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 microm versus 350-450 microm). The developed method included rapid step-wise introduction of microencapsulated hepatocytes to vitrification solution (40v/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 microencapsulated 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.

Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum.

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

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.