Overcoming mass and heat transfer limitations in vitrification of large diffusion-limited tissues via volumetric...

Long-term preservation of large, viable biological tissues remains a major hurdle in transplantation and regenerative medicine, particularly for dense, non-perfusable tissues that rely solely on diffusion for cryoprotectant (CPA) transport and heat transfer. Ice-free vitrification offers a promising solution but requires sufficient CPA penetration and rapid and spatially uniform rewarming to prevent devitrification, recrystallization, and thermomechanical damage. Here, we present a volumetric radiofrequency electromagnetic (RF-EM) rewarming platform integrated with diffusion-guided CPA loading to enable vitrification of large diffusion-limited tissues. Using porcine articular cartilage as a representative dense, avascular tissue model, micro-computed tomography-guided diffusion modeling and experimental optimization achieved >85% CPA penetration without cytotoxicity. Finite element thermal modeling and experimental validation demonstrated that RF-EM volumetric rewarming achieved warming rates exceeding 100 °C min^-1while reducing temperature gradients in 10 ml tissue samples to <10 °C, compared with >70 °C during conventional convective warming, thereby preventing thermal fracture and devitrification. When combined with optimized loading protocols, the integrated transport and rewarming approach preserved >90% cellular viability and supported metabolic recovery, with metabolic activity exceeding 80% by day 1 in 3 ml cartilage discs and exceeding 70% by day 3 in 10 ml cartilage pieces, followed by an increasing trend over the 5-day culture period. The approach also maintained extracellular matrix composition with >90% glycosaminoglycan retention, and retained native mechanical properties comparable to fresh controls. These findings demonstrate that volumetric RF-EM rewarming, coupled with diffusion-guided CPA loading, provides an engineering solution to mass and heat transfer limitations in vitrification of porcine cartilage tissues up to 10 ml, while modeling results suggest its potential scalability to larger tissue volumes, with potential applications in tissue banking, transplantation, and regenerative medicine.