First human cell-based cultivation system for the syphilis spirochete Treponema pallidum

Treponema pallidum subsp. pallidum (T. pallidum), the etiological agent of syphilis, was first isolated from infected humans in the early 20th century and, for decades, could only be propagated in laboratory rabbits. In 2018, a reproducible in vitro cultivation system was established using rabbit epithelial Sf1Ep cells and a complex culture medium, allowing stable growth of T. pallidum. While in vitro cultivation has revolutionized T. pallidum research, further humanization of this system is necessary to study host–pathogen interactions. We evaluated three human foreskin fibroblast cell lines (HFF1, HFFC, and MoNa) for their ability to support in vitro growth of T. pallidum as an alternative to rabbit Sf1Ep cells. All human cell lines displayed typical fibroblast morphology and showed growth rates comparable to or slower than Sf1Ep cells, a feature favorable for treponemal propagation. Eight available T. pallidum strains representing the Nichols-like (DAL-1, Madras, London, Haiti B) and the SS14-like clusters (Mexico A, SS14, Grady, Philadelphia 1) sustained growth on human fibroblast cells for ten weeks. Notably, DAL-1 cultures have been continuously maintained on human fibroblasts for over one year. Cultivation on human fibroblasts resulted in slower overall growth of T. pallidum compared to rabbit cells. Nevertheless, Nichols-like strains retained their higher replication rates relative to SS14-like strains, consistent with observations from rabbit-based systems. Human foreskin fibroblasts can serve as effective cells for support of T. pallidum growth in vitro. This approach represents a step toward humanized culture conditions, enabling further investigation of the pathogenesis of syphilis.