Influence of cell culture configuration on the post-cryopreservation viability of primary rat hepatocytes.

Cryopreservation has been identified as a necessary barrier to overcome in the production of tissue engineered products for clinical application. Liver engineering and bioartificial liver assisting devices are on the forefront of tissue engineering research due to its high demand and clinical potential. In this study we propose that the cryopreservation of primary mammalian hepatocytes yields better results when these cells are in a tissue-like culture configuration since cell attachment is essential for cell survival in this cell type. We used two different tissue-engineered culture configurations: monolayers and spheroid culture; and two different concepts of cryopreservation, namely vitrification and freezing. Cell suspensions were also cryopreserved using both approaches and results were compared to the engineered cultures. Both engineered configurations and suspension were cryopreserved using both conventional freezing (cooling at 1 °C/minute using 10% DMSO in foetal calf serum) and vitrification (using 40% ethylene glycol 0.6 m sucrose supplemented with 9% Ficoll). These two approaches differ on the degree of mechanical stress they inflict on the material to be cryopreserved. The maintenance of cell-to-cell and the integrity of the actin cytoskeleton were assessed using scanning electron microscopy and immunohistochemistry respectively. Results showed that while there was no significant difference between the degree of integrity shown between vitrified and control engineered cultures, the same did not happen to the frozen engineered constructs. The disruption of the cytoskeletal structure correlated with increased levels of apoptotic markers. With cryopreserved suspensions there was evidence of disruption of the cytoskeletal structure. This study concluded that cell-to-cell contact is beneficial in the maintenance of viability post-cryopreservation and that the vitrification approach was far superior to those of conventional freezing when applied to 2D and 3D hepatocyte based engineered cultures.