Method for preparing sheeted taste bud organoids

Abstract

The present invention relates to a method for manufacturing a flattened taste bud organoid using epithelial cells of the anterior part of the tongue, a flattened taste bud organoid manufactured by the said method, a cell sheet preparation containing the said organoid, and a screening method for a taste stimulating substance. Unlike organoids formed through 3D culture, the flattened taste bud organoid produced by the manufacturing method of the present invention has the taste bud stimulation site exposed, so it can be used for screening taste stimulating substances and studying taste cell expression by reproducing the epithelial layer of oral tissue and the aggregate layer of taste cells and confirming the cellular response to taste stimulation.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing a flattened taste bud organoid, a flattened taste bud organoid manufactured by the said method, a cell sheet preparation comprising the said organoid, and a method for screening a taste stimulating substance. Background Technology

[0003] Taste buds are primary gustatory organs that first form sensory information regarding taste stimuli, and they are located on the surface of the tongue, the soft palate, and the pharynx and larynx within the oral cavity. Among these, dozens to hundreds of taste buds are distributed in the circumvallate papillae and similar foliate papillae located in the posterior part of the tongue, while only one or two taste buds exist in the pneumatopodiform papillae located in the anterior part of the tongue. Therefore, while circumvallate and foliate papillae can generate strong taste information, pneumatopodiform papillae exhibit more diverse response patterns to taste stimuli, thereby forming taste information of various qualities. Taste buds are composed of dozens of cells and can be classified into Type 1, Type 2, Type 3, and Type 4 taste cells based on gene expression, taste stimulus expression, and morphological classification.

[0004] Stem cell research using conventional 2D culture techniques is prone to losing the inherent properties of the cells and characteristics of the tissue unit, and animal models are unsuitable for generalizing tissue regeneration due to biological differences from human organs. To overcome these problems, the production of organoids using 3D culture techniques is emerging.

[0005] Organoids, which can be generated by embedding pluripotent stem cells and adult stem cells in an extracellular matrix-like matrix and culturing them in three dimensions, are aggregates of cells that possess the specific structures and functions of the organ to be mimicked by artificially manipulating the differentiation potential of the stem cells. A method for forming taste bud organoids by culturing taste bud stem cells located around taste buds using organoid culture methods has been introduced, and these can be differentiated into Type 1, Type 2, Type 3, and Type 4 taste cells, which are the constituent cells within the taste bud. However, regarding tissue reproduction through organoids, organs with lumens, such as salivary glands and lacrimal glands, can form their original polarity well when converted into organoids; whereas the intestinal epithelium, such as the oral mucosa and taste buds, lacks a lumen and has an apical region exposed to the outside of the body, resulting in a polarity opposite to that of the original tissue when organoids are formed through 3D culture.

[0006] Against this backdrop, the inventors have diligently researched a method to form an organoid in which the taste bud stimulation area is exposed through the flattening of taste bud epithelial cells, and as a result, established a method for manufacturing a flattened taste bud organoid. Furthermore, by confirming that a flattened taste bud organoid derived from the anterior tongue epithelium formed by this method can reproduce the epithelial surface of oral tissue, the present invention has been completed. Prior art literature

[0008] Republic of Korea Published Patent Application No. 10-2021-0078704 The problem to be solved

[0009] The present invention aims to solve the aforementioned problem and other related problems.

[0010] One exemplary object of the present invention is to provide a method for manufacturing a flattened taste bud organoid comprising the following steps:

[0011] (a) A step of culturing anterior tongue epithelial cells to obtain a culture containing taste bud organoids;

[0012] (b) a step of removing the extracellular matrix from the culture;

[0013] (c) a step of obtaining a single cell by adding an enzyme to the culture from which the extracellular matrix has been removed and applying physical pressure;

[0014] (d) a step of obtaining a suspension by suspending a single-cell pellet obtained by centrifuging the culture containing the single cell above in a medium;

[0015] (e) a step of seeding the above suspension into a culture vessel; and

[0016] (f) A step of obtaining flattened taste bud organoids by adding a medium containing growth factors to the culture vessel and then culturing.

[0017] Another exemplary objective of the present invention is to provide a flattened taste bud organoid produced by the above-described manufacturing method.

[0018] Another exemplary objective of the present invention is to provide a cell sheet composition comprising the flattened taste bud organoid.

[0019] Another exemplary objective of the present invention is to provide a screening method for taste stimulants, comprising the steps of: treating the flattened taste bud organoid with a target substance; and determining whether the target substance is a taste stimulant.

[0020] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0022] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0024] As one embodiment for achieving the above objective, the present invention provides a method for manufacturing a flattened taste bud organoid comprising the following steps.

[0025] (a) A step of culturing anterior tongue epithelial cells to obtain a culture containing taste bud organoids;

[0026] (b) a step of removing the extracellular matrix from the culture;

[0027] (c) a step of obtaining a single cell by adding an enzyme to the culture from which the extracellular matrix has been removed and applying physical pressure;

[0028] (d) a step of obtaining a suspension by suspending a single-cell pellet obtained by centrifuging the culture containing the single cell above in a medium;

[0029] (e) a step of seeding the above suspension into a culture vessel; and

[0030] (f) A step of obtaining flattened taste bud organoids by adding a medium containing growth factors to the culture vessel and then culturing.

[0031] The term "taste bud" in the present invention refers to a cell distributed in the oral cavity, such as the tongue or soft palate, which can perform the role of sensing taste upon contact with food, and specifically is known to be able to sense sweet, bitter, salty, sour, and umami tastes.

[0032] The term "organoid" in the present invention refers to an in vitro three-dimensional cellular cluster composed of primary tissue, tissue subunits, or single cells (e.g., stem cells). Organoids have the characteristics of being capable of self-renewal and self-organization, and can realize phenotypes and functions similar to original tissues, so they may also be referred to as "small organ-like structures" or "organ analogs."

[0033] The term "flattened taste bud organoid" in the present invention refers to an organoid formed through 3D culture in a flattened form. Through flattening, a form in which the taste bud stimulation area is exposed can be secured, making it suitable for studying cellular responses to taste stimulation and contributing to basic research on taste cell expression by establishing a platform for screening taste stimulating substances.

[0034] The term "culture" in the present invention refers to the proliferation, growth, maintenance, and differentiation of cells, two-dimensional or three-dimensional aggregates thereof, tissues, or parts of tissues separated from a living organism 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 the term "composition for culture" is a concept that includes "composition for proliferation," "composition for growth," "composition for maintenance," and "composition for differentiation induction."

[0035] In the present invention, the anterior tongue epithelial cells can be obtained by treating the tongue tissue with a dispase enzyme to separate the epithelial lamellae and extracting cells therefrom.

[0036] In the present invention, the culture of step (a) may be sequentially cultured in a first medium composition comprising R-spondin 1, Noggin, FGF2, IGF1, SAG (Smoothed agonist), and Y-27632, and a second medium composition comprising R-spondin 1, Noggin, FGF2, IGF1, SAG (Smoothed agonist), CHIR99021, and SU5402.

[0037] In the present invention, the extracellular matrix may be Matrigel or collagen, but is not limited thereto.

[0038] In the present invention, if the extracellular matrix of step (b) is Matrigel, the extracellular matrix can be removed by treating the culture with cold DMEM / F12 medium.

[0039] In the present invention, if the extracellular matrix of step (b) is collagen, the extracellular matrix can be removed by treating the culture with collagenase.

[0040] In one embodiment of the present invention, the method may further include the step of incubating at 37°C for 10 to 30 minutes, specifically 15 to 25 minutes, more specifically 15 to 20 minutes, after treating the collagenase.

[0041] In the present invention, the cells in the suspension of step (e) are 1 x 10 4 Pieces up to 1 x 10 8 It can be one, specifically 1 x 10 5 Pieces up to 1 x 10 7 It can be 1 x 10 5 Pieces up to 1 x 10 6 It may be one, but is not limited to this.

[0042] In the present invention, the growth factor may be one or more selected from EGF and Y27632, but is not limited thereto.

[0043] The above EGF is an epidermal growth factor that influences proliferation, differentiation, and self-replication by regulating intracellular signaling pathways, and the concentration of EGF in the culture medium composition can be appropriately selected to efficiently proliferate and differentiate taste bud constituent cells.

[0044] The above Y-27632 (dihydrochloride) is a compound that inhibits the Rho / Rock signaling pathway and has the structure of Chemical Formula 1 below, and the concentration of Y-27632 in the medium composition can be appropriately selected to efficiently proliferate and differentiate taste bud constituent cells.

[0045] [Chemical Formula 1]

[0046]

[0047] In the present invention, the enzyme is used to obtain a single cell of taste bud cells and keratinocytes from an organoid with the extracellular matrix removed, and may be, for example, Tryple, Trypsin, EDTA, or Accutase, and specifically may be Tryple, but is not limited thereto.

[0048] In the present invention, the culture of step (f) may be performed for 1 to 10 days, specifically for 2 to 8 days or 3 to 7 days, and more specifically for 4 to 7 days, but is not limited thereto.

[0049] In the present invention, the culture of step (f) can be performed at 35°C to 45°C, specifically at 35°C to 38°C, but is not limited thereto.

[0050] In the present invention, a step of culturing at 20°C to 30°C after the cultivation of step (f) may be further included, and specifically, a step of culturing at 20°C to 25°C may be further included, but is not limited thereto.

[0051] In the present invention, the surface of the culture vessel may be coated with N-isopropylacrylamide (PIPAAm). At temperatures of 37°C or higher, the coating layer is hydrophobic, so cell attachment and growth are possible, but at temperatures of 32°C or lower, the coating layer changes to hydrophilic, combines with water, and swells, allowing the cells to detach from the vessel.

[0052] In another embodiment for achieving the above objective, the present invention provides a flattened taste bud organoid produced by the above manufacturing method.

[0053] In the present invention, the flattened taste bud organoid may be in a form in which the first to third taste cells and keratinocytes are exposed to the surface of the organoid.

[0054] Specifically, in an embodiment of the present invention, a flattened taste bud organoid was prepared using the above-described manufacturing method, and when the prepared organoid was observed through immunofluorescence staining, it was confirmed that type 1, 2, and 3 taste bud cells and keratinocytes were distributed.

[0055] In another embodiment for achieving the above objective, the present invention provides a cell sheet composition comprising the flattened taste bud organoid.

[0056] In the present invention, the cell sheet material may be formed in the form of a sheet containing a flattened taste bud organoid.

[0057] In the present invention, the cell sheet material reproduces the epithelial layer of the oral cavity and the aggregate layer of taste cells, so that the cellular response to taste stimulation can be confirmed.

[0058] In the present invention, the cell sheet material can be attached to an oral lesion site and used to prevent or treat oral lesions.

[0059] As another embodiment for achieving the above objective, the present invention comprises the step of treating the planarized taste bud organoid with a target substance; and

[0060] A screening method for taste stimulating substances is provided, comprising the step of determining whether the above-mentioned target substance is a taste stimulating substance.

[0061] The flattened taste bud organoid of the present invention is formed from the anterior epithelium of the tongue (including pyloric papillae) containing a group of cells that respond to salty taste, and is not formed from the posterior part of the tongue (including circumvallate papillae and phyllodes papillae) where said group of cells is lacking; therefore, it contains taste bud cells in which a protein capable of detecting salty taste is expressed. Accordingly, when the flattened taste bud organoid of the present invention is used as a cell sensor, screening for various taste stimuli including salty taste is possible.

[0062] In the present invention, it is possible to determine whether the target substance is a taste stimulant by measuring the change in the expression amount of the taste-sensing protein of the target substance treated with the flattened taste bud organoid.

[0063] In the present invention, the taste-sensing protein may include TRPM5, etc., but is not limited thereto.

[0064] In one embodiment of the present invention, the response of the taste bud organoid to the target substance can be determined by measuring the change in cell activity using electrophysiological techniques (e.g., patch clamp), calcium imaging, etc., thereby determining whether the target substance is a taste stimulant. Effects of the invention

[0066] Unlike organoids formed through 3D culture, the flattened taste bud organoid produced by the manufacturing method of the present invention has the taste bud stimulation site exposed, so it can be used for screening taste stimulating substances and studying taste cell expression by reproducing the epithelial layer of oral tissue and the aggregate layer of taste cells and confirming the cellular response to taste stimulation.

[0067] 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 a person skilled in the art from the description below. Brief explanation of the drawing

[0069] A brief description of each drawing is provided to help to better understand the drawings cited in this specification. Figure 1 illustrates the process of fabricating an anterior tongue organoid. Figure 2 shows the results of confirming a flattened taste bud organoid (cell sheet) prepared by the manufacturing method of the present invention (a: appearance of cells immediately after seeding in an upcell dish, b: flattened taste bud organoid formed after culture for 4 to 7 days). Figure 3 shows (a) the detachment from a culture vessel and (b) the cutting into appropriate sizes and immersing in a culture medium of a flattened taste bud organoid prepared by the method of the present invention. Figure 4 shows the results of confirming first to third taste bud cells and keratinocytes distributed in a flattened taste bud organoid prepared by the method of the present invention through immunofluorescence staining (a: confirmation of type 1, 2, and 3 taste bud cells, b: confirmation of keratinocytes). Specific details for implementing the invention

[0070] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0072] Example 1: Preparation of anterior tongue organoid

[0073] A tongue was collected from a mouse, and dispase II enzyme (2 mg / mL) (Roche, cat #4942078001) diluted in physiological saline was injected into the space between the epithelium and the subepithelial connective tissue to induce expansion (② in Fig. 1). The expanded tongue tissue was then cultured in the diluted dispase II enzyme solution at 37°C for 30 minutes. The epithelial layer was exfoliated from the subepithelial connective tissue to separate the epithelial lamellae, which were then finely cut into the smallest possible area using sterile surgical scissors, a scalpel, or other tools in a trypsin / EDTA (Gibco, cat #25200072) solution. The finely cut tissue was cultured at 37°C for 30 minutes. Subsequently, the culture medium of the finely cut tissue was filtered by continuous pipetting, and cells were extracted from the tissue by applying pressure to form a single-cell suspension. The cell pellet obtained by centrifuging this suspension was embedded in an extracellular matrix, such as Matrigel or collagen, in appropriate proportions and cultured. The culture medium was cultured for 4 days in a medium based on the basic culture medium (DMEM / F12 + 1x GlutaMax + 1x HEPES + 1x N2 supplement + 1x B27 supplement + 1x penicillin / streptomycin) to which the first medium composition (20% R-spondin 1, 10% Noggin, 50 ng / mL FGF2, 50 ng / mL IGF1 (BioLegend, cat #590908), 1 μM SAG (Abcam, cat #ab142160), 10 μM Y-27632 (Cayman chemical, cat # Cay10005583-50)) was added, and then the second medium composition (20% R-spondin 1.The medium was replaced with a medium supplemented with 10% Noggin, 50 ng / mL FGF2, 50 ng / mL IGF1, 1 μM SAG, 3 μM CHIR99021 (Sigma, cat #SML1046-5MG), and 10 μM SU5402 (Sigma, cat #SML0443-5MG), and cultured for a total of 7 to 10 days while replacing the medium every 2 to 3 days to obtain a culture containing organoid cell aggregates.

[0075] Example 2: Preparation of flattened taste bud organoids

[0076] The extracellular matrix was removed from the culture obtained in Example 1. When Matrigel was used as the extracellular matrix, cold DMEM / F12 medium (Gibco, #11320033) was added to the culture vessel; when collagen was used, collagenase was added and the culture was treated at 37°C for 15 to 20 minutes to remove the extracellular matrix. Subsequently, Tryple (Gibco, cat #12604013) was added to the culture, and continuous pressure was applied using a glass pipette for at least 5 minutes to rupture the organoid into a single-cell form. Afterward, a single-cell pellet was obtained by centrifugation at 450g RCF and 4°C for 4 minutes and 30 seconds. The pellet was then dispersed in a medium heated to 37°C (a medium to which the first medium composition was added based on the basic culture medium prepared in Example 1) and 1 x 10 6 Canine cells were seeded into upcell dishes surface-coated with PIPAAm. Subsequently, a medium containing 50 ng / mL of EGF and 10 μM of Y27632 was added, and the cells were cultured at 37°C for 4 to 7 days to produce flattened taste bud organoids (cell sheet materials) with a cell confluency (percentage of culture dish surface covered by adherent cells) of 90% or higher (Fig. 2, a: appearance of cells immediately after seeding into the upcell dish, b: flattened taste bud organoid formed after 4 to 7 days of culture).

[0077] Subsequently, the dish containing the culture was transferred to an environment of 20°C to 25°C, and the culture medium was replaced with the basic culture medium prepared in Example 1. After being left for 20 to 30 minutes, the cell sheet material detached from the container, and the cell sheet was separated using a pipette or membrane (Fig. 3, a: detachment of the cell sheet material from the container, b: the sheet cut to an appropriate size and immersed in the culture medium).

[0079] Example 3: Morphological evaluation of flattened taste bud organoids

[0080] The flattened taste bud organoid prepared in Example 2 was observed via immunofluorescence analysis. Specifically, the sheet desorbed in Example 2 was immersed in a 4% PFA solution and left at room temperature for 10 minutes. Subsequently, it was washed three times by immersing it in a 0.2% PBST washing solution and leaving it at room temperature for 5 minutes. A blocking solution was prepared by adding 5% donkey serum to PBST, and the sheet was immersed in it and left at room temperature for 30 minutes. Afterward, a primary antibody for detecting taste cells was added to a new blocking solution, the sheet was placed in it, and it was left at 4°C for 24 hours. After immersing it in a 0.2% PBST washing solution and washing it three times at room temperature for 10 minutes, a secondary antibody was added to the blocking solution, the sheet was placed in it, and it was left at room temperature in a light-blocked environment for 2 hours. After washing, the sheet was spread onto a slide using microforceps, mounting solution was applied, and the sheet was covered with a coverslip. The immunofluorescence-stained sheet was then examined using a confocal microscope.

[0081] As a result, it was confirmed to be a monolayer cell sheet containing differentiated type 1, 2, and 3 taste cell groups and keratinocyte groups (Fig. 4, a: confirmation of type 1, 2, and 3 taste bud cells, b: confirmation of keratinocytes).

[0083] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A method for preparing a flattened taste bud organoid comprising the following steps: (a) culturing anterior tongue epithelial cells embedded in an extracellular matrix to obtain a culture containing a taste bud organoid; (b) removing the extracellular matrix from the culture; (c) adding an enzyme to the culture from which the extracellular matrix has been removed and applying physical pressure to obtain a single cell; (d) suspending a single-cell pellet obtained by centrifuging the culture containing the single cell in a medium to obtain a suspension; (e) seeding the suspension into a culture vessel whose surface is coated with poly(N-isopropylacrylamide) (PIPAAm). and (f) a step of adding a medium containing growth factors to the culture vessel and culturing to obtain a flattened taste bud organoid, wherein the flattened taste bud organoid is in the form of a single-layer cell sheet in which type 1, type 2, and type 3 taste cells and keratinocytes are exposed on the surface of the organoid. Claim 2 A method of manufacturing according to claim 1, wherein the anterior tongue epithelial cells are obtained by treating tongue tissue with a dispase enzyme to separate epithelial lamellae and extracting cells therefrom. Claim 3 A method for manufacturing, wherein the culture of step (a) is performed by sequentially culturing the anterior tongue epithelial cells in a first medium composition comprising R-spondin 1, Noggin, FGF2, IGF1, SAG (Smoothed agonist), and Y-27632, and a second medium composition comprising R-spondin 1, Noggin, FGF2, IGF1, SAG (Smoothed agonist), CHIR99021, and SU5402. Claim 4 A method for manufacturing, wherein in the case where the extracellular matrix of step (b) is Matrigel, the culture is treated with cold DMEM / F12 medium to remove the extracellular matrix. Claim 5 A method of manufacturing according to claim 1, wherein if the extracellular matrix of step (b) is collagen, the culture is treated with collagenase to remove the extracellular matrix. Claim 6 A manufacturing method according to claim 5, further comprising the step of incubating at 37°C for 10 to 30 minutes after treating the collagenase. Claim 7 In claim 1, the cells in the suspension of step (e) are 1 x 10 4 Pieces up to 1 x 10 8 Individual, manufacturing method. Claim 8 A method of manufacturing according to claim 1, wherein the growth factor is one or more selected from EGF and Y27632. Claim 9 A method of preparation according to claim 1, wherein the enzyme is Tryple, Trypsin, EDTA, or Accutase. Claim 10 A manufacturing method according to claim 1, wherein the culture of step (f) is performed for 1 to 10 days. Claim 11 A manufacturing method according to claim 1, wherein the culture of step (f) is performed at 35℃ to 45℃. Claim 12 A manufacturing method according to claim 11, further comprising the step of culturing at 20°C to 30°C after the cultivation of step (f). Claim 13 A flattened taste bud organoid manufactured by the method of any one of claims 1 to 12, wherein the organoid is a flattened taste bud organoid in which type 1, type 2, and type 3 taste cells and keratinocytes are exposed on the surface of the organoid. Claim 14 delete Claim 15 A cell sheet preparation comprising the flattened taste bud organoid of claim 13. Claim 16 A screening method for taste stimulants comprising: a step of treating a target substance in a flattened taste bud organoid of claim 13; and a step of determining whether the target substance is a taste stimulant. Claim 17 A screening method for taste stimulating substances according to claim 16, wherein the above determination is made by measuring the change in the expression amount of a taste-sensing protein of a target substance treated with a flattened taste bud organoid.

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