KR102547900B1 - Preparation method of lacrimal gland and use thereof - Google Patents

Abstract

The present invention relates to a method for preparing lacrimal gland organoids. In addition, the present invention relates to a lacrimal gland organoid prepared by the above preparation method, a preparation for preventing or treating dry eye syndrome containing the lacrimal gland organoid, and a screening method for a substance for preventing or treating dry eye syndrome using the lacrimal gland organoid. .
The lacrimal gland organoid prepared according to the present invention has high tissue similarity to human lacrimal tissue and excellent tear secretion ability, and in particular, when transplanted into a living body, it can regenerate damaged lacrimal tissue to normalize tear secretion ability. Therefore, the method for preparing lacrimal organoids according to the present invention can be used to prepare lacrimal organoids having a similar shape and function to living tissue, and the prepared lacrimal organoids can be used to treat dry eye symptoms such as Sjögren's syndrome and dry cornea. It can be used as a treatment substance for, or can be usefully utilized as a method of screening the treatment substance for dry eye syndrome.

Description

Manufacturing method of lacrimal gland organoid and its use {PREPARATION METHOD OF LACRIMAL GLAND AND USE THEREOF}

The present invention relates to a method for preparing lacrimal gland organoids. In addition, the present invention relates to a lacrimal gland organoid prepared by the above preparation method, a preparation for preventing or treating dry eye syndrome containing the lacrimal gland organoid, and a screening method for a substance for preventing or treating dry eye syndrome using the lacrimal gland organoid. .

Dry eye disease (Dry eye disease) is a relatively common disease suffered by about 11 to 22% of the world's population, half of which are severe dry eye disease patients with structural and functional damage to the lacrimal gland. Dry eye syndrome is defined as a multifactorial disease of the ocular surface mainly caused by physical damage and functional disturbance of the lacrimal gland and characterized by a lack of tear film stability. In addition, it is accompanied by symptoms such as tear film instability, hyper osmolarity, ocular surface inflammation and damage, and neurosensory abnormalities. The primary cause of dry eye syndrome is trauma or dysfunction of the lacrimal gland, and systemic autoimmune diseases such as Sjogren's syndrome, rheumatoid arthritis, and systemic lupus erythematosus are also considered secondary causes of dry eye syndrome. there is.

On the other hand, the lacrimal gland, an organ that produces tears, is composed of three types of cells: acini cells, ductal cells, and myoepithelial cells. The acinar cells are filled with secretory granules and produce tears. It is composed of serous cells that produce the back. Myoepithelial cells surround the basal surface of acinar cells and pancreatic duct cells, and these cells exert pressure on the acinar cells to expel fluid into the duct. These lacrimal glands can be damaged for a variety of reasons. For example, it is known that it is also damaged by radiation for cancer treatment (S S Batth et al., Br J Radiol. 2013 Dec, 86(1032): 20130459.), and dry eye syndrome caused by damage to the tear gland is cured. It is difficult, and in severe cases, the cornea may dry out and severely deteriorate vision.

As a currently developed treatment method for dry eye syndrome, artificial tear eye drops that supply moisture and additional lubrication to the ocular surface are used, and anti-inflammatory drugs and topical immunosuppressants are also used to solve chronic inflammatory symptoms. However, these steroid-based drugs used as therapeutic agents have short-term effects, but have severe side effects and no efficacy in completely damaged lacrimal tissue.

Accordingly, an organoid preparation capable of regenerating damaged lacrimal glands has emerged as a new strategy. Organoids are aggregates of cells differentiated from specific stem cells and can be used for regeneration of damaged tissues because they have structural and histological characteristics very similar to actual tissues in the body. In particular, since organoids can be cultured in vitro while maintaining tissue characteristics, they are highly anticipated in the art as they can replace cell and animal experiments.

In this regard, in the art, research is being actively conducted to utilize organoids having a form similar to actual tissues. For example, a study using mesenchymal stem cells through two-dimensional culture as a treatment for lacrimal gland regeneration has been conducted (Samantha You et al., Invest Ophthalmol Vis Sci. 2011 Apr 4, 52(5):2087-94 .). However, the therapeutic effect of the manufactured stem cell therapy for dry eye syndrome has not yet been verified.

Accordingly, the present inventors conducted various studies to develop lacrimal gland organoids that treat dry eye syndrome by regenerating damaged lacrimal tissue. As a result, a method for producing human lacrimal gland organoids was developed through 3-dimensional culture using Matrigel and a medium in which specific culture components were combined, and the organoids prepared by the method have a similar tissue shape to actual lacrimal tissue, The present invention has been completed as it has been experimentally proven that the tear secretion ability is similar and that damaged tear tissue can be regenerated, especially when transplanted in vivo.

Each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

In addition, terms not specifically defined in this specification should be understood to have meanings commonly used in the technical field to which the present invention belongs. In addition, unless otherwise defined in context, singular includes plural, and plural includes singular.

One aspect of the present invention provides a method for preparing a lacrimal gland organoid comprising the following steps: a) isolating cells from lacrimal tissue isolated from a subject; and b) culturing the cells isolated in step a) in Matrigel.

The term "organoid" refers to a cell mass having a 3D three-dimensional structure. It is defined as a reduced and simplified version of an organ that is manufactured through an artificial culture process without collection, acquisition, or collection from animals, etc.

Step a) is a step of isolating cells from the lacrimal gland tissue isolated from the subject, and is a step of obtaining lacrimal gland-derived stem cells.

The term "individual" includes all subjects who have never had dry eye syndrome, those who are likely to have it, those who have it, or those who have been cured after it has occurred, and may include, without limitation, humans or any non-human animals. can The non-human animals may be vertebrates such as primates, dogs, cows, horses, pigs, rodents such as mice, rats, hamsters, guinea pigs, and the like. In this specification, the "individual" may be used interchangeably with "subject" or "patient".

The cells separated from the lacrimal tissue may be separated through a process of cutting the lacrimal tissue and an enzymatic degradation process. Specifically, the cutting includes both physical cutting and mechanical cutting, and may be performed by a general tissue cutting method known in the art. In addition, the enzymatic degradation may be performed under general enzymatic degradation conditions known in the art, for example selected from the group consisting of dispase Ⅱ, DNase Ⅰ and collagenase Ⅱ This can be done using one or more enzymes.

Step b) is a step of culturing the cells isolated in step a) in Matrigel, and by culturing the lacrimal gland-derived cells or stem cells in a three-dimensional manner using Matrigel, lacrimal gland organoids having a tissue-like shape are formed. It is a step to

The term "matrigel" refers to a protein complex (product name of BD Bioscience) extracted from sarcoma cells of EHS (Engelbreth-Holm-Swarm) mice. The matrigel is an extracellular matrix (ECM) such as laminin, collagen, heparan sulfate proteoglycan, and fibroblast growth factor (FGF), epithelial cells Epiderma growth factor (EFG), insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), platelet-derived growth factor factor; PDGF).

In the present invention, the matrigel may be attached to the culture plate and have a dome shape. When the organoid or Matrigel containing the same is attached to a culture plate and cultured, it has various advantages in terms of culture efficiency compared to floating culture without attachment. For example, in the case of long-term culture of 5 days or more, it is essential to replace the culture medium every 2 to 3 days. In the case of floating culture, organoids are highly likely to be lost when the medium is replaced, but they are attached to the culture plate and fixed. In this case, there is no loss of organoids during medium replacement, and the medium replacement time can be shortened.

In the present invention, the culture is Wnt, R-Spondin, FGF-2, N2, Nicotinamide, A83-01, Noggin, EGF, Insulin, Dexametasone ), Y-27632, and DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing N-Acetyl-L-cysteine.

Conventional techniques in the art for organoid production methods use at least two types of culture media having different components. For example, in the initial stage of culture, a growth medium for growing the size of organoids was used, and then, after the growth medium was removed, a differentiation medium for differentiating organoids into specific tissues was used. However, conventional methods for preparing organoids using two or more types of culture media having different components have disadvantages such as high cost and complicated manufacturing processes.

In addition, due to the nature of organoids, the production results vary significantly depending on the culture conditions. As an example of culture conditions, culture conditions such as culture day, culture temperature, culture medium, and culture substrate are important factors in determining the characteristics of organoids. It plays the biggest role in growing to have characteristics similar to the desired tissue or organ.

In the present invention, it was confirmed that organoids cultured in different media conditions show very different characteristics in terms of similarity to real tissues or viability, and are very similar in shape and function to real tissues, and in particular, have increased viability, resulting in therapeutic substances. Alternatively, an optimal culture medium condition capable of obtaining a sufficient number of organoids that can be utilized for screening of a therapeutic substance was established. Furthermore, when using the above culture medium, it was confirmed that growth and differentiation of organoids could be successfully induced even when only one kind of culture medium was used instead of two or more kinds of culture medium.

Specifically, the method for preparing lacrimal gland organoids according to the present invention includes Wnt, R-spondin, FGF-2, N2, nicotinamide, A83-01, Noggin, EGF, insulin, dexamethasone, Y -27632, a culture medium containing N-acetyl-L-cysteine may be used. The Wnt may be obtained from Wnt conditioned media, or Wnt conditioned media. In addition, the R-spondin may be obtained from an R-spondin conditioned medium, or may be R-spondin.

Preferably, Wnt may be used at 40 to 60 v/v%, more specifically at 45 to 55 v/v%, and most specifically at 50 v/v%; R-spondin may be used at 1 to 20 v/v%, more specifically at 5 to 15 v/v%, and most specifically at 10 v/v%; FGF-2 may be used at 10 to 30 ng/ml, more specifically 15 to 25 ng/ml, most specifically 20 ng/ml; N2 may be used from 0.1 to 2X, more specifically from 0.5 to 1.5X, and most specifically from 1X; nicotinamide may be used at 1 to 20 mM, more specifically at 5 to 15 mM, and most specifically at 10 mM; A83-01 can be used at 300 to 700 nM, more specifically at 400 to 500 nM, and most specifically at 500 nM; Noggin can be used at 1 to 200 ng/ml, more specifically 50 to 150 ng/ml, and most specifically 100 ng/ml; EGF may be used at 10 to 30 ng/ml, more specifically 15 to 25 ng/ml, and most specifically 20 ng/ml; Insulin can be used at 1 to 20 μg/ml, more specifically 5 to 15 μg/ml, most specifically 10 μg/ml; dexamethasone may be used at 0.1 to 2 μM, more specifically 0.5 to 1.5 μM, most specifically 1 μM; Y-27632 can be used at 1 to 20 μM, more specifically 5 to 15 μM, most specifically 10 μM; and N-acetyl-L-cysteine at 100 to 1,000 μM, more specifically 250 to 750 μM, most specifically 500 μM.

In addition, the culture of step b) may be performed in 1 to 19 passages.

The term "passage" refers to a process of transferring some or all of the cells in culture or organoids formed therefrom to a new culture environment (plate), and is performed to obtain organoids in a desired amount. As the number of passages increases, a larger amount of organoids with the same characteristics can be obtained, but since the cells may be damaged during the process of transferring the organoids to a new culture plate, an organoid culture method capable of increasing the number of passages development was urgently needed. In the present invention, it was confirmed that organoids could be cultured from 1 to 19 passages without damaging the organoids when using the above-described medium conditions.

Specifically, the first passage may be performed for 1 to 20 days. During the culture period of 1 to 20 days, the size increases, and at the same time, it has histological characteristics similar to real tissues, and eventually it is possible to perform functions similar to real tissues.

In the present invention, the lacrimal gland organoids are lysozyme (LYZ), alpha-smooth muscle actin (α-SMA), aquaporin 5 (AQP5), E-cadherin (E-CAD) And it may be to specifically express one or more selected from the group consisting of Vimentin (VIM). The lysosome, α-SMA, aquaporin 5, E-cadherin, and vimentin proteins are proteins specifically observed during differentiation and development of lacrimal tissue from stem cells, and are used as markers of lacrimal gland organoids (Zaki Hakami et al., Eur J Oral Sci. 2020 Oct, 128(5):379-385; Shubha Tiwari et al., PLoS One. 2012, 7(1):e29458.).

In addition, the lacrimal gland organoids may include secretory granules.

The term "secretory granules" refers to organelles that store secreted substances and control secretion, mainly neuroendocrine cells, granulocytes, a type of white blood cell, and acinar cells present in lacrimal glands (acini). It is known to exist in cells such as cell) and serous cell. Specifically, the secretory granules may produce tears, store them, and secrete them.

In addition, the lacrimal gland organoid may increase Ca ²⁺ absorption, β-hexosaminidase expression, or secretome secretion when treated with a substance for preventing or treating dry eye syndrome. At this time, specific examples of the substance for preventing or treating dry eye syndrome may be at least one selected from the group consisting of pilocarpine, cevimeline, ciclosporin, and rebamipid. , as long as it is used for the treatment of dry eye syndrome, but is not limited thereto.

Specifically, in order to secrete tears from the lacrimal gland, first, Ca ²⁺ present outside the cell is absorbed into the cell through the Ca ²⁺ ion channel of the cell membrane constituting the lacrimal gland. This Ca ²⁺ absorption and concentration increase into the cell induces tear secretion, but dry eye patients such as Sjogren's syndrome do not have Ca ²⁺ channels in the cell membrane, so the Ca ²⁺ signaling pathway is not properly performed. (Imada T et al., Sci Rep. 2017, 7(1):6965; Garcia-Posadas L et al., Am J Pathol. 2020, 190(10):2067-2079.; Imada T et al. ., Sci Rep. 2017;7(1):6965.). In addition, beta-hexosaminidase, a type of lysosomal protein, is known to be mainly contained in lysosomes of tears or blood plasma, and serves to protect the eye by decomposing pathogens or toxic substances on the ocular surface. (Sofia V Andersson et al., Glycobiology. 2005 Mar, 15(3):211-20.). In addition, the secretome is a set of proteins expressed in cells and secreted into the extracellular space, and is known to include cytokines, growth factors, junction molecules, proteases, extracellular matrix proteins, and the like. The secretome of the present invention is a protein constituting extracellular organelles, exosomes, vesicles, and the like, and may be a set of proteins involved in tear production.

In the present invention, the lacrimal gland organoid may be for preventing or treating dry eye syndrome.

The term "dry eye syndrome" refers to a disease caused by lack of tears or excessive evaporation of tears, in which the surface of the eyeball is easily damaged because tears do not dry or flow. Dry eye due to tear gland damage, dry eye due to damage to the ocular surface, dry eye due to aging, dry eye due to irradiation, dacryocystitis, dacryocystitis, lacrimal stenosis, Sjogren's syndrome, alacrima , Keratoconjunctivitis sicca, Stevens Johnson syndrome, and graft versus host disease, but not limited to one or more selected from the group consisting of, but not limited thereto, if the disease causes dry eye all included

Another aspect of the present invention provides a lacrimal gland organoid prepared by the above production method.

Another aspect of the present invention provides a preparation for preventing or treating dry eye syndrome, including the lacrimal gland organoid prepared by the above preparation method.

Another aspect of the present invention provides a method for preventing or treating dry eye syndrome, comprising transplanting the lacrimal gland organoid prepared according to the above method to a subject.

In the lacrimal gland organoid, preparation for preventing or treating dry eye syndrome, and method for preventing or treating dry eye syndrome according to the present invention, each term has the same meaning as described in the method for preparing the lacrimal gland organoid, unless otherwise specified.

The lacrimal gland organoid according to the present invention has a histological composition and function very similar to that of the human lacrimal gland. Therefore, since it can regenerate damaged lacrimal glands when administered in vivo, it can be used as a preventive or therapeutic agent related to lacrimal gland damage, such as dry eye syndrome, and can also be used as an alternative to animal models of lacrimal gland-related diseases.

The term "treatment" refers to any activity in which the symptoms of dry eye syndrome are improved or cured by transplantation or administration of the lacrimal gland organoids according to the present invention. In addition, the term “prevention” refers to any action that suppresses or delays the symptoms of dry eye syndrome by implantation or administration of the lacrimal gland organoids according to the present invention.

In the present invention, the lacrimal gland organoids are lysozyme (LYZ), alpha-smooth muscle actin (α-SMA), aquaporin 5 (AQP5), E-cadherin (E-CAD) and Vimentin (VIM), and may specifically express one or more selected from the group consisting of, and the lacrimal gland organoid may include secretory granules. In addition, the lacrimal gland organoid may increase Ca ²⁺ absorption, β-hexosaminidase expression, or secretome secretion when treated with a substance for preventing or treating dry eye syndrome. In this case, the substance for preventing or treating dry eye syndrome may be at least one selected from the group consisting of pilocarpine, cevimeline, ciclosporin, and rebamipid.

In addition, the lacrimal gland organoid may be used to prevent or treat dry eye syndrome, which includes dry eye syndrome caused by tear gland damage, dry eye syndrome caused by damage to the ocular surface, dry eye syndrome caused by radiation, and Sjogren's syndrome (Sjogren's syndrome). syndrome), alacrima, keratoconjunctivitis sicca, Stevens Johnson syndrome, and graft versus host disease.

Another aspect of the present invention provides a method for screening a substance for preventing or treating dry eye syndrome, comprising the following steps: a) treating the lacrimal gland organoid with a candidate substance for preventing or treating dry eye syndrome; and b) comparing the tear secretion ability of the lacrimal gland organoids treated with the candidate substance with that of a control group.

In the method for screening a substance for preventing or treating dry eye syndrome according to the present invention, each term has the same meaning as described in the method for preparing lacrimal gland organoids unless otherwise specified.

In the present invention, step a) is a step of treating the lacrimal gland organoid with a candidate substance for preventing or treating dry eye syndrome.

The term "candidate" means a substance expected to be able to treat dry eye syndrome. Specifically, any substance expected to directly or indirectly improve or ameliorate dry eye syndrome can be used without limitation, and includes all expected therapeutic substances such as compounds, genes, or proteins.

Treatment of the candidate material may be performed using a method known in the art. As a specific example, the candidate substance may be treated with the lacrimal gland organoid and cultured together, or the candidate substance may be treated by administering the lacrimal gland organoid into a living body, but is not limited thereto, and those skilled in the art of the present invention You can use any method that suits your purpose.

In addition, the step b) is a step of comparing the tear secretion ability of the lacrimal gland organoids treated with the candidate substance with that of the control group.

The term "control group" refers to lacrimal gland organoids not treated with a candidate substance or lacrimal gland organoids treated with a substance for preventing or treating dry eye syndrome, in order to compare tear secretion ability with lacrimal gland organoids treated with the candidate substance. used

The tear secretion ability may be confirmed through an increase in Ca ²⁺ absorption, beta-hexosaminidase expression, or secretome secretion. Therefore, step b) may be a step of determining a substance for preventing or treating dry eye syndrome when the amount of Ca ²⁺ absorption, beta-hexosaminidase expression, or secretome secretion of the organoid increases.

The Ca ²⁺ absorption, beta-hexosaminidase expression or secretome secretion play an important role in the production and secretion of tears. First, in order to secrete tears from the lacrimal gland, Ca ²⁺ present outside the cell is absorbed into the cell through the Ca ²⁺ ion channel of the cell membrane constituting the lacrimal gland. This Ca ²⁺ absorption and concentration increase into the cell induces tear secretion, but dry eye patients such as Sjogren's syndrome do not have Ca ²⁺ channels in the cell membrane, so the Ca ²⁺ signaling pathway is not properly performed. (Imada T et al., Sci Rep. 2017, 7(1):6965; Garcia-Posadas L et al., Am J Pathol. 2020, 190(10):2067-2079.; Imada T et al. ., Sci Rep. 2017;7(1):6965.). In addition, beta-hexosaminidase, a type of lysosomal protein, is known to be mainly contained in lysosomes of tears or blood plasma, and serves to protect the eye by decomposing pathogens or toxic substances on the ocular surface. (Sofia V Andersson et al., Glycobiology. 2005 Mar, 15(3):211-20.). In addition, the secretome is a set of proteins expressed in cells and secreted into the extracellular space, and is known to include cytokines, growth factors, junction molecules, proteases, extracellular matrix proteins, and the like. The secretome of the present invention is a protein constituting extracellular organelles, exosomes, vesicles, and the like, and may be a set of proteins involved in tear production.

In the present invention, when the lacrimal gland organoid is treated with pilocarpine, a known preventive or therapeutic substance for dry eye syndrome, the rate and amount of Ca ²⁺ absorption in the organoid are increased, and beta-hexosaminidase in the organoid is increased. It was confirmed that the expression level of and the secretome secretion level of organoids increased. Therefore, a candidate substance that increases Ca ²⁺ absorption, beta-hexosaminidase expression, or secretome secretion when treated with the lacrimal gland organoid can be determined to be a substance for preventing or treating dry eye syndrome.

The Ca ²⁺ absorption capacity, beta-hexosaminidase expression capacity, or secretome secretion capacity may be measured using methods known to those skilled in the art. Specifically, Western blot, co-immunoprecipitation assay (Co-Immunoprecipitation assay), ELISA (Enzyme Linked Immunosorbent Assay), tissue immunostaining (Immunostaining), Flow cytometry analysis, fluorescence-based assays, electron microscopic analysis, etc. may be used, but are not limited thereto, and those skilled in the art may use a method suitable for the purpose of the present invention. There will be.

The lacrimal gland organoid prepared according to the present invention has high tissue similarity to human lacrimal tissue and excellent tear secretion ability, and in particular, when transplanted into a living body, it can regenerate damaged lacrimal tissue to normalize tear secretion ability.

Therefore, the method for preparing lacrimal organoids according to the present invention can be used to prepare lacrimal organoids having a similar shape and function to living tissue, and the prepared lacrimal organoids can be used to treat dry eye symptoms such as Sjögren's syndrome and dry cornea. It can be used as a treatment substance for, or can be usefully utilized as a method of screening the treatment substance for dry eye syndrome.

1 is a schematic diagram showing a method for preparing lacrimal gland organoids according to the present invention.
Figure 2 relates to the formation and growth characteristics of lacrimal gland organoids in each medium. A is an image showing the degree of growth of organoids prepared in each medium according to passage (10× magnification, scale bar 500 μm). , B is a graph showing the number of viable passages of organoids prepared in each medium, and C is an image showing the morphology of organoids prepared in the medium of Experimental Group 5 over the course of culture days (scale bar: 100 μm).
3 is an image showing the degree of growth of normal lacrimal gland organoids or Sjogren's syndrome lacrimal gland organoids according to passage (10× magnification, scale bar 500 μm).
Figure 4 relates to the similarity of the prepared normal lacrimal gland organoid with the lacrimal gland tissue, A is an image showing the histochemical staining results of H&E, Alcian Blue, PAS and Masson's Trichrome (40 × magnification, scale bar 100 μm), B is an image showing the results of transmission electron microscopy (TEM) analysis (scale bar 5 μm), C is lysozyme, α-SMA (Alpha-smooth muscle actin), aquaporin 5 (Aquaporin 5), E-cadherin (E-Cadherin) and vimentin (Vimentin) This is an image showing the results of immunofluorescence staining (scale bar 50 μm).
Figure 5 relates to the similarity of prepared normal lacrimal gland organoids or Sjogren's syndrome lacrimal gland organoids with lacrimal tissue, A is histochemical staining of H&E, Alcian Blue, PAS and Masson's Trichrome Image showing the result (40× magnification, scale bar 100 μm), B is Lysozyme, α-SMA (Alpha-smooth muscle actin), Aquaporin 5, E-Cadherin And it is an image showing the result of immunofluorescence staining for Vimentin protein (100× magnification, scale bar 50 μm).
6 is a graph showing the results of analysis of the AQP5 protein expression level, which relates to the similarity of the prepared normal lacrimal gland organoids or Sjögren's syndrome lacrimal gland organoids with lacrimal tissue.
Figure 7 relates to the secretory function of prepared normal lacrimal gland organoids, A is an image showing the degree of Ca ²⁺ uptake before and after treatment with pilocarpine (10× magnification, scale bar 100 μm) , B is a graph quantifying the degree of Ca ²⁺ absorption before and after treatment with pilocaprine, C is a graph of the MAG assay analysis results showing the expression level of β-hexosaminidase, D is It is an image showing a transmission electron microscope (TEM) analysis result for a secretome (scale bar 10 μm).
Figure 8 relates to the results of proteomic analysis of proteins secreted from the prepared normal lacrimal gland organoids, where A is a heat map for the proteomic analysis results, B is a Venn diagram for secreted protein types, and C is a secreted protein. D is a heat map of DEPs (differentially expressed proteins).
Figure 9 relates to the regenerative effect of the prepared normal lacrimal gland organoids on damaged lacrimal tissue, A is a schematic diagram showing an experimental schedule for confirming the regenerative effect, B is immunofluorescence staining of lacrimal tissue transplanted with lacrimal gland organoids This is an image showing the result (40× magnification, scale bar 100 μm).

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of human lacrimal gland organoids

As human lacrimal organoids, lacrimal gland organoids exhibiting normal functions and lacrimal gland organoids simulating dry eye syndrome, which have the characteristics of Sjogren's syndrome, were prepared.

Specifically, as shown in FIG. 1, lacrimal gland tissue was collected from a normal person or a patient with Sjogren's syndrome, washed, cut into small pieces, and then mixed with a dissociation buffer (0.125 mg/ml dispase II, 0.1 mg/ml DNase I, 0.125 mg/ml DNase I, DMEM/F12 containing 0.125 mg/ml collagenase II and 1 v/v% penicillin-streptomycin) and incubated at 37° C. for 60 minutes with shaking at 150 rpm to separate lacrimal gland-derived cells. Then, the separated lacrimal gland-derived cells were centrifuged, suspended in a culture medium at a density of 5×10 ⁵ cells/ml in a pellet state, mixed with Matrigel at a ratio of 1:1, and plated in a 48-well plate at 20 It was dispensed by μl. At this time, the Matrigel has a dome shape and includes the cell suspension, which was prepared by polymerization at 37° C. for 60 minutes. Thereafter, 300 μl of lacrimal gland organoid culture medium was added to each well, and the medium was exchanged once every 2-3 days and cultured for 20 days per passage.

In addition, in order to maximize the production efficiency of organoids, various combinations of compositions and ratios of organoid culture media were tested (Table 1). At this time, R-Spondin conditioned media was added to the medium of all experimental groups of Experimental groups 1 to 5 at a concentration of 10 v / v%, and to the medium of the other experimental groups except for Experimental group 2, Wnt conditioned medium ( Wnt Conditioned media) was added at a concentration of 50 v/v%.

experimental group 1 experimental group 2 experimental group 3 experimental group 4 experimental group 5 FGF2 5 ng/mL 5 ng/mL 20 ng/mL 20 ng/mL 20 ng/mL FGF10 10 ng/mL 10 ng/mL - 10 ng/mL - N2 1X - 1X 1X 1X B27 1X 1X - - - nicotinamide 10 mM 10 mM - - 10 mM A83-01 5 µM 5 µM - - 500 nM Noggin 100 ng/mL 100 ng/mL - - 100 ng/mL EGF 25 ng/mL 25 ng/mL 20 ng/mL 20 ng/mL 20 ng/mL SB202190 3 µM 3 µM - 3 µM - Prostaglandin E2 1 µM 1 µM - 1 µM - insulin 10 μg/mL - 10 μg/mL 10 μg/mL 10 μg/mL Dexamethasone 1 µM - 1 µM 1 µM 1 µM Y-27632 10 µM - 10 µM 10 µM 10 µM N-acetyl-L-cysteine 500 µM - 500 µM 500 µM 500 µM

As a result, as can be seen in A and B of FIG. 2, among the five media described in Table 1, FGF-2 (20 ng / ml), N2 (1X), nicotinamide (10 mM), A83-01 (500 nM), Noggin (100 ng/ml), EGF (20 ng/ml), Insulin (10 μg/ml), Dexamethasone (1 μM), Y-27632 (10 μM), N-acetyl-L-cysteine (500 μM), it was confirmed that organoids were efficiently prepared when cultured in the experimental group 5 medium containing DMEM/F12.

Specifically, it was confirmed that organoids were not well formed in the culture medium of Experimental Groups 1 to 4 even when the passage was 0, and organoids did not survive and were mostly killed when the passage increased. On the other hand, in the case of culturing with the experimental group 5 medium, the number of organoids rapidly increased as the number of passages increased, and when the number of passages reached 3, it was confirmed that the number increased by about 8 times or more compared to the case when the number of passages reached 0. In particular, it was confirmed that they could survive until passage 19 (Fig. 2A and B).

In addition, as can be seen in FIG. 2C, the lacrimal gland organoids cultured in the medium of Experimental Group 5 increase in size as the culture day elapses. It was confirmed that lacrimal gland organoids of the form were formed and increased in size to about 150 μm in diameter until 15 days after the start of culture.

In addition, as shown in FIG. 3, the number of organoids formed in the Sjögren's syndrome lacrimal gland organoids cultured in the medium of Experimental Group 5 also increased rapidly as the number of passages increased, which was confirmed to be the same as that of normal lacrimal gland organoids.

Through the above results, it was confirmed that lacrimal gland organoids could be efficiently prepared when the medium of Experimental Group 5 was used.

Example 2. Tissue similarity of human lacrimal gland organoids

In this example, it was confirmed whether the human lacrimal gland organoid prepared in Example 1 was histologically similar to the actual human lacrimal gland.

Specifically, for the human lacrimal gland organoids prepared using the medium of Experimental Group 5 according to Example 1 and the lacrimal tissue collected from actual humans, H&E, Alcian Blue, PAS and Masson's Trichrome ), etc. Histochemical staining was performed. In addition, transmission electron microscopy (TEM) analysis was performed to confirm the intracellular structure of lacrimal gland organoids, and lysozyme (LYZ), α-SMA (Alpha-smooth muscle actin) known to be specifically expressed in lacrimal tissue , Aquaporin 5 (AQP5), E-Cadherin (E-CAD) and Vimentin (Vimentin; VIM) protein immunofluorescence staining was performed using antibodies.

As a result, as can be seen in A to C of FIG. 4, through histochemical staining results, it was confirmed that the prepared lacrimal gland organoid exhibited a histologically very similar shape to human lacrimal gland tissue (FIG. 4 A), and TEM Through the image results, it was confirmed that there are organelles that can be identified in human lacrimal tissue, and that there are also many secretory granules known as characteristics of acinar cells (FIG. 4B), and immunofluorescence staining Through the results, Lysozyme (LYZ), α-SMA (Alpha-smooth muscle actin), Aquaporin 5 (AQP5), E-Cadherin (E-CAD) and Vimentin ; VIM) was found to be very similar to that of human lacrimal tissue (Fig.

In addition, as shown in A and B of FIG. 5, through histochemical and immunofluorescence staining results, the prepared normal lacrimal gland organoids and Sjögren's syndrome lacrimal gland organoids were collected from normal people and Sjögren's syndrome patients, respectively, and histologically It shows a very similar form (Fig. 5 A), lysosome (Lysozyme), α-SMA (Alpha-smooth muscle actin), aquaporin 5 (Aquaporin 5), E-cadherin (E-Cadherin) and vimentin ( Vimentin) was also confirmed to be very similar to the expression patterns of lacrimal gland specific markers (FIG. 5B).

Furthermore, as can be seen in FIG. 6, each normal or Sjögren organoid is a membrane protein present in salivary glands, lacrimal glands, etc., and it is confirmed that the protein expression level of AQP5 (aquaporin 5), which is known to perform a secretory function, is very similar to that of actual lacrimal tissue. did

Through the above results, since the human lacrimal gland organoid prepared by the manufacturing method according to the present invention is histologically very similar to the actual human lacrimal gland, it can be used for the treatment of human lacrimal gland-related diseases such as dry eye syndrome, and can also be used for the treatment of drugs. It was confirmed that it can also be used for screening.

Example 3. Tear secretion ability of human lacrimal gland organoids

In this example, it was confirmed whether the human lacrimal gland organoid prepared in Example 1 could properly mimic the tear secretion function of the actual human lacrimal gland tissue.

Specifically, human lacrimal organoids prepared using the medium of Experimental Group 5 according to Example 1 and pilocarpine 1 μg/ml was processed. Thereafter, the degree of Ca ²⁺ absorption was confirmed with the Fluo-4 Calcium Imaging Kit (Thermo Fisher Scientific) to confirm the ability to induce tear secretion, and the NAG (N-acetyl-β-glucosaminidase) assay kit (MilliporeSigma) was used. The expression level of β-hexosaminidase, which is known to be present in tears secreted from the lacrimal gland, was confirmed, and through TEM (Transmission electron microscopy) image analysis, a set of proteins secreted from cells constituting organoids was confirmed. The secretome was confirmed. Ca ²⁺ absorption into cells and increase in concentration induce tear secretion, but dry eye patients such as Sjogren's syndrome do not have Ca ²⁺ channels in the cell membrane, so the Ca ²⁺ signaling pathway does not work properly. (Imada T et al., Sci Rep. 2017, 7(1):6965; Garcia-Posadas L et al., Am J Pathol. 2020, 190(10):2067-2079.; Imada T et al., Sci Rep. 2017;7(1):6965.).

As a result, as can be seen in A to D of FIG. 7 , when the prepared lacrimal gland organoids are treated with pilocaprine, Ca ²⁺ absorption is achieved within 5 seconds and the amount of absorption is increased by about 2 times or more. (A and B in FIG. 7), it was confirmed that the expression level of beta-hexosaminidase increased over time, increasing by about 1.5 times after 24 hours compared to the expression level at 2 hours (Fig. 7 C). In addition, the secretome is secreted from the cells constituting the organoid, and when the secretome is secreted, a phenomenon in which the cell gap is widened, thereby confirming that tear secretion can be performed normally (FIG. 7D).

Through the above results, the human lacrimal gland organoid prepared by the manufacturing method according to the present invention can perform the secretion function of the actual human lacrimal gland tissue to a very similar extent, and thus is useful for the treatment of human lacrimal gland-related diseases such as dry eye syndrome. It was confirmed that it can be used.

Example 4. Analysis of proteins secreted from human lacrimal gland organoids

In this example, by analyzing the types of proteins secreted from the human lacrimal gland organoids prepared in Example 1, it was confirmed that they functioned the same as the actual human lacrimal glands.

Specifically, 1 μg/ml of pilocarpine, which is used for the treatment of dryness, was added to human lacrimal gland organoids prepared using the medium of Experimental Group 5 according to Example 1 and lacrimal tissue collected from actual humans. processed. Thereafter, proteomics analysis was performed on the secretome secreted from the lacrimal gland organoids.

As a result, as can be seen in A to D of FIG. 8, when the prepared lacrimal gland organoids are treated with pilocaprin, the amount of secreted protein increases and the types of proteins also diversify (FIG. 8A), about 120 It was confirmed that as many proteins were newly synthesized and secreted (FIG. 8B). In particular, most of the secreted proteins are located in the extracellular region, vesicle, extracellular region part, extracellular space, extracellular organelle, and extracellular space. It was confirmed that they were expressed for tear production as proteins constituting extracellular vesicles, extracellular exosomes, and the like (FIG. 8C). In addition, proteins whose expression level increases by 20 or more include RPS8, RPL5, RPL12, CTTN, CCT2, ADH5, CAPZB, ACLY, PGLS, HNRNPC, WDR1, CAPG, COL6A1, CTSB, RPSA, RAN, SET, HNRNPAB, etc. It was confirmed that they belonged to moths or vesicles (Fig. 8D).

Through the above results, the human lacrimal gland organoid prepared by the manufacturing method according to the present invention can perform the secretion function of the actual human lacrimal gland tissue to a very similar extent, and thus is useful for the treatment of human lacrimal gland-related diseases such as dry eye syndrome. It was confirmed that it can be used.

Example 5. Therapeutic effect of human lacrimal gland organoids

In this example, the therapeutic effect of the human lacrimal gland organoid prepared in Example 1 on lacrimal gland damage was confirmed.

Specifically, human lacrimal gland organoids prepared using the medium of Experimental Group 5 according to Example 1 were transplanted into a mouse dry eye syndrome model (1×10 ⁴ cells/15 μl/lacrimal gland), and 2 weeks later, the engrafted organoids It was confirmed whether the damaged lacrimal gland was regenerated by the organoid by checking the level of expression of AQP5, a specific marker of lacrimal gland cells, in the tissue of the organoid. At this time, the lacrimal gland organoid was prepared using lacrimal gland-derived stem cells collected from GFP-Tg mice in order to easily track it even after transplantation into the mouse body, and the dry eye syndrome mouse model uses Concanavalin A It was manufactured.

As a result, as can be seen in FIG. 9 , it was confirmed that the lacrimal gland organoids expressing GFP were well engrafted to the lacrimal tissue even after 2 weeks of transplantation. In particular, it was confirmed that the expression level of AQP5, a specific protein marker for lacrimal gland cells, was remarkably increased by about 3-fold in the organoid-transplanted tissue compared to the lacrimal gland damaged tissue.

Through the above results, the human lacrimal gland organoid prepared by the manufacturing method according to the present invention can efficiently induce regeneration of damaged lacrimal tissue, and thus can be usefully used for the treatment of human lacrimal gland-related diseases such as dry eye syndrome. confirmed that there is

Claims (25)

A method for producing lacrimal gland organoids, comprising the following steps:
a) isolating cells from lacrimal tissue isolated from the subject; and
b) culturing the cells isolated in step a) with a medium containing Wnt, A83-01 and Noggin, but not containing FGF10. According to claim 1,
Wherein the cells of step a) are isolated by cutting and enzymatically digesting the lacrimal tissue. According to claim 1,
The medium of step b) is attached to the culture plate to have a dome shape, the manufacturing method. According to claim 1,
Wherein the culture of step b) is performed in 1 to 19 passages. According to claim 4,
Wherein the first passage is performed for 1 to 20 days. According to claim 1,
The lacrimal gland organoid is one selected from the group consisting of lysozyme, alpha-smooth muscle actin (α-SMA), aquaporin 5, E-cadherin, and vimentin. A manufacturing method that specifically expresses the abnormality. According to claim 1,
The method of manufacturing the lacrimal gland organoid comprising a secretory granule. According to claim 1,
The lacrimal gland organoid is a production method in which the amount of Ca ²⁺ absorption, the amount of beta-hexosaminidase expression or the amount of secretome secretion increases when a substance for preventing or treating dry eye syndrome is treated. According to claim 8,
The method for preventing or treating dry eye syndrome is at least one selected from the group consisting of pilocarpine, cevimeline, ciclosporin and rebamipid. According to claim 1,
The lacrimal gland organoid is for the prevention or treatment of dry eye syndrome, the manufacturing method. According to claim 10,
The dry eye symptoms include dry eye syndrome caused by tear gland damage, dry eye syndrome caused by damage to the ocular surface, dry eye syndrome caused by aging, dry eye syndrome caused by irradiation, lacrimal inflammation, dacryocystitis, lacrimal duct stricture, Sjogren's syndrome, and murmur. At least one selected from the group consisting of alacrima, keratoconjunctivitis sicca, Stevens Johnson syndrome and graft versus host disease, a manufacturing method. delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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