Enhancing the cryopreservation of ex vivo 3D tumor models using vitrification strategies

Abstract

Microdissected tumor tissue explants (MDTs) are promising ex vivo models for oncology research but remain limited by poor preservation and the rapid loss of viability following resection. Here, we systematically optimized MDT cryopreservation using prostate-derived 49F and ovarian TOV112D tumor models. Before cryopreservation, we evaluated the effects of antioxidant supplementation, pre-cooling, and a short recovery period. MDTs were then preserved by either conventional slow-freezing or vitrification using ultra-rapid cooling. Following thawing, tissue morphology, apoptosis, and proliferative capacity were assessed relative to fresh controls. Vitrification improved morphological preservation and reduced apoptosis compared with slow-freezing, although recovery of proliferation differed between tumor models. Antioxidant supplementation enhanced post-thaw proliferation at optimal concentrations but induced toxicity at higher doses. Pre-cooling and a short pre-cryopreservation recovery period further improved post-thaw outcomes. Combining these parameters produced an optimized vitrification protocol that preserved up to 98% of the proliferative capacity of 49F MDTs and 69% of that of TOV112D MDTs relative to fresh controls. These findings establish optimized vitrification as a reproducible, high-yield strategy for preserving MDTs for downstream ex vivo oncology applications.