Cell culture is a widely used in vitro tool that enhances our understanding of cell biology, disease mechanisms, drug responses, and the development of tissue engineering. However, there are a number of important drawbacks to conventional two-dimensional (2D) cultures, such as the loss of polarity, altered cell shape, and disruption of cell-extracellular matrix connections. Alternatively, organoids are tissue-engineered, cell-based in vitro models derived from stem cells that can self-organize and differentiate into three-dimensional (3D) structures, recapitulating the morphology and functions of their in vivo counterparts. Bisphenol A (BPA), a ubiquitous industrial chemical, has recently gained recognition as an environmental hazard. Previous research has demonstrated that BPA negatively affects the integrity of the intestinal barrier by triggering programmed cell death and suppressing cell growth in human colonic epithelial cell lines. However, a 2D-based cellular study cannot represent its exposure to multicellular organs. This work investigates the impact of BPA on the structure and function of the intestinal barrier. We examine the effect of BPA on the proliferation and tight junction gene expression with two models: the HT-29 colon cancer cell line and an intestine organoid model and morphological changes of intestinal organoid (I/O). The proliferation was increased in a dose-dependent manner with I/O, but at the same concentration, BPA does not increase the significant number of HT-29 cell respectively. Proliferation-related gene and tight junction gene expression pattern was similar between HT-29 and I/O other than Claudin-4. Therefore, this study offers a more precise depiction of the functional and morphological alterations caused by BPA in comparison to traditional 2D cell cultures.

Tight junctions (TJs) form continuous intercellular contacts in intercellular junctions. TJs involve integral proteins such as occludin (OCLN) and claudins (CLDNs) as well as peripheral proteins such as zona occludens-1 (ZO-1) and junctional adhesion molecules (JAMs). TJs control paracellular transportation across cell-to-cell junctions. Although TJs have been studied for several decades, comparison of the transcriptional-translational levels of these molecules in canine organs has not yet been performed. In this study, we examined uterine expression of CLDNs, OCLN, junction adhesion molecule-A, and ZO-1 in canine. Expression levels of canine uterine TJ proteins, including CLDN1, 2, 4, 5, JAM-A, ZO-1, and OCLN, were measured using reverse transcription PCR, real-time PCR, and Western blotting, whereas TJs distribution was determined by immunohistochemistry. The mRNA and protein expression levels of OCLN, CLDN-1, 4, JAM-1, and ZO-1 were identified in the uterus. Immunohistochemistry demonstrated that TJs were localized to the endometrium and/or myometrium of the uterus. Our results show that canine TJ proteins, including CLDNs, OCLN, JAM-A, and ZO-1, were expressed in the canine uterus. Taken together, these proteins may perform unique physiological roles in the uterus. Therefore, these findings may serve as a basis for further studies on TJ proteins and their roles in the physiological or pathological condition of the canine uterus.