Set Up and Utilization of a Three-Dimensional In Vitro Bioreactor System for Human Intestinal Studies and Microbial Co-Cultures

Sara E. Rudolph, Eesha Bethi, Lilianne Iglesias-Ledon, Hirunika U. Kumarasinghe, Omar Da'darah, Gautham K. Salgam, Karolinny C. Vieira, Ying Chen, David L. Kaplan
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Abstract

The study of human intestinal physiology and host-microbe interactions is crucial for understanding gastrointestinal health and disease. Traditional two-dimensional cell culture models lack the complexity of the native intestinal environment, limiting their utility in studying intestinal biology. Here, we present a detailed protocol for the set up and utilization of a three-dimensional (3D) in vitro bioreactor system for human intestinal studies and bacterial co-culture. This article outlines the design and assembly of the bioreactor system, scaffold fabrication, bacterial culture techniques, analysis methods, and troubleshooting tips. By providing step-by-step instructions, the goal is to enable other laboratories to utilize physiologically relevant tissue models of the human intestine, incorporating key features, such as nutrient flow, multiple human cell types, 3D architecture, and microbial communities. The incorporation of commensal bacteria into the bioreactor system allows for the investigation of complex host-microbe interactions, providing insight into gastrointestinal health and pathology. This article serves as a comprehensive resource for scientists seeking to advance their understanding of intestinal biology toward the development of novel therapeutic strategies for gastrointestinal disorders. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Scaffold design

Basic Protocol 2: Intestinal cell culture: Caco2 cells

Basic Protocol 3: Intestinal cell culture: organoids

Basic Protocol 4: Bioreactor design and set up

Basic Protocol 5: Bacteria in 3D bioreactor set up

Basic Protocol 6: Bacteria and drug dosing

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建立和利用三维体外生物反应器系统进行人体肠道研究和微生物共培养
研究人类肠道生理学以及宿主与微生物之间的相互作用对于了解肠道健康和疾病至关重要。传统的二维细胞培养模型缺乏原生肠道环境的复杂性,限制了其在研究肠道生物学方面的实用性。在此,我们介绍了建立和利用三维体外生物反应器系统进行人体肠道研究和细菌共培养的详细方案。本文概述了生物反应器系统的设计和组装、支架制作、细菌培养技术、分析方法和故障排除技巧。通过提供分步说明,目的是让其他实验室能够利用与生理相关的人体肠道组织模型,并结合营养流、多种人体细胞类型、三维结构和微生物群落等关键特征。在生物反应器系统中加入共生细菌可以研究宿主与微生物之间复杂的相互作用,从而深入了解胃肠道的健康和病理情况。这篇文章为科学家们提供了全面的资源,帮助他们加深对肠道生物学的理解,从而开发出治疗胃肠道疾病的新策略。© 2024 Wiley Periodicals LLC.Basic Protocol 1: Scaffold designBasic Protocol 2: Intestinal cell culture:基本程序 3:肠细胞培养:器官组织基本程序 4:生物反应器的设计和设置基本程序 5:三维生物反应器中细菌的设置基本程序 6:细菌和药物剂量
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