A scalable long-term in vitro model of human colonic epithelium with continuous barrier function

Human in vitro models of differentiated intestinal epithelium are valuable research tools for discovery and translational medicine. Primary adult colonic epithelium barrier function is difficult to assess in a 2-dimensional (2D) in vitro format for more than a few days due to rapid cellular turnover. We developed a transwell model of the colonic epithelium that maintains proliferative and differentiated cell types, allowing long-term culture.

Using 3-dimensional (3D) primary human colonoids, we seeded dissociated epithelium onto 96-well transwell membranes and added different media that was growth factor specific to apical or basolateral transwell chambers. Barrier integrity was assessed using a variety of stimuli and TEER was continuously monitored in real time for up to 2 weeks, followed by quantitative single-cell image analysis using high-content imaging and deep-learning-based cell segmentation.

Epithelium cultured up to 30 days sustained a time-dependent differentiation, leading to the establishment of a monolayer with proliferative stem/progenitor and differentiated cell types. While monitoring continuous TEER across multiple donor organoids, barrier challenge assays recorded donor-specific responses to IFN-γ, TNF-α, Clostridium difficile toxin B (TcdB), and ethylenediaminetetraacetic acid (EDTA). Endpoint immunofluorescence analysis of tight junctions and whole cell staining identified unique cellular features among treatment groups which significantly correlated with TEER, including cell-cell orientation (e.g., adjacent cell mean angle).

We demonstrate a high-throughput in vitro model of the homeostatic human colonic epithelium that maintains a long-term functional barrier. This model can be used to investigate complex epithelial barrier responses over time.