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

Microdissected tumor tissue explants (MDTs) represent promising ex vivo models for oncology research but remain limited by poor preservation methods and limited viability once resected. We systematically evaluated cryopreservation as a solution to this problem by combining strategies across two ovarian cancer models, comparing slow-freeze and vitrification while integrating antioxidants, temperature modulation, and pre-cryopreservation culture. Vitrification consistently preserved morphology and reduced apoptosis more effectively than slow-freeze, though proliferation outcomes varied by cell line. Supplementation with a defined antioxidant mix (A3) improved proliferation but required careful dosing to avoid toxicity. Pre-cooling enhanced recovery depending on the cell line, while a short resting period prior to freezing further improved survival independent of culture platform. By combining optimized parameters, our protocol preserved up to 98% (49F) and 69% (TOV112D) of proliferative capacity relative to fresh controls. These findings establish vitrification, when rationally optimized, as a reproducible and high-yield strategy for MDT cryopreservation over traditional slow-freezing methods, enabling more reliable use of explant models in personalized medicine and research.