Composition for endometrial regeneration containing uterus-derived decellularized extracellular matrix and method for producing same

Abstract

The present invention relates to a composition for endometrial regeneration containing a uterus-derived decellularized extracellular matrix and a method for producing the same. The composition is characterized by containing a uterus-derived decellularized extracellular matrix (UdECM), and has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium.

Description

[Technical Field]

[0001] The present invention relates to a uterine tissue-specific extracellular matrix that can improve the regeneration, receptivity, and implantation ability of the endometrium, and in particular to a composition for endometrial regeneration containing a uterine-derived decellularized extracellular matrix (UdECM) and a method for producing the same. [Background technology]

[0002] The uterus is one of the main female reproductive organs and is composed of several tissue layers: the endometrium, myometrium, and perimetria.

[0003] Approximately 10% of female infertility cases are due to problems related to uterine health. Healthy endometrial tissue is extremely important in terms of successful embryo implantation and pregnancy maintenance. In particular, endometrial thickness is one of the main characteristics that has been used as an indicator of endometrial receptivity. Numerous studies have shown that a sufficiently thick endometrium is essential for successful implantation and pregnancy. In other words, if the endometrium is too thin (less than 7 mm), it becomes difficult to maintain pregnancy.

[0004] For this reason, various research efforts are being conducted on treatment methods to improve endometrial thickness. Despite various treatment strategies, including hormone therapy, intrauterine injection of growth factors, and the use of vasoactive substances, clinically meaningful effects have yet to be achieved, and endometrial regeneration remains a challenging aspect of female infertility treatment.

[0005] To overcome these limitations, implantation of tissue-specific extracellular matrix (ECM) components or collagen- or hyaluronic acid-based biomaterials has been used to regenerate the endometrium. However, such single ECM-based biomaterials have the limitation that they are unable to replicate the tissue-specific extracellular matrix, which supports the tissue-specific microenvironment and cell interactions based on a unique combination of various proteins. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Republic of Korea Publication Patent No. 10-2021-0018639 (February 18, 2021) Summary of the Invention [Problem to be solved by the invention]

[0007] In order to solve the above problems, the present invention aims to provide a composition for endometrial regeneration that can improve the regeneration, receptivity, and implantation ability of the endometrium.

[0008] Another object of the present invention is to provide a composition for endometrial regeneration, which contains collagens, glycoproteins, and proteoglycans, increases the expression of steroid hormone receptors upon treatment with the hormones, and has the functions of regenerating the endometrium and promoting angiogenesis.

[0009] Furthermore, another object of the present invention is to provide a more excellent responsiveness to steroid hormones and to further increase the receptivity of the endometrium. [Means for solving the problem]

[0010] In order to achieve the above-mentioned objectives, one embodiment of the present invention is a composition for endometrial regeneration comprising a uterine-derived decellularized extracellular matrix (UdECM).

[0011] In one embodiment of the present invention, the uterus may be derived from a porcine.

[0012] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be a decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimetria.

[0013] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM).

[0014] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM) isolated from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimeter.

[0015] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM) separated from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimeter, after treating the tissue with a surfactant.

[0016] In one aspect of the present invention, the composition for endometrial regeneration containing the decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) is preferably intended for use in patients with uterine adhesions (Uter inesynechiae).

[0017] In one embodiment of the present invention, the composition for endometrial regeneration according to the present invention may contain collagens, glycoproteins, and proteoglycans.

[0018] In one aspect of the present invention, the composition for endometrial regeneration according to the present invention may increase the expression of receptors for steroid hormones upon treatment with the hormones, and may have endometrial regeneration and angiogenesis-promoting functions.

[0019] Another embodiment of the present invention is a method for producing a composition for endometrial regeneration, comprising the steps of preparing uterine tissue (Whole Uterus) including the endometrium and myometrium, treating the prepared uterine tissue with a first surfactant, separating the endometrium from the uterine tissue treated with the first surfactant, and treating the separated endometrium with a second surfactant.

[0020] In one embodiment of the present invention, the first surfactant may be sodium dodecyl sulfate (SDS).

[0021] In one embodiment of the present invention, the second surfactant may be Triton (trade name) X-100.

[0022] Another embodiment of the present invention is a bio-ink composition comprising uterine derived decellularized extracellular matrix (UdECM).

[0023] Another embodiment of the present invention is a composition for culturing uterine organoids, comprising a uterine-derived decellularized extracellular matrix (UdECM).

[0024] Another embodiment of the present invention is a composition for fabricating an organ-on-a-chip comprising uterine derived decellularized extracellular matrix (UdECM).

[0025] Specific details of other embodiments are included in the detailed description and drawings. [Effects of the Invention]

[0026] The composition for endometrial regeneration according to the present invention is characterized by containing a uterus-derived decellularized extracellular matrix (UdECM), and has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium.

[0027] The composition for endometrial regeneration according to the present invention contains collagens, glycoproteins, and proteoglycans, and when treated with steroid hormones, increases the expression of the hormone receptors, thereby having the functions of regenerating the endometrium and promoting angiogenesis.

[0028] Furthermore, the composition for endometrial regeneration according to the present invention has superior responsiveness to steroid hormones, and has the effect of further increasing the receptivity of the endometrium. [Brief explanation of the drawings]

[0029] [Figure 1] 1 shows the results of the density of matrisome proteins obtained by performing proteomic analysis on Endo-UdECM and Whole-UdECM according to one embodiment of the present invention. [Figure 2] 1 is a heat map showing specific components of concentrated proteoglycans, collagens, and glycoproteins contained in Endo-UdECM and Whole-UdECM according to an embodiment of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the time course of treatment with each hormone when evaluating steroid hormone responsiveness in vitro using Endo-UdECM and Whole-UdECM according to one embodiment of the present invention. [Figure 4]The results of evaluating the steroid hormone responsiveness shown in Figure 3 are shown in Figure 4A, which shows the morphological differentiation of human endometrial matrix cells in response to ovarian steroid hormones (estrogen (E2) and progesterone (P4)) (Figure 4A), a shape index calculated from the prototype of embryonic stem body morphology that changes in response to steroid hormones (Figure 4B), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showing the expression of estrogen receptors (ESR) in endometrial stromal cells (ESCs) encapsulated with E2 and P4, respectively (Figure 4C), qRT-PCR analysis showing the expression of progesterone receptors (PGR) in ESCs encapsulated with E2 and P4, respectively (Figure 4D), and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression of decidualization markers (prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1)) in encapsulated ESCs in response to E2, P4, and cAMP (Figures 4E and 4F). [Figure 5] The injection effect of Endo-UdECM and Whole-UdECM according to one embodiment of the present invention was examined in a thin endometrium model. The hematoxylin and eosin (H&E) stained image (FIG. 5A) and endometrial thickness (FIG. 5B) after treatment in the thin endometrium model were shown. The immunostaining of ITGβ3 and osteopontin (OPN) in the thin endometrium treated with saline or UdECM (endometrium or uterine tissue) was also shown (FIG. 5C). Protein expression visualized by diaminobenzidine (DAB) color intensity and quantification of Gβ3 and OPN staining results (Figure 5D, E). Co-immunofluorescent staining of CD31 (red) and Ki67 (green) in a thin endometrial model treated with UdECM (endometrium or uterine tissue) (Figure 5F). Comparison of Ki67-positive cells (Figure 5G, H), total blood vessel count (Figure 5J), and CD31-positive intensity (Figure 5K) in each group. [Figure 6] 1 shows the results of gene ontology analysis of thin endometrial models treated with Endo-UdECM and Whole-UdECM according to one embodiment of the present invention. [Figure 7]7A and 7B show the results of in vitro validation of the efficacy of Endo-UdECM and Whole-UdECM according to one embodiment of the present invention on human endometrial tissue, including co-immunofluorescence staining of Ki67 (green) and CD31 (red) in human endometrial tissue (patient H sample (A) and patient S sample (B)) cultured in Endo-UdECM- or Whole-UdECM-containing medium compared to control medium (Figure 7A and B). Also shown are representative images of immunostaining for OPN in human endometrial tissue cultured in Endo-UdECM- or Whole-UdECM-containing medium compared to control medium (Figure 7C), and a comparison of OPN expression in each group (Figure 7D). DETAILED DESCRIPTION OF THE INVENTION

[0030] Since the present invention can be modified in various ways and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description section, but it should be understood that this is not intended to limit the present invention to the specific embodiments, and that it includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0031] In describing the present invention, detailed descriptions of known technologies related to the present invention will be omitted if it is recognized that such descriptions may obscure the gist of this disclosure.

[0032] The terms used in this disclosure are merely used to describe particular embodiments and are not intended to limit the scope of the present invention. The singular expressions include plural expressions unless the context clearly dictates otherwise.

[0033] In this disclosure, the terms "comprises," "includes," or "having" are to be understood as specifying only the presence of a feature, number, step, operation, component, part, or combination thereof stated in the specification, but not as precluding the possible presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0034] The terms "first," "second," and the like used in this disclosure may be used to describe multiple components, but the components are not limited by the terms. The terms "first," "second," and the like are used only to distinguish one component from another.

[0035] One embodiment of the present invention is a composition for endometrial regeneration comprising uterine derived decellularized extracellular matrix (UdECM).

[0036] The present invention relates to a uterine tissue-specific extracellular matrix that can improve the regeneration, receptivity, and implantation ability of the endometrium, and in particular to a composition for endometrial regeneration containing uterine-derived decellularized extracellular matrix (UdECM) and a method for producing the same.

[0037] As used herein, "uterus-derived decellularized extracellular matrix (UdECM)" refers to an extracellular matrix obtained by decellularizing human or animal uterine tissue.

[0038] In particular, the uterus-derived decellularized extracellular matrix (UdECM) may be a porcine uterus-derived extracellular matrix (P-UdECM). The type of pig and the location of the uterine tissue are not particularly limited, and various decellularization methods known in the art can be used. For example, the present inventors have previously developed a method (Korean Patent Registration No. 10-2282073) that involves decellularizing porcine uterine tissue, freeze-drying the decellularized tissue, pulverizing it, adding a protein-digesting enzyme and an acid to the pulverized tissue, and dissolving it under stirring. A basic solution is then added to the resulting solution to adjust the pH, thereby obtaining a porcine uterus-derived decellularized extracellular matrix (P-UdECM).

[0039] Uterine-derived decellularized extracellular matrix (UdECM) actually contains tissue-specific extracellular matrix components, and therefore can provide a physical, mechanical, and biochemical environment for the tissue, making it highly effective in promoting differentiation into uterine tissue cells and tissue-specific functionality.

[0040] The term "decellularized extracellular matrix" refers to a natural support for cell growth prepared through decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix may be a mixture of structural and nonstructural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. The extracellular matrix may be of various forms in mammals and may contain approximately 90% collagen. Extracellular matrices derived from various biological tissues may differ in overall structure and composition due to the specific roles required for each tissue.

[0041] The composition for endometrial regeneration according to the present invention is characterized by containing such a uterus-derived decellularized extracellular matrix (UdECM).

[0042] Previously, implantation of biomaterials based on individual extracellular matrix (ECM) components of specific tissues or collagen or hyaluronic acid has been used for endometrial regeneration. However, such single ECM-based biomaterials have the limitation that they are difficult to replicate tissue-specific extracellular matrices that support tissue-specific microenvironments and cell interactions based on unique combinations of various proteins.

[0043] Needless to say, the present inventors realized that the most effective method for regenerating the endometrium is to use a decellularized extracellular matrix derived from the uterus. Therefore, they prepared a decellularized extracellular matrix derived from uterine tissue (Whole-Uterus Decellularized Extracellular Matrix; Whole-UdECM) and then isolated specific tissue layers of the uterus to prepare an endometrium-derived decellularized extracellular matrix (Endo-UdECM). They then performed in vivo and in vitro analyses of the proteome, steroid hormone responsiveness, and therapeutic efficacy of each UdECM, verifying its clinical efficacy and tissue-specific properties, leading to the completion of the present invention.

[0044] Accordingly, as can be seen from the examples and experimental examples described below, the composition for endometrial regeneration according to the present invention is characterized by containing a uterus-derived decellularized extracellular matrix (UdECM), and has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium.

[0045] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be a decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimetria.

[0046] Generally, uterine tissue is composed of three membranes. Therefore, in a specific part of the uterus, uterine tissue may include the endometrium, myometrium, and perimetrium. The present invention may be a composition comprising an extracellular matrix obtained by decellularizing uterine tissue including the endometrium, myometrium, and perimetrium.

[0047] The composition for endometrial regeneration according to the present invention contains collagens, glycoproteins, and proteoglycans, and when treated with steroid hormones, increases the expression of the hormone receptors, thereby having the functions of regenerating the endometrium and promoting angiogenesis.

[0048] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM).

[0049] The endometrium-derived decellularized extracellular matrix (Endo-UdECM) may be an extracellular matrix that has been subjected to a decellularization process after the endometrium has been separated from human or animal uterine tissue, or may be an extracellular matrix that has been subjected to a decellularization process after the endometrium has been separated from the uterine tissue during the process of decellularizing the uterine tissue.

[0050] As an example, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM) isolated from uterine tissue (Whole Uterus), which includes the endometrium, myometrium, and perimetrium.

[0051] Thus, a composition containing endometrium-derived decellularized extracellular matrix (Endo-UdECM) has superior responsiveness to steroid hormones, can further enhance the endometrial receptivity, and can exert even more effective effects, particularly in patients with endometrial hyperplasia.

[0052] In one embodiment of the present invention, the uterus-derived decellularized extracellular matrix may be endometrium-derived decellularized extracellular matrix (Endo-UdECM) separated from uterine tissue (Whole Uterus), which includes the endometrium, myometrium, and perimetrium, after treating the tissue with a surfactant.

[0053] That is, the endometrium-derived decellularized extracellular matrix (Endo-UdECM) may be an extracellular matrix that has undergone a decellularization process after the endometrium has been separated from the uterine tissue during the process of decellularizing the uterine tissue. However, it is preferable to separate the endometrium from the uterine tissue after surfactant treatment during the decellularization process.

[0054] The present inventors have conducted research into uterine tissue and endometrial decellularization protocols based on a surfactant combination containing sodium dodecyl sulfate (SDS) and Triton® X-100. They found that the most effective way to obtain Endo-UdECM separately was to separate the swollen endometrial layer from the uterine muscle layer after SDS treatment and then proceed with the decellularization process. After SDS treatment of uterine tissue, the endometrium swells within the myometrium. Therefore, by separating the endometrium after SDS treatment, we were able to obtain decellularized endometrial extracellular matrix while effectively isolating the endometrium without tissue loss.

[0055] In one embodiment of the present invention, the composition for endometrial regeneration containing the decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) is preferably used for patients with uterine adhesions (Uterine synechiae).

[0056] As shown in the experimental examples below, the inventors tested the in vitro efficacy of Endo-UdECM and Whole-UdECM on human endometrial tissue, and found that decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) was superior in expressing the endometrial receptive marker (OPN) in tissue from patients with uterine adhesions.

[0057] In one embodiment of the present invention, a composition for endometrial regeneration containing endometrium-derived decellularized extracellular matrix (Endo-UdECM) is preferably used for patients with endometrial hyperplasia.

[0058] As shown in the experimental examples below, the inventors tested the in vitro efficacy of Endo-UdECM and Whole-UdECM on human endometrial tissue, and found that endometrium-derived decellularized extracellular matrix (Endo-UdECM) was superior in expressing the endometrial receptive marker (OPN) in tissue from patients with endometrial hyperplasia.

[0059] The above-described composition for endometrial regeneration according to the present invention essentially comprises a uterus-derived decellularized extracellular matrix (UdECM), and may optionally further comprise other biomaterials such as collagen and / or hyaluronic acid.

[0060] Such biomaterials may be encapsulated in the vacant spaces of uterine decellularized extracellular matrix (UdECM) from which cells have been removed by decellularization. This encapsulated uterine decellularized extracellular matrix (UdECM) does not lose its functionality over time and can retain excellent endometrial regeneration function even when cultured for long periods of time.

[0061] An embodiment of the present invention may further include a hydrogel.

[0062] That is, the composition for endometrial regeneration according to the present invention essentially contains a decellularized extracellular matrix (UdECM) derived from the uterus, and may additionally and selectively contain a hydrogel.

[0063] The hydrogel is not particularly limited and may include various hydrogels known in the art, for example, natural hydrogels such as collagen and agarose, or synthetic hydrogels such as polylactic-co-glycolic acid (PLGA).

[0064] In the present invention, the hydrogel has the function of supporting and holding the uterus-derived decellularized extracellular matrix (UdECM) and / or other biomaterials, and therefore has the effect of further enhancing the endometrial regeneration efficacy of the extracellular matrix.

[0065] According to this, one aspect of the present invention may be a hydrogel mixed with the uterine-derived decellularized extracellular matrix (UdECM) of the present invention and / or other biomaterials. Furthermore, the uterine-derived decellularized extracellular matrix (UdECM) and / or other biomaterials may be mixed and encapsulated in a hydrogel. In this case, the extracellular matrix (UdECM) and / or other biomaterials are mixed and / or fused at close range, which has the effect of maximizing responsiveness and receptivity to steroid hormones.

[0066] Another embodiment of the present invention is a method for producing a composition for endometrial regeneration, comprising the steps of preparing uterine tissue (Whole Uterus) including the endometrium and myometrium, treating the prepared uterine tissue with a first surfactant, separating the endometrium from the uterine tissue treated with the first surfactant, and treating the separated endometrium with a second surfactant.

[0067] The uterine tissue (whole uterine tissue) may include the endometrium and myometrium, or may optionally include the perimetrium in addition to the endometrium. Uterine tissue can be obtained from the uterus of mammals, including humans and animals.

[0068] The method may further include a step of cutting the prepared uterine tissue into pieces of an appropriate size and washing the pieces with water or distilled water.

[0069] Subsequently, the uterine tissue may be treated with a first surfactant. The first surfactant is not particularly limited, and a wide variety of surfactants known in the art can be used. For example, sodium dodecyl sulfate (SDS) or Triton (trade name) X-100 can be used, with SDS being preferred.

[0070] The present invention then involves a step of separating the endometrium from the uterine tissue treated with the first surfactant. That is, the separation of the endometrium is performed after the uterine tissue is treated with the first surfactant. As described above, after the uterine tissue is treated with the surfactant, the endometrium swells in the myometrium, so separating the endometrium after the surfactant treatment can most effectively separate the endometrium without losing tissue.

[0071] Subsequently, the method may further include a step of washing the separated endometrium again with water or distilled water.

[0072] The method may then include a step of treating the separated endometrium with a second surfactant. The second surfactant is not particularly limited, and a wide variety of surfactants known in the art can be used. For example, sodium dodecyl sulfate (SDS) or Triton (trade name) X-100 can be used, with Triton (trade name) X-100 being preferred.

[0073] This may be followed by a further step of washing the endometrium treated with the second surfactant again with water or distilled water.

[0074] It is also possible to further carry out a process of adding a protein-digesting enzyme and / or an acid to the endometrium and dissolving it under stirring, and then adding a basic solution to the dissolved solution to adjust the pH.

[0075] Another embodiment of the present invention is a bio-ink composition comprising uterine derived decellularized extracellular matrix (UdECM).

[0076] That is, according to the present invention, a composition containing a uterus-derived decellularized extracellular matrix (UdECM) has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium, and therefore such a composition can also be used as a three-dimensional bioink for producing a uterine tissue replica.

[0077] According to the present invention, a culture environment substantially identical to that of the human uterus can be constructed using a uterus-derived decellularized extracellular matrix as a base. This differs from other decellularized compositions of uterine tissue that have been reported so far, and has the effect of being able to most closely replicate the human uterine environment outside the body.

[0078] The bio-ink composition of the present invention may further include various biodegradable materials, bioactive materials, hydrogels, crosslinkers, etc., known in the art.

[0079] Another embodiment of the present invention is a composition for culturing uterine organoids, comprising a uterine-derived decellularized extracellular matrix (UdECM).

[0080] That is, according to the present invention, a composition containing a uterus-derived decellularized extracellular matrix (UdECM) has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium, and therefore such a composition can also be used as a composition for culturing uterine organoids.

[0081] The term "organoid" refers to a micro-organ produced by culturing cells derived from tissues or omnipotent stem cells in a 3D form, similar to an artificial organ. Organoids are three-dimensional tissue analogs containing organ-specific cells that develop from stem cells and self-organize (or self-pattern) in a manner similar to that found in vivo, and can develop into specific tissues through the patterning of specific elements (e.g., growth factors).

[0082] Uterine organoids cultured in the composition of the present invention have excellent endometrial regeneration, receptivity, and implantation abilities, and can be developed into a structure that is almost identical to the actual endometrium.

[0083] Another embodiment of the present invention is a composition for fabricating an organ-on-a-chip, comprising uterine derived decellularized extracellular matrix (UdECM).

[0084] That is, the composition containing the uterus-derived decellularized extracellular matrix (UdECM) according to the present invention has the effect of improving the regeneration, receptivity, and implantation ability of the endometrium, and therefore, such a composition can also be used as a composition for fabricating organ chips and / or bioprinted organs. The term "bioprinted organ" refers to an organ formed by 3D or bioprinting techniques.

[0085] Existing Matrigel-based culture systems are extracts derived from animal cancer tissues, and have large batch-to-batch variations, are unable to replicate the actual uterine environment, and are poor in differentiation and development efficiency into uterine organoids. In contrast, the composition of the present invention can create an environment nearly identical to that of endometrial tissue, making it suitable for culturing uterine organ chips and / or bioprinted organs.

[0086] Another embodiment of the present invention is a method for culturing endometrium, comprising the step of culturing endometrium in the above-described composition for endometrial regeneration.

[0087] The specific method or process for culturing the endometrium in the composition for endometrial regeneration is not particularly limited. The culturing refers to a process of maintaining and growing the endometrium under suitable conditions, and suitable conditions may refer to, for example, the temperature at which the endometrium is maintained, the availability of nutrients, the CO content in the atmosphere, and the cell density.

[0088] The present invention will be better understood from the following examples, which are intended to be merely illustrative of the present invention and are not intended to limit the scope of protection defined by the appended claims.

[0089] Example 1: Preparation of Uterine-Derived Decellularized Extracellular Matrix Using pig uteri, we produced decellularized extracellular matrix derived from uterine tissue (Whole-UdECM) and decellularized extracellular matrix derived from endometrium (Endo-UdECM).

[0090] To date, no studies have been conducted demonstrating differences in the structure and function of the uterine tissue as a whole and the specific part of the uterine tissue, the endometrium.

[0091] The present inventors undertook a study of uterine tissue and endometrial decellularization protocols based on a surfactant combination containing sodium dodecyl sulfate (SDS) and Triton™ X-100.

[0092] The specific decellularization process is shown in Table 1 below.

[0093] [Table 1] The process of decellularizing Whole-UdECM and Endo-UdECM derived from porcine uterus JPEG2025534151000002.jpg181170

[0094] The overall process for obtaining uterine tissue (Whole Uterus)-derived decellularized extracellular matrix (Whole-UdECM) and endometrium-derived decellularized extracellular matrix (Endo-UdECM) is similar. However, to obtain Endo-UdECM separately, the swollen endometrial layer was separated from the uterine muscle layer after the SDS treatment, and then the decellularization process was carried out.

[0095] Specifically, the decellularization processes for Endo-UdECM and Whole-UdECM were performed in the same manner up to step 4 in Table 1. Separation of Endo-UdECM and Whole-UdECM was performed differently in step 5. After treating uterine tissue with SDS, the endometrium swells in the myometrium. Therefore, it was found that separating the endometrium after SDS treatment was advantageous for preventing tissue loss. After this, the swelled endometrial tissue was removed separately, and the decellularization processes were carried out separately from step 6.

[0096] Through this decellularization process, different compositions for endometrial regeneration containing Endo-UdECM and Whole-UdECM, respectively, were prepared.

[0097] Experimental Example 1: Comparison of components of decellularized extracellular matrix Proteomics analysis was carried out on Whole-UdECM and Endo-UdECM prepared according to Example 1.

[0098] Proteome analysis was performed as follows. 10 mg of lyophilized Whole-UdECM and Endo-UdECM were prepared and then added to 10 μg of Lys-C / trypsin and 700 μL of 50 mM triethylammonium bicarbonate buffer (TEAB) and incubated at 37°C for 16 hours. The samples were then desalted using a SEK-PAC C18 and lyophilized again. The samples were then redissolved in 100 μL of 0.1% formic acid and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0099] Proteins detected through LC-MS analysis were quantified and identified using Sequest HT in proteome discoverer 2.4 (Thermo Fisher Scientific) software. Each identified protein was classified using the UniprotKB database, and matrisome analysis was performed using the Human Matrisome Reference (Atlas).

[0100] FIG. 1 shows the results of proteomic analysis of Endo-UdECM and Whole-UdECM according to one embodiment of the present invention, showing the density of matrisome proteins.

[0101] As a result of the analysis, it was confirmed that, from the perspective of matrisomes, Endo-UdECM contains a greater amount of glycoproteins than Whole-UdECM, but contains a smaller amount of collagen (see Figure 1).

[0102] FIG. 2 is a heat map showing the specific components of enriched proteoglycans, collagens, and glycoproteins contained in Endo-UdECM and Whole-UdECM according to one embodiment of the present invention.

[0103] Upon closer examination of the protein composition, we confirmed that both Whole-UdECM and Endo-UdECM contained COL type VI as the most abundant collagen protein, and fibrinogen (FGA, FGB, FGG) as the most abundant glycoprotein (Figure 2). COL type VI is an essential component for the formation of the human endometrium and is highly expressed in the endometrium during the menstrual cycle prior to embryo implantation. Fibrinogen also plays an important role in maternal vascular remodeling.

[0104] These results suggest that a composition comprising Endo-UdECM and Whole-UdECM according to the present invention can provide a complex microenvironment for reproducing endometrial function.

[0105] Experimental Example 2: In vitro evaluation of Endo-UdECM and Whole-UdECM for steroid hormone responsiveness The response of human endometrial stromal cells (hESCs) to steroid hormones is important for endometrial receptivity and the maintenance of pregnancy.

[0106] Therefore, to confirm whether the composition of the present invention can reproduce the functions of such cells, hESCs were encapsulated in Whole-UdECM and Endo-UdECM prepared according to Example 1, as well as in a collagen hydrogel for comparison, and their responsiveness to steroid hormones was then examined.

[0107] First, three experimental groups were prepared by encapsulating hESCs at a concentration of 50,000 cells / ml in 10 mg / ml Endo-UdECM, Whole-UdECM, or collagen hydrogel.

[0108] Steroid hormone treatment was carried out in three ways: Three types of steroid hormones were administered to induce decidualization corresponding to the first 7 days of the menstrual cycle.

[0109] 1) Two days after encapsulating the cells in the hydrogel, each experimental group was treated with 10 nM E2 (β-estradiol, Sigma-Aldrich, E8875) to observe the expression of estrogen and progesterone receptors, respectively.

[0110] 2) Two days after encapsulating the cells in the hydrogel, each experimental group was treated with 10 nM E2 + 1 uM P4 (progesterone, Sigma-Aldrich, P8783) to observe the expression of estrogen and progesterone receptors, respectively.

[0111] 3) To induce decidualization, each experimental group was treated with a combination of 10 nM E2, 1 uM P4, and 1 uM cAMP (8-bromoadenosine 3',5'-cyclic monophosphate, Sigma-Aldrich, B7880) for 7 days.

[0112] The time course for each hormone treatment is shown in Figure 3. Figure 3 is a schematic diagram showing the time course for each hormone treatment when evaluating steroid hormone responsiveness in vitro using Endo-UdECM and Whole-UdECM according to one embodiment of the present invention.

[0113] The results are shown in Figure 4. Figure 4 shows the evaluation results of steroid hormone responsiveness shown in Figure 3, including the morphological differentiation of human endometrial matrix cells in response to ovarian steroid hormones (estrogen (E2) and progesterone (P4)) (Figure 4A), a shape index calculated from the original shape of embryonic stem body morphology that changes in response to steroid hormones (Figure 4B), qRT-PCR analysis showing the expression of estrogen receptor (ESR) in ESCs encapsulated with E2 and P4, respectively (Figure 4C), qRT-PCR analysis showing the expression of progesterone receptor (PGR) in ESCs encapsulated with E2 and P4, respectively (Figure 4D), and RT-PCR analysis results for the expression of decidualization markers (PRL and IGFBP-1) in encapsulated ESCs in response to E2, P4, and cAMP (Figures 4E and 4F).

[0114] Seven days after hormone treatment, hESCs cultured in Endo-UdECM showed a significant change from a conical cell shape to a circular shape, indicating improved endometrial receptivity (Figure 4A, B). Furthermore, we observed significant increases in the expression of reproductive hormone receptors estrogen (ESR) and progesterone (PGR) in both the Endo-UdECM and Whole-UdECM groups (Figure 4C, D). Furthermore, we confirmed that the expression of PRL and IGFBP-1, markers of hormone receptivity, was significantly increased in the Endo-UdECM group (Figure 4E, F).

[0115] Experimental Example 3: Effect of direct intrauterine injection of Endo-UdECM and Whole-UdECM in a thin endometrium model An animal model with a thin endometrium (disease model) was created by injecting 95% ethanol (EtOH) into the uterus of female mice during estrus. Specifically, 40 μl of 95% ethanol was injected into one uterine wall using a 31G insulin syringe, and 40 μl of saline was injected into the contralateral uterine wall in the same manner as for the normal control group. After 4 days of estrus (one estrous cycle) following the ethanol and saline injection, the thin endometrium models were divided into an Endo-UdECM injection group and a Whold-UdECM injection group, and each UdECM was injected into the uterus to evaluate the therapeutic effects. The saline injection group served as a control group.

[0116] The results are shown in Figure 5. Figure 5 shows the injection effects of Endo-UdECM and Whole-UdECM according to one embodiment of the present invention injected into a thin endometrium model, and shows hematoxylin and eosin (H&E) staining images (Figure 5A) and endometrial thickness (Figure 5B) after treatment in the thin endometrium model, immunostaining for ITGβ3 and OPN in the thin endometrium treated with saline or UdECM (inner or whole) (Figure 5C), protein expression visualized by DAB color intensity and quantification of ITGβ3 and OPN staining results (Figures 5D and 5E), co-immunofluorescent staining for CD31 (red) and Ki67 (green) in the thin endometrium treated with UdECM (inner or whole) (Figure 5F), and comparison of Ki67-positive cells (Figure 5G and H), total blood vessel count (Figure 5J), and CD31-positive intensity (Figure 5K) in each group.

[0117] When Endo-UdECM and Whole-UdECM were injected into the thin endometrium model, we confirmed that endometrial thickness significantly increased, unlike the saline injection group (Figure 5A and B). Integrin β3 and osteopontin (OPN), markers of endometrial receptivity, significantly increased in the Endo-UdECM-treated group, whereas in the Whole-UdECM-treated group, only the increase in OPN was significant, and the integrin β3 level did not change significantly (Figure 5D and E).

[0118] The main characteristics of endometrial pathology are endometrial ischemic pathology and proliferation of the damaged endometrium. Therefore, in this experiment, we investigated whether Endo-UdECM and Whole-UdECM could promote angiogenesis in the endometrium and normalize (promote) endometrial cell proliferation. First, the Whole-UdECM treatment group showed a higher number of cells expressing the cell proliferation marker Ki67 in both the epithelium and stromal layer than the control group (saline treatment group). In contrast, the Endo-UdECM treatment group showed an increase in Ki67-positive cells exclusively in the epithelium, suggesting that tissue-specific cell proliferation can be promoted (Figure 5F, G, H). Furthermore, the angiogenesis marker CD31 was significantly increased in both the Endo-UdECM and Whole-UdECM treatment groups, suggesting that both UdECMs can promote angiogenesis (Figure 5F, J, K).

[0119] Experimental Example 4: Investigation of the common therapeutic mechanism between Endo-UdECM and Whole-UdECM The regulatory mechanism of UdECM was investigated through gene ontology analysis of thin endometrial models treated with Endo-UdECM and Whole-UdECM, respectively.

[0120] Figure 6 shows the results of gene ontology analysis of thin endometrial models treated with Endo-UdECM and Whole-UdECM according to one embodiment of the present invention. Gene-gene interaction network analysis of DEGs classified as cellular component GO terms for Endo-UdECM-treated vs. saline-treated uterus, and Whole-UdECM-treated vs. saline-treated uterus, revealed a common GO term (0042567).

[0121] Both Endo-UdECM and Whole-UdECM treatment conditions expressed common GO terms for insulin-like growth factor ternary complex, including IGF1 and IGFBP3. In particular, IGF1 expression was significantly lower in all UdECM treatments compared to saline treatment, whereas IGFBP3 expression was higher in UdECM treatments. Based on this, we hypothesized that the primary mechanism underlying the therapeutic efficacy of UdECM is downregulation of IGF1 and upregulation of IGFBP3.

[0122] Therefore, in this experimental example, it was hypothesized that injection of UdECM+IGF1 would weaken the efficacy of UdECM, whereas treatment with IGFBP3 alone would have therapeutic efficacy approximately similar to that of UdECM.

[0123] To demonstrate this, we administered IGF1 at a concentration of 1 mg / ml together with Endo-UdECM or Whole-UdECM to a mouse model of thin endometrium. Furthermore, to verify the efficacy of IGFBP3, we administered IGFBP3 alone at a concentration of 1 mg / ml and then examined the therapeutic efficacy of each experimental group.

[0124] The experimental results showed that the UdECM + IGF1 mixed experimental group showed a significant decrease in endometrial thickness and epithelial thickness compared to the UdECM alone treatment group, confirming that IGF1 reduces the therapeutic efficacy of UdECM. Additionally, in the IGFBP3 treatment group, the recovery of endometrial thickness and endometrial epithelial thickness were similar to those in the UdECM alone treatment group, confirming that IGFBP3 plays a role similar to that of the therapeutic efficacy of UdECM.

[0125] Experimental Example 5: In vitro efficacy of Endo-UdECM and Whole-UdECM on human endometrial tissue Human endometrial samples were collected under IRB approval (approval number: 2020-10-007) at CHA College of Medicine, Bundang CHA Hospital (Bundang, Seongnam-si, Gyeonggi-do, Republic of Korea). Endometrial samples were cut into 1-2 mm pieces. 3 The cells were sliced ​​into 1000 ml and cultured on a cell culture chamber slide (SPL 30108) in the following medium.

[0126] Culture medium composition: DMEM / F12 + FBS 20% (v / v) + L-glutamine 1% (v / v)

[0127] After this, to verify the in vitro efficacy of each UdECM, 0.1% Endo-UdECM or 0.1% Whole-UdECM was added to the culture medium, and the pH was adjusted to 6.0 by adding 1 mol-1 sodium hydroxide (NaOH) or hydrogen chloride (HCl).

[0128] The efficacy of Endo-UdECM and Whole-UdECM was verified by comparing the expression of CD31, an angiogenesis marker, and Ki67, a cell proliferation marker, respectively.

[0129] Figure 7 shows the results of in vitro validation of the efficacy of Endo-UdECM and Whole-UdECM according to one embodiment of the present invention on human endometrial tissue, including co-immunofluorescent staining of Ki67 (green) and CD31 (red) in human endometrial tissue (patient H sample (A) and patient S sample (B)) cultured in Endo-UdECM- or Whole-UdECM-containing medium compared to control medium (Figure 7A, B), representative images of immunostaining for OPN in human endometrial tissue cultured in Endo-UdECM- or Whole-UdECM-containing medium compared to control medium (Figure 7C), and a comparison of OPN expression in each group (Figure 7D).

[0130] In Patient H, a patient with endometrial hyperplasia, CD31 expression was confirmed to be increased in both UdECM treatment groups. In contrast, in Patient S, a patient with uterine adhesions, Ki67 expression was confirmed to be increased in both UdECM treatment groups. Furthermore, OPN, an endometrial receptivity marker, was significantly increased in Patient H treated with Endo-UdECM and in Patient S treated with Whole-UdECM. These findings suggest that Endo-UdECM and Whole-UdECM each have distinct efficacies for endometrial hyperplasia and uterine adhesions. These results suggest that tissue-specific dECM compositional regulation is essential for successful endometrial regeneration and improved fertility, allowing for personalized treatments tailored to each patient and disease.

[0131] The applicant has described preferred embodiments of the present invention above, but these embodiments are merely one embodiment that realizes the technical idea of ​​the present invention, and any changes or modifications that realize the technical idea of ​​the present invention should be construed as falling within the scope of the present invention.

Claims

1. A composition for endometrial regeneration comprising a uterus-derived decellularized extracellular matrix (UdECM).

2. The composition for endometrial regeneration according to claim 1, wherein the uterus is derived from a pig (porcine).

3. The composition for endometrial regeneration according to claim 1, wherein the uterus-derived decellularized extracellular matrix is ​​a decellularized extracellular matrix (Whole-UdECM) derived from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimetrium.

4. The composition for endometrial regeneration according to claim 1, wherein the uterus-derived decellularized extracellular matrix is ​​endometrium-derived decellularized extracellular matrix (Endo-UdECM).

5. The composition for endometrial regeneration according to claim 1, wherein the uterus-derived decellularized extracellular matrix is ​​an endometrium-derived decellularized extracellular matrix (Endo-UdECM) isolated from uterine tissue (Whole Uterus) including the endometrium, myometrium, and perimetrium.

6. The composition for endometrial regeneration according to claim 1, wherein the uterus-derived decellularized extracellular matrix is ​​an endometrium-derived decellularized extracellular matrix (Endo-UdECM) separated from uterine tissue (Whole Uterus) including endometrium, myometrium, and perimetrium, after treating the uterine tissue (Whole Uterus) with a surfactant.

7. 7. The composition for endometrial regeneration according to claim 1, characterized in that it contains collagens, glycoproteins and proteoglycans.

8. Increase expression of receptors for steroid hormones upon treatment with said hormones; The composition for endometrial regeneration according to claim 7, characterized in that it has endometrial regeneration function and angiogenesis promoting function.

9. Providing uterine tissue (Whole Uterus) including the endometrium and myometrium; treating the prepared uterine tissue with a first surfactant; separating the endometrium from the first surfactant-treated uterine tissue; treating the separated endometrium with a second surfactant; A method for producing a composition for endometrial regeneration, comprising:

10. The method for producing a composition for endometrial regeneration according to claim 9, wherein the first surfactant is sodium dodecyl sulfate (SDS).

11. The method for producing a composition for endometrial regeneration according to claim 9, wherein the second surfactant is Triton (trade name) X-100.

12. A bioink composition comprising a uterus-derived decellularized extracellular matrix (UdECM).

13. A composition for culturing uterine organoids, comprising a uterine-derived decellularized extracellular matrix (UdECM).

14. A composition for fabricating an organ-on-a-chip, comprising a uterus-derived decellularized extracellular matrix (UdECM).

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