# Protocol for modeling the repair of intestinal damage by co-culturing mesenchymal stromal/stem cells and intestinal organoids

## TL;DR

This paper presents a detailed protocol for studying how mesenchymal stem cells help repair intestinal damage when co-cultured with intestinal organoids.

## Contribution

A novel and adaptable co-culture system to investigate MSC-organoid interactions in intestinal repair.

## Key findings

- The protocol enables assessment of MSC effects on organoid growth and survival after injury.
- The system supports both short-term and long-term evaluation of repair processes.
- Tools like OrganoSeg and OrganoAna allow for detailed data quantification.

## Abstract

Mesenchymal stem/stromal cells (MSCs) are known for their regenerative properties. This protocol describes a co-culture system for investigating molecular interactions between MSCs and intestinal epithelial organoids following injury. We outline steps for assessing the immediate effects of MSCs on organoid growth and survival, as well as a model for evaluating longer term responses. The workflow is adaptable and can be readily modified to examine MSC interactions with additional cell types or in different injury contexts.

For complete information on the use and execution of this protocol, please refer to Yetkin-Arik et al.

•Procedures for the co-culture of human MSCs and human duodenal epithelial organoids•Steps for light microscopy-based readout of MSC effects on organoids exposed to injury•Detailed steps for data quantification using OrganoSeg and OrganoAna tools•System compatible with downstream analyses using standard cell biology methods

Procedures for the co-culture of human MSCs and human duodenal epithelial organoids

Steps for light microscopy-based readout of MSC effects on organoids exposed to injury

Detailed steps for data quantification using OrganoSeg and OrganoAna tools

System compatible with downstream analyses using standard cell biology methods

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Mesenchymal stem/stromal cells (MSCs) are known for their regenerative properties. This protocol describes a co-culture system for investigating molecular interactions between MSCs and intestinal epithelial organoids following injury. We outline steps for assessing the immediate effects of MSCs on organoid growth and survival, as well as a model for evaluating longer term responses. The workflow is adaptable and can be readily modified to examine MSC interactions with additional cell types or in different injury contexts.