# Construction of a Duck Intestinal Organoid Culture System: From Crypt Isolation to Medium Optimization

## TL;DR

This study creates a reliable method to grow duck intestinal organoids in the lab, which can help improve poultry health and research.

## Contribution

The first reproducible duck intestinal organoid culture system is developed, optimized for stem cell isolation and growth conditions.

## Key findings

- EGTA was most effective for isolating duck intestinal crypts and improving organoid survival.
- Suspension culture outperformed other methods for organoid growth and differentiation.
- Adding duckling serum and retinol significantly enhanced organoid formation and differentiation.

## Abstract

Understanding how the intestine grows and functions is important for animal health and production. Organoids are miniature three-dimensional structures grown in the lab that can mimic real intestinal tissues. They serve as useful tools for studying gut development, disease mechanisms, treatment strategies, and how microbes interact with the host. While organoid culture systems for chickens are well developed, similar systems for ducks are still lacking. In this study, we established a culture method better suited to duck intestines by optimizing the isolation of intestinal stem cells and refining the growth conditions. We found that a solution called ethylene glycol tetraacetic acid was most effective for isolating duck intestinal crypts, and that suspension culture helped the organoids survive, grow, and develop better than other methods. We also discovered that adding natural factors such as serum, retinol, and retinoic acid significantly improved the formation and early differentiation of the organoids. This work establishes the first reproducible model of duck intestinal organoids, highlighting its potential as a foundation for further research and practical applications in poultry science.

Intestinal organoids possess self-organizing capacity and recapitulate essential features of intestinal architecture and function, making them powerful models for investigating development, disease mechanisms, pharmacological testing, and host–microbe interactions. Although standardized protocols for chicken intestinal organoids have been established, a defined culture system for ducks has not been available. In this study, we optimized crypt isolation procedures and culture medium composition to establish a reproducible system tailored to duck intestinal stem cells. Among various digestive solutions, ethylene glycol tetraacetic acid (EGTA) achieved the highest crypt isolation efficacy and organoid survival. Suspension culture resulted in better survival, proliferation, and differentiation of intestinal stem cells than air–liquid interface and embedding methods (p < 0.05). Immunofluorescence and real-time PCR indicated the presence of multiple epithelial lineages, including stem cells, Paneth cells, enterocytes, goblet cells, and enteroendocrine cells. Media supplemented with CHIR99021 and LDN193189 (CL) supported growth comparable to that of media with EGF, Noggin, and R-spondin 1 (ENR). Duckling serum and specific factors, such as SB203580 and retinol, further improved organoid formation and promoted differentiation. While long-term passaging and expansion remain technically challenging, this work provides the first duck intestinal organoid model and lays the foundation for future applications in avian intestinal research, including nutrition, disease modeling, and intervention strategies.