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This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 μg/106 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 μg/106 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.