A method for constructing a human endometrial organoid aging model

Abstract

This invention provides a method for constructing a human endometrial organoid aging model, belonging to the field of biological model technology. Based on human endometrial organoids, this invention utilizes hydrogen peroxide (H2O2) stimulation to induce a human endometrial organoid aging model. This human endometrial organoid aging model clearly exhibits multiple cellular aging phenotypes, including slowed organoid growth rate, decreased cell proliferation index (Ki67), enhanced SA-β-gal activity, upregulated expression of cell cycle inhibitors P16 and P21, and significantly upregulated gene expression levels of aging-associated secretory phenotype (SASP) components. It can provide an effective tool for screening anti-aging drugs for women, high-throughput and high-content screening, personalized treatment strategy development, and mechanistic research.

Description

Technical Field

[0001] This invention belongs to the field of biological model technology, specifically relating to a method for constructing a human endometrial organoid aging model. Background Technology

[0002] A woman's fertility declines significantly with age, and previous research has largely focused on age-related ovarian dysfunction, particularly the decline in egg quality and quantity. However, recent studies have increasingly demonstrated that uterine physiological aging is a key factor influencing pregnancy outcomes. The endometrium, as a crucial site for embryo implantation and maintaining pregnancy, directly determines pregnancy success. Research shows that the endometrium in older women undergoes a series of changes at the molecular and cellular levels, including decreased cell proliferation, accumulation of cellular aging markers (such as SA-β-gal, P16, and P21), increased aging-associated secretory phenotypes (SASP), elevated oxidative stress levels, weakened DNA damage repair capacity, and reduced responsiveness to sex hormones. These aging characteristics may lead to decreased endometrial receptivity, resulting in implantation failure, recurrent miscarriage, and adverse pregnancy outcomes commonly seen in older pregnancies. Defective decidualization of the endometrium is considered one of the important pathophysiological mechanisms. Decidualization is the process by which endometrial stromal cells undergo morphological and functional transformation under the influence of progesterone and local signals, providing a suitable microenvironment for embryo implantation and placental formation. The aging of the endometrium in older women may impair its normal decidualization ability, thereby affecting the establishment and maintenance of pregnancy.

[0003] For a long time, the challenge in studying endometrial aging and its impact on pregnancy has been the lack of an ideal in vitro model. Traditional two-dimensional cell culture systems cannot fully simulate the complex cellular composition, three-dimensional structure, and physiological microenvironment of the endometrium, making it difficult to accurately reflect the aging process at the tissue level. Organoid technology, as a revolutionary three-dimensional cell culture method, has emerged. Organoids can form miniature organs with structures, cellular compositions, and some physiological functions similar to in vivo tissues and organs through a self-organization process. However, although progress has been made in the research of human endometrial organoids and organoid aging models, combining the two to construct a model that can accurately simulate human endometrial aging and be used for the study of decidualization defects in advanced maternal pregnancy remains a gap. In particular, there is a lack of effective in vitro tools for understanding the mechanisms of decreased decidualization capacity of the endometrium during aging in older women and for screening therapeutic strategies that can reverse or delay endometrial aging. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing a human endometrial organoid aging model. Using human endometrial organoids as the object, the method simulates the accelerated cellular aging process by treating them with hydrogen peroxide (H2O2) as an inducer. The resulting aging model can simulate the aging characteristics of the endometrium in older women in terms of morphology, cell proliferation, expression of aging markers, SASP secretion, and response to hormones.

[0005] This invention provides a method for constructing a human endometrial organoid aging model, comprising the following steps:

[0006] Constructing human endometrial organoids;

[0007] The human endometrial organoids were treated with H2O2 to obtain an aging model of human endometrial organoids.

[0008] Preferably, the concentration of H2O2 used for treatment is 45~55 nM.

[0009] Preferably, the concentration of H2O2 used in the treatment is 50 nM.

[0010] Preferably, the treatment time of H2O2 is 110~125 h.

[0011] Preferably, during the H2O2 treatment, the human endometrial organoids are cultured in a culture medium containing H2O2.

[0012] The culture medium containing H2O2 was changed every 48 hours;

[0013] The culture density of the human endometrial organoids is 100-200 organoids / 20 μL.

[0014] Preferably, the method for constructing human endometrial organoids involves enzymatic digestion of pretreated human endometrial tissue, followed by solid-liquid separation and purification of the resulting enzymatic hydrolysate to obtain single-cell or glandular cell clusters of human endometrial tissue.

[0015] Human endometrial single cells or glandular cell clusters were cultured in three dimensions using extracellular matrix gel as a medium to obtain human endometrial organoids.

[0016] Preferably, during the three-dimensional culture, the number of human endometrial single cells or glandular cell clusters contained in each 20 μL or 50 μL of extracellular matrix gel is 1 × 10⁻⁶. 4 ~5×10 4 .

[0017] Preferably, the enzyme solution used for enzyme digestion is RPMI 1640 culture medium containing 10% FBS at a volume percentage concentration of 0.5-2 mg / mL dispersant, 0.05-0.1 mg / mL DNase I, and 0.5-2 mg / mL type I or type II collagenase.

[0018] This invention provides a method for constructing a human endometrial organoid aging model. First, a human endometrial organoid maintaining a three-dimensional structure is constructed. Then, the human endometrial organoid is stimulated with H2O2 to induce an aging model, resulting in the human endometrial organoid exhibiting multiple cellular aging phenotypes, including slowed organoid growth, decreased cell proliferation index (Ki67), enhanced SA-β-gal activity, upregulated expression of cell cycle inhibitory proteins P16 and P21, and significantly upregulated gene expression levels of aging-associated secretory phenotype (SASP) components (such as IL-6 and CCL-2). These core aging characteristics make this aging model a powerful tool for studying the mechanisms of endometrial aging. Therefore, this invention provides a rapid, controllable, and reproducible in vitro aging-inducing method based on H2O2 stimulation, while ensuring a multi-dimensional aging phenotype assessment system, guaranteeing a comprehensive, objective, and quantitative assessment of the aging state. Meanwhile, the aging model constructed in this invention is based on human endometrial organoids, providing an in vitro model that more closely resembles the in vivo physiological environment. This allows for in-depth analysis of molecular and cellular changes in the endometrial aging process in older women, particularly its role in decidualization defects during advanced pregnancy. Furthermore, the aging model can serve as a highly efficient screening platform for testing and evaluating potential anti-aging drugs or treatments, accelerating the development of personalized intervention strategies for reproductive aging-related diseases and geriatric syndromes. In addition, the construction method provided by this invention opens new avenues for research on biological aging, particularly reproductive system aging, helping to discover new aging biomarkers, predict adverse outcomes during the aging process, and offer new possibilities for future precision medicine and personalized treatment. As can be seen, this invention, by constructing a highly biomimetic, fully functional human endometrial organoid model that can accurately simulate aging, and combining it with advanced analytical techniques, effectively overcomes the limitations of existing research models in simulating the complexity of human physiological and pathological processes, evaluating drug effects, and conducting high-throughput screening. It provides an unprecedented, reliable, and efficient tool for studying the mechanism of endometrial aging and its resulting decidualization defects in advanced pregnancy, as well as for screening anti-aging drugs and developing treatment plans for women, and has significant scientific value and clinical application prospects. Attached Figure Description

[0019] Figure 1These are representative morphological images of human endometrial organoids. Image A shows the morphology of the organoids on day 3 of P3 culture (left image scale bar: 500 μm, right image scale bar: 200 μm); Image B shows the morphology of the organoids on day 6 of P3 culture (left image scale bar: 500 μm, right image scale bar: 200 μm); Image C shows the human endometrial organoids with typical glandular three-dimensional structure after HE staining (left image: 40 ×, right image: 200 ×).

[0020] Figure 2 Immunofluorescence staining results of epithelial cell markers in human endometrial tissue and human endometrial organoids. A shows the co-expression of CK7 (green) and E-cad (red) in endometrial tissue (top image) and human endometrial organoids (bottom image) using immunofluorescence staining. Note: DAPI (blue) was used for nuclear counterstaining. Top image bar: 50 μm, bottom image bar: 250 μm. B shows the co-expression of LAM (green) and EPCAM (red) in endometrial tissue (top image) and human endometrial organoids (bottom image) using immunofluorescence staining. Note: DAPI (blue) was used for nuclear counterstaining. Top image bar: 50 μm, bottom image bar: 250 μm.

[0021] Figure 3 The images show the morphological observations of human endometrial organoids after treatment with different concentrations of H2O2. Image A shows bright-field images of human endometrial organoids exposed to different concentrations of H2O2; scale bar: 500 μm. Image B shows bright-field images of human endometrial organoids from the CTR and SEN groups at 48 h, 72 h, 96 h, and 120 h after the addition of H2O2-containing culture medium; scale bar: 1000 μm.

[0022] Figure 4 The results of Ki67 immunofluorescence staining to assess organoid proliferation capacity are shown in Figure A. Figure A shows the staining images (100 μm) of DAPI (blue), Ki67 (red), and CK7 (green) in CTR group organoids (top image) and the staining images (100 μm) of DAPI (blue), Ki67 (red), and CK7 (green) in SEN group organoids (bottom image). Figure B is a bar chart of quantitative analysis of the proportion of Ki67-positive cells, showing mean ± SD. p <0.01;

[0023] Figure 5The results of SA-β-gal staining for assessing organoid aging levels are shown in Figure A (top image): SA-β-gal staining images (100 μm) of CTR organoids and SEN organoids (bottom image); Figure B is a bar chart of quantitative analysis of the proportion of SA-β-gal positive cells, showing mean ± SD. p <0.05;

[0024] Figure 6 The results show the expression levels of cell cycle inhibitory proteins and SASP factors, including the relative expression levels of P16 (A), P21 (B), IL-6 (C), and CCL-2 (D) mRNA detected by RT-qPCR; Note: Bar charts are presented as mean ± SD, * p <0.05, *** p <0.001; Detailed Implementation

[0025] This invention provides a method for constructing a human endometrial organoid aging model, comprising the following steps:

[0026] Constructing human endometrial organoids;

[0027] The human endometrial organoids were treated with H2O2 to obtain an aging model of human endometrial organoids.

[0028] This invention first constructs human endometrial organoids.

[0029] In this invention, the preferred method for constructing human endometrial organoids is to perform enzymatic digestion on human endometrial tissue that has undergone impurity removal and pretreatment, and to perform solid-liquid separation and purification on the resulting enzymatic hydrolysate to obtain human endometrial single cells or glandular cell clusters.

[0030] Human endometrial single cells or glandular cell clusters were cultured in three dimensions using extracellular matrix gel as a medium to obtain human endometrial organoids.

[0031] In this invention, the pretreatment method for removing impurities from the human endometrial tissue preferably involves removing excess adipose tissue, muscle layer, and fibrous connective tissue from the human endometrial tissue, retaining only the functional layer and / or basal layer tissue with organoid formation potential, and further breaking it into uniform fragments. The particle size of the fragments is preferably 1~2 mm³.

[0032] In this invention, the enzyme solution used for enzyme digestion is preferably RPMI 1640 culture medium containing 0.5-2 mg / mL dispersant II, 0.05-0.1 mg / mL DNase I, and 0.5-2 mg / mL type I or type II collagenase, at a volume percentage concentration of 10% FBS. Alternatively, it can be RPMI 1640 culture medium containing 1-1.5 mg / mL dispersant II, 0.06-0.08 mg / mL DNase I, and 1-1.5 mg / mL type I or type II collagenase, at a volume percentage concentration of 10% FBS. The enzyme digestion temperature is preferably 36-38°C, and can be 37°C. The enzyme digestion time is preferably 30-90 min, can be 45-80 min, and can also be 50-60 min. Shaking is preferably performed during enzyme digestion. The enzyme digestion is preferably carried out in a constant temperature shaker or water bath.

[0033] In this invention, the preferred method for solid-liquid separation of the obtained enzymatic hydrolysate is filtration. The filtration is preferably performed using a cell sieve. The cell sieve is preferably passed first through a 70 μm pore size sieve, and then through a 40 μm pore size sieve. After filtration, the cell suspension is preferably collected for purification. The purification method preferably includes treatment with red blood cell lysis buffer or differential centrifugation to remove red blood cells and dead cell debris. After obtaining human endometrial single cells or glandular cell clusters, precise cell counting is preferably performed using a hemocytometer or automated cell counter, and / or cell viability and purity are detected using trypan blue staining.

[0034] After obtaining human endometrial single cells or glandular cell clusters, the present invention performs three-dimensional culture of the human endometrial single cells or glandular cell clusters in extracellular matrix gel as a medium to obtain human endometrial organoids.

[0035] In this invention, during the three-dimensional culture, the number of endometrial single cells or glandular cell clusters preferably contained in every 20 μL or 50 μL of extracellular matrix gel is 1 × 10⁻⁶. 4 ~5×10 4 It can be 2×10 4 ~4×10 4 It can also be 3×10 4The extracellular matrix (Matrigel) serves as a three-dimensional culture scaffold, providing necessary physical support and biochemical signals for organoids to mimic the in vivo extracellular matrix environment. The extracellular matrix is ​​preferably a commercially available growth factor-reduced matrix, purchased from Corning, catalog number 356231. The preferred method for three-dimensional culture involves mixing and incubating the endometrial single cells or glandular cell clusters with the extracellular matrix. After the extracellular matrix solidifies, it is placed in a complete culture medium for three-dimensional culture. The preferred incubation conditions are 36–38°C at 5% CO2 for 15–30 min, or 37°C at 5% CO2 for 20–25 min. The complete culture medium is preferably based on AdvancedDMEM / F12 and also includes the following components: B27 supplement (1:50 dilution), N-2 supplement (1:100 dilution), antibiotic Primocin (100 μg / mL), antioxidant N-Acetyl-L-cysteine ​​(1.25 mM), amino acid L-glutamine (2 mM), epidermal growth factor (EGF, 50 ng / mL), fibroblast growth factor 10 (FGF10, 100 ng / mL), R-spondin 1 (500 ng / mL), Noggin (100 ng / mL), HGF (50 ng / mL), A83-01 (ALK5 inhibitor, 500 nM), and nicotinamide (10 mM). All culture medium components are of cell culture grade and are prepared and filtered under strict aseptic conditions (0.22 μm filter membrane) in a laminar flow hood. The complete culture medium successfully cultured human endometrial organoids and maintained their long-term viability. The preferred culture environment for the human endometrial organoids was a humidified incubator at 37°C and 5% CO2, with freshly prepared complete culture medium replaced every 2-3 days. During culture, when the human endometrial organoids grew to approximately 300-500 μL in diameter, or when the density of human endometrial organoids in the culture wells became too high, passage was performed to maintain their long-term stable growth and expand the culture scale. The passage culture method for the human endometrial organoids preferably included mechanical passage and / or enzymatic digestion passage. The mechanical passage method preferably involved repeatedly and gently dispersing the organoid clumps using a sterile pipette tip to break them into appropriately sized fragments or small cell clusters, and then re-embedding them in extracellular matrix gel at a ratio of 13:1:6 for culture.The enzymatic digestion and passage method preferably uses Cell Recovery Solution (Corning, 354253) to separate the organoids from the matrix gel, followed by digestion using TrypLE Express dissociation enzyme (Invitrogen, 12604-013) at 37°C for 5 min to completely dissociate them into single cells or small cell clusters. Subsequently, the cells are collected by centrifugation and, as needed, re-embedded in fresh matrix gel for culture. Through the above-described refined and standardized method for constructing and maintaining human endometrial organoids long-term, this invention can stably and efficiently obtain human endometrial organoids with good morphology, structure, and function. This lays a solid and reliable foundation for the subsequent construction of human endometrial aging models and their application in the study of decidualization defects in advanced maternal pregnancy and drug screening.

[0036] In this invention, the preferred concentration of H2O2 is 45-55 nM, and can be 50 nM. The preferred treatment time of H2O2 is 110-125 h, and can be 115-120 h. During H2O2 treatment, the human endometrial organoids are preferably cultured in a culture medium containing H2O2. The culture medium containing H2O2 is preferably changed every 48 h. The preferred culture density of the human endometrial organoids is 100-200 organoids / 20 μL.

[0037] In this invention, the construction method uses human endometrial organoids as a model, with cells derived from epithelial cells (especially glandular cells) in human endometrial tissue. These cells, through precise isolation and culture techniques, can self-organize into three-dimensional organoids with structures similar to in vivo endometrial glands within a specific three-dimensional culture scaffold. Specifically, the organoids are hollow spherical in shape and contain at least one cell type capable of expressing epithelial cell markers such as E-cadherin, CK7, LAM, and EPCAM. This organoid system allows for long-term stable in vitro passage culture, maintaining its basic morphological structure and functional stability, laying a solid foundation for subsequent aging induction and functional studies.

[0038] The human endometrial organoid aging model constructed by the method described in this invention exhibits one or more distinct cellular senescence phenotypes. These phenotypes include, but are not limited to: significantly slowed overall organoid growth rate, retarded volume growth, significantly reduced cell proliferation index (Ki67), significantly enhanced SA-β-gal activity, significantly upregulated mRNA expression levels of cell cycle repressor proteins P16 and P21, significantly upregulated gene expression levels of senescence-associated secretory phenotype (SASP) components (such as IL-6, CCL-2, etc.), and decreased mitochondrial membrane potential. Macroscopically, the cells in senescent organoids may increase in volume and become flattened, and the overall organoid structure may exhibit disorder or degeneration.

[0039] This invention provides the application of the human endometrial organoid aging model constructed by the above method in the development, screening or evaluation of drugs for treating decidualization of the uterus in older pregnant women, drugs for uterine aging or drugs for treating recurrent miscarriage.

[0040] In this invention, the construction method constructs a highly biomimetic, fully functional human endometrial organoid model that can accurately simulate aging. Combined with advanced analytical techniques, it effectively overcomes the limitations of existing research models in simulating the complexity of human physiological and pathological conditions, evaluating drug effects, and conducting high-throughput screening. It provides an unprecedented, reliable, and efficient tool for studying the mechanism of endometrial aging and its resulting decidualization defects in advanced pregnancy, and has significant scientific value and clinical application prospects.

[0041] The following detailed description of the method for constructing a human endometrial organoid aging model provided by the present invention, with reference to specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0042] Example

[0043] 1. Construction method of human endometrial organoids

[0044] The foundation for establishing a human endometrial organoid aging model lies in constructing stable, long-lasting human endometrial organoids that can respond to hormonal stimulation. This construction method aims to efficiently and reliably isolate and culture organoids with three-dimensional structures and some physiological functions from human endometrial tissue, providing a solid in vitro experimental platform for subsequent aging model construction and drug safety studies.

[0045] 1.1 Methods for obtaining human endometrial tissue samples and preparing cells

[0046] ① The acquisition of endometrial tissue samples must strictly adhere to ethical guidelines and procedures. Samples must be obtained from individuals approved by the ethics committee and from whom the patient has given full informed consent. After acquisition, samples should be immediately placed in cold, sterile phosphate-buffered saline (PBS, preferably calcium and magnesium-free) or basal culture medium containing antibiotics, and transported to the laboratory as soon as possible for further processing.

[0047] ② In a biosafety cabinet, the obtained endometrial tissue undergoes meticulous pretreatment and enzymatic digestion. First, excess adipose tissue, muscle layer, and fibrous connective tissue are removed from the tissue sample, ensuring that only the functional layer and / or basal layer of the endometrium with organoid formation potential are retained. Subsequently, the purified endometrial tissue is cut into uniformly sized small fragments (e.g., 1–2 mm³) and transferred to a digestion solution containing a digestive enzyme mixture for enzymatic digestion. The digestive enzyme mixture contains 2 mg / mL type I collagenase and 2 mg / mL dispersant (Dispase II) in RPMI 1640 culture medium containing 10% FBS. Enzymatic digestion is carried out at 37°C in a shaker or water bath for 60 min, during which the digestion is observed every 20 min until the tissue fragments are completely dissociated into a single-cell suspension or a mixture containing glandular structures.

[0048] ③ After enzymatic digestion, the digestion products need to be filtered and purified. First, the digested cell suspension is filtered through a cell sieve (preferably first through a 70 μm sieve, then through a 40 μm sieve). Then, the filtered cell suspension is transferred to a centrifuge tube and centrifuged at 600 g for 6 min to collect the cell pellet. To further remove red blood cells and dead cell debris, cell purification can be performed using red blood cell lysis buffer or differential centrifugation as needed. Finally, the purified cell pellet is resuspended in an appropriate amount of basal culture medium, and the obtained live cells are accurately counted using a hemocytometer or automated cell counter. Cell viability and purity are assessed using methods such as trypan blue staining.

[0049] 1.2 Three-dimensional culture and long-term maintenance of endometrial organoids

[0050] Three-dimensional culture of endometrial organoids is the core step of this invention. First, the isolated and purified endometrial cells (or glandular cell clusters) are cultured at an optimized cell density (typically 5 × 10⁻⁶). 450 μL of extracellular matrix gel (50 viable cells per well) is resuspended in ice-cold, highly concentrated extracellular matrix gel (commercial growth factor-depleted matrix gel, such as Corning, 356231). Then, under aseptic conditions, this cell-matrix mixture is carefully and evenly added dropwise to the bottom of the wells of a pre-chilled, low-adhesion culture plate using a pre-chilled pipette tip, avoiding air bubbles. Immediately after addition, the culture plate is transferred to a cell culture incubator at 37°C and 5% CO2 and incubated for 20 min to allow the matrix gel to completely solidify. Once solidified, pre-warmed endometrial organoid complete culture medium is immediately and carefully added to each well. The complete culture medium is based on Advanced DMEM / F12 and also includes the following components: B27 supplement (1:50 dilution), N-2 supplement (1:100 dilution), antibiotic Primocin (100 μg / mL), antioxidant N-Acetyl-L-cysteine ​​(1.25 mM), amino acid L-glutamine (2 mM), epidermal growth factor (EGF, 50 ng / mL), fibroblast growth factor 10 (FGF10, 100 ng / mL), R-spondin 1 (500 ng / mL), Noggin (100 ng / mL), HGF (50 ng / mL), A83-01 (ALK5 inhibitor, 500 nM), and nicotinamide (10 mM). All culture medium components are of cell culture grade and are prepared and filtered under strict aseptic conditions in a clean bench (0.22 μm filter membrane).

[0051] Routine culture of organoids is conducted in a humidified incubator at 37°C and 5% CO2. Freshly prepared endometrial organoid complete culture medium should be carefully replaced every 2-3 days. During culture, the growth morphology, size, structure, budding status, and contamination of the organoids should be observed regularly using an inverted microscope, and their growth curves should be recorded. When the organoids grow to approximately 300-500 μm in diameter, or when the density of organoids in the culture wells becomes too high, subculturing is necessary to maintain long-term stable growth and expand the culture scale. The subculturing method can be flexibly selected according to the characteristics of the organoids and experimental requirements.

[0052] Mechanical passage: Organoid clumps are repeatedly and gently agitated using sterile pipette tips to disperse them into appropriately sized fragments or small cell clusters. These dispersed organoid fragments are then re-embedded in fresh substrate gel at a ratio of 1:3 to 1:6 for culture.

[0053] Enzymatic digestion and passage: Organoids were isolated from the matrix gel using Cell Recovery Solution (Cell Recovery Solution, Corning, 354253) and digested using TrypLE Express dissociation enzyme (Invitrogen, 12604-013) at 37°C for 5 min to completely dissociate them into single cells or small cell clusters. Cells were then collected by centrifugation and, as needed, re-embedded in fresh matrix gel for culture.

[0054] 2. Methods for constructing an endometrial organoid aging model

[0055] The H2O2-induced aging model was constructed using young, well-grown, and cultured endometrial organoids (e.g., passaged 3-5 times). Before H2O2 treatment, the organoids were removed from the matrix gel and resuspended at an appropriate density (e.g., 150 organoids / 20 μL) in the gel and seeded into 48-well plates. After the endometrial organoids had stabilized (2-3 days), different concentrations of H2O2 (e.g., 10 nM, 50 nM, 100 nM, 200 nM, 400 nM) were added to the complete culture medium, while a control group (without H2O2 treatment) was also included. The endometrial organoids were cultured in H2O2-containing medium for 120 h, with medium changes every 48 h during the culture period. After H2O2 treatment, the induced organoids were comprehensively evaluated at different time points.

[0056] After determining the optimal H2O2 stimulation concentration, endometrial organoids were cultured in a medium containing the optimal H2O2 concentration for different times (e.g., 48 h, 72 h, 96 h, and 120 h) to comprehensively evaluate the induced organoids. A control group was also established, consisting of endometrial organoids cultured in a medium without H2O2 for the aforementioned different times.

[0057] 3. Characterization of the hydrogen peroxide (H2O2)-induced aging model

[0058] A control group (CTR) and an aging model group (SEN) were set up. SEN was the endometrial organoid aging model constructed in step 2 above, and CTR consisted of endometrial organoids treated according to the "Method for Constructing the Endometrial Organoid Aging Model". Both groups of organoids were cultured under the same basic culture conditions. Organoid samples were collected at predetermined time points for subsequent phenotypic analysis.

[0059] 3.1 Methods for assessing aging phenotypes:

[0060] ①Observation of organoid growth status and morphology:

[0061] Monitoring of growth rate and volume changes: After H2O2 treatment, bright-field images of each group of organoids were observed and captured every 24 hours under an inverted microscope. The overall structure of the organoids was observed under the inverted microscope to check for integrity, clarity of edges, and signs of disintegration or degeneration. Simultaneously, the morphology of cells within the organoids was observed, noting any aging-related changes such as increased cell volume and flattening.

[0062] ② Cell proliferation index detection (Ki67):

[0063] After the collected organoids were fixed and permeabilized, immunofluorescence staining was performed using Ki67 antibody. Simultaneously, DAPI was used for nuclear counterstaining, and endometrial epithelial markers (such as E-cadherin) could be used for co-staining to confirm cell type. The cells were observed under a fluorescence microscope, and multiple fields of view were randomly selected for imaging. The number of Ki67-positive cells and the total number of cell nuclei were counted, and the proportion of Ki67-positive cells was calculated.

[0064] ③ Detection of aging-related β-galactosidase (SA-β-gal) activity:

[0065] Endometrial organoids fixed using a commercially available SA-β-gal staining kit were stained. At pH 6.0, β-galactosidase in senescent cells hydrolyzed the X-gal substrate to produce a blue product. After staining, the proportion of blue-positive (senescent) cells was observed and counted under a light microscope.

[0066] ④ Detection of cell cycle repressor protein (P16, P21) mRNA expression levels:

[0067] Total RNA was extracted from organoids collected from each group and cDNA was synthesized via reverse transcription. Using cDNA as a template, real-time quantitative PCR (RT-qPCR) was performed using primers specific to the human P16 (CDKN2A) and P21 (CDKN1A) genes, as well as primers for internal reference genes (such as GAPDH). The results were analyzed by comparing Ct values ​​and using 2... -ΔΔCt The relative expression levels of P16 and P21 mRNA were calculated using the following steps: Total RNA was extracted from endometrial organoids using the RNeasy Mini Kit. Each group was treated with Buffer RLT containing 1% β-mercaptoethanol, thoroughly mixed by pipetting, and then an equal volume of 70% anhydrous ethanol was added. After mixing, the RNA was transferred to an RNA binding column for purification. The purified RNA was analyzed for RNA content and quality using a NanoDrop micro-spectrophotometer. RNA samples meeting the experimental requirements were classified as A... 260 / A 280The absorbance ratio ranged from 1.8 to 2.1. Next, a 20 μL reverse transcription system was prepared according to the HiFiScript cDNA Synthesis Kit instructions, including 4.0 μL 5× RT Buffer, 4.0 μL dNTP Mix, 2.0 μL Primer Mix, 2.0 μL DTT, 1.0 μL HiFiScript, and 7.0 μL RNA sample. After thorough mixing, the reaction was incubated at 42°C for 15 min followed by 85°C for 5 min for reverse transcription. The resulting cDNA sample was aliquoted and stored at -20°C for later use. HieffUNICON was used. ® The SYBR Green Master Mix qPCR kit was used to perform qRT-PCR on cDNA samples. A 20 μL reaction mixture was prepared according to the manufacturer's instructions, containing 2.0 μL cDNA template, 7.2 μL DEPC water, 0.4 μL 10M forward primer, 0.4 μL 10 μM reverse primer, and 10 μL SYBR Green PCR Master Mix. After thorough mixing, the reaction mixture was placed in a PCR instrument and incubated at 95°C for 2 min. This was followed by a two-step cycle of 95°C for 10 s and 60°C for 30 s for 40 cycles. 2 -ΔΔCt The relative expression level of the target gene was calculated using a method shown in Table 1.

[0068] Table 1 Primer sequences used for qRT-PCR experiments

[0069]

[0070] ⑤ Detection of gene expression levels of aging-associated secretory phenotype (SASP) components:

[0071] RNA was extracted and analyzed by RT-qPCR using a method similar to that described above for detecting cell cycle repressor protein mRNA. Specific primers targeting key SASP components (IL-6, CCL-2) were selected to detect their relative mRNA expression levels.

[0072] 4. Construction and identification results of human endometrial organoids

[0073] This embodiment successfully isolated cells from human endometrial tissue and constructed endometrial organoids with stable morphology and function.

[0074] 4.1 Morphological characteristics of organoids

[0075] See results Figure 1The morphology of organoids on day 3 of P3 generation culture: Under a light microscope, the endometrial organoids have a clear lumen and are surrounded by a ring of tightly packed, hollow spherical structures formed by a single layer of columnar cells (see...). Figure 1 (A). Morphology of organoids on day 6 of P3 generation culture: Under an optical microscope, the morphology of the organoids remained unchanged, but their volume increased, with some reaching a diameter of 200 μm (see [reference]). Figure 1 (B) Endometrial organoids stained with hematoxylin and eosin (HE) showed a typical glandular-like three-dimensional structure, exhibiting multiple ring-shaped or tubular configurations. The central cavity was clearly visible, and the rim was surrounded by a single layer of tightly packed cells. The light staining of the cavity contents suggested the accumulation of secretions. The outer cytoplasm, after hematoxylin-eosin double staining, showed a significant contrast between the purple cytoplasm and the deep purple-red nucleus. The nucleus was regularly shaped with a clearly identifiable nucleolus, and the nucleoplasm-to-cytoplasm ratio was consistent with the characteristics of normal epithelial cells (see [link to article]). Figure 1 (C) As can be seen, under an inverted microscope, the successfully constructed endometrial organoids exhibit a typical three-dimensional hollow spherical structure with regular morphology and clear boundaries. These organoids can grow stably in the Matrigel three-dimensional matrix and form complex tubular structures.

[0076] 4.2 Organoid cell composition and biomarker expression

[0077] To identify the cell type of the constructed organoids, immunofluorescence staining was performed to detect the expression of biomarkers in endometrial epithelial cells. Results are shown below. Figure 2 The results showed that the organoids could mimic the expression of epithelial cell markers Cytokeratin 7 (CK7), E-cadherin (E-cad), Epithelial cell adhesion molecule (EPCAM), and basement membrane marker Laminin (LAM) in in vivo endometrial tissue. These cells were arranged in a regular pattern, forming the main cystic structure of the organoids. These results suggest that the organoids are primarily of epithelial origin.

[0078] 4.3 Construction and Characterization Results of the Endometrial Organoid Aging Model

[0079] 4.3.1 Construction and Characterization of H2O2-Induced Aging Model

[0080] Normal endometrial organoids were treated with different concentrations of H2O2 (e.g., 10 nM, 50 nM, 100 nM, 200 nM, 400 nM), and the results are shown in [Figure number missing]. Figure 3Compared to CTR, treatment with 10 nM H2O2 caused organoids to begin developing black clumps; treatment with 50 nM H2O2 further shrank the organoids, reduced their volume, and significantly weakened their proliferation; while treatments with 100 nM, 200 nM, and 400 nM H2O2 resulted in organoid death and inability to grow, thus determining the optimal exposure concentration to be 50 nM. Subsequently, normal endometrial organoids were treated with 50 nM H2O2 for different durations (e.g., 48 h, 72 h, 96 h, and 120 h), ultimately determining the optimal senescence-inducing conditions as: 50 nM H2O2 treatment for 120 h, with fluid changes every 48 h. Under these conditions, the H2O2-treated organoids exhibited typical senescence phenotypes.

[0081] ① Increased early apoptosis in organoids: Endometrial organoids were treated with 50 nM H2O2 for 120 h, with the medium changed every 48 h and morphological changes of the organoids captured every 24 h. The results are shown in the table below. Figure 3 In the B group, compared with CTR, H2O2 treatment for 48 h did not significantly change the growth status of organoid cells; after 72 h, the organoid volume increased, indicating normal proliferation; after 96 h, the organoids gradually aggregated, folded, and shrank, indicating that organoid proliferation began to weaken; after 120 h, the morphology of the organoids further deteriorated, cell death and aggregation became more severe (blue arrows), and the structural integrity of the organoids decreased significantly. In contrast, the CTR group of endometrial organoids maintained good morphological and structural integrity throughout the observation period, without obvious cell death or morphological changes, indicating that organoids can maintain normal growth and development in the absence of hydrogen peroxide treatment.

[0082] ② Decreased cell proliferation capacity: Cell proliferation index was assessed by Ki67 immunofluorescence staining. The results showed that the proportion of Ki67-positive cells in the SEN group organoids was significantly lower than that in the CTR group. Figure 4 (A, B) indicates that the cell proliferation capacity of organoids in the SEN group was significantly impaired.

[0083] ③ Increased SA-β-gal activity: SA-β-gal staining results showed that the proportion of SA-β-gal-positive (blue staining) cells in the endometrial organoids of the SEN group was significantly higher than that of the CTR group. This is a classic biomarker of cellular senescence. Figure 5 (A, B)

[0084] ④ Upregulation of cell cycle inhibitory proteins and increased secretion of SASP factor: Results obtained from RT-qPCR experiments (see...) Figure 6The results showed that the mRNA expression levels of cell cycle repressor proteins P16 and P21, as well as senescence-associated secretory phenotype (SASP) factors IL-6 and CCL2, were significantly higher in the SEN group organoids than in the CTR group organoids. These results indicate that, compared to the CTR group, the SEN group organoids exhibited upregulation of cell cycle repressor proteins and increased secretion of SASP factors, suggesting a more significant cellular senescence state.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a human endometrial organoid aging model, characterized in that, Includes the following steps: Constructing human endometrial organoids; the method for constructing human endometrial organoids involves enzymatic digestion of pretreated human endometrial tissue, followed by solid-liquid separation and purification of the resulting enzymatic hydrolysate to obtain human endometrial single cells or glandular cell clusters. Human endometrial single cells or glandular cell clusters were cultured in three dimensions using extracellular matrix gel as a medium to obtain human endometrial organoids. The human endometrial organoids were treated with H2O2 to obtain an aging model of human endometrial organoids. The human endometrial organoids were cultured in a medium containing H2O2. The culture medium containing H2O2 was changed every 48 hours; the concentration of H2O2 was 45-55 nM; and the treatment time of H2O2 was 110-125 hours. The culture density of the human endometrial organoids is 100-200 organoids / 20 μL.

2. The construction method according to claim 1, characterized in that, The concentration of H2O2 used in the treatment was 50 nM.

3. The construction method according to claim 1, characterized in that, During the three-dimensional culture, the number of human endometrial single cells or glandular cell clusters contained in each 20 μL or 50 μL extracellular matrix gel was 1 × 10⁻⁶. 4 ~5×10 4 .

4. The construction method according to claim 1, characterized in that, The enzyme solution used for the enzyme digestion is RPMI 1640 culture medium containing 10% FBS at a volume percentage concentration of 0.5-2 mg / mL dispersant, 0.05-0.1 mg / mL DNase I, and 0.5-2 mg / mL type I or type II collagenase.

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