A contractility-competent immortalized human sweat gland myoepithelial line with dual epithelial-mesenchymal characteristics.

[OBJECTIVE] To isolate and culture primary human eccrine sweat gland myoepithelial cells (MECs) and establish novel immortalized myoepithelial cells (iMECs), thereby providing essential materials for sweat gland biology research.

[METHODS] Sweat gland secretory coils were microdissected from upper eyelid skin specimens obtained post-blepharoplasty. MECs were isolated through differential trypsinization combined with adhesion-based purification. Lentiviral vectors encoding SV40T were used to generate iMECs, with stable clones selected via Blasticidin resistance. Comprehensive characterization encompassed proliferation kinetics assessed through CCK-8 and EdU assays, immortalization validation via RT-PCR detection of SV40T expression, genetic authentication using Short tandem repeat (STR) profiling and karyotyping, phenotypic analysis through immunofluorescence staining for CK7, CK19, α-SMA, and Vimentin markers, and ultrastructural evaluation by transmission electron microscopy.

[RESULTS] Both MECs and iMECs exhibited a mesenchymal-like spindle morphology. iMECs demonstrated superior proliferative capacity, sustaining 50 passages with 2-fold higher EdU incorporation versus primary MECs (P < 0.0001). Genetic authentication confirmed stable diploid karyotype (46, XX) and unique STR profile, excluding cross-contamination with existing cell lines. Phenotypic characterization revealed persistent co-expression of epithelial (CK7/CK19) and mesenchymal (α-SMA/Vimentin) markers in iMECs across passages, while maintaining negative expression for CEACAM5, CK14, CK17, and P63. Ultrastructural analysis validated the preservation of contractile myofilaments and intercellular junction complexes (desmosomes/zonula adherens) through transmission electron microscopy (TEM).

[CONCLUSION] We demonstrate a reproducible methodology for isolating functionally competent MECs and establishing a phenotypically stable immortalized sweat gland myoepithelial line. The iMECs retain native biomarker profiles and ultrastructural features while overcoming primary cell senescence limitations, providing a transformative resource for glandular regeneration studies and sweat secretion pathophysiology modeling.