Irineja Cubela

The intestinal mucosal barrier contains microbial organisms within the lumen while preserving the ability to absorb nutrients. Dietary, microbial, and other exposures shaped human barrier evolution and continue to impact disease susceptibility. Here, we established engineered barrier models of the human small intestine and colon composed of a multilineage epithelium, mucus layer, accessible microbial compartment and autologous tissue-resident immune cells. The epithelium has crypt- and villus-like topological domains, with stem cells differentiating into absorptive and secretory lineages with region-specific identities. Secreted mucins accumulate apically, forming a dense mucus layer separating the epithelium from colonizing commensal and pathogenic bacteria. Intestinal memory T cells integrate into and interact with the epithelium. We use the engineered intestinal tissues to identify an epithelial gene regulatory network underlying response to Salmonella Typhimurium infection, and uncover epithelial-immune-pathogen crosstalk coordinating cytokine release and epithelial damage. Overall, this work allows for the modular integration of epithelial, microbial, and immune compartments providing a versatile system for studying human intestinal physiology and pathologies.

Abstract Lung-resident immune cells, spanning both innate and adaptive compartments, preserve the integrity of the respiratory barrier, but become pathogenic if dysregulated 1 . Current in vitro organoid models aim to replicate interactions between the alveolar epithelium and immune cells but have not yet incorporated lung-specific immune cells critical for tissue residency 2 . Here we address this shortcoming by describing human lung alveolar immuno-organoids (LIO) that contain an autologous tissue-resident lymphoid compartment, primarily composed of tissue-resident memory T cells (TRMs). Additionally, we introduce lung alveolar immuno-organoids with myeloid cells (LIOM), which include both TRMs and a macrophage-rich alveolar myeloid compartment. The resident immune cells formed a stable immune-epithelial system, frequently interacting with the epithelium and promoting a regenerative alveolar transcriptomic profile. To understand how dysregulated inflammation perturbed the respiratory barrier, we simulated T-cell-mediated inflammation in LIOs and LIOMs and used single-cell transcriptomic analyses to uncover the molecular mechanisms driving immune responses. The presence of innate cells induced a shift in T cell identity from cytotoxic to immunosuppressive, reducing epithelial cell killing and inflammation. Based on insights obtained with bulk RNA-seq data from the phase 3 IMpower150 trial, we tested whether LIOM cultures could model clinically-relevant but poorly understood pulmonary side effects caused by immunotherapies such as the checkpoint inhibitor atezolizumab 3 . We observed a decrease in immunosuppressive T cells and identified gene signatures that matched the transcriptomic profile of patients with drug-induced pneumonitis. Given its effectiveness in capturing outcomes and mechanisms associated with a prevalent pulmonary disease, this system unlocks opportunities for studying a wide range of immune-related pathologies in the lung.

Organoid research offers valuable insights into human biology and disease, but reproducibility and scalability remain significant challenges, particularly for epithelial organoids. Here, we present an integrated microfluidic platform that addresses these limitations by enabling high-throughput generation of uniform hydrogel microparticles embedded with primary-derived human adult intestinal stem cells. Our platform includes a cell distribution system for homogeneous cell encapsulation and a microfluidic oil-removal module for efficient particle transfer to aqueous media. We demonstrate the successful culture and differentiation of both healthy- and tumor-derived intestinal organoids within these microparticles, achieving high homogeneity and reproducibility. This integrated microfluidic approach holds promise for scalable and standardized organoid production, with potential applications in drug screening, disease modeling, and personalized medicine.

Organoids have revolutionized the study of human adult stem cells, offering new insights into complex biological systems. Alveolar epithelial type 2 cells (AT2) have been difficult to grow in vitro as they spontaneously differentiate into alveolar epithelial type 1 cells. However, when cultured as 3D organoids they retain typical AT2 markers. Since organoid models are epithelium-only structures, they lack immune cells, specifically the tissue-resident immune cells which are crucial for studying immune-related diseases and drug-induced adverse events. To address this gap, we have developed a fully autologous immunocompetent human alveolar organoid model derived from fresh tissue resections. From the same resection we isolated AT2 cells for alveolar organoids, tissue-resident memory T cells and alveolar myeloid cells. The various cell types were shown to retain their functionality upon cryopreservation. The different compartments were then combined in a 3D co-culture presenting an innovative in vitro model that enables the investigation of tissue-resident immune responses specifically focusing on the role of the myeloid compartment upon T cell stimulation. This novel in vitro approach allows exploring relevant cellular interactions by single-cell RNA-sequencing and imaging revealing that the alveolar myeloid cells affect the treatment response by directly communicating with other cell types present in the culture condition. Additionally, we leveraged this immunocompetent model to study clinically relevant adverse events induced by targeted therapies and immune checkpoint inhibitors. Our findings demonstrate the potential of this model to provide valuable insights into tissue-specific immune responses, paving the way for improved therapeutic strategies and personalized medicine.