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

Cuttitta, A. J.; Meyer, S. R.; Frum, T.; Gunuguntla, B.; Wu, A.; Huang, S.; Morgan, J.; Dame, M. K.; Sexton, J. Z.; Spence, J. R.

doi:10.1101/2025.09.24.677447

bioRxiv · 10.1101/2025.09.24.677447

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

Cuttitta, A. J.·Meyer, S. R.·Frum, T.·Gunuguntla, B.·Wu, A.·Huang, S.·Morgan, J.·Dame, M. K.·Sexton, J. Z.·Spence, J. R.

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Abstract

Background & AimsHuman 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. MethodsUsing 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. ResultsEpithelium 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-{gamma}, TNF-, 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). ConclusionsWe 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.

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Cuttitta, A. J., Meyer, S. R., Frum, T., Gunuguntla, B., Wu, A., Huang, S., Morgan, J., Dame, M. K., Sexton, J. Z., Spence, J. R.. 2025-09-25. A scalable long-term in vitro model of human colonic epithelium with continuous barrier function. https://doi.org/10.1101/2025.09.24.677447

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