An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells

Area of Science:

• Gastroenterology
• Cell Biology
• Biotechnology

Background:

• The intestinal epithelium's cell cycle is crucial for tissue homeostasis.
• Existing methods are insufficient for analyzing cell cycle phases in primary human intestinal epithelial cells (IECs).

Purpose of the Study:

• To develop and validate a novel experimental platform for real-time cell cycle analysis in live, non-immortalized human IECs.
• To enable detailed investigation of cell cycle regulation within the intestinal epithelium.

Main Methods:

• Development of two fluorescent cell cycle reporter lines (PIP-FUCCI and PIP-H2A) in primary human IECs.
• Design and 3D printing of a collagen press for optimal chamber slide preparation and imaging.
• Establishment of a computational pipeline for semi-automated cell cycle phase analysis.
• Live imaging of cell cycle dynamics in response to mitochondrial respiration inhibition (oligomycin).

Main Results:

• The PIP-FUCCI construct allows single-image cell cycle phase assignment.
• The PIP-H2A construct enables semi-automated quantification of cell cycle phase lengths.
• Inhibition of mitochondrial respiration with oligomycin was shown to lengthen the G1 phase.
• Oligomycin differentially extended S and G2/M phases in heterogeneous IEC populations.

Conclusions:

• The developed platform provides a robust method for studying cell cycle dynamics in primary human IECs.
• This technology facilitates future research into pharmaceutical impacts on the intestinal epithelium and cell cycle regulation.
• The findings highlight the sensitivity of IEC cell cycle phases to mitochondrial function.