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

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.

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.

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).

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.