Immunocompetent Human 3D Organ-Specific Hormone-Responding Vaginal Mucosa Model of HIV-1 Infection.

The lack of appropriate experimental models often limits our ability to investigate the establishment of infections in specific tissues. To reproduce the structural and spatial organization of vaginal mucosae to study human immunodeficiency virus type-1 (HIV-1) infection, we used the self-assembly technique to bioengineer tridimensional vaginal mucosae using human cells extracted from HIV-1-negative healthy pre- and postmenopausal donors. We produced a stroma, free of exogenous material, that can be adapted to generate near-to-native vaginal tissue with the best complexity obtained with seeded epithelial cells on the organ-specific stroma. The autologous engineered tissues had mechanical properties close to native mucosa and shared similar glycogen production, which declined in reconstructed tissues of the postmenopausal donor. The in vitro-engineered tissues were also rendered immune competent by adding human monocyte-derived macrophages (MDMs) on the epithelium or in the stroma layers. The model was infected with HIV-1, and viral replication and transcytosis were observed when immunocompetent reconstructed vaginal mucosa tissue has incorporated MDMs into the stroma and infected with free HIV-1 green fluorescent protein (GFP) viral particles. These data illustrate a natural permissiveness of immunocompetent untransformed human vaginal mucosae to HIV-1 infection. This model offers a physiological tool to explore viral load, HIV-1 transmission in an environment that may contribute to the virus propagation, and new antiviral treatments in vitro. Impact statement This study introduces an innovative immunocompetent three-dimensional human organ-specific vaginal mucosa free of exogenous material for in vitro modeling of human immunodeficiency virus type-1 (HIV-1) infection. The proposed model is histologically close to native tissue, especially by presenting glycogen accumulation in the epithelium's superficial cells, responsive to estrogen, and able to sustain a monocyte-derived macrophage population infected or not by HIV-1 during ∼2 months.

Urothelial cell expansion and differentiation are improved by exposure to hypoxia.

Cells obtained from a patient's biopsy have to be expanded after extraction to produce autologous tissues, but standard cell culture conditions often limit their growth or lifespan and could induce early and inadequate cell differentiation. Moreover, it has previously been reported that the air-liquid interface, that induces maturation of the urothelium, stimulated inadequate differentiation associated with aberrant keratin-14 expression. The aim of this study was to test the benefits of hypoxia during expansion of urothelial cells and maturation of the bladder epithelium in the context of tissue engineering. Bladder mucosa substitutes were reconstructed using the self-assembly method with urothelial cells (UCs) expanded in normoxia or hypoxia. Hypoxia improved UCs expansion until passage P7, whereas normoxic conditions limited the use of UCs to passage P4. Maturation of the urothelium was also compared in normoxic vs. hypoxic conditions. Using laminin V, p63, Ki-67, keratin-5 and -14, Claudin-4 and zonula occludens protein-1, we show a better organization of the basal UC layer in hypoxia despite a thinner intermediate layer. Finally, barrier function was assessed by permeation tests. Cell culture in hypoxia allowed the generation of bioengineered urological tissue closer to native bladder characteristics, which represents a promising avenue to circumvent the lack of adequate tissues for reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.