The Rho kinase (ROCK) pathway, which regulates the actomyosin cytoskeleton, is a crucial target for improving the cryopreservation of glioblastoma organoids (GBOs). GBOs are 3D, tissue-like modeling systems of the deadliest primary brain tumor in adults, glioblastoma (GBM). Previously, we determined ROCK inhibitor (ROCKi) Y-27632 enhanced GBM stem cell expansion, and it is negligible in our novel self-assembled, animal product-free (i.e., no fetal bovine serum and exogenous extracellular matrix (ECM) hydrogels) system. ECM is the biopolymers that makeup the space between cells that play a vital role in structural integrity and cell fate. Here, we aim to determine the role of ROCK in maintaining the cell-ECM interaction during and post-cryopreservation to strengthen the cellular cytoskeleton and maintain cell-ECM interactions. We tested this with three different DMSO-based cryopreservation agents: 10% DMSO, MED (1% methyl cellulose + 10% ethylene glycol + 10% DMSO), and commercially available Bambanker™. Preliminary results indicate that Bambanker™ and 10% DMSO maintain cellular viability and structural integrity, with studies of ECM expression currently ongoing. These findings elucidate the role of the ROCK pathway in self-secreted ECM during cryopreservation. Overall, this work will enable long-term storage and widespread distribution of physiologically relevant GBOs to accelerate research and therapeutic development for GBM.

ALEXANDRA AVERA

The University of Alabama

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