Fluidigm Based Single-cell Gene Expression Library Preparation from Patient-derived Small Intestinal Organoids

Area of Science:

• Gastroenterology and Hepatology
• Stem Cell Biology
• Biotechnology

Background:

• Human intestinal stem cells are crucial for tissue regeneration and disease modeling.
• Current methods for culturing intestinal stem cells can be complex and require optimization.
• Patient-derived organoids offer a powerful tool for personalized medicine and research.

Purpose of the Study:

• To provide a detailed protocol for isolating and culturing human intestinal stem cells into organoids from patient biopsy samples.
• To describe a method for dissociating these organoids into single cells for advanced analyses.
• To establish a robust in-vitro assay system for intestinal stem cell research.

Main Methods:

• Crypt isolation from patient intestinal biopsy specimens.
• Culture of isolated crypts to form human intestinal organoids.
• Dissociation of organoids into single cells for single-cell analysis.

Main Results:

• Successful generation of human intestinal organoids from patient biopsies.
• Demonstration of organoid cell dissociation into a single-cell suspension.
• Establishment of a reproducible protocol for in-vitro intestinal stem cell culture.

Conclusions:

• This protocol enables the generation of patient-derived human intestinal organoids.
• The described methods facilitate subsequent single-cell analyses of organoid cultures.
• The protocol provides a valuable tool for establishing stable in-vitro intestinal stem cell assay systems.