Vitreous cryopreservation of cell-biomaterial constructs involving encapsulated hepatocytes

We put forward a new strategy for cryopreservation, namely vitrification or ice-free preservation, of cell biomaterial constructs for tissue-engineering applications. In this study, for a period of 6 days, we tested vitrified and control hepatocytes entrapped at 2 different cell densities (1.5 x 10(6) and 5 x 10(6) cells/mL) in 2 types of engineered collagen matrices (M- and G-collagen) as models to evaluate efficacy and universality of the developed vitrification method. The nature of collagens caused differences in capsule sizes (100-200 mu m versus 350-450 mu m). The developed method included rapid step-wise introduction of micro-encapsulated hepatocytes to vitrification solution (40 v/v% ethylene glycol 0.6 M sucrose in medium) and their direct immersion in liquid nitrogen. Vitrification did not affect viability and functions of the micro-encapsulated hepatocytes, which exhibited trends similar to those of untreated controls in the decline of their functions and the rate of cell death during continuous culture, irrespective of physical and chemical properties of the biomaterial and cell density. For control and vitrification, the percentage of live cells varied from 80.3% +/- 0.9% to 82.3% +/- 1.4% in capsules formed by M- collagen, from 82.8% +/- 1.1% to 85.0% +/- 3.3% in capsules formed by G-collagen with cells entrapped at low density, and from 84.4% +/- 1.3% to 86.8% +/- 0.6% in capsules formed by G-collagen with cells entrapped at high density (p > 0.05). Within the same day, the maximum relative change in cell viability and functions between control and vitrification was 4% and 16%, respectively. The developed vitrification approach, which is an alternative to freezing, can be applied to other tissue-engineered constructs with comparable sizes, various cell numbers, and various properties of the biomaterials involved.