Organoid and Primary Culture Core

CORE 2: ORGANOID AND HUMAN CELL CULTURE CORE SUMMARY The Organoid and Human Cell Culture Core is a critical component of the Gladstone PhAIge Therapy Center, designed to accelerate the clinical development of phage-based therapeutics targeting ESKAPE pathogens, with a particular focus on Klebsiella pneumoniae (Kp). Antimicrobial resistance is a growing global health crisis, contributing to over a million deaths annually. Phage therapy represents a promising solution to this challenge, especially as the discovery of antibiotics with novel mechanisms of action continues to lag. However, the lack of advanced preclinical models that adequately replicate human physiology and host responses hinders the translation of phage therapy to clinical use. The Organoid and Human Cell Culture Core will address this unmet need by developing and implementing cutting-edge ex vivo human cell culture systems, including 3D human airway organoids (HAOs), 2D air-liquid interface (ALI) cultures, and other primary cell models. These models offer significant advantages over traditional in vitro models by closely mimicking the complexity of human lung tissues, including cellular diversity, barrier integrity, and innate immune responses, enabling more precise evaluation of phage efficacy and safety in a clinically relevant context. The Core will accomplish this goal in two specific aims. Aim 1 focuses on establishing robust infection models for Kp in 3D HAOs and 2D ALI cultures and leveraging high-throughput sequencing-based assays to assess phage adsorption, entry efficiency, and specificity to support Project 1. By identifying phages with high on-target effects against bacteria and minimal off-target effects on human cells, this aim will streamline the selection of optimal candidates for clinical development. Aim 2 will evaluate mammalian host responses to phage therapy, including bacterial clearance, epithelial damage, host cell viability, Kp strain variation, and inflammatory cytokine expression, a critical step to advance the most promising phage cocktails to future clinical trials. The Core will also incorporate patient-derived organoids, including those from individuals with cystic fibrosis, to investigate how pre-existing epithelial dysfunction affects therapeutic outcomes. The Core leverages Dr. Melanie Ott's recognized expertise in organoid technology, with a proven track record of using HAOs to model infectious diseases, including SARS-CoV-2, HCV, HIV, and influenza. This foundational expertise, applied to Kp infections, will help develop rigorous quantitative assays to evaluate phage efficacy and safety independent from human donor bias. By integrating human models into the Center's research pipeline in Projects 1-2 and the Advanced Computational Core, the Organoid and Human Cell Culture Core will accelerate the translation of phage therapies from bench to bedside.