A physiologically relevant, estradiol‐17β [E2]‐responsive in vitro tissue‐engineered model of the vaginal epithelium for vaginal tissue research

Abstract

Aims: There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research.Methods: Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air-liquid interface.Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins.Results: Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17β [E 2 ] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E 2 concentrations (100-400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression. Conclusion:In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT.

Estradiol-17β [E2] stimulates wound healing in a 3D in vitro tissue-engineered vaginal wound model

“…Decellularized sheep vaginal matrices were seeded with a co-culture of vaginal epithelial cells and fibroblasts expanded in Green’s media at a seeding density of 6 × 10 5 /model and 3 × 10 5 /model respectively as previously reported. 6 The average size of each TE vaginal model was about 1.0 × 1.5 ± 0.1 cm 2 . The models were cultured for 3 days in submerged conditions and then partial-thickness (PT) incisional wounds were made using a sterile surgical grade scalpel blade (Swann Morton ® Scalpel Blades no.10, Sheffield, UK) on the upper surface of individual models.…”

“…Primary sheep vaginal epithelial cells and fibroblasts were isolated as previously reported 6 and cultured on decellularized sheep vaginal tissues (prepared using the detergent method) to develop the tissue-engineered models. Vaginal tissues were decellularized using a detergent mix method.…”

“…Ki67 and α-SMA expression in TE wound vaginal models with and without E 2 was measured using immunohistofluorescence techniques as described previously. 6 Briefly, slides were deparaffinized in a graded series of ethanol and treated with a trypsin/CaCl 2 solution for antigen retrieval in a humidified chamber. Slides were washed and tissue sections were permeabilized by adding 0.5% v/v Tween20 followed by incubation with a serum-free protein blocking buffer (ab64226; Abcam).…”

“…Another reason for looking at the effects of estrogen on wound healing was that we have recently shown the ability of estrogen to stimulate 3D tissue formation in this vaginal model. 6 …”

“… 13 However, these 2D culture systems are unable to recapitulate the complexity of in vivo environment due to their simplicity of design and in particular their lack of stromal components. Accordingly, we used a TE vaginal model which we recently developed based on sheep vaginal tissues and cells 6 and here, we subjected it to partial incisional wounding. We followed its ability to heal over 3 weeks looking at changes both in the epithelium and stroma and in the absence and presence of a physiological concentration of estradiol-17β [E 2 ].…”