CAR-T cell therapy has demonstrated curative potential across hematologic malignancies and increasingly is investigated for solid tumor and autoimmune disease treatment. However, manufacturing complexity, high viral vector costs, and extended vein-to-vein timelines limit patient access. Biomaterial scaffold-based manufacturing platforms—including the alginate-based DryDux MASTER systems— have emerged as promising tools to compress CAR-T manufacturing while dramatically improving viral transduction efficiency. However, the physical parameters governing scaffold-mediated transduction, the material requirements for this enhancement, and the compositional flexibility of these all-in-one platforms remain poorly characterized. This dissertation systematically addresses these gaps to establish rational design principles for biomaterial-based CAR-T manufacturing.First, I demonstrate that viral transduction is fundamentally a concentration-mediated phenomenon. Both droplet and scaffold-based transduction are governed primarily by total cell-virus concentration at fixed volume rather than multiplicity of infection alone—a finding with direct implications for virus utilization and manufacturing cost. DryDux scaffolds achieve approximately 8-fold greater transduction efficiency over simple mixing. Mechanistic investigation using calcium-free scaffolds revealed that rapid viral adhesion within the scaffold matrix is a primary driver of this enhancement, pointing toward inherent material effects rather than physical scaffold architecture alone driving this phenomenon.Second, this material-dependent adhesion finding motivated a systematic screen of diverse biomaterial scaffolds for transduction enhancement capacity. Transduction enhancement is associated with distributed surface porosity, liquid absorption, and, preferentially, negative polymer charge. Critically, synthetic polymers fail to mediate transduction despite maintaining porosity, implicating inherent material chemistry as a necessary parameter alongside physical architecture.Third, I demonstrate that the MASTER all-in-one activation and transduction platform is robust to component modification. Factor-by-factor analysis of alginate molecular weight, calcium crosslinking, azide modification, antibody presentation, and cytokine formulation reveals that calcium crosslinking and chemical conjugation are unnecessary for ex vivo production, enabling a simplified formulation—TDUCTS—that reduces manufacturing complexity without compromising CAR-T cell yield, transduction efficiency, or memory phenotype.Collectively, this work provides mechanistic insight into scaffold-mediated transduction and establishes actionable design principles for simplified, efficient, and broadly accessible CAR-T manufacturing platforms.
Micah James Mallory (Fri,) studied this question.