The use of vitrification to preserve primary rat hepatocyte monolayer on collagen-coated poly(ethylene-terephthalate) surfaces for a hybrid liver support system.

We developed a scaled-up procedure for vitrifying hepatocytes for hybrid liver support system applications. Hepatocyte monolayer cultured on collagen-coated polyethylene terephthalate (PET) discs constituted the basic module for a hybrid liver support system. Freshly isolated rat hepatocytes were seeded on collagen-coated PET discs with a diameter of 33 mm at a density of 5x10(6) cells per disc, and were cultured for 24 h before cryopreservation. The total duration of procedure starting from exposure to low concentrations of cryoprotectants up to cryostorage is 10 min. Vitrification of the modules was achieved by using two vitrification solutions sequentially with first vitrification solution containing two cryoprotectants, ethylene glycol (EG) and sucrose, while second vitrification solution contained additionally polymer, Ficoll. Direct exposure to liquid nitrogen vapours was followed by immersion into liquid nitrogen. Recovery procedure for vitrified modules included their warming in 1m sucrose at temperature of 38-39 degrees C followed by subsequently washing in sucrose-based solutions of decreased concentration within 15 min at room temperature. Viability, structural characteristics, and functions of cells were preserved by vitrification. Hepatocytes in the post-vitrified and warmed monolayer maintained differentiated hepatocyte characteristics both structurally and functionally. In average, protein synthesis measured as albumin production was 181.00+/-33.46 ng/million cells and 166.10+/-28.11 ng/million cells, for control and vitrified modules respectively. Urea production was, in average, 1.52+/-0.40 ng/million cells and 1.36+/-0.31 ng/million cells for a 7 day culture respectively, with no significant statistical difference between the control and vitrified modules.