KR20240120263A - Producing method of esophageal organoid and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing esophageal organoids and their use. It provides a medium and a production method for producing esophageal organoids that reproduce the stratified squamous epithelium of the esophagus, and utilizes the medium for long-term passage of esophageal organoids. It enables cultivation and is used as a drug processing platform.

Description

Method for producing esophageal organoids and uses thereof {PRODUCING METHOD OF ESOPHAGEAL ORGANOID AND USES THEREOF}

The present invention relates to methods for producing esophageal organoids and their uses.

The esophagus is an animal organ that extends from the pharynx to the stomach, and passes food that enters the pharynx to the stomach through peristalsis. In particular, the wall of the esophagus surrounds the lumen and is composed of a mucosa, a submucosa, two muscular layers made of muscle fibers (muscularis propria), and an adventitia made of connective tissue. The mucous membrane is a stratified squamous epithelium structure composed of several layers of squamous epithelial cells. The basal cells are located at the bottom, and the top is composed of differentiated cells made up of several layers of flat thin cells. .

Meanwhile, the esophagus is prone to damage and disease due to esophagitis, achalasia, esophageal cancer, etc., so it can be of great help in the development of regenerative medicine treatments, and it is necessary to verify the effectiveness and screen for side effects when treating various drugs. Do this.

Organoids are three-dimensional mini structures (hundreds of micrometers to several millimeters) formed through self-renewal and differentiation from stem cells. They are also called mini organs because they reproduce the histological characteristics and physiological functions of the mother organ. Therefore, the development of esophageal organoids that reproduce the stratified squamous epithelium, the histological structure of the esophagus, will greatly contribute to the study of esophageal disease mechanisms, disease modeling, and evaluation of drug effectiveness and toxicity.

US published patent US 2021-0189349 A1 (published on June 24, 2021)

The purpose of the present invention is to provide a medium composition for culturing esophageal organoids.

Another object of the present invention is to provide an esophageal organoid with a stratified squamous epithelium structure and a method for producing the same.

Another object of the present invention is to provide a method for long-term subculture of esophageal organoids with a stratified squamous epithelial structure.

Another object of the present invention is to provide a method for screening anticancer drug toxicity using the esophageal organoids.

In order to achieve the above object, the present invention provides a basic medium for stem cell culture containing epidermal growth factor (EGF), R-spondin1, and noggin. Provided is a medium composition for culturing esophageal organoids having a stratified squamous epithelium structure containing the active ingredient.

In addition, the present invention includes the steps of a) extracting basal cells from isolated esophageal epithelial tissue; b) adding the medium composition to the basal cells extracted in step a) to suspend them and mixing them with matrigel; and c) culturing the mixed mixture and supplying the medium composition when the Matrigel hardens.

Additionally, the present invention provides an esophageal organoid having a stratified squamous epithelial structure prepared according to the above method.

In addition, the present invention provides a long-term subculture method of esophageal organoids with a stratified squamous epithelial structure, comprising the step of disassembling the prepared esophageal organoids into single cells and culturing them every 10 to 20 days.

In addition, the present invention includes the steps of disassembling the esophageal organoid into single cells and isolating esophageal epithelial cells; Culturing the esophageal epithelial cells in the medium composition; and treating the cultured culture with a candidate drug and then analyzing whether esophageal organoids are formed.

The present invention relates to a method for producing esophageal organoids and their uses. The produced esophageal organoids are stratified squamous epithelium histologically very similar to the esophagus by three-dimensionally culturing esophageal epithelial cells containing esophageal stem cells. Due to its structure, it can be used as a model that can replace cell and animal experiments for disease modeling and drug screening. In addition, the esophageal organoids allow stable long-term subculture, allowing a large amount of cells to be obtained from a small amount of tissue.

Figure 1 shows an overview of the esophageal organoid production method system.
Figure 2 shows the results of culturing esophageal organoids in esophageal organoid culture medium (A) and the results of subculture (B and C).
Figure 3 shows the results of histological confirmation of the stratified squamous epithelial structure of esophageal organoids through H&E staining.
Figure 4 shows the results of confirming the expression pattern of epithelial cell markers in esophageal organoids.
Figure 5 shows the results of confirming the use of esophageal organoids for drug screening.

The present inventors sought to develop regenerative medicine treatments for damage and disease caused by esophagitis, achalasia, esophageal cancer, etc., which are derived from esophageal epithelial cells like the esophagus and have a structure similar to stratified squamous epithelium. The present invention has been completed.

The present invention is a stratified squamous cell comprising basal media for stem cell culture containing epidermal growth factor (EGF), R-spondin1, and noggin as active ingredients. Provided is a medium composition for culturing esophageal organoids with an epithelial structure.

Preferably, the composition includes Wnt-3a, B27, N2, N-acetylcysteine, glutamax, Y-27632, HGF, FGF, VEGF, antibiotic-antifungal agent and Neureglin. ) may further include any one or more selected from the group consisting of, but is not limited thereto.

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

Preferably, the antibiotic-antifungal agent may be two or more selected from the group consisting of penicillin, streptomycin, gentamicin, nystatin, and amphotericin, but is limited thereto. It doesn't work.

Preferably, the esophageal organoid may be capable of continuous subculture, but is not limited thereto.

As used herein, “organoid” refers to a cell aggregate with a 3D three-dimensional structure derived from esophageal epithelial cells containing stem cells obtained from humans, animals, etc., and is a primary organ manufactured through an artificial culture process. It refers to a model similar to . The origin of the cells constituting it is not limited.

As used herein, “esophagus” refers to an organ composed of the esophageal mucosa including the esophageal epithelium, the submucosa, two muscle layers made of muscle fibers, and an outer membrane made of connective tissue.

As used herein, “esophageal organoid” refers to esophageal organoids that are derived from esophageal epithelial cells, such as the esophagus with a stratified squamous epithelium structure, and include esophageal organoids that exhibit a structure similar to stratified squamous epithelium.

As used herein, “medium” refers to a culture medium that contains the necessary components for cells to grow, survive, and differentiate into organoids in vitro, and that enables this, and is used in the organoid field. and all conventional media suitable for differentiation. Depending on the type of cell or the technical level of the field, the type of medium and culture conditions can be changed. The medium used for esophageal organoid culture may generally be a minimal culture medium containing a carbon source, a nitrogen source, and trace elements.

As used herein, “passage” means separating organoids into single cells or organoids in order to continuously culture them in a healthy state for a long period of time and then transferring them to a new culture vessel to continue culturing the organoids. Dividing a cell group or changing a culture vessel once is called the first passage.

In addition, the present invention includes the steps of a) extracting basal cells from isolated esophageal epithelial tissue; b) adding the medium composition to the basal cells extracted in step a) to suspend them and mixing them with matrigel; and c) culturing the mixed mixture and supplying the medium composition when the Matrigel hardens.

Preferably, the esophageal organoids may have an average particle diameter of 50 to 500 μm, but are not limited thereto.

Preferably, the esophageal organoid contains one or more basal cell markers selected from the group consisting of cytokeratin 14, cytokeratin 5, cytokeratin 1, and loricrin, or Differentiated cell markers may be expressed, but are not limited thereto.

“Differentiation” within this specification refers to a phenomenon in which the structure or function of a cell is specified in a tissue-specific manner while the cell divides, proliferates, and grows. In other words, it refers to the process by which biological cells, tissues, etc. acquire the appropriate form and function to perform the role required for a specific organ. For example, not only the process by which esophageal stem cells transform into esophageal epithelial cells, but also the process by which hematopoietic stem cells become red blood cells, The process of changing into white blood cells, platelets, etc., that is, everything that causes progenitor cells to express specific differentiation traits can be included in differentiation.

Additionally, the present invention provides an esophageal organoid having a stratified squamous epithelial structure prepared according to the above method.

In addition, the present invention provides a method for long-term subculture of esophageal organoids with a stratified squamous epithelial structure, comprising the step of disassembling the esophageal organoids into single cells and culturing them every 10 to 20 days.

In addition, the present invention includes the steps of disassembling the esophageal organoid into single cells and isolating esophageal epithelial cells; Culturing the esophageal epithelial cells in the medium composition; and treating the cultured culture with a candidate drug and then analyzing whether esophageal organoids are formed.

Preferably, the anticancer drug may be one or more selected from the group consisting of cisplatin, paclitaxel, 5-fluorouracil (5-FU), and irinotecan, but is not limited thereto.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

< Example 1> Esophagus of organoids Formation medium preparation

The esophageal organoid culture medium includes the following composition. DMEM/F12 or Advanced DMEM/F12 with 1X penicillin-streptomycin, 1X N2, 1X B27, 1X glutamax, 1 μM N-acetylcysteine, 1 μM Y- 27632, 50 ng/ml EGF, 100 μg/ml R-spondin1 and 100 μg/ml noggin, 20 ng/ml HGF, 20 ng/ml FGF, 50 ng/ml Wnt3a and 20 ng/ml Neureglin was mixed, and the concentration of these components can be adjusted between 1/10 and 10 times the suggested concentration, and other components can be added.

< Example 2> 3D esophagus of humans and animals organoid manufacturing

1. Esophagus from human and animal esophageal tissue basal cell separation

After thoroughly washing a patient's esophagus specimen or an animal's esophagus with phosphate buffered saline (PBS), cut one wall of the empty tube lengthwise and flatten it. Dispase (Stem cell, #07913) was added to DMEM (Dulbecco's modified eagle medium; Gibco, #12430112) in a flat open tube, and incubated at 37°C in a cell incubator supplied with 5% carbon dioxide for 1 to 2 hours. Enzymatically dissociated.

To isolate epithelial cells from enzymatically dissociated human and animal esophageal tissue, only the esophageal epithelial tissue was removed using forceps while observing under a stereomicroscope. The cleanly peeled epithelial tissue was placed in 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.

Enzymatically dissociated human and animal esophageal epithelial tissue was loosened well by pipetting to extract esophageal basal cells, and then PBS containing 2% fetal bovine serum (FBS) was added and 0.25% trypsin/0.05% EDTA. was neutralized. Afterwards, the basal cells separated from the epithelial tissue were passed through a 40 μm cell strainer (SPL, #93040), centrifuged for 5 minutes, and the supernatant was removed. The centrifuged esophageal basal cells were suspended using Advanced DMEM/F12 medium, and the cell number was measured.

2. 3D view of the esophagus organoid produce

Esophageal organoids were produced by three-dimensionally culturing the esophageal basal cells extracted above for 7 to 14 days. Specifically, 1,000 to 10,000 esophageal basal cells were suspended in 50 μl of esophageal organoid culture medium, mixed with Matrigel (Coring, #356231) at a 1:1 ratio, and i) placed in a 24-well plate in a dome shape. After dispensing, culture for 30 to 60 minutes in a cell incubator supplied with 5% carbon dioxide at 37°C, and when Matrigel hardens, add esophageal organoid culture media; or ii) After dispensing onto a 24-well cell culture transwell (Corning, #3470), culture it for 30 to 60 minutes at 37°C in a cell incubator supplied with 5% carbon dioxide. When the Matrigel hardens, an esophagus is placed at the bottom of the cell culture transwell. Organoid culture medium was supplied.

As a result, as shown in Figures 2A and 2B, rat esophageal organoids grew to a size of 50 to 200 μm in the esophageal organoid culture medium.

3. Esophagus of organoids long time subculture

The esophageal organoids produced above can be grown by subculturing several times over a long period of time. Esophageal organoids grown in esophageal organoid culture medium were disassembled into single cells every 14 days and subjected to long-term subculture. Specifically, dispase (Stem cell, #07913) was added to DMEM (Dulbecco's modified eagle medium; Gibco, #12430112) and incubated with Matrigel for 1 to 2 hours in a cell incubator supplied with 5% carbon dioxide at 37°C. The organoids generated within were enzymatically dissociated. After trypsin treatment, the above process was repeated to form the next generation of organoids. The results of long-term subculture are as shown in Figures 2C and 2D.

< Experiment example 1> Esophagus of organoids Confirmation of structural and histological biomimeticity

The esophagus has a stratified squamous epithelial structure composed of one or more epithelial cell layers on the upper part of the lamina propria. In the basal layer, there are actively dividing basal cells with strong stem cell characteristics, and cells divided and created from the basal cells move to the upper layer and differentiate. As it approaches the surface, it shows structural characteristics of increasingly differentiated, flat, keratin-rich cells.

In order to confirm whether the esophageal organoid produced in Example 2 reproduced the histological structure of the actual esophagus, H&E staining and immunofluorescence staining were performed.

1. H&E staining

After fixing the esophageal organoids in 4% paraformaldehyde solution (fromaldehyde solution, Sigma, #47608), the esophageal organoids were O.C.T. in a cryo mold (15ⅹ15ⅹ5 mm, Tissue-tek, #4566). It was mixed with compound (O.C.T. compound, Scigen, #4583) and solidified using dry ice. Afterwards, the solidified esophageal organoids were cut to a thickness of 10 μm using a frozen tissue slicer (Leica, #CM3050S) to prepare slides and stored at -20°C. The prepared slides were left at room temperature for 5 minutes, washed lightly with PBS, and then H&E stained using a hematoxylin and eosin stain kit (Vector laboratories, #H-3502). Specifically, staining with hematoxylin for 5 minutes at room temperature, washing twice with distilled water for 15 seconds, staining with bluing reagent for 10 to 15 seconds at room temperature, and washing twice with distilled water for 15 seconds. , immersed in 100% ethanol for 10 seconds, stained with eosin at room temperature for 2 to 3 minutes, washed with 100% ethanol for 10 seconds, and dehydrated in 100% ethanol three times for 1 to 2 minutes. Afterwards, it was covered, stored at room temperature, and observed under a microscope.

As a result, as shown in Figure 3, a structure that seemed to be surrounded by growth rings of the stratified squamous epithelium of the esophagus was always observed in H&E-stained esophageal organoids.

2. Immunofluorescent staining

After fixing the esophageal organoids in 4% paraformaldehyde solution (fromaldehyde solution, Sigma, #47608), the esophageal organoids were O.C.T. in a cryo mold (15 × 15 × 5 mm, Tissue-tek, #4566). It was mixed with compound (O.C.T. compound, Scigen, #4583) and solidified using dry ice. Afterwards, the solidified esophageal organoids were cut to a thickness of 10 μm using a frozen tissue slicer (Leica, #CM3050S) to prepare slides and stored at -20°C. The prepared slides were left at room temperature for 5 minutes, washed lightly with PBS, and then washed with 0.1% Triton , #9048-46-8) was blocked at room temperature for 1 hour. Afterwards, the slides were incubated overnight at 4°C with primary antibodies diluted in antibody diluent solution (REAL antibody diluent solution, Agilent, #S2022). Slides were washed three times using PBS containing 0.1% Triton did. After washing three times using PBS, the slide was incubated with DAPI (Sigma, #10236276001) solution diluted in PBS for 10 minutes at room temperature. The slides were washed three times using PBS and observed under a fluorescence microscope.

As a result, according to Figure 4, it was confirmed that keratin 5, a marker of the esophageal basal layer, and loricrin, a marker of the suprabasal layer of differentiated cells, were stained at different locations in the esophageal organoid. did. This indicates that the esophageal organoid structure shows a basal layer and suprabasal layer structure similar to the esophageal epithelium.

< Experimental example 2> Esophagus Organoids Confirmation of usability as a drug screening platform

We examined the use of esophageal organoids as a drug toxicity evaluation platform. Esophageal epithelial cells isolated into single cells were cultured in organoid formation medium as described in Example 2 above. Thereafter, the cells were treated with the anticancer drugs Cispatin and Paclitaxel at concentrations of 1 to 5 μM and 1 to 10 nM, respectively. Drug treatment was continued throughout the organoid formation period, and the medium was renewed once every 2 to 3 days.

As a result, according to Figure 5, when the drug was treated, the formation of esophageal organoids was observed to decrease, confirming that esophageal organoids can be used for drug effectiveness and toxicity evaluation and screening.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (12)

A stratified squamous epithelial structure containing basal media for stem cell culture containing epidermal growth factor (EGF), R-spondin1, and noggin as active ingredients. A medium composition for culturing esophageal organoids. The method of claim 1, wherein the composition includes Wnt-3a, B27, N2, N-acetylcysteine, glutamax, Y-27632, HGF, FGF, VEGF, antibiotic-antifungal agent, and neuregulin. A medium composition characterized in that it further comprises at least one selected from the group consisting of (Neureglin). In claim 1, the basal media for stem cell culture is a medium composition characterized in that any one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM/F12, and Advanced DMEM/F12. The method of claim 2, wherein the antibiotic-antifungal agent is at least two selected from the group consisting of penicillin, streptomycin, gentamicin, nystatin, and amphotericin. A medium composition that does. The medium composition according to claim 1, wherein the esophageal organoids are capable of continuous subculture. a) extracting basal cells from isolated esophageal epithelial tissue;
b) adding the medium composition according to any one of claims 1 to 5 to the basal cells extracted in step a) to suspend them and then mixing them with matrigel; and
c) culturing the mixed mixture and supplying the medium composition according to any one of claims 1 to 5 when Matrigel hardens; Method for producing esophageal organoids with a stratified squamous epithelium structure comprising a. The method of claim 6, wherein the esophageal organoids have an average particle diameter of 50 to 500 μm. The method of claim 6, wherein the esophageal organoid is one or more basal cells selected from the group consisting of cytokeratin 14, cytokeratin 5, cytokeratin 1, and loricrin. A method characterized by expressing a marker or differentiated cell marker. An esophageal organoid having a stratified squamous epithelial structure prepared according to the method of any one of claims 6 to 8. A long-term subculture method of esophageal organoids with a stratified squamous epithelial structure, comprising the step of disassembling and culturing the esophageal organoids according to claim 9 into single cells every 10 to 20 days. A step of disassembling the esophageal organoid according to claim 9 into single cells and isolating esophageal epithelial cells;
Culturing the esophageal epithelial cells in the medium composition according to any one of claims 1 to 5; and
An anticancer drug toxicity screening method comprising the step of treating the cultured culture with a candidate drug and then analyzing whether esophageal organoids are formed. The screening method according to claim 11, wherein the anticancer drug is at least one selected from the group consisting of cisplatin, paclitaxel, 5-fluorouracil (5-FU), and irinotecan.