Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy.

Area of Science:

• Gastroenterology
• Immunology
• Cell Biology

Background:

• Increased intestinal epithelial cell (IEC) death is a hallmark of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD).
• Cytokines like TNF-α and IFN-γ contribute to IEC death, potentially worsening intestinal barrier defects and inflammation.
• Understanding cytokine-induced cytotoxicity is crucial for elucidating IBD immunopathogenesis.

Purpose of the Study:

• To develop a practical assay for quantifying cytokine-induced cytotoxicity in patient-derived colonic organoids.
• To utilize the Bliss independence model for calculating a coefficient of perturbagen interaction (CPI).
• To assess synergistic, additive, or antagonistic interactions between cytokine combinations in IBD models.

Main Methods:

• Employing patient-derived colonic organoids as a model system.
• Utilizing SYTOX Green fluorescent dye and live fluorescence microscopy to detect cell death.
• Applying open-source image analysis software for quantification and the Bliss independence model for CPI calculation.

Main Results:

• A reproducible method for measuring cytokine-induced IEC death in colonic organoids was established.
• The study successfully calculated the coefficient of perturbagen interaction (CPI) for cytokine combinations.
• The CPI effectively distinguished between synergistic, additive, and antagonistic effects of combined cytokines.

Conclusions:

• This protocol provides a robust method to investigate cytotoxic effects of cytokines and other perturbagens on IBD-relevant organoids.
• The CPI offers a quantitative measure to characterize interactions between different agents affecting IECs.
• This approach can advance the understanding of disease mechanisms and therapeutic strategies in IBD.