Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

Assessing donor-to-donor variability in human intestinal organoid cultures.

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high-confidence data using human primary cultures through thorough characterization of variability.

Human physiomimetic model integrating microphysiological systems of the gut, liver, and brain for studies of neurodegenerative diseases.

Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ- and organ-immune-specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson's disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell-derived cerebral MPS in a systemically circulated common culture medium containing CD4+ regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo-like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD.

Gut-Liver Physiomimetics Reveal Paradoxical Modulation of IBD-Related Inflammation by Short-Chain Fatty Acids.

Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 T cells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFAs exacerbated CD4+ T cell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.