A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis

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It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Methods In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify and evaluate the viability of isolated ectopic endometrial cells. Results Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37% and 93.49%, respectively. Conclusion Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis. Adenomyosis Cell culture Flow cytometry EpCAM/CD326 CD10 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Adenomyosis is a benign uterine disorder characterized by the presence of heterotopic endometrial glands and stroma in the myometrium and reactive fibrosis of the surrounding smooth muscles cells of the myometrium [ 1 ]. The pathogenesis of this condition involves sex steroid hormone abnormalities, inflammation, fibrosis and neuroangiogenesis; however, the specific mechanisms involved have yet to be fully elucidated [ 2 ]. Uterine adenomyosis is a common chronic disorder that is frequently encountered in women of reproductive-age, causing heavy menstrual bleeding, intense pelvic pain, and infertility [ 3 ]. The symptomatology of this condition is not specific and may overlap with other gynecological disorders, including endometriosis and leiomyomas [ 4 ]. Because adenomyosis is a common benign gynecological disorder that affects many women of reproductive-age and causes pelvic pain and infertility, and the precise etiology as well as the mechanisms leading to the disorder are still not clearly determined, so it is very important that researchers aim to identify the specific mechanisms involved in the pathogenesis of this condition [ 5 , 6 ]. Thus far, our knowledge of the cellular localization of the abnormalities associated with adenomyosis has been heavily dependent on immunohistochemistry and has not been verified by other methods because it is difficult to perform primary culture of the cellular components of adenomyosis [ 7 ]. Most of the current research on adenomyosis is based on eutopic endometrium cells which are easy to acquire and culture. Although ectopic stromal cells of human adenomyosis have been described in some articles, there is no established process for primary cell culture. In a previous study, Guan et al. [ 8 ] reported the primary culture and identification of human adenomyosis cell; however, the ectopic epithelial and stromal cells of human adenomyosis foci could not be isolated and cultured thereafter. Thus far, there have been no methods reported that can efficiently isolate and culture ectopic epithelial and stromal cells from human adenomyosis lesions with high purity. The classical diagnosis of adenomyosis relies on the identification of heterotopic endometrial glands and stromal cells within the myometrium [ 9 ]. There is strong evidence that ectopic epithelial and stromal cells play an important role in the development of uterine adenomyosis. To better investigate human adenomyosis, it is very important that we develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells. Primary cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical research [ 10 ]. In such methods, primary cells are obtained from isolated tissues and are then subjected to in vitro culture procedures; however, there are several disadvantages associated with primary cell culture. Furthermore, primary cells derived from non-cancerous tissues have a finite lifespan and reduced proliferation ability when cultured in vitro [ 11 ]. Historically, epithelia have proved more difficult to culture than many other cell types, and only very limited culture was possible [ 12 ]. However, primary cells retain the same characteristics as those of the original tissue, thus facilitating studies in many research areas, including medical and cell biology research, that otherwise would be far more complicated [ 13 ]. Moreover, primary cells represent the normal physiological condition of human cells [ 10 ]. Consequently, primary cell acquisition has attracted increasing levels of attention [ 14 ]. In the present study, hematoxylin and eosin staining was used to identify adenomyosis lesions of different histopathological types. In a previous study, Pistofidis et al. analyzed cases of adenomyosis that had been treated laparoscopically; on the basis of intraoperative and histopathology findings, four distinct types of the disease were identified: diffuse, sclerotic, nodular and cystic adenomyosis [ 6 ]. In this study, we performed flow cytometry to analyze the epithelial and stromal cells and to assist with identification and the evaluation of purity and viability. Here, we describe the use of epithelial cell adhesion molecule (EpCAM/CD326), a pan-epithelial carcinoma-associated antigen expressed in most carcinomas, as a cell surface marker [ 15 ]. Due to vimentin is a mesenchymal marker that is also expressed in mesoderm-derived epithelia, such as the endometrial epithelium [ 16 ]. So we finally determined that primary ectopic epithelial cells were labelled with Ep-CAM while stromal cells were labelled with CD10. In addition, histopathology was combined with preoperative magnetic resonance imaging for analysis. Recent advances in imaging techniques have had a significant impact on the detection of uterine adenomyosis and imaging criteria are now part of the diagnostic workup along with histopathological features [ 17 ]. Transvaginal ultrasonography (TVUS) and magnetic resonance imaging (MRI) are increasingly becoming the dominant diagnostic tools for adenomyosis [ 18 ]. Finally, clinical MRI imaging of adenomyosis was combined with histopathology to identify an extensive area of high-signal intensity myometrial spots on T2-weighted MRI with more ectopic endometrial tissue. Despite major advances in adenomyosis over the last few decades, adenomyosis research and effective drug discovery still suffer from the lack of an ideal cellular model. In this study, we propose a novel and simple protocol for the isolation and culture of ectopic epithelial and stromal cells from human adenomyosis lesions. This method isolated cells with high purity and good viability; moreover, our results were highly reproducible. The culture of ectopic epithelial and stromal cells is of great significance for studying the pathogenesis of human adenomyosis. This method is simple, inexpensive and enables large amounts of cells to be obtained for analysis. 2. Material and Methods 2.1. Patients and sample collection We recruited patients who were diagnosed with adenomyosis from the Women's Hospital, School of Medicine, Zhejiang University between September 2022 to March 2023. Patient age ranged from 34 to 50 years. The inclusion criteria were as follows: premenopausal patients with adenomyosis, accompanied by dysmenorrhea or heavy menstrual bleeding, and were not treated with no hormones or intrauterine devices for the 6 months before surgery. Human adenomyosis lesion samples were obtained from patients who underwent the removal of adenomyosis lesions or hysterectomy surgeries. The diagnosis of adenomyosis was confirmed by pathology. Adenomyosis lesion tissue collection was approved by the Human Ethics Committee of Women's Hospital, School of Medicine, Zhejiang University (Approval number: IBR-20220283-R). For all participants, informed consent was obtained prior to surgery. 2.2. Preparation of reagents and culture medium For this protocol, four reagents are required: 0.2% collagenase Ⅰ, 12% DMEM/F12 complete medium, 10% MEM complete medium and 0.05% trypsin-EDTA. To prepare 0.2% collagenase Ⅰ, 100 mg of type I collagenase powder (BioFroxx; Einhausen, Germany) was dissolved in 50 ml of PBS (BioSharp; Beijing, China ) solution, mixed, and finally stored at 4°C for 1 month. To prepare 12% DMEM/F12 complete medium, DMEM/F12 medium (Gibco, Thermo Fisher; Waltham, MA, USA ) was added into a sterile 50-ml centrifuge tube. Then, 12% fetal bovine serum (Gibco, Thermo Fisher; Waltham, MA, USA) and 50 U/mL penicillin, and 50 µg/mL streptomycin (Gibco, Thermo Fisher; Waltham, MA, USA) was added and mixed well. The fully configured 12% DMEM/F12 medium could be stored at 4°C or 1 month. To prepare 10% MEM complete medium, 10% fetal bovine serum and 50 U/mL penicillin, and 50 µg/mL streptomycin was added to MEM medium (Keyi; Hangzhou, China). To prepare 0.05% trypsin-EDTA, 0.5% trypsin-EDTA 10× (Gibco, Thermo Fisher; Waltham, MA, USA) was diluted 1:10 in PBS (BioSharp; Beijing, China). All of these solutions could be stored at 4°C for 1 month; except for trypsin, all solutions were filtered with a 0.22-µm sterile mesh. 2.3. Preparation and digestion of specimens 2.3.1. Preparation All steps were performed in a laminar flow hood using sterile disposable material. Fresh ectopic endometrial tissue was collected from human adenomyosis lesions under sterile conditions, and ectopic endometrial tissue was collected, as shown in Fig. 1 A and 1 B. The method used to collect samples of ectopic endometrial tissue of adenomyosis is shown in Fig. 1 C. Specifically, a sharp knife (No. 11) was vertically inserted at the junction of the ectopic endometrial lesion and myometrium of adenomyosis and then rotated clockwise for two or three circles along the endometrial surface according to the size of the lesion. The collected sample was then placed in a 50-ml sterile centrifuge tube containing Dulbecco's Modified Eagle Medium (DMEM)/F12 containing 1% penicillin and streptomycin (Gibco, Thermo Fisher; Waltham, MA, USA). Then, specimens were transported from the operating room to the laboratory in an ice box. Specimens were kept at 4°C for no more than 4 hours. The tissue was washed in sterilized twice in declined Mg/Ca PBS (BioSharp; Beijing, China) and then centrifuged at 1000 rpm for 5 minutes. Then, the supernatant was discarded and the washed tissue samples were transferred to 10-cm petri dishes, as shown in Fig. 1 D. 2.3.2. Digestion The tissue samples were cut into smaller fragments less than 1 mm wide in a 10-cm petri dish, using sterile eye scissors. The tissue fragments were then transferred to a sterile 50-ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The samples were then digested using pre-configured 0.2% type Ⅰ collagenase (volume ratio 1:3) and slowly shaken on a constant temperature shaker at 37℃ for 30–60 minutes. The digestion time was set according to the amount of tissue sample. The degree of tissue digestion was observed every 10 minutes after 30 minutes. We observed digestion and terminated this step when tissue fragments disappeared or became flocculent; the dissociation solution became cloudy as cells detached. Digestion was terminated by adding an equal volume of 12% DMEM/F12 complete medium. The dissociation solution was then blended with a 5-ml sterile pasteurized dropper, and gently blown for 1–2 minutes to digest the tissue sample and to generate a single cell suspension. 2.4. Cells isolation and culture 2.4.1. Primary cells isolation and culture The cell suspension was passed first through 100- and 40-µm nylon meshes to separate primary ectopic epithelial cells and stromal cells. The epithelial cells remained in the upper layer of the 40-µm mesh; therefore the 40-µm sterile cell filter was inverted on the pipe orifice of the sterile 50-ml centrifuge tube, washed with PBS, and ectopic epithelial cells were collected. Separated ectopic epithelial and stromal cells were centrifuged at 1000 rpm for 7 minutes to remove the supernatant solution and the cell pellet was resuspended in 12% DMEM/F12 complete medium. Cells were cultured at 37˚C in a humidified incubator containing 5% CO 2 . After the cells were adherent, the medium was changed after 24 h for stromal cells and 48 h for epithelial cells. The growth and morphological characteristics of the primary cells were observed daily under an inverted microscope (Inverted Microscope BGI-V53; magnification, 10 x 500 µm). Eutopic stromal cells from adenomyosis were isolated and cultured in the same manner as described above. 2.4.2. Cell culture of human endometrial adenocarcinoma Human endometrial adenocarcinoma cells (HEC-1B cells) were purchased from CBTCCCAS (Cell Bank, Type Culture Collection, Chinese Academy of Sciences) (catalog number: TCHu115) and then cultured in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS), 50 u/ml penicillin, and 50 µg/ml streptomycin. Our methodology was the same as that utilized in our previous study [ 19 ]. The medium was completely changed every 2–3 days. Cells were sub-cultured every 3–4 days, or when they reached 90% confluency. 2.5. Hematoxylin and eosin staining To verify whether our findings were correct, hematoxylin and eosin staining were performed on adenomyosis lesions of different histopathological types, and then observed under a microscope. Specifically, different histopathological tissue types of adenomyosis lesions were collected (10×10×10 mm 3 in size), fixed in 4% paraformaldehyde for 48 h at room temperature, embedded in paraffin, and cut into 4-µm-thick sections. Each section was then deparaffinized, hydrated, washed, and stained with hematoxylin–eosin (H&E) using a commercial kit (Beyotime; Shanghai, China). Sections were sealed with neutral resin, using a microtome. Images were then captured under a microscope (Nikon TE2000-S; Nikon) and analyzed using ImageScope software. 2.6. Flow cytometry The primary cells were cultured for 2–3 days and the human endometrial adenocarcinoma cells were verified to be in good condition. The medium was then removed and the cells were washed three times with sterile PBS. Then, the cells were incubated with a sufficient volume of 0.05% trypsin-EDTA solution to cover the cell layer, and then incubated at 37℃ until cell detachment. The progress of cell detachment was observed under a light microscope; typically for 3–8 minutes. Digestion was terminated by adding a double volume of 12% DMEM/F12 complete medium; then, the cells were transferred into a 15-ml tube. After centrifugation at 1000 rpm for 5 minutes at room temperature, the supernatant was removed and resuspended with PBS to prepare a single cell suspension. Then, 100 µl of single-cell suspension was added into each 1.5-ml microcentrifuge tube. A negative control was set for each group. Except for the blank control group, we then added anti-human Cytokeratin-FITC (Invitrogen, Thermo Fisher; Waltham, MA, USA), anti-human Ep-CAM-PE (CD326 Invitrogen, Thermo Fisher; Waltham, MA, USA), and anti-human CD10 PE-Cy7 (Biolegend; San Diego, California, USA) antibodies to the corresponding microcentrifuge tube and incubated in the dark for 30 minutes at 4°C. The cells were then centrifuged at 2000 rpm for 5 min at room temperature and the unconjugated antibody was removed. The cells were then washed in PBS. Stained cells were then resuspended in 500 µl of PBS and 0.5 µl of SYTOX AADvanced Dead Cell Stain Kit was added to a live/dead single dye tube and triple-dyed tubes to identify live cells. After 5–10 minutes, samples were detected by flow cytometry (BD FACSVerse); data was analyzed by BD FACSuite software. 3. Results 3.1. The discovery and collection of ectopic endometrial tissue from human adenomyosis lesions Ectopic endometrial tissue is constituted by glands and stroma and exists as foci of variable sizes, located haphazardly in the myometrium [ 2 ]. Previous studies suggested that the ectopic endometrium plays an important role in the existence and proliferation of the myometrium. Therefore, there is an urgent need to identify ectopic endometrial tissue in adenomyosis lesions. The discovery of ectopic endometrial tissue in adenomyosis and the ability to culture ectopic epithelial and stromal cells will contribute significantly to the study of adenomyosis. In the process of sample collection and primary cell culture, we continued to adapt and improve, and finally detected ectopic endometrial tissue in human adenomyosis lesions which were located haphazardly in the myometrium (Fig. 1 ). In addition, we found that a scalpel with a size 11 blade was the best option for collecting ectopic endometrial tissue from adenomyosis (Fig. 1 C). Specimen collection is shown in Fig. 1 D; these specimens were used for cell culture, thus significantly increasing the number, purity and viability of both ectopic epithelial and stromal cells. 3.2. Analysis results of HE staining of different histopathological types of human adenomyosis The types of adenomyosis described in most previous articles were divided into two types: diffuse and focal. According to the typing described by Pistofidis et al. [6], in this study, only diffuse, sclerotic and cystic adenomyosis were described. To further validate and determine our present findings, we performed hematoxylin and eosin staining on adenomyosis lesions of different histopathological types; this was followed by microscopy (Figure 2). HE staining was performed on the ectopic endometrium and surrounding myometrium tissues. Microscopically, obvious ectopic endometrial glands and stromal cells were observed in the myometrium (Figure 2E, F); these structures were significantly more abundant than the sclerotic type and cystic adenomyosis. Of these, cystic adenomyosis featured a significant amount of hemosiderin (Figure 2A, B). These results showed that we had located the soft tissue of adenomyosis which protruded slightly from the surface of the lesion. These structures were distributed in the myometrium as islets and represented ectopic endometrial tissue in human adenomyosis lesions. 3.3. Primary cell culture of ectopic epithelial and stromal cells from human adenomyosis lesions Ectopic endometrium tissue was collected from human adenomyosis lesions for primary cells isolation and culture; then, we observed their morphology. On day 1 after the first inoculation, the stromal cells were completely adherent, and the epithelial cells were partially adherent. Compared to other cell types, the stromal cells appeared to be of a uniformly small size (Fig. 3 B). However, the epithelial cells varied in size and grew in clusters (Fig. 3 A). On days 1–3 of culture, the epithelial cells were round or oval and slowly spread outwards. On day 5 after the first inoculation, the epithelial cells had become flattened. The stromal cells gradually became larger with the extension of inoculation time, followed by protrusion of pseudopodia; subsequently, the cells formed a single-layer adherent spindle and adopted a polygonal appearance. There were many more stromal cells than epithelial cells. 3.4. Identification of primary cell surface markers Flow cytometry is a commonly used research and diagnostic tool that uses fluorophore conjugated antibodies as probes to identify, characterize, and/or isolate cell populations [ 20 ]. We performed flow cytometry to confirm the epithelial and stromal phenotype of the cultured cells. Moreover, human endometrial adenocarcinoma cells (HEC-1B cells) and primary eutopic stromal cells of adenomyosis were used as positive controls. Generally, the primary endometrium of uterine cells was characterized by a profile that was EpCAM+, cytokeratin+, vimentin + or CD10+. We also detected cytokeratin for comparison; analysis revealed only minimal expression of the epithelial cell marker Cytokeratin (5.71%) in human endometrial adenocarcinoma cells (HEC-1B cells) (Fig. 4 A). We also found that HEC-1B cells expressed high levels of Ep-CAM/CD326 (99.7%) (Fig. 4 B). CD10, a broadly applied stromal cell marker, was selected for confirmation. Analysis showed that CD10 was robustly expressed by eutopic stromal cells in human adenomyosis (a positive rate of 98.41%) (Fig. 4 D). After verification, we finally determined that primary ectopic epithelial cells were labelled with Ep-CAM while stromal cells were labelled with CD10. Furthermore, flow cytometry was performed on cultured ectopic epithelial cells from adenomyosis lesions; this revealed a positive rate of 4.23% for cytokeratin and 80.54% for EpCAM/CD326 (Fig. 4 C). 3.5. Identification, purity, and viability analysis of primary cells from human adenomyosis lesions The identification, purity, and viability of the ectopic epithelial and stromal cells cultured from human adenomyosis lesions was demonstrated by flow cytometry, and then compared with cells cultured from a small piece of adenomyosis lesion tissue. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58% (Fig. 5 B). In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37% and 93.49%, respectively (Fig. 5 C). Due to the contamination of uterine smooth muscle cells and fibrocytes, the purity and viability of cells cultured from one piece of adenomyosis lesion tissue were significantly lower than those of ectopic endometrial tissues (Fig. 5 A). These results showed that the primary cell culture of ectopic endometrium tissue was better than the traditional culture method involving a small piece of adenomyosis lesion tissue in terms of cell number, purity and viability. 3.6. MRI findings of human adenomyosis exhibited a certain correlation with histopathology The diagnosis of adenomyosis used to be determined solely by histology after hysterectomy; however, with the advent of modern imaging technology, adenomyosis is increasingly being diagnosed in a non-invasive fashion [ 21 , 22 ]. Typical adenomyosis MRI images appear as an ill-demarcated low-signal intensity area on T2-weighted images and represent smooth muscle hyperplasia (Fig. 6 A, C). In addition, intramyometrial cysts and small high-signal intensity areas referring to ectopic endometrium may also be detected on T2-weighted MRI [ 2 ] (Fig. 6 A). Hyperintense myometrial foci are direct imaging evidence of the structures visualized by histopathology and represent islets of the heterotopic endometrium in the myometrium. They are visualized on MRI as T2 bright foci, varying in size from 2 to 7 mm, abnormally located in the myometrium [ 23 ] (Fig. 6 A). We then compared preoperative MRI images with intraoperative ex vivo adenomyosis tissue. We found that an extensive area of high-signal intensity myometrial spots were visible on T2-weighted MRI images with more ectopic endometrial tissue (Fig. 6 A, B). In contrast, the MRI images showed low signal intensity area in the myometrium (Fig. 6 C), there was no obvious macroscopic ectopic endometrial tissue in intraoperative specimens; according to a previous study, the histopathological classification of this tissue was sclerotic. In the ‘sclerotic’ type, lesions present as an irregular thickening of the myometrium in which the tissue is hard and adopts an off-white pale and fibrotic appearance [ 6 ] (Fig. 6 D). Furthermore, HE staining images revealed the presence of ectopic endometrium glands and stromal cells in the myometrium (Fig. 2 C, D). 4. Discussion At present, the exact etiology of adenomyosis remains unknown. However, the most widely accepted theories propose that endometrial glands directly invade the myometrium, thus resulting in angiogenesis of the spiral vessels along with hyperplasia and hypertrophy of the adjacent smooth muscle tissue [ 24 ]. The isolation and culture of ectopic epithelial and stromal cells from human adenomyosis lesions is the first step to accomplish when studying adenomyosis. The present study describes the first isolation and culture of primary ectopic epithelial and stromal cells from adenomyosis lesions. In addition, we were able to culture primary ectopic epithelial and stromal cells with high purity and good viability by performing simple and operable methods. In general, when obtained from diverse tissue sources, ectopic epithelial and stromal cells exhibit significant differences in yield and purity. During our initial attempts, we used a small piece of adenomyosis lesion tissue for digestion and culture; however, only a small number of primary ectopic epithelial and stromal cells were acquired. Furthermore, due to the long digestion time involved, cell viability was weakened and there was an increased risk of contamination by fibroblasts and smooth muscle cells. To overcome these problems, after continuous observation and research, we finally identified ectopic endometria tissue in adenomyosis lesions and collected this tissue for culture. When processing and digesting specimens, we employed type I collagenase; according to the literature, collagenase I, a less potent enzyme, causes less damage to primary cells during the digestion process when compared to other enzymes [ 14 ]. Furthermore, we used HE staining and flow cytometry to verify our results; results were consistent. In this study, we developed a simple and optimal purification technique for primary cells. Initially, we attempted to isolate primary ectopic epithelial and stromal cells by flow cytometry; however, it proved difficult to continue to culture after sorting cells in this manner. Moreover, this method extended the time prior to culture initiation; moreover, the technique was expensive, lead to the loss of epithelial and stromal cells, and was associated with a lower success rate. However, the purity of the sorted cells exceeded 95%. Because the former approach was difficult and expensive, we considered that epithelial cells and stromal cells adopted different sizes and could therefore be separated from each other by cell mesh filtration. The results of flow cytometry showed that the purity of cells exceeded 90% and that the purity of stromal cells reached 97%. We were able to harvest a large number of ectopic epithelial and stromal cells during a short period of time if a sufficient amount of tissue was used. Eventually, we developed simple purification and culture technique for primary ectopic epithelial and stromal cells from human adenomyosis lesions. In the present study, we observed that ectopic epithelial cells had underwent adherence later than stromal cells. If the culture medium was changed too early, we observed unnecessary cell loss. According to the results of the present study, the optimal time for the first medium change was 2 days after inoculation. We also found that ectopic epithelial cells could not expand normally; nor could they proliferate and achieve higher confluency; therefore, it was not possible to passage these cells. In most instances, flow cytometry can be used to detect cell surface markers expressed on epithelial and stromal cells. EpCAM is a promising biomarker that is highly expressed on epithelial cancer cells but expressed at lower levels on normal epithelial cells [ 15 ]. Following verification, we found that cytokeratin was only expressed at low levels on ectopic epithelial cells while Ep-CAM was expressed stably and at high levels. Flow cytometry analysis showed that ectopic epithelial and stromal cells stably expressed Ep-CAM (93.74%) and CD10(97.1%). The successful identification of ectopic epithelial and stromal cell surface markers is critical if we are to investigate the molecular mechanisms underlying adenomyosis. There are also limitations to this study that need to be considered. Due to the different histopathological types of adenomyosis, not every ex vivo specimen could allow for the satisfactory collection of ectopic endometrium tissue. As with the sclerotic adenomyosis described earlier, there was no obvious ectopic endometrium tissue in some cases; instead, we observed large areas of fibrotic hard tissue. However, ectopic endometrial glands and stromal cells were still visible under microscopy following HE staining. Despite major advances in adenomyosis over recent decades, hysterectomy would not be a treatment option for young women wishing to preserve their child-bearing capacity. Furthermore, adenomyosis research and effective drug discovery still suffer from the lack of an ideal cellular model. In the past, the diagnosis of adenomyosis was mainly based on pathological examination after hysterectomy. At present, most researchers believe that vaginal ultrasound and MRI are good non-invasive diagnostic tools for the diagnosis of adenomyosis. In addition, in this study, we combined clinical preoperative MRI imaging to compare intraoperative ex vitro adenomyosis lesions. We successfully observed multiple punctate T2W1 high signal intensity regions within the low-signal intensity area on MRI. Furthermore, there were more ectopic endometria tissues in intraoperative specimens, and cultured cells showed good viability. A previous case report described a case with severe uterine adenomyosis. Prior to treatment, MRI showed an enlarged uterus with diffuse and disseminated adenomyosis. Multiple regions of T2W1 high signal intensity were evident on MRI. After 12 weeks of GnRH antagonist therapy (a daily dose of 200 mg linzagolix), a significant reduction was observed in uterine size and the size of the adenomyotic lesions [ 4 ]. In addition, T2W1 high density signal atrophy disappeared on MRI images. From a clinical perspective, an ideal imaging technique should not only diagnose adenomyosis accurately; it should also facilitate the selection of the best treatment modality [ 2 ]. Finally, this study concluded that ectopic endometrial tissue plays an important role in the occurrence and development of adenomyosis. In most instances, T2W1 high signal intensity within the low-signal intensity area on MRI images revealed more ectopic endometrial tissues. Controlling ectopic endometrial tissue or causing the ectopic endometrial tissue to undergo atrophy can effectively control and treat adenomyosis. 5. Conclusion Our newly developed method is a simple, efficient and economical technique to establish primary ectopic epithelial and stromal cells cultures with high purity and good viability from human adenomyosis lesion. Moreover, this technique provides a new experimental model for studying the pathogenesis of human adenomyosis. Declarations Authors’ contributions Zhou Fang : Conceptualization, Writing – original draft, Data curation, Methodology, Writing – review & editing, Validation, Resources. Jianzhang Wang and Tiantian Li : Conceptualization, Methodology, Writing – review & editing, Validation, Supervision. Meichen Yin and Yangying Peng : Data curation, Methodology, Resources, Validation. Xinmei Zhang : Supervision, Conceptualization, Writing – review & editing, Project administration, Validation. All the authors read the submitted version and approved it. Acknowledgements This work was funded by National Key R&D Program of China (Grant number: 2022YFC2704003), National Natural Science Foundation of China (Grant numbers: 81974225, 82001518 and 82171636). Conflict of interest statement The authors report no conflict of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References T. Harada, Y.M. Khine, A. Kaponis, T. Nikellis, G. Decavalas, F. Taniguchi, The Impact of Adenomyosis on Women's Fertility, Obstet Gynecol Surv, 71 (2016) 557-568, http://dx.doi.org/10.1097/OGX.0000000000000346. C. Chapron, S. Vannuccini, P. Santulli, M.S. Abrão, F. Carmona, I.S. Fraser, S. Gordts, S.-W. Guo, P.-A. Just, J.-C. Noël, G. Pistofidis, T. Van den Bosch, F. Petraglia, Diagnosing adenomyosis: an integrated clinical and imaging approach, Hum Reprod Update, 26 (2020) 392-411, http://dx.doi.org/10.1093/humupd/dmz049. J. 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Joplin, Isolation and culture of biliary epithelial cells, Gut, 35 (1994) 875-878. F.A. Abade Dos Santos, C.L. Carvalho, I. Almeida, T. Fagulha, F. Rammos, S.C. Barros, M. Henriques, T. Luís, M.D. Duarte, Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures, Cells, 10 (2021), http://dx.doi.org/10.3390/cells10082100. Z. Hu, Y. Chen, M. Gao, X. Chi, Y. He, C. Zhang, Y. Yang, Y. Li, Y. Lv, Y. Huang, X. Deng, Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I, Cell Prolif, 56 (2023) e13320, http://dx.doi.org/10.1111/cpr.13320. Y. Zhang, J. An, M. Liu, N. Li, W. Wang, H. Yao, N. Li, X. Yang, Y. Sun, N. Xu, L. Wu, Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma, Am J Reprod Immunol, 84 (2020) e13251, http://dx.doi.org/10.1111/aji.13251. D.F. O, T. Roskams, K. Van den Eynde, A. Vanhie, D.P. Peterse, C. Meuleman, C. Tomassetti, K. Peeraer, T.M. D'Hooghe, A. Fassbender, The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis, Reprod Sci, 24 (2017) 242-251, http://dx.doi.org/10.1177/1933719116653677. M. Bazot, E. Daraï, Role of transvaginal sonography and magnetic resonance imaging in the diagnosis of uterine adenomyosis, Fertil Steril, 109 (2018) 389-397, http://dx.doi.org/10.1016/j.fertnstert.2018.01.024. S.-W. Guo, G. Benagiano, M. Bazot, In Search of an Imaging Classification of Adenomyosis: A Role for Elastography?, J Clin Med, 12 (2022), http://dx.doi.org/10.3390/jcm12010287. Q. Yu, J. Wang, T. Li, X. Xu, X. Guo, S. Ding, L. Zhu, G. Zou, Y. Chen, X. Zhang, RON Mediates Tumor-Promoting Effects in Endometrial Adenocarcinoma, Biomed Res Int, 2021 (2021) 2282916, http://dx.doi.org/10.1155/2021/2282916. L.R. Bonser, K.D. Koh, K. Johansson, S.P. Choksi, D. Cheng, L. Liu, D.I. Sun, L.T. Zlock, W.L. Eckalbar, W.E. Finkbeiner, D.J. Erle, Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets, Am J Respir Cell Mol Biol, 64 (2021) 308-317, http://dx.doi.org/10.1165/rcmb.2020-0149MA. L. Fedele, S. Bianchi, M. Dorta, L. Arcaini, F. Zanotti, S. Carinelli, Transvaginal ultrasonography in the diagnosis of diffuse adenomyosis, Fertil Steril, 58 (1992) 94-97. A.S. Mark, H. Hricak, L.W. Heinrichs, M.R. Hendrickson, M.L. Winkler, J.A. Bachica, J.E. Stickler, Adenomyosis and leiomyoma: differential diagnosis with MR imaging, Radiology, 163 (1987) 527-529. M. Zhang, M. Bazot, M. Tsatoumas, M.G. Munro, C. Reinhold, MRI of Adenomyosis: Where Are We Today?, Can Assoc Radiol J, 74 (2023) 58-68, http://dx.doi.org/10.1177/08465371221114197. V. Celli, M. Dolciami, R. Ninkova, G. Ercolani, S. Rizzo, M.G. Porpora, C. Catalano, L. Manganaro, MRI and Adenomyosis: What Can Radiologists Evaluate?, Int J Environ Res Public Health, 19 (2022), http://dx.doi.org/10.3390/ijerph19105840. Cite Share Download PDF Status: Published Journal Publication published 23 Oct, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Reviewers agreed at journal 25 Aug, 2023 Reviewers invited by journal 28 Jul, 2023 Editor invited by journal 27 Jul, 2023 Editor assigned by journal 26 Jul, 2023 First submitted to journal 26 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3194957","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":222292525,"identity":"048fae84-1c84-4790-a3a2-aaca9ae4c2b8","order_by":0,"name":"Zhou Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie2PIQ7CQBBFp9mEmoHaNdxhkiYLhKZnoSFZheAITbAcgGPgGlxhAqqAReBIMJiViAoWUJiWOhL2if9HzEtmAByOH4QAn5UDCVuGoriZ4i2metxAsSnQbLy0TukFezZQnpPMb/MtolyAz9tllTJID1oCXpPVrKOHEzp3ALU+VR62nisAycmSUYUTugqQqKoVxtDYfCt9Yi+tVXZIEkYvJbzAV0qBSkLO4WqGypuTHrdqfzkW9rCSu1lgh3sZxYHPu0rliSjf3ZKvrFv/UE2TbYfD4fgfHvcNSqu0tz8qAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0001-8367-6043","institution":"Women's Hospital School of Medicine Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Fang","suffix":""},{"id":222292526,"identity":"f886d5d2-445e-47ef-8051-523976c22089","order_by":1,"name":"Jianzhang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianzhang","middleName":"","lastName":"Wang","suffix":""},{"id":222292527,"identity":"4dffe1d3-d0a0-4214-b191-104b0e75acc4","order_by":2,"name":"Tiantian Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Li","suffix":""},{"id":222292528,"identity":"8316bf86-041f-408a-9e10-ca155142e250","order_by":3,"name":"Meichen Yin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Meichen","middleName":"","lastName":"Yin","suffix":""},{"id":222292529,"identity":"814a0fd7-55f8-43a0-947f-db2e76044abf","order_by":4,"name":"Yangying Peng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yangying","middleName":"","lastName":"Peng","suffix":""},{"id":222292530,"identity":"9c0181c0-9184-4948-af42-10dadf9c8ebf","order_by":5,"name":"Xinmei Zhang","email":"","orcid":"https://orcid.org/0000-0001-7122-6435","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xinmei","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-07-22 16:03:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3194957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3194957/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00404-023-07254-8","type":"published","date":"2023-10-23T15:02:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":40972109,"identity":"ba1cb032-ea97-4148-b83f-83d74700d209","added_by":"auto","created_at":"2023-08-02 17:34:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5344300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe detection of ectopic endometrium in human adenomyosis lesions. \u003c/strong\u003e(A) and (B) show adenomyosis lesions of following surgical removal from patients. The blue arrow indicates islet tissue protruding from the surface; this represents the ectopic endometrium tissue within adenomyosis lesions that needed to be collected for analysis. The yellow arrow in (B) refers to the uterine cavity. (C) The method used to collect ectopic endometrium tissue from human adenomyosis lesions with a sharp knife blade (No. 11). (D) Samples of ectopic endometrium tissue from human adenomyosis lesions after collection and washing.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/2629a53e7fcf8804bc5a9c88.png"},{"id":40970991,"identity":"5ba7b756-2141-4856-87bf-60e484533201","added_by":"auto","created_at":"2023-08-02 17:26:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14563514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHE staining images of different histopathological types in human adenomyosis lesions. \u003c/strong\u003eThe image on the right shows a magnified image of the rectangle shown on the left. \u0026nbsp;(A, B) representative H\u0026amp;E images of cystic adenomyosis (scale bars: 2 mm and 200 µm). (C, D) representative H\u0026amp;E images of sclerotic adenomyosis (scale bars: 2 mm and 200 µm). (E, F) represent H\u0026amp;E images of diffuse adenomyosis (scale bars: 2 mm and 200 µm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/70038afd54725f41b4752ba1.png"},{"id":40970990,"identity":"79b05636-49cd-402e-9637-58bdc8f905de","added_by":"auto","created_at":"2023-08-02 17:26:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14994719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characteristics of human adenomyosis ectopic epithelial and stromal cells (10×).\u003c/strong\u003e (A, C, E, G) The left column shows typical epithelial cell morphology on days 1–3 and 5 of culture after the first inoculation. (B, D, F, H) Plots of stromal cell morphology at the same time after inoculation. Scale bar = 500 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/a4cfae14aabe717f72eb76b5.png"},{"id":40970988,"identity":"67c5ef6a-7ee9-48e8-8be6-659d263d0ce6","added_by":"auto","created_at":"2023-08-02 17:26:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1600848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of surface markers on primary ectopic epithelial and stromal cells. \u003c/strong\u003eNumbers represent the percentage cells in the gate. (A) The proportion of cells was 5.71% in human endometrial adenocarcinoma cells (HEC-1B cells); (B) Ep-CAM was highly expressed in human endometrial adenocarcinoma cells (the proportion of HEC-1B cells that were positive for Ep-CAM was 99.7%). (C) Only a small proportion (4.23%) of ectopic epithelial cells of adenomyosis lesions expressed cytokeratin. However, 80.58% of cells expressed high levels of EpCAM/CD326. (D) CD10 was robustly expressed by eutopic stromal cells in human adenomyosis (the proportion of cells that were positive for CD10 was 98.41%).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/b713180523ebbf553875fc86.png"},{"id":40970985,"identity":"bfe85591-1fd4-4746-958a-0490ddffdd42","added_by":"auto","created_at":"2023-08-02 17:26:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2019377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative gating strategy for isolating and culturing ectopic epithelial and stromal cells. \u003c/strong\u003eTypically, we gated all events, including single cells, then live cells for both ectopic epithelial and stromal cells. Finally, we performed cell purity analysis. (A) The results of flow cytometry experiments involving primary cells from adenomyosis lesions. Among others, 69.95% of live cells in the gating were further analyzed; the purity of ectopic epithelial and stromal cells were 2.8% and 27.12%, respectively. (B) Purity, viability and phenotype of isolated and cultured ectopic epithelial cells. Analysis showed that the purity and viability were 93.74% and 84.92%, respectively. (C) Flow cytometry and CD10 staining detected the purity and viability of isolated ectopic stromal cells; the purity and viability were for 97.1% and 96.05%, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/30d9cf7a28241bc17d3e6237.png"},{"id":40970987,"identity":"50efb65a-9560-4b39-8e29-72ca2aa810d3","added_by":"auto","created_at":"2023-08-02 17:26:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3318470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological characteristics of ectopic lesions corresponding to MRI images acquired from adenomyosis patients.\u003c/strong\u003e (A) MRI characteristics of adenomyosis, which presents as an extensive area of high signal intensity in the myometrium on T2-weighted MRI (white arrow). (B) The corresponding intraoperative tissue map of an \u003cem\u003eex vivo\u003c/em\u003e lesion shows a large amount of ectopic endometria tissues. (C) The MRI characteristics of adenomyosis, which presents as low-signal intensity on T2-weighted MRI (white arrow). (D) Intraoperative tissue diagram of a patient with adenomyosis; the tissue was hard with a fibrotic appearance.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/f36b58d190e9a6b213fa5e18.png"},{"id":45453854,"identity":"260a2510-a08d-40a1-a297-55522f4ee713","added_by":"auto","created_at":"2023-10-30 15:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7518969,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3194957/v1/d46726e3-b855-4b8c-a8d9-b3d0b1501b92.pdf"}],"financialInterests":"","formattedTitle":"A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdenomyosis is a benign uterine disorder characterized by the presence of heterotopic endometrial glands and stroma in the myometrium and reactive fibrosis of the surrounding smooth muscles cells of the myometrium [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathogenesis of this condition involves sex steroid hormone abnormalities, inflammation, fibrosis and neuroangiogenesis; however, the specific mechanisms involved have yet to be fully elucidated [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Uterine adenomyosis is a common chronic disorder that is frequently encountered in women of reproductive-age, causing heavy menstrual bleeding, intense pelvic pain, and infertility [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The symptomatology of this condition is not specific and may overlap with other gynecological disorders, including endometriosis and leiomyomas [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Because adenomyosis is a common benign gynecological disorder that affects many women of reproductive-age and causes pelvic pain and infertility, and the precise etiology as well as the mechanisms leading to the disorder are still not clearly determined, so it is very important that researchers aim to identify the specific mechanisms involved in the pathogenesis of this condition [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus far, our knowledge of the cellular localization of the abnormalities associated with adenomyosis has been heavily dependent on immunohistochemistry and has not been verified by other methods because it is difficult to perform primary culture of the cellular components of adenomyosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Most of the current research on adenomyosis is based on eutopic endometrium cells which are easy to acquire and culture. Although ectopic stromal cells of human adenomyosis have been described in some articles, there is no established process for primary cell culture. In a previous study, Guan et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] reported the primary culture and identification of human adenomyosis cell; however, the ectopic epithelial and stromal cells of human adenomyosis foci could not be isolated and cultured thereafter. Thus far, there have been no methods reported that can efficiently isolate and culture ectopic epithelial and stromal cells from human adenomyosis lesions with high purity. The classical diagnosis of adenomyosis relies on the identification of heterotopic endometrial glands and stromal cells within the myometrium [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. There is strong evidence that ectopic epithelial and stromal cells play an important role in the development of uterine adenomyosis. To better investigate human adenomyosis, it is very important that we develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells.\u003c/p\u003e \u003cp\u003ePrimary cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical research [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In such methods, primary cells are obtained from isolated tissues and are then subjected to \u003cem\u003ein vitro\u003c/em\u003e culture procedures; however, there are several disadvantages associated with primary cell culture. Furthermore, primary cells derived from non-cancerous tissues have a finite lifespan and reduced proliferation ability when cultured \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Historically, epithelia have proved more difficult to culture than many other cell types, and only very limited culture was possible [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, primary cells retain the same characteristics as those of the original tissue, thus facilitating studies in many research areas, including medical and cell biology research, that otherwise would be far more complicated [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, primary cells represent the normal physiological condition of human cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, primary cell acquisition has attracted increasing levels of attention [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, hematoxylin and eosin staining was used to identify adenomyosis lesions of different histopathological types. In a previous study, Pistofidis et al. analyzed cases of adenomyosis that had been treated laparoscopically; on the basis of intraoperative and histopathology findings, four distinct types of the disease were identified: diffuse, sclerotic, nodular and cystic adenomyosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, we performed flow cytometry to analyze the epithelial and stromal cells and to assist with identification and the evaluation of purity and viability. Here, we describe the use of epithelial cell adhesion molecule (EpCAM/CD326), a pan-epithelial carcinoma-associated antigen expressed in most carcinomas, as a cell surface marker [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to vimentin is a mesenchymal marker that is also expressed in mesoderm-derived epithelia, such as the endometrial epithelium [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. So we finally determined that primary ectopic epithelial cells were labelled with Ep-CAM while stromal cells were labelled with CD10. In addition, histopathology was combined with preoperative magnetic resonance imaging for analysis. Recent advances in imaging techniques have had a significant impact on the detection of uterine adenomyosis and imaging criteria are now part of the diagnostic workup along with histopathological features [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Transvaginal ultrasonography (TVUS) and magnetic resonance imaging (MRI) are increasingly becoming the dominant diagnostic tools for adenomyosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Finally, clinical MRI imaging of adenomyosis was combined with histopathology to identify an extensive area of high-signal intensity myometrial spots on T2-weighted MRI with more ectopic endometrial tissue.\u003c/p\u003e \u003cp\u003eDespite major advances in adenomyosis over the last few decades, adenomyosis research and effective drug discovery still suffer from the lack of an ideal cellular model. In this study, we propose a novel and simple protocol for the isolation and culture of ectopic epithelial and stromal cells from human adenomyosis lesions. This method isolated cells with high purity and good viability; moreover, our results were highly reproducible. The culture of ectopic epithelial and stromal cells is of great significance for studying the pathogenesis of human adenomyosis. This method is simple, inexpensive and enables large amounts of cells to be obtained for analysis.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Patients and sample collection\u003c/h2\u003e \u003cp\u003eWe recruited patients who were diagnosed with adenomyosis from the Women's Hospital, School of Medicine, Zhejiang University between September 2022 to March 2023. Patient age ranged from 34 to 50 years. The inclusion criteria were as follows: premenopausal patients with adenomyosis, accompanied by dysmenorrhea or heavy menstrual bleeding, and were not treated with no hormones or intrauterine devices for the 6 months before surgery. Human adenomyosis lesion samples were obtained from patients who underwent the removal of adenomyosis lesions or hysterectomy surgeries. The diagnosis of adenomyosis was confirmed by pathology. Adenomyosis lesion tissue collection was approved by the Human Ethics Committee of Women's Hospital, School of Medicine, Zhejiang University (Approval number: IBR-20220283-R). For all participants, informed consent was obtained prior to surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of reagents and culture medium\u003c/h2\u003e \u003cp\u003eFor this protocol, four reagents are required: 0.2% collagenase Ⅰ, 12% DMEM/F12 complete medium, 10% MEM complete medium and 0.05% trypsin-EDTA. To prepare 0.2% collagenase Ⅰ, 100 mg of type I collagenase powder (BioFroxx; Einhausen, Germany) was dissolved in 50 ml of PBS (BioSharp; Beijing, China ) solution, mixed, and finally stored at 4\u0026deg;C for 1 month. To prepare 12% DMEM/F12 complete medium, DMEM/F12 medium (Gibco, Thermo Fisher; Waltham, MA, USA ) was added into a sterile 50-ml centrifuge tube. Then, 12% fetal bovine serum (Gibco, Thermo Fisher; Waltham, MA, USA) and 50 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin (Gibco, Thermo Fisher; Waltham, MA, USA) was added and mixed well. The fully configured 12% DMEM/F12 medium could be stored at 4\u0026deg;C or 1 month. To prepare 10% MEM complete medium, 10% fetal bovine serum and 50 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin was added to MEM medium (Keyi; Hangzhou, China). To prepare 0.05% trypsin-EDTA, 0.5% trypsin-EDTA 10\u0026times; (Gibco, Thermo Fisher; Waltham, MA, USA) was diluted 1:10 in PBS (BioSharp; Beijing, China). All of these solutions could be stored at 4\u0026deg;C for 1 month; except for trypsin, all solutions were filtered with a 0.22-\u0026micro;m sterile mesh.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation and digestion of specimens\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Preparation\u003c/h2\u003e \u003cp\u003eAll steps were performed in a laminar flow hood using sterile disposable material. Fresh ectopic endometrial tissue was collected from human adenomyosis lesions under sterile conditions, and ectopic endometrial tissue was collected, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. The method used to collect samples of ectopic endometrial tissue of adenomyosis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. Specifically, a sharp knife (No. 11) was vertically inserted at the junction of the ectopic endometrial lesion and myometrium of adenomyosis and then rotated clockwise for two or three circles along the endometrial surface according to the size of the lesion. The collected sample was then placed in a 50-ml sterile centrifuge tube containing Dulbecco's Modified Eagle Medium (DMEM)/F12 containing 1% penicillin and streptomycin (Gibco, Thermo Fisher; Waltham, MA, USA). Then, specimens were transported from the operating room to the laboratory in an ice box. Specimens were kept at 4\u0026deg;C for no more than 4 hours. The tissue was washed in sterilized twice in declined Mg/Ca PBS (BioSharp; Beijing, China) and then centrifuged at 1000 rpm for 5 minutes. Then, the supernatant was discarded and the washed tissue samples were transferred to 10-cm petri dishes, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Digestion\u003c/h2\u003e \u003cp\u003eThe tissue samples were cut into smaller fragments less than 1 mm wide in a 10-cm petri dish, using sterile eye scissors. The tissue fragments were then transferred to a sterile 50-ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The samples were then digested using pre-configured 0.2% type Ⅰ collagenase (volume ratio 1:3) and slowly shaken on a constant temperature shaker at 37℃ for 30\u0026ndash;60 minutes. The digestion time was set according to the amount of tissue sample. The degree of tissue digestion was observed every 10 minutes after 30 minutes. We observed digestion and terminated this step when tissue fragments disappeared or became flocculent; the dissociation solution became cloudy as cells detached. Digestion was terminated by adding an equal volume of 12% DMEM/F12 complete medium. The dissociation solution was then blended with a 5-ml sterile pasteurized dropper, and gently blown for 1\u0026ndash;2 minutes to digest the tissue sample and to generate a single cell suspension.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cells isolation and culture\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Primary cells isolation and culture\u003c/h2\u003e \u003cp\u003eThe cell suspension was passed first through 100- and 40-\u0026micro;m nylon meshes to separate primary ectopic epithelial cells and stromal cells. The epithelial cells remained in the upper layer of the 40-\u0026micro;m mesh; therefore the 40-\u0026micro;m sterile cell filter was inverted on the pipe orifice of the sterile 50-ml centrifuge tube, washed with PBS, and ectopic epithelial cells were collected. Separated ectopic epithelial and stromal cells were centrifuged at 1000 rpm for 7 minutes to remove the supernatant solution and the cell pellet was resuspended in 12% DMEM/F12 complete medium. Cells were cultured at 37˚C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After the cells were adherent, the medium was changed after 24 h for stromal cells and 48 h for epithelial cells. The growth and morphological characteristics of the primary cells were observed daily under an inverted microscope (Inverted Microscope BGI-V53; magnification, 10 x 500 \u0026micro;m). Eutopic stromal cells from adenomyosis were isolated and cultured in the same manner as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Cell culture of human endometrial adenocarcinoma\u003c/h2\u003e \u003cp\u003eHuman endometrial adenocarcinoma cells (HEC-1B cells) were purchased from CBTCCCAS (Cell Bank, Type Culture Collection, Chinese Academy of Sciences) (catalog number: TCHu115) and then cultured in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS), 50 u/ml penicillin, and 50 \u0026micro;g/ml streptomycin. Our methodology was the same as that utilized in our previous study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The medium was completely changed every 2\u0026ndash;3 days. Cells were sub-cultured every 3\u0026ndash;4 days, or when they reached 90% confluency.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Hematoxylin and eosin staining\u003c/h2\u003e \u003cp\u003eTo verify whether our findings were correct, hematoxylin and eosin staining were performed on adenomyosis lesions of different histopathological types, and then observed under a microscope. Specifically, different histopathological tissue types of adenomyosis lesions were collected (10\u0026times;10\u0026times;10 mm\u003csup\u003e3\u003c/sup\u003e in size), fixed in 4% paraformaldehyde for 48 h at room temperature, embedded in paraffin, and cut into 4-\u0026micro;m-thick sections. Each section was then deparaffinized, hydrated, washed, and stained with hematoxylin\u0026ndash;eosin (H\u0026amp;E) using a commercial kit (Beyotime; Shanghai, China). Sections were sealed with neutral resin, using a microtome. Images were then captured under a microscope (Nikon TE2000-S; Nikon) and analyzed using ImageScope software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Flow cytometry\u003c/h2\u003e \u003cp\u003eThe primary cells were cultured for 2\u0026ndash;3 days and the human endometrial adenocarcinoma cells were verified to be in good condition. The medium was then removed and the cells were washed three times with sterile PBS. Then, the cells were incubated with a sufficient volume of 0.05% trypsin-EDTA solution to cover the cell layer, and then incubated at 37℃ until cell detachment. The progress of cell detachment was observed under a light microscope; typically for 3\u0026ndash;8 minutes. Digestion was terminated by adding a double volume of 12% DMEM/F12 complete medium; then, the cells were transferred into a 15-ml tube. After centrifugation at 1000 rpm for 5 minutes at room temperature, the supernatant was removed and resuspended with PBS to prepare a single cell suspension. Then, 100 \u0026micro;l of single-cell suspension was added into each 1.5-ml microcentrifuge tube. A negative control was set for each group. Except for the blank control group, we then added anti-human Cytokeratin-FITC (Invitrogen, Thermo Fisher; Waltham, MA, USA), anti-human Ep-CAM-PE (CD326 Invitrogen, Thermo Fisher; Waltham, MA, USA), and anti-human CD10 PE-Cy7 (Biolegend; San Diego, California, USA) antibodies to the corresponding microcentrifuge tube and incubated in the dark for 30 minutes at 4\u0026deg;C. The cells were then centrifuged at 2000 rpm for 5 min at room temperature and the unconjugated antibody was removed. The cells were then washed in PBS. Stained cells were then resuspended in 500 \u0026micro;l of PBS and 0.5 \u0026micro;l of SYTOX AADvanced Dead Cell Stain Kit was added to a live/dead single dye tube and triple-dyed tubes to identify live cells. After 5\u0026ndash;10 minutes, samples were detected by flow cytometry (BD FACSVerse); data was analyzed by BD FACSuite software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The discovery and collection of ectopic endometrial tissue from human adenomyosis lesions\u003c/h2\u003e \u003cp\u003eEctopic endometrial tissue is constituted by glands and stroma and exists as foci of variable sizes, located haphazardly in the myometrium [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies suggested that the ectopic endometrium plays an important role in the existence and proliferation of the myometrium. Therefore, there is an urgent need to identify ectopic endometrial tissue in adenomyosis lesions. The discovery of ectopic endometrial tissue in adenomyosis and the ability to culture ectopic epithelial and stromal cells will contribute significantly to the study of adenomyosis. In the process of sample collection and primary cell culture, we continued to adapt and improve, and finally detected ectopic endometrial tissue in human adenomyosis lesions which were located haphazardly in the myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, we found that a scalpel with a size 11 blade was the best option for collecting ectopic endometrial tissue from adenomyosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Specimen collection is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; these specimens were used for cell culture, thus significantly increasing the number, purity and viability of both ectopic epithelial and stromal cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Analysis results of HE staining of different histopathological types of human adenomyosis\u003c/h2\u003e \u003cp\u003eThe types of adenomyosis described in most previous articles were divided into two types: diffuse and focal. According to the typing described by Pistofidis et al. [6], in this study, only diffuse, sclerotic and cystic adenomyosis were described. To further validate and determine our present findings, we performed hematoxylin and eosin staining on adenomyosis lesions of different histopathological types; this was followed by microscopy (Figure 2). HE staining was performed on the ectopic endometrium and surrounding myometrium tissues. Microscopically, obvious ectopic endometrial glands and stromal cells were observed in the myometrium (Figure 2E, F); these structures were significantly more abundant than the sclerotic type and cystic adenomyosis. Of these, cystic adenomyosis featured a significant amount of hemosiderin (Figure 2A, B). These results showed that we had located the soft tissue of adenomyosis which protruded slightly from the surface of the lesion. These structures were distributed in the myometrium as islets and represented ectopic endometrial tissue in human adenomyosis lesions.\u0026nbsp;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Primary cell culture of ectopic epithelial and stromal cells from human adenomyosis lesions\u003c/h2\u003e \u003cp\u003eEctopic endometrium tissue was collected from human adenomyosis lesions for primary cells isolation and culture; then, we observed their morphology. On day 1 after the first inoculation, the stromal cells were completely adherent, and the epithelial cells were partially adherent. Compared to other cell types, the stromal cells appeared to be of a uniformly small size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, the epithelial cells varied in size and grew in clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On days 1\u0026ndash;3 of culture, the epithelial cells were round or oval and slowly spread outwards. On day 5 after the first inoculation, the epithelial cells had become flattened. The stromal cells gradually became larger with the extension of inoculation time, followed by protrusion of pseudopodia; subsequently, the cells formed a single-layer adherent spindle and adopted a polygonal appearance. There were many more stromal cells than epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Identification of primary cell surface markers\u003c/h2\u003e \u003cp\u003eFlow cytometry is a commonly used research and diagnostic tool that uses fluorophore conjugated antibodies as probes to identify, characterize, and/or isolate cell populations [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We performed flow cytometry to confirm the epithelial and stromal phenotype of the cultured cells. Moreover, human endometrial adenocarcinoma cells (HEC-1B cells) and primary eutopic stromal cells of adenomyosis were used as positive controls. Generally, the primary endometrium of uterine cells was characterized by a profile that was EpCAM+, cytokeratin+, vimentin\u0026thinsp;+\u0026thinsp;or CD10+. We also detected cytokeratin for comparison; analysis revealed only minimal expression of the epithelial cell marker Cytokeratin (5.71%) in human endometrial adenocarcinoma cells (HEC-1B cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We also found that HEC-1B cells expressed high levels of Ep-CAM/CD326 (99.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). CD10, a broadly applied stromal cell marker, was selected for confirmation. Analysis showed that CD10 was robustly expressed by eutopic stromal cells in human adenomyosis (a positive rate of 98.41%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). After verification, we finally determined that primary ectopic epithelial cells were labelled with Ep-CAM while stromal cells were labelled with CD10. Furthermore, flow cytometry was performed on cultured ectopic epithelial cells from adenomyosis lesions; this revealed a positive rate of 4.23% for cytokeratin and 80.54% for EpCAM/CD326 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Identification, purity, and viability analysis of primary cells from human adenomyosis lesions\u003c/h2\u003e \u003cp\u003eThe identification, purity, and viability of the ectopic epithelial and stromal cells cultured from human adenomyosis lesions was demonstrated by flow cytometry, and then compared with cells cultured from a small piece of adenomyosis lesion tissue. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37% and 93.49%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Due to the contamination of uterine smooth muscle cells and fibrocytes, the purity and viability of cells cultured from one piece of adenomyosis lesion tissue were significantly lower than those of ectopic endometrial tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results showed that the primary cell culture of ectopic endometrium tissue was better than the traditional culture method involving a small piece of adenomyosis lesion tissue in terms of cell number, purity and viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. MRI findings of human adenomyosis exhibited a certain correlation with histopathology\u003c/h2\u003e \u003cp\u003eThe diagnosis of adenomyosis used to be determined solely by histology after hysterectomy; however, with the advent of modern imaging technology, adenomyosis is increasingly being diagnosed in a non-invasive fashion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Typical adenomyosis MRI images appear as an ill-demarcated low-signal intensity area on T2-weighted images and represent smooth muscle hyperplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C). In addition, intramyometrial cysts and small high-signal intensity areas referring to ectopic endometrium may also be detected on T2-weighted MRI [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Hyperintense myometrial foci are direct imaging evidence of the structures visualized by histopathology and represent islets of the heterotopic endometrium in the myometrium. They are visualized on MRI as T2 bright foci, varying in size from 2 to 7 mm, abnormally located in the myometrium [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then compared preoperative MRI images with intraoperative \u003cem\u003eex vivo\u003c/em\u003e adenomyosis tissue. We found that an extensive area of high-signal intensity myometrial spots were visible on T2-weighted MRI images with more ectopic endometrial tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). In contrast, the MRI images showed low signal intensity area in the myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), there was no obvious macroscopic ectopic endometrial tissue in intraoperative specimens; according to a previous study, the histopathological classification of this tissue was sclerotic. In the \u0026lsquo;sclerotic\u0026rsquo; type, lesions present as an irregular thickening of the myometrium in which the tissue is hard and adopts an off-white pale and fibrotic appearance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, HE staining images revealed the presence of ectopic endometrium glands and stromal cells in the myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAt present, the exact etiology of adenomyosis remains unknown. However, the most widely accepted theories propose that endometrial glands directly invade the myometrium, thus resulting in angiogenesis of the spiral vessels along with hyperplasia and hypertrophy of the adjacent smooth muscle tissue [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The isolation and culture of ectopic epithelial and stromal cells from human adenomyosis lesions is the first step to accomplish when studying adenomyosis. The present study describes the first isolation and culture of primary ectopic epithelial and stromal cells from adenomyosis lesions. In addition, we were able to culture primary ectopic epithelial and stromal cells with high purity and good viability by performing simple and operable methods.\u003c/p\u003e \u003cp\u003eIn general, when obtained from diverse tissue sources, ectopic epithelial and stromal cells exhibit significant differences in yield and purity. During our initial attempts, we used a small piece of adenomyosis lesion tissue for digestion and culture; however, only a small number of primary ectopic epithelial and stromal cells were acquired. Furthermore, due to the long digestion time involved, cell viability was weakened and there was an increased risk of contamination by fibroblasts and smooth muscle cells. To overcome these problems, after continuous observation and research, we finally identified ectopic endometria tissue in adenomyosis lesions and collected this tissue for culture. When processing and digesting specimens, we employed type I collagenase; according to the literature, collagenase I, a less potent enzyme, causes less damage to primary cells during the digestion process when compared to other enzymes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, we used HE staining and flow cytometry to verify our results; results were consistent.\u003c/p\u003e \u003cp\u003eIn this study, we developed a simple and optimal purification technique for primary cells. Initially, we attempted to isolate primary ectopic epithelial and stromal cells by flow cytometry; however, it proved difficult to continue to culture after sorting cells in this manner. Moreover, this method extended the time prior to culture initiation; moreover, the technique was expensive, lead to the loss of epithelial and stromal cells, and was associated with a lower success rate. However, the purity of the sorted cells exceeded 95%. Because the former approach was difficult and expensive, we considered that epithelial cells and stromal cells adopted different sizes and could therefore be separated from each other by cell mesh filtration. The results of flow cytometry showed that the purity of cells exceeded 90% and that the purity of stromal cells reached 97%. We were able to harvest a large number of ectopic epithelial and stromal cells during a short period of time if a sufficient amount of tissue was used. Eventually, we developed simple purification and culture technique for primary ectopic epithelial and stromal cells from human adenomyosis lesions.\u003c/p\u003e \u003cp\u003eIn the present study, we observed that ectopic epithelial cells had underwent adherence later than stromal cells. If the culture medium was changed too early, we observed unnecessary cell loss. According to the results of the present study, the optimal time for the first medium change was 2 days after inoculation. We also found that ectopic epithelial cells could not expand normally; nor could they proliferate and achieve higher confluency; therefore, it was not possible to passage these cells.\u003c/p\u003e \u003cp\u003eIn most instances, flow cytometry can be used to detect cell surface markers expressed on epithelial and stromal cells. EpCAM is a promising biomarker that is highly expressed on epithelial cancer cells but expressed at lower levels on normal epithelial cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Following verification, we found that cytokeratin was only expressed at low levels on ectopic epithelial cells while Ep-CAM was expressed stably and at high levels. Flow cytometry analysis showed that ectopic epithelial and stromal cells stably expressed Ep-CAM (93.74%) and CD10(97.1%). The successful identification of ectopic epithelial and stromal cell surface markers is critical if we are to investigate the molecular mechanisms underlying adenomyosis.\u003c/p\u003e \u003cp\u003eThere are also limitations to this study that need to be considered. Due to the different histopathological types of adenomyosis, not every \u003cem\u003eex vivo\u003c/em\u003e specimen could allow for the satisfactory collection of ectopic endometrium tissue. As with the sclerotic adenomyosis described earlier, there was no obvious ectopic endometrium tissue in some cases; instead, we observed large areas of fibrotic hard tissue. However, ectopic endometrial glands and stromal cells were still visible under microscopy following HE staining.\u003c/p\u003e \u003cp\u003eDespite major advances in adenomyosis over recent decades, hysterectomy would not be a treatment option for young women wishing to preserve their child-bearing capacity. Furthermore, adenomyosis research and effective drug discovery still suffer from the lack of an ideal cellular model. In the past, the diagnosis of adenomyosis was mainly based on pathological examination after hysterectomy. At present, most researchers believe that vaginal ultrasound and MRI are good non-invasive diagnostic tools for the diagnosis of adenomyosis. In addition, in this study, we combined clinical preoperative MRI imaging to compare intraoperative \u003cem\u003eex vitro\u003c/em\u003e adenomyosis lesions. We successfully observed multiple punctate T2W1 high signal intensity regions within the low-signal intensity area on MRI. Furthermore, there were more ectopic endometria tissues in intraoperative specimens, and cultured cells showed good viability. A previous case report described a case with severe uterine adenomyosis. Prior to treatment, MRI showed an enlarged uterus with diffuse and disseminated adenomyosis. Multiple regions of T2W1 high signal intensity were evident on MRI. After 12 weeks of GnRH antagonist therapy (a daily dose of 200 mg linzagolix), a significant reduction was observed in uterine size and the size of the adenomyotic lesions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, T2W1 high density signal atrophy disappeared on MRI images. From a clinical perspective, an ideal imaging technique should not only diagnose adenomyosis accurately; it should also facilitate the selection of the best treatment modality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Finally, this study concluded that ectopic endometrial tissue plays an important role in the occurrence and development of adenomyosis. In most instances, T2W1 high signal intensity within the low-signal intensity area on MRI images revealed more ectopic endometrial tissues. Controlling ectopic endometrial tissue or causing the ectopic endometrial tissue to undergo atrophy can effectively control and treat adenomyosis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur newly developed method is a simple, efficient and economical technique to establish primary ectopic epithelial and stromal cells cultures with high purity and good viability from human adenomyosis lesion. Moreover, this technique provides a new experimental model for studying the pathogenesis of human adenomyosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eZhou Fang\u003c/strong\u003e: Conceptualization, Writing \u0026ndash; original draft, Data curation, Methodology, Writing \u0026ndash; review \u0026amp; editing, Validation, Resources. \u003cstrong\u003eJianzhang Wang and Tiantian Li\u003c/strong\u003e: Conceptualization, Methodology, Writing \u0026ndash; review \u0026amp; editing, Validation, Supervision. \u003cstrong\u003eMeichen Yin and Yangying Peng\u003c/strong\u003e: Data curation, Methodology, Resources, Validation. \u003cstrong\u003eXinmei Zhang\u003c/strong\u003e: Supervision, Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Project administration, Validation. All the authors read the submitted version and approved it.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was funded by National Key R\u0026amp;D Program of China (Grant number: 2022YFC2704003), National Natural Science Foundation of China (Grant numbers: 81974225, 82001518 and 82171636).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eT. Harada, Y.M. Khine, A. Kaponis, T. Nikellis, G. Decavalas, F. Taniguchi, The Impact of Adenomyosis on Women\u0026apos;s Fertility, Obstet Gynecol Surv, 71 (2016) 557-568, http://dx.doi.org/10.1097/OGX.0000000000000346.\u003c/li\u003e\n \u003cli\u003eC. Chapron, S. Vannuccini, P. Santulli, M.S. Abr\u0026atilde;o, F. Carmona, I.S. Fraser, S. Gordts, S.-W. Guo, P.-A. Just, J.-C. No\u0026euml;l, G. Pistofidis, T. Van den Bosch, F. Petraglia, Diagnosing adenomyosis: an integrated clinical and imaging approach, Hum Reprod Update, 26 (2020) 392-411, http://dx.doi.org/10.1093/humupd/dmz049.\u003c/li\u003e\n \u003cli\u003eJ. Donnez, C.A. Stratopoulou, M.-M. Dolmans, Uterine Adenomyosis: From Disease Pathogenesis to a New Medical Approach Using GnRH Antagonists, Int J Environ Res Public Health, 18 (2021), http://dx.doi.org/10.3390/ijerph18199941.\u003c/li\u003e\n \u003cli\u003eO. Donnez, J. Donnez, Gonadotropin-releasing hormone antagonist (linzagolix): a new therapy for uterine adenomyosis, Fertil Steril, 114 (2020) 640-645, http://dx.doi.org/10.1016/j.fertnstert.2020.04.017.\u003c/li\u003e\n \u003cli\u003eA. Camboni, E. Marbaix, Ectopic Endometrium: The Pathologist\u0026apos;s Perspective, Int J Mol Sci, 22 (2021), http://dx.doi.org/10.3390/ijms222010974.\u003c/li\u003e\n \u003cli\u003eG. Pistofidis, E. Makrakis, O. Koukoura, N. Bardis, P. Balinakos, V. Anaf, Distinct types of uterine adenomyosis based on laparoscopic and histopathologic criteria, Clin Exp Obstet Gynecol, 41 (2014) 113-118.\u003c/li\u003e\n \u003cli\u003eS.E. Bulun, S. Yildiz, M. Adli, J.-J. Wei, Adenomyosis pathogenesis: insights from next-generation sequencing, Hum Reprod Update, 27 (2021) 1086-1097, http://dx.doi.org/10.1093/humupd/dmab017.\u003c/li\u003e\n \u003cli\u003e关永格, 李坤寅, 何昱雯, 宋阳, 子宫腺肌病病灶细胞的培养与鉴定, 中国妇幼健康研究, (2013) 827-829+838.\u003c/li\u003e\n \u003cli\u003eM. Habiba, G. Benagiano, Classifying Adenomyosis: Progress and Challenges, Int J Environ Res Public Health, 18 (2021), http://dx.doi.org/10.3390/ijerph182312386.\u003c/li\u003e\n \u003cli\u003eP. Zafari, A. Rafiei, F. Faramarzi, S. Ghaffari, A.H. Amiri, M. Taghadosi, Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis, Rev Assoc Med Bras (1992), 67 (2021) 1654-1658, http://dx.doi.org/10.1590/1806-9282.20210706.\u003c/li\u003e\n \u003cli\u003eY. Wang, S. Chen, Z. Yan, M. Pei, A prospect of cell immortalization combined with matrix microenvironmental optimization strategy for tissue engineering and regeneration, Cell Biosci, 9 (2019) 7, http://dx.doi.org/10.1186/s13578-018-0264-9.\u003c/li\u003e\n \u003cli\u003eR. Joplin, Isolation and culture of biliary epithelial cells, Gut, 35 (1994) 875-878.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eF.A. Abade Dos Santos, C.L. Carvalho, I. Almeida, T. Fagulha, F. Rammos, S.C. Barros, M. Henriques, T. Lu\u0026iacute;s, M.D. Duarte, Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures, Cells, 10 (2021), http://dx.doi.org/10.3390/cells10082100.\u003c/li\u003e\n \u003cli\u003eZ. Hu, Y. Chen, M. Gao, X. Chi, Y. He, C. Zhang, Y. Yang, Y. Li, Y. Lv, Y. Huang, X. Deng, Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I, Cell Prolif, 56 (2023) e13320, http://dx.doi.org/10.1111/cpr.13320.\u003c/li\u003e\n \u003cli\u003eY. Zhang, J. An, M. Liu, N. Li, W. Wang, H. Yao, N. Li, X. Yang, Y. Sun, N. Xu, L. Wu, Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma, Am J Reprod Immunol, 84 (2020) e13251, http://dx.doi.org/10.1111/aji.13251.\u003c/li\u003e\n \u003cli\u003eD.F. O, T. Roskams, K. Van den Eynde, A. Vanhie, D.P. Peterse, C. Meuleman, C. Tomassetti, K. Peeraer, T.M. D\u0026apos;Hooghe, A. Fassbender, The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis, Reprod Sci, 24 (2017) 242-251, http://dx.doi.org/10.1177/1933719116653677.\u003c/li\u003e\n \u003cli\u003eM. Bazot, E. Dara\u0026iuml;, Role of transvaginal sonography and magnetic resonance imaging in the diagnosis of uterine adenomyosis, Fertil Steril, 109 (2018) 389-397, http://dx.doi.org/10.1016/j.fertnstert.2018.01.024.\u003c/li\u003e\n \u003cli\u003eS.-W. Guo, G. Benagiano, M. Bazot, In Search of an Imaging Classification of Adenomyosis: A Role for Elastography?, J Clin Med, 12 (2022), http://dx.doi.org/10.3390/jcm12010287.\u003c/li\u003e\n \u003cli\u003eQ. Yu, J. Wang, T. Li, X. Xu, X. Guo, S. Ding, L. Zhu, G. Zou, Y. Chen, X. Zhang, RON Mediates Tumor-Promoting Effects in Endometrial Adenocarcinoma, Biomed Res Int, 2021 (2021) 2282916, http://dx.doi.org/10.1155/2021/2282916.\u003c/li\u003e\n \u003cli\u003eL.R. Bonser, K.D. Koh, K. Johansson, S.P. Choksi, D. Cheng, L. Liu, D.I. Sun, L.T. Zlock, W.L. Eckalbar, W.E. Finkbeiner, D.J. Erle, Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets, Am J Respir Cell Mol Biol, 64 (2021) 308-317, http://dx.doi.org/10.1165/rcmb.2020-0149MA.\u003c/li\u003e\n \u003cli\u003eL. Fedele, S. Bianchi, M. Dorta, L. Arcaini, F. Zanotti, S. Carinelli, Transvaginal ultrasonography in the diagnosis of diffuse adenomyosis, Fertil Steril, 58 (1992) 94-97.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eA.S. Mark, H. Hricak, L.W. Heinrichs, M.R. Hendrickson, M.L. Winkler, J.A. Bachica, J.E. Stickler, Adenomyosis and leiomyoma: differential diagnosis with MR imaging, Radiology, 163 (1987) 527-529.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eM. Zhang, M. Bazot, M. Tsatoumas, M.G. Munro, C. Reinhold, MRI of Adenomyosis: Where Are We Today?, Can Assoc Radiol J, 74 (2023) 58-68, http://dx.doi.org/10.1177/08465371221114197.\u003c/li\u003e\n \u003cli\u003eV. Celli, M. Dolciami, R. Ninkova, G. Ercolani, S. Rizzo, M.G. Porpora, C. Catalano, L. Manganaro, MRI and Adenomyosis: What Can Radiologists Evaluate?, Int J Environ Res Public Health, 19 (2022), http://dx.doi.org/10.3390/ijerph19105840.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adenomyosis, Cell culture, Flow cytometry, EpCAM/CD326, CD10","lastPublishedDoi":"10.21203/rs.3.rs-3194957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3194957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eAlthough adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition has yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify and evaluate the viability of isolated ectopic endometrial cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37% and 93.49%, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis.\u003c/p\u003e","manuscriptTitle":"A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-02 17:26:33","doi":"10.21203/rs.3.rs-3194957/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-08-25T12:48:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-28T10:57:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Archives of Gynecology and Obstetrics","date":"2023-07-27T20:32:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-26T14:46:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2023-07-26T09:31:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bcf400cb-3195-4eba-9c20-ff5af27348cc","owner":[],"postedDate":"August 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-30T15:04:24+00:00","versionOfRecord":{"articleIdentity":"rs-3194957","link":"https://doi.org/10.1007/s00404-023-07254-8","journal":{"identity":"archives-of-gynecology-and-obstetrics","isVorOnly":false,"title":"Archives of Gynecology and Obstetrics"},"publishedOn":"2023-10-23 15:02:09","publishedOnDateReadable":"October 23rd, 2023"},"versionCreatedAt":"2023-08-02 17:26:33","video":"","vorDoi":"10.1007/s00404-023-07254-8","vorDoiUrl":"https://doi.org/10.1007/s00404-023-07254-8","workflowStages":[]},"version":"v1","identity":"rs-3194957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3194957","identity":"rs-3194957","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}