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Adenomyosis, as a clinically impactful disease, causes significant pelvic pain and heavy menstrual bleeding. Adenomyosis remains understudied due to the paucity of translational research tools and model systems. This study aimed to create a telomerase-transformed epithelial-like cell line derived from the eutopic endometrium of a subject with focal adenomyosis. De-identified endometrium was processed via mechanical and enzymatic digestion. Epithelial and stromal populations were separated via selective adhesion, followed by fluorescence-activated cell sorting with an epithelial cellular adhesion molecule (EpCAM). EpCAM+ cells were efficiently immortalized with the human telomerase reverse transcriptase gene. Analyses confirmed that the cells were human, without mycoplasma contamination, and exhibited a unique 16-marker short tandem repeat profile. Cytogenetic analysis on G-banded metaphase spreads indicated polyploidy with multiple chromosomal rearrangements. The line, designated as tAEC21, expressed epithelial markers cytokeratin-5 and N-cadherin but not stromal marker CD10. Cells avidly responded to tumor necrosis factor-alpha stimulation by upregulating interleukin-6, C-X-C motif chemokine ligand 8, C-C motif chemokine ligand 2, and mucin 1 gene expression. In a heterotypic, three-dimensional spheroid model, tAEC21 displayed a biologically relevant pattern by assembling into an epithelial shell around the stromal cell core. In two-dimensional monolayers, tAEC21 cells were negative for estrogen and progesterone receptors, while as 3D spheroids, tAEC21 exhibited strong positivity for estrogen but not progesterone receptors. This new epithelial-like, adenomyosis-derived cell line, tAEC21, will be an impactful research resource.<\/jats:p>","DOI":"10.1101\/2025.06.16.660025","type":"posted-content","created":{"date-parts":[[2025,6,17]],"date-time":"2025-06-17T07:15:11Z","timestamp":1750144511000},"source":"Crossref","is-referenced-by-count":0,"title":["Establishment and Characterization of a New Immortalized Human Adenomyosis Epithelial-Like Cell Line, tAEC21"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7324-8427","authenticated-orcid":false,"given":"Yuliya","family":"Klymenko","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8664-1794","authenticated-orcid":false,"given":"Jessica L.","family":"Kersey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2518-390X","authenticated-orcid":false,"given":"Hunter D.","family":"Quigley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0727-3971","authenticated-orcid":false,"given":"Shannon M.","family":"Hawkins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025061804150871000_2025.06.16.660025v1.1","doi-asserted-by":"publisher","DOI":"10.1097\/01.AOG.0000428646.67925.9a"},{"key":"2025061804150871000_2025.06.16.660025v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpobgyn.2006.01.006"},{"key":"2025061804150871000_2025.06.16.660025v1.3","doi-asserted-by":"publisher","DOI":"10.1111\/aogs.14337"},{"key":"2025061804150871000_2025.06.16.660025v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.fertnstert.2018.01.006"},{"key":"2025061804150871000_2025.06.16.660025v1.5","doi-asserted-by":"crossref","unstructured":"Borghese G , Doglioli M , Orsini B , Raffone A , Neola D , Travaglino A , Rovero G , Del Forno S , de Meis L , Locci M . 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