 보호하도록 하였다.After that, the dish containing the culture was transferred to an environment of 20°C to 25°C, the medium was removed, a transport membrane (Thermo, cat #174904) was placed on it, and 0.1 mL of medium ((DMEM/F12 + 1x GlutaMax + 1x HEPES + 1x N2 supplement + 1x B27 supplement + 1x penicillin/streptomycin) was sprayed on the membrane to prevent the flattened organoids from drying. After leaving it for 10 minutes, the cell sheet was detached from the dish and attached to the membrane. The edge of the membrane was held using microforceps and the sheet was slowly lifted. The membrane was placed on a sterilized silicone pad, the medium was added to separate the sheet from the membrane, and the cell sheet was cut to fit the oral lesion. The fabricated cell sheet was reattached to a new membrane and transferred to the oral lesion area, and moisture was treated to ensure that the cell sheet It was allowed to take root in the lesion while being detached from the membrane. Afterwards, moisture was removed and a conventional oral patch product (Curatic, TBM Co., Ltd.) was attached to the site of take root to protect the cell sheet while it took root in the lesion.

실시예 3: 평면화 구강점막 오가노이드의 생착 평가Example 3: Evaluation of engraftment of planarized oral mucosal organoids

면역형광염색을 통해 실시예 2에서 구강병변 부위에 생착시킨 평면화 구강점막 오가노이드(세포 시트제)가 구강병변에 잘 생착되는지 확인하였다. 구체적으로, 실시예 2에서 생착 부위에 부착시킨 세포 시트제를 4% PFA 용액에 담군 후 10분 실온에 두었다. 이후 0.2% PBST 세척 용액에 담군 후 5분 실온에 두는 방식으로 3번 세척하였다. PBST에 5% 당나귀 세럼(donkey serum)을 첨가하여 블로킹 용액을 제조하여 시트를 담군 후 30간 실온에 두었다. 새 블로킹 용액에 시트의 각질세포를 검출하는 1차 항체를 첨가한 뒤 시트를 넣고 4℃에 24시간 두었다. 이후 0.2% PBST 세척 용액에 담군 후 10분 실온에서 3번 세척하고, 블로킹 용액에 2차 항체를 첨가하고 시트를 넣고 차광하여 실온에 2시간 두었다.Immunofluorescence staining was used to confirm whether the planarized oral mucosal organoids (cell sheets) that were implanted in the oral lesion site in Example 2 were implanted well in the oral lesion site. Specifically, the cell sheets that were attached to the implantation site in Example 2 were immersed in a 4% PFA solution and left at room temperature for 10 minutes. Then, they were washed three times by immersing them in a 0.2% PBST washing solution and leaving them at room temperature for 5 minutes. A blocking solution was prepared by adding 5% donkey serum to PBST, the sheets were immersed in it, and left at room temperature for 30 minutes. A primary antibody that detects keratinocytes in the sheets was added to the new blocking solution, the sheets were placed, and left at 4°C for 24 hours. After that, they were immersed in a 0.2% PBST washing solution, washed three times for 10 minutes at room temperature, a secondary antibody was added to the blocking solution, the sheets were placed, and kept in a light-blocking state at room temperature for 2 hours.

세척 후 마이크로포셉을 이용해 시트제를 슬라이드에 펼쳐놓고 마운팅 용액(mounting solution)을 떨어트린 후 커버 슬립으로 덮어준 뒤 공초점 현미경으로 면역형광염색된 시트제를 확인하였다.After washing, the sheet was spread on a slide using microforceps, a mounting solution was dropped, and the sheet was covered with a cover slip. The immunofluorescently stained sheet was then observed using a confocal microscope.

그 결과, 병변 부위에 세포 시트제가 잘 생착되었음을 확인하였다(도 3, a: 10 배율, b: 25 배율).As a result, it was confirmed that the cell sheet was well established at the lesion site (Fig. 3, a: 10x magnification, b: 25x magnification).

이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts rather than the above detailed description.

Claims (15)

다음의 단계를 포함하는, 평면화 구강점막 오가노이드 제조방법:
(a) 세포외기질에 포매되어 있는 구강점막 상피세포를 배양하여 구강점막 오가노이드를 함유한 배양물을 수득하는 단계;
(b) 상기 배양물에서 세포외기질을 제거하는 단계;
(c) 상기 세포외기질이 제거된 배양물에 효소를 첨가하고 물리적 압력을 가하여 단일세포를 수득하는 단계;
(d) 상기 단일세포를 함유한 배양물을 원심분리하여 얻은 단일세포펠렛을 배지에 현탁하여 현탁액을 수득하는 단계;
(e) 상기 현탁액을 배양 용기에 시딩(seeding)하는 단계; 및
(f) 상기 배양 용기에 성장인자를 포함한 배지를 첨가한 후 배양하여 평면화 구강점막 오가노이드를 수득하는 단계.
A method for producing planarized oral mucosal organoids, comprising the following steps:
(a) a step of culturing oral mucosal epithelial cells embedded in an extracellular matrix to obtain a culture containing oral mucosal organoids;
(b) a step of removing the extracellular matrix from the culture;
(c) a step of adding an enzyme to a culture from which the extracellular matrix has been removed and applying physical pressure to obtain single cells;
(d) a step of obtaining a suspension by suspending a single cell pellet obtained by centrifuging a culture containing the single cell in a medium;
(e) a step of seeding the suspension into a culture vessel; and
(f) A step of adding a medium containing growth factors to the culture vessel and culturing it to obtain a flat oral mucosal organoid.
 제1항에 있어서,
상기 구강점막 상피세포는 구강점막 조직에 디스파제(dispase) 효소를 처리하여 상피층판을 분리하고, 이로부터 세포를 추출하여 얻어지는 것인, 제조방법.
In the first paragraph,
A manufacturing method wherein the above oral mucosal epithelial cells are obtained by treating oral mucosal tissue with dispase enzyme to separate the epithelial layer and extracting cells therefrom.
 제1항에 있어서,
상기 (a)단계의 배양은 EGF, Y27632 및 A8301을 포함하는 배지에서 배양되는 것인, 제조방법.
In the first paragraph,
A manufacturing method, wherein the culturing in step (a) is performed in a medium containing EGF, Y27632 and A8301.
 제1항에 있어서,
상기 (b)단계에서 상기 배양물에 콜라게나제(collagenase)를 처리하여 세포외기질을 제거하는, 제조방법.
In the first paragraph,
A manufacturing method in which the extracellular matrix is removed by treating the culture with collagenase in the step (b).
 제4항에 있어서,
상기 콜라게나제를 처리한 후 10분 내지 30분간 37℃에서 인큐베이션하는 단계를 더 포함하는, 제조방법.
In paragraph 4,
A manufacturing method further comprising a step of incubating at 37°C for 10 to 30 minutes after treating the collagenase.
 제1항에 있어서,
상기 (e) 단계의 현탁액 내 세포가 1 x 104 개 내지 1 x 108 개인, 제조방법.
In the first paragraph,
A manufacturing method wherein the number of cells in the suspension of step (e) is 1 x 10 4 to 1 x 10 8 .
 제1항에 있어서,
상기 성장인자는 EGF 및 Y27632 중 선택되는 하나 이상인, 제조방법.
In the first paragraph,
A manufacturing method, wherein the growth factor is at least one selected from EGF and Y27632.
 제1항에 있어서,
상기 효소는 트리플(Tryple), 트립신(Trypsin), EDTA 또는 아큐테이즈(Accutase)인, 제조방법.
In the first paragraph,
A manufacturing method wherein the enzyme is Tryple, Trypsin, EDTA or Accutase.
 제1항에 있어서,
상기 (f)단계의 배양은 5일 내지 15일간 수행하는 것인, 제조방법.
In the first paragraph,
A manufacturing method wherein the culturing in step (f) is performed for 5 to 15 days.
 제1항에 있어서,
상기 (f)단계의 배양은 35℃ 내지 45℃에서 수행되는 것인, 제조방법.
In the first paragraph,
A manufacturing method wherein the culturing in step (f) is performed at 35°C to 45°C.
 제10항에 있어서,
상기 (f)단계의 배양 이후 20℃ 내지 30℃에서 배양하는 단계를 더 포함하는, 제조방법.
In Article 10,
A manufacturing method further comprising a step of culturing at 20°C to 30°C after the culturing in step (f).
 제1항 내지 제11항 중 어느 한 항의 제조방법으로 제조된, 평면화 구강점막 오가노이드.
A planarized oral mucosal organoid manufactured by any one of the manufacturing methods of claims 1 to 11.
 제12항의 평면화 구강점막 오가노이드를 포함하는 세포 시트제.
A cell sheet comprising a flat oral mucosal organoid of claim 12.
 제12항의 평면화 구강점막 오가노이드를 유효성분으로 포함하는, 구강점막 질환 예방 또는 치료용 의약외품 조성물.
A pharmaceutical composition for preventing or treating oral mucosal diseases, comprising the flat oral mucosal organoid of Article 12 as an effective ingredient.
 제14항에 있어서,
상기 구강점막 질환은 구내염, 아프타성 구내염, 편평태선, 약물에 의한 접촉성 병소, 천포창 및 점막 유천포창으로 이루어진 군에서 선택되는 1종 이상인, 의약외품 조성물.In Article 14,
A pharmaceutical composition, wherein the oral mucosal disease is at least one selected from the group consisting of stomatitis, aphthous stomatitis, lichen planus, drug-induced contact lesions, pemphigoid, and mucosal pemphigoid.

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Patent event code: PA02011R01I

Patent event date: 20230615

Comment text: Patent Application

2024-12-24 PG1501 Laying open of application