Establishment of endometrial organoids
This study was conducted in accordance with the Declaration of Helsinki. All analyses involving patient and human samples adhered to the guidelines and procedures of the Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The Institutional Review Board (IRB number:2023-KY-011-02), approved the appropriate licenses and protocols, and informed consent was obtained from all participating patients. Endometrial samples were collected from premenopausal women (n = 3) undergoing routine hysterectomy for benign uterine conditions. These women had not taken any hormonal drugs in the three months prior to tissue collection. The endometrial samples were histologically classified as normal proliferative by pathologists at Beijing Obstetrics and Gynecology Hospital. Fresh endometrial tissue was collected during surgeries and processed immediately to ensure the preservation of cellular characteristics. The tissue was enzymatically digested at 37 °C for 45 min using collagenase II (1 mg/mL, Sigma, C2-28-100MG) and collagenase IV (1 mg/mL, Sigma, C4-28-100MG), in combination with a ROCK inhibitor (10µM, Y27632, MCE, HY-10071/CS-0131) to facilitate single-cell dissociation and prevent cell aggregation. The collagenase concentration and digestion time were optimized to ensure effective tissue dissociation while maintaining cell viability. After enzymatic digestion, the digested tissue was filtered through a 100 μm cell strainer to remove undigested tissue fragments, and the resulting single-cell suspension was collected. The entire dissociation process was carried out in a sterile environment, and cell viability was regularly checked using trypan blue exclusion assay to ensure the high quality of the dissociated cells. To ensure proper cell encapsulation for organoid culture, the cell suspension was resuspended in Matrigel (70%, Corning, 356255, no phenol red) and plated in a 24-well plate. The use of Matrigel provides a suitable three-dimensional environment for organoid formation, mimicking the in vivo tissue architecture. Three drops of this Matrigel-cell suspension were plated into each well of a 24-well plate, which was overlaid with 500µL of media containing DMEM/F12 (HyClone, SH30272.01), B27 (1X, Gibco, 17504-044), N2 supplement (1X, Gibco, 17502-048), 500nM A83-01 (MCE, HY-10432), 250 µg/mL EGF (MCE, HY-P7109), 250 µg/mL Rspondin-1 (MCE, HY-P7114), and 100 µg/mL Noggin (MCE, HY-P7051A). Organoids formed within 3–4 days and were passaged according to their growth and confluence, which were monitored using both an optical microscope and a fluorescence microscope (OLYMPUS).
Generation and propagation of Apical-Out endometrial organoids (AO-EOs)
To generate apical-out endometrial organoids (AO-EOs), the growth medium was first aspirated, and cell recovery solution was added to the culture. The organoid-cell mixture was then transferred into Eppendorf tubes using wide-bore 1 mL pipette tips. The tubes were gently rotated at 4 °C for 1 h to allow complete dissociation. Following this, the organoids were pelleted by centrifugation at 100 g for 5 min and washed with DMEM/F12 to remove the recovery solution. The washed organoids were resuspended in a medium supplemented with the ROCK inhibitor Y27632, which supports cell survival and prevents anoik is in suspension cultures. These organoids were then seeded into ultra-low attachment plates (Corning, Cat. No. 3473) to establish a suspension culture. To avoid organoid clumping, gentle agitation using a pipette was performed twice daily. The growth medium was replaced every other day to maintain optimal conditions for organoid proliferation and apical-out orientation.
Establishment of ALI culture system
Organoids were first removed from Matrigel and dissociated into single cells using TrypLE (Gibco, 12604021). To ensure cell viability, the cell suspension was diluted in trypan blue, and the live cells were counted using a hemocytometer. A total of 5 × 10⁵ viable cells were seeded onto the porous membranes of 12-well Transwell inserts (0.4 μm pore size, Thermo Fisher, 141002), with 250µL of culture medium added to the apical side of the insert and 500µL to the basolateral chamber. After 72 h of culture, the medium in the apical compartment was carefully aspirated to establish the ALI culture, leaving only 500µL of medium in the basolateral chamber. The basolateral medium was replaced every three days to maintain optimal cell growth and differentiation.
Hormone treatment of organoids
To examine the hormone responsiveness of EOs following passaging, 10,000 cells per Matrigel droplet were plated in 24-well plates (3 droplets per well) and allowed to establish into organoids over 3 d in organoid medium. We chose 10nM E2 (estradiol) and the corresponding concentration of P4 (progesterone) based on previous studies that demonstrated these levels effectively simulate the hormonal environment necessary for the physiological functions of endometrial cells in vitro17. EOs were then treated with either 10nM estradiol (MCE, HY-B0141) or control for 3 d. Following this, organoids were treated with either 10nM E2 and 1µM P4 (Progesterone, MCE, HY-N0437) (E2 + P4), 10nM E2, for a further 4 d. Each treatment was performed in triplicate wells with a fourth well for each treatment used for histology purposes.
Co-culture of organoids with bacteria
Escherichia coli (ATCC-25922) was obtained from the Microecology Laboratory of Beijing Obstetrics and Gynecology Hospital and routinely cultured on LB agar plates at 37 °C. For bacterial growth, isolated colonies were picked from agar plates and inoculated into LB broth (Solarbio, L8291), followed by incubation at 37 °C with shaking. EOs were removed from Matrigel by treating them with cell recovery solution and then washed thoroughly with DMEM/F12 medium. The E. coli concentration was measured using a spectrophotometer (OD600). The bacteria were used in their exponential phase to ensure maximum viability and infectivity. We used an MOI of 1 for the infection experiments. The EOs were subsequently resuspended in organoid culture medium. To initiate the co-culture, EOs were combined with bacterial cells in a tissue culture incubator and rotated for 1 h to promote bacterial interaction. After the incubation, excess bacterial cells were removed by washing the organoids with PBS multiple times to minimize non-adherent bacteria. The co-cultured organoids were then incubated for a recovery period of 24 h, with fresh medium added every other day. At predetermined timepoints, 500µL of the culture medium supernatant was collected for ELISA to quantify cytokine or protein levels related to bacterial infection.
Total RNA isolation and real-time quantitative PCR
Total RNA was extracted using the Trizol reagent (RNA Extraction Kit, AG11701, Accurate Biology Co., Ltd.) according to the manufacturer’s protocol. The isolated RNA was then reverse transcribed into complementary DNA (cDNA) using the Reverse Transcription Kit (AG11728, Accurate Biology Co., Ltd.). Real-time quantitative PCR (qPCR) was conducted using SYBR Green dye (AG11701, Accurate Biology Co., Ltd.) on an ABI7500 system. The gene expression levels were calculated using the comparative threshold cycle (Ct) method. Primers for the genes of interest are listed in Table 1. Each reaction was performed in triplicate to ensure reproducibility and minimize variation.
RNA-seq analysis
Total RNA was extracted from EOs using Trizol reagent according to standard protocols. RNA integrity was assessed with the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), ensuring high-quality RNA for downstream applications. The quality control parameters included an A260/A280 ratio ≥ 1.8, A260/A230 ratio ≥ 2.0, and an RNA Integrity Number (RIN) ≥ 8.0. For RNA sequencing, 1 µg of RNA was used for library preparation. mRNA was enriched using oligo(dT) beads, fragmented, and reverse transcribed to generate cDNA. The cDNA was then subjected to end repair, adapter ligation, and amplification to construct the sequencing library. The library’s quality was assessed using the Agilent Bioanalyzer and Qubit Fluorometer, and only libraries with a size range of 300–500 bp were selected for sequencing. RNA sequencing was performed on the Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA) with paired-end sequencing (2 × 150 bp). Raw data were processed with FastQC for quality control, and reads were aligned to the reference genome using STAR. Differential expression analysis was conducted using DESeq2, with differentially expressed genes (DEGs) identified based on a fold change > 2 and a false discovery rate (FDR) < 0.05. KEGG pathway analysis was performed using the clusterProfiler R package with an FDR threshold of 0.05 to assess the statistical significance of enriched pathways. The primers used for qPCR are listed in Table 1.
ELISA assay
The ELISA assay utilized antibodies specific to human TNF-α (NeoBioscience, ECC102a), IL-8 (NeoBioscience, EHC008.96), and IFN-γ (NeoBioscience, EHC102g.96), which were immobilized in 96-well plates according to the manufacturer’s instructions. Organoid-conditioned medium and protein standards were added to the wells, allowing the target proteins to bind to the immobilized antibodies for 2.5 h at room temperature. Following this incubation, biotinylated secondary antibodies were added and allowed to incubate for an additional hour. After incubation, the wells were washed four times with 1× wash solution to remove unbound material. The absorbance at 450 nm was measured using a microplate reader. All experiments were performed in triplicate to ensure statistical reliability.
Statistical analysis
Significant differences among the three experimental groups were determined using one-way analysis of variance (ANOVA) for comparisons involving three sets of data and t-tests for comparisons involving two sets of data. All statistical analyses were performed using GraphPad Prism 9, and the data are presented as mean ± standard error of the mean (SEM). Differences were considered statistically significant at P < 0.05.
