Redox shielding unlocks damage-free photopolymerization for GelMA 3D co-cultures mimicking pancreatic tumor complexity

Developing physiologically relevant 3D tumor models is critical to study cell-stroma interactions driving cancer progression. In pancreatic ductal adenocarcinoma (PDAC), pancreatic stellate cells (PSCs) are key stromal actors that acquire pro-tumoral functions upon activation into cancer-associated fibroblasts (CAFs). We aimed to create a 3D co-culture model combining PSCs (or CAFs) and tumor organoids (or spheroids) that reproduces the tumor microenvironment while preserving the viability of fragile stroma cells. Gelatin methacryloyl (GelMA) was selected as a biocompatible scaffold with mechanical properties comparable to pancreatic tissue. However, fragile stromal cells, such as PSCs, displayed extensive photopolymerization-induced cell death when embedded in GelMA, preventing long-term culture. We developed a simple preconditioning protocol with an antioxidant (N-acetylcysteine, NAC) that protects sensitive cells prior to GelMA encapsulation. This approach allowed long-term 3D culture of healthy PSCs and CAFs, alone or in co-culture with tumor spheroids or organoids. Cells self-organized, maintained physiological morphology, secreted extracellular matrix, and formed stromal networks around tumor structures, closely mimicking the PDAC tissue architecture. This straightforward and reproducible approach enables sensitive stromal cells to thrive in 3D GelMA, providing a robust platform to study tumor-stroma crosstalk and to support drug testing in PDAC-relevant models.