Development and characterization of a novel immortalized human vaginal fibroblast cell line for advanced applications in reproductive health

Abstract

Background: Reproductive health issues related to the vagina, face significant challenges due to the lack of standardized research models. Vaginal fibroblasts, which constitute approximately 55% of the vaginal wall's cellular composition, are crucial for tissue repair, remodelling, and reproductive health. These fibroblasts have broad applications in regenerative medicine and gynaecological treatments. Despite their importance, current research relies primarily on epithelial cells or primary vaginal fibroblasts, but primary fibroblasts are limited by their short lifespan, donor-to-donor variability, and susceptibility to senescence. Immortalized fibroblast lines offer a solution by extending the lifespan and enabling reproducible studies. However, a well-characterized immortalized human vaginal fibroblast line has not been established, highlighting the need for novel models to better understand and address vaginal-associated conditions.

Methods: Primary human vaginal fibroblasts were immortalized via the lentiviral transfection of human telomerase reverse transcriptase. The resulting cell line was characterized through histological, immunofluorescent, RT-qPCR and flow cytometry analyses. Proliferation, senescence, gene expression, hormone responsiveness and genomic stability were assessed via quantitative polymerase chain reaction, transcriptome sequencing, gene set enrichment analysis, short tandem repeat profiling, and karyotype analysis.

Results: The immortalized human vaginal fibroblasts (ihVFs) retained typical spindle-shaped fibroblast morphology and fibroblast-specific marker expression. Compared with primary vaginal fibroblasts, ihVF exhibited significantly reduced senescence, maintained sustained growth through extended culture passages, and preserved genetic stability. Transcriptome sequencing revealed high gene expression similarity between immortalized and primary fibroblasts, with no significant alterations in oncogenic pathways. PCR and immunofluorescent analyses revealed that ihVFs are responsive to estrogen and progesterone stimulation. Short tandem repeat analysis confirmed the novelty of the immortalized cell line, with no overlap with existing cell databases.

Conclusions: The novel ihVF cell line retains key phenotypic, functional, and genetic characteristics of primary vaginal fibroblasts, providing a stable, reproducible, and physiologically relevant model for reproductive health research. This cell line addresses the limitations of primary fibroblasts and has broad applications in tissue engineering, gynaecological disorder research, and drug screening, advancing our understanding of vaginal fibroblast biology and therapeutic interventions.

Keywords: Immortalized cell line; Reproductive health; Tissue engineering; Vaginal fibroblasts; hTERT.

Fig. 1

Schematic diagram of the preparation and workflow for developing ihVFs. The diagram highlights key steps, including cell isolation, lentiviral transfection, immortalization, and subsequent analyses

Fig. 2

Characterization of the vaginal tissue structure and validation of immortalized human vaginal fibroblasts (ihVFs) with hTERT overexpression. (A) Schematic diagram illustrating the structural layers of the vaginal wall. (B) H&E-stained section of vaginal tissue showing its structural organization. (C) IHC staining of vaginal tissue with a vimentin antibody. (D) Microscopy image showing the spindle-shaped morphology of primary human vaginal fibroblasts (phVFs). (E) Flow cytometry analysis; representative histograms showing the expression of surface markers on ihVF. (F) RT‒qPCR results showing relative hTERT mRNA expression in ihVF and phVF. (G) Immunofluorescence images showing hTERT protein localization in phVF and ihVF

Fig. 3

Identification of ihVF by immunofluorescence staining. ihVFs were stained for vimentin, FSP-1, α-SMA, AE1/AE3 and FGF-1 to validate their phenotype. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm

Fig. 4

Senescence and proliferation analysis of ihVF and phVF at different passages. (A) Morphology of ihVF and phVF at passage 15 under bright-field microscopy. phVF shows SA-β-gal-positive staining (blue), whereas ihVF exhibits typical fibroblast morphology. Scale bar: 100 μm. (B) Percentage of SA-β-gal-positive cells at passage 15. (C–E) Proliferation analysis using CCK-8 assay at passages 5 (C), 10 (D), and 20 (E). (F) Proliferation comparison of ihVFs between passages 20 and 30

Fig. 5

Comprehensive analysis of the impact of immortalization on the global gene expression profile of ihVF compared phVF. (A) Gene expression correlation scatter plot. (B) Venn diagram of genes expressed in different groups of samples. (C-G) GSEA of selected gene ontologies. (H) Summary of NESs and statistical significance across GO terms

Fig. 6

Hormone responsiveness assays. (A) IF staining of ER and PR in ihVFs. Nuclei are stained with DAPI (blue), ER and PR markers are labelled in red. Scale bar = 100 μm. (B) Relative mRNA expression levels of ER-related and PR-related target genes under different stimulation conditions

Fig. 7

Genomic stability and authenticity of the phVF and ihVF cell lines were assessed by STR profiling and karyotypic analysis. (A) STR profiling of ihVF-P20 across 21 loci. (B) G-banding analysis of phVF-P3. (C) G-banding analysis of ihVF-P20