Boone, L. A. | Search bioRxiv

bioRxiv2025

High-Throughput Human Gut Immune Co-Culture Model for Evaluating Inflammatory Bowel Disease Anti-Inflammatory Therapies

Current treatments for inflammatory bowel disease (IBD) are often ineffective long-term, as many patients ultimately become unresponsive to anti-inflammatory drugs. The need for improved therapeutics is urgent. Animal models utilized for drug development are limited by interspecies variability and poor translatability. However, most in vitro models lack the sophistication to model the key interplay of the immune system with the intestinal epithelium in line with the known role of the immune system in the etiology of the disease. To address this gap, we developed a primary intestinal epithelial cell co-culture system to incorporate elements of innate immune signaling. This system models immune-epithelial interactions using RepliGut(R) - Planar Transverse Colon cultured on a Transwell system with THP-1 derived macrophages in a receiver compartment of a 96-well plate. Epithelial barrier integrity and cell viability were maintained in co-culture with unstimulated macrophages. However, similar to the pathology associated with IBD, epithelial integrity was compromised in co-culture with LPS + IFN-{gamma} pre-stimulated macrophages as evidenced by declining TEER and cell viability and increased inflammatory cytokine release. Cotreatment with anti-inflammatory IBD therapeutics adalimumab or tofacitinib mitigated these effects, demonstrating the models ability to replicate key inflammatory responses and prevention. Reproducibility and scalability of the model system further position the model for high-throughput screening of anti-inflammatory drugs, improving drug discovery, and accelerating the translation of new IBD therapies into clinical practice. HighlightsO_LICo-culture model: RepliGut(R) - Planar Transverse Colon with THP-1 derived macrophages C_LIO_LIHigh throughput and human-relevant model C_LIO_LI"Healthy" co-culture resembling healthy intestine C_LIO_LI"Inflamed" co-culture mimicking IBD innate inflammatory signaling C_LIO_LIPotential to screen anti-inflammatory drugs relevant to IBD C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/654072v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@101a675org.highwire.dtl.DTLVardef@157605borg.highwire.dtl.DTLVardef@1770c97org.highwire.dtl.DTLVardef@dbc9c0_HPS_FORMAT_FIGEXP M_FIG Gut-immune co-culture model simulating healthy and inflamed intestine. The immune co-culture model consists of mature differentiated primary human transverse colon epithelial cells cultured on a 96-well Transwell(R) plate with macrophage differentiated THP-1 cells (THP-1m) cultured in the receiver plate. In this configuration, the THP-1m are located basally to the epithelial cells, allowing for apical treatment in the transwell and basal treatment in the receiver plate. In the unstimulated state, intestinal cells and immune cells maintain a stable co-culture. Upon stimulation with LPS and IFN-y, both cell types initiate an inflammatory response that results in release of cytokines, loss of intestinal barrier integrity, and cytotoxicity. C_FIG

immunology↗

bioRxiv2024

High-Throughput Assay for Predicting Diarrhea Risk Using a 2D Human Intestinal Stem Cell-Derived Model

Gastrointestinal toxicities (GITs) are the most prevalent adverse events (AE) reported in clinical trials, often resulting in dose-limitations that reduce drug efficacy and delay development and treatment optimization. Preclinical animal models do not accurately replicate human GI physiology, leaving few options for early detection of GI side effects prior to human studies. Development of an accurate model that predicts GIT earlier in drug discovery programs would better support successful clinical trial outcomes. Chemotherapeutics, which exhibit high rates of clinical GIT, frequently target mitotic cells. Therefore, we hypothesized that a model utilizing proliferative cell populations derived from human intestinal crypts would predict the occurrence of clinical GITs with high accuracy. Here, we describe the development of a multiparametric assay utilizing the RepliGut(R) Planar system, an intestinal stem cell-derived platform cultured in an accessible high throughput Transwell format. This assay addresses key physiological elements of GIT by assessing cell proliferation (EdU incorporation), cell abundance (DAPI quantification), and barrier function (TEER). Using this approach, we demonstrate that primary proliferative cell populations reproducibly respond to marketed chemotherapeutics at physiologic concentrations. To determine the ability of this model to predict clinical diarrhea risk, we evaluated a set of 30 drugs with known clinical diarrhea incidence in three human donors, comparing results to known plasma drug concentrations. This resulted in highly accurate predictions of diarrhea potential for each endpoint (balanced accuracy of 91% for DAPI, 90% for EdU, 88% for TEER) with minimal variation across human donors. In vitro toxicity screening using primary proliferative cells may enable improved safety evaluations, reducing the risk of AEs in clinical trials and ultimately lead to safer and more effective treatments for patients.

pharmacology and toxicology↗