A foundational approach to culture and analyze malnourished organoids

ABSTRACT

The gastrointestinal (GI) epithelium plays a major role in nutrient absorption, barrier formation, and innate immunity. The development of organoid-based methodology has significantly impacted the study of the GI epithelium, particularly in the fields of mucosal biology, immunity, and host-microbe interactions. Various effects on the GI epithelium, such as genetics and nutrition, impact patients and alter disease states. Thus, incorporating these effects into organoid-based models will facilitate a better understanding of disease progression and offer opportunities to evaluate therapeutic candidates. One condition that has a significant effect on the GI epithelium is malnutrition, and studying the mechanistic impacts of malnutrition would enhance our understanding of several pathologies. Therefore, the goal of this study was to begin to develop methodology to generate viable malnourished organoids with accessible techniques and resources that can be used for a wide array of mechanistic studies. By selectively limiting distinct macronutrient components of organoid media, we were able to successfully culture and evaluate malnourished organoids. Genetic and protein-based analyses were used to validate the approach and confirm the presence of known biomarkers of malnutrition. Additionally, as proof-of-concept, we utilized malnourished organoid-derived monolayers to evaluate the effect of malnourishment on barrier formation and the ability of the bacterial pathogen Shigella flexneri to infect the GI epithelium. This work serves as the basis for new and exciting techniques to alter the nutritional state of organoids and investigate the related impacts on the GI epithelium.

KEYWORDS:

• alkaline phosphatase
• gastrointestinal
• environmental enteric dysfunction
• interleukin-8
• malnutrition
• M cells
• mucus
• organoids
• Shigella
• transepithelial electrical resistance

Acknowledgments

We would like to thank Dr. Maureen Leonard, Dr. Alba Miranda-Ribera, Ms. Victoria Kenyon, Ms. Tina Tran, and Ms. Rosiane Lima for their assistance with this project, as well as Dr. Alessio Fasano, Dr. Jason Harris, Dr. Christopher Moran, and Dr. Harland Winter for their thoughtful discussions. The following authors have current affiliations: M.P., Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Washington University School of Medicine in St. Louis, St. Louis, Missouri; J.C., Medical Student at Drexel University College of Medicine, Philadelphia, Pennsylvania; and S.S., Center for Scientific Review, National Institutes of Health, Bethesda, Maryland. Disclaimer: this article was prepared while Dr. Stefania Senger was employed at Massachusetts General Hospital. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. The authors declare no competing interests.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Additional information

Funding

Support for the authors includes Massachusetts General Hospital’s Executive Committee on Research Interim Support Funding (ISF) awards 2020A003037 and 2022A009041, National Institute of Allergy and Infectious Diseases grant R21AI146405, and the National Institute of Diabetes and Digestive and Kidney Diseases grant Nutrition Obesity Research Center at Harvard (NORCH) 2P30DK040561-26. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.