(182o) Cryopreservation of Self-Assembled Glioblastoma Organoids (GBOs)

Tumor organoids are advanced 3D in vitro multicellular model systems that bridge the gap between simplistic 2D cultures and complex in vivo tumor systems. These models, often derived through stem cell differentiation, replicate the highly organized and heterogeneous microenvironments of tumors. However, biomanufacturing tumor organoids, particularly for the most aggressive primary brain tumor glioblastoma, faces challenges in reproducibility and clinical relevance. We recently developed a scalable method to overcome these issues by biomanufacturing self-assembled glioblastoma organoids (GBOs) to study the role of endogenously secreted extracellular matrix. Scaling this production to large quantities of GBOs has led to new challenges in cryopreserving these GBOs due to their complex structures, sensitivity to cryogenic processes, and diffusion limitations. In this study, we evaluated the efficacy of three cryoprotective agents (CPAs) for cryopreserving GBOs: the commercially available Bambanker™, Bambanker™ supplemented with Y27632 (a ROCK inhibitor), and MEDY (a mixture of methylcellulose, ethylene glycol, DMSO, and Y27632). A pre-exposure period to each CPA was optimized, and GBOs were frozen for 3 days. Pre-cryopreservation and post-thaw studies on GBO dissociated total cell number, viability, and structural stability via imaging and compressive modulus. Results indicated that the addition of ROCKi was essential with a pre-exposure period of 1 hour yielding the highest structural integrity and cell viability for 1 mm GBOs. Our findings highlight the critical role of ROCK inhibition in preserving the viability and mechanical properties of larger GBOs, providing valuable insights for advancing organoid biobanking protocols and their application in translational research.