Advancing Cellular and Organ Model Metabolic Analysis with Hyperpolarized ¹³C-NMR

As cellular models progress in complexity, and metabolic measurement techniques advance, integrating complex cellular models with advanced metabolic assays becomes a promising avenue for gaining deeper insights into cellular function, disease progression, and therapeutic responses. However, this integration remains a significant challenge, as traditional methods like mass spectrometry and ¹H NMR are limited in their real-time capabilities. Dissolution dynamic nuclear polarization (dDNP) NMR addresses many of these challenges by enabling non-invasive, longitudinal metabolic analysis, though its application in advanced in vitro models requires overcoming technical barriers.

This dissertation explores the application of dDNP NMR for real-time metabolic analysis in various in vitro models. The research demonstrates the feasibility of integrating dDNP NMR with biological systems ranging from simple suspension cultures to complex microfluidic chip platforms. Through four distinct studies, this work highlights the advantages of real-time metabolic monitoring for understanding immune cell function, bacterial infections, and cancer metabolism.

Study 1 examines the metabolic adaptation of CAR-T cells during their 21-day expansion, revealing metabolic shifts in glycolysis and oxidative phosphorylation using hyperpolarized [U-¹³C,²H] glucose. Study 2 investigates intracellular bacterial metabolism during Shigella infection, demonstrating real-time pathogen-specific metabolic signatures using hyperpolarized [2-¹³C] pyruvate. Study 3 applies dDNP NMR to a 3D hydrogel-based cancer model, enabling longitudinal metabolic tracking and identifying early metabolic shifts in response to therapy. Study 4 integrates dDNP NMR with microfluidic technology to create a physiologically relevant system for real-time non-invasive metabolic flux analysis.

This dissertation establishes a framework for advancing metabolic studies with dDNP NMR, providing new opportunities to study cellular function in health and disease. The findings contribute to the development of improved in vitro models for metabolic research, with potential applications in immunotherapy, infectious disease studies, and cancer treatment.