Development and Characterization of an Immortalized Human Dermal Fibroblast Cell Line That Retains Primary Cell Functionality

Background and Purpose

Dermal fibroblasts (DFs) are indispensable for skin biology, playing a critical role in extracellular matrix production, wound healing, and skin regeneration. Primary DFs have long been the gold-standard model system for studying skin biology; however, donor variability and the limited lifespan of primary cells restrict their potential. While the traditional method of immortalizing primary cells through SV40 can overcome these restrictions, it often leads to the loss of normal cell biofunction. Here, we present a new method for fully immortalizing human DFs that preserves the physiological attributes of the original primary cells, thereby providing an unlimited source of cells that will help improve reproducibility in toxicology research.

Methods

We generated a clonal cell line that was immortalized by stably expressing hTERT and mutant CDK4 (CDK4R24C) in normal human primary DFs. The cell morphology, growth rate, cell markers, and functionality of the immortalized cell line were analyzed and compared to that of the parental primary cells.

Results

The fully immortalized cell line hTERT HDFa (ATCC CRL-4066) was continuously cultured to 111 population doublings without any signs of replicative senescence. hTERT HDFa cells had an apparently normal human female karyotype and a fibroblast-like morphology. Both primary DFs and hTERT HDFa expressed TE-7 and could be differentiated into myofibroblasts. Similar to the parental primary cells, the hTERT HDFa cells responded to drug treatment, such as Chlorhexidine (CHX), in a time- and dose-dependent manner. Further, in vitro 3-D skin structures were formed when hTERT HDFa cells were co-cultured with primary or immortalized keratinocytes (ATCC PCS-200-010 or ATCC CRL-4048).

Conclusions

The hTERT HDFa cell line overcomes the limitations of primary cells while still retaining the functional characteristics of primary cells. This novel cell line provides a robust and reproducible in vitro cell model for skin disease, skin aging, wound healing, cosmetic, and toxicology studies.