Comparison of Culturing Methods of Primary Vaginal Fibroblasts.

Abstract

Importance: Vaginal fibroblast function is altered in people with pelvic organ prolapse. Thus, it is important to study vaginal fibroblasts to better understand the pathophysiology of prolapse.

Objective: This study aimed to compare 3 culturing methods of primary vaginal fibroblasts.

Study design: This was an in vitro study. Patients who were undergoing surgery for vaginal prolapse were recruited. Excess vaginal epithelial tissue that would have otherwise been discarded was collected. The vaginal fibroblasts from each participant were cultured via (1) 3-hour digest, (2) coverslip, and (3) gelatin-coat methods. Differences in the efficiency of cell isolation, expression of known fibroblast-associated genes, and cellular function were compared between the 3 methods using one-way analysis of variance and Tukey test for post hoc pairwise comparisons (P < 0.05).

Results: Five patients with pelvic organ prolapse were recruited. Fibroblasts cultured via the 3-hour digest method became confluent within 3-5 days in a 100-mm dish compared to 2-3 weeks in a 6-well dish for the coverslip and gelatin-coat methods. Cells from all culture methods expressed similar amounts of vimentin and α smooth muscle actin. There were no significant differences in morphology; gene expression levels of MMP1, MMP2, ACTA2, COL1A1, COL3A1, and LOXL1 on qPCR; cell viability; proliferation; and migration between the 3 culturing methods.

Conclusion: Culturing primary vaginal fibroblasts via the 3-hour digest, coverslip, and gelatin-coat methods similarly resulted in reliable primary vaginal fibroblast growth and function.