Accessible luminal interface of bovine rectal organoids generated from cryopreserved biopsy tissues

by Minae Kawasaki, Yoko M. Ambrosini

Developing precise species-specific in vitro models that closely resemble in vivo intestinal tissues is essential for advancing our understanding of gastrointestinal physiology and associated diseases. This is especially crucial in examining host-pathogen interactions, particularly in bovines, a known reservoir for microbes and pathogens posing substantial public health threats. This research investigated the viability of producing bovine rectal organoids from cryopreserved tissues. We compared two cryopreservation methods with a traditional technique using fresh tissues, evaluating their effectiveness through growth rates, long-term viability, and comprehensive structural, cellular, and genetic analyses. These assessments utilized phase-contrast imaging, immunofluorescence imaging, and RT-qPCR assays. Additionally, the study developed a sophisticated method for forming a functional epithelial barrier from organoid-derived bovine rectal monolayers, incorporating a wide range of epithelial cells. This methodology employed transepithelial electrical resistance (TEER), parallel artificial membrane permeability assay (P_app), confocal microscopy, and advanced imaging techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings decisively show that bovine rectal organoids can be effectively generated from cryopreserved biopsy tissues. Moreover, we formulated a robust and optimized protocol for creating functional rectal monolayers from these organoids. This significant progress is particularly relevant given the susceptibility of the bovine rectum to various enteric pathogens of public health concern, marking a vital step forward in veterinary and biomedical research. The creation of accurate species specific in vitro models that faithfully mimic in vivo intestinal tissues is critical for enhancing our understanding of gut physiology and related pathologies. This is particularly relevant in studying the interactions between hosts and microbes or pathogens with significant public health risks where bovine can be the major reservoir.

Study protocol for a prospective, multicentre, phase II trial on endoscopic treatment using two fully covered self-expandable metallic stents for benign strictures after hepaticojejunostomy

Introduction

The current endoscopic treatment for postoperative benign hepaticojejunostomy anastomotic stricture (HJAS) has a high technical success rate and is highly effective in the short term. However, long-term results have shown a high rate of stenosis recurrence, which indicates an insufficient response to treatment. Three prospective studies on fully covered self-expandable metallic stent (FC-SEMS) treatment for benign HJAS used the stenosis resolution rate as the primary endpoint, and no study has yet used the long-term non-stenosis rate (at 12 months) as the primary endpoint.

Methods and analysis

We launched the ‘saddle-cross study’, which will be conducted as a multicentre, prospective intervention of endoscopic treatment using two modified FC-SEMSs (BONASTENT M-Intraductal) that have been improved for benign stenosis in patients with benign HJAS, with the long-term non-restenosis rate (at 12 months) as the primary endpoint. This study aims to evaluate the long-term non-restenosis rate (at 12 months) and safety of the saddle-cross technique for benign HJAS. We plan to enrol 50 participants.

Ethics and dissemination

This study has been approved by the Certified Review Board of the National Cancer Center, Japan (CRB3180009). The results will be reported at various conferences and published in international peer-reviewed journals.