Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function

Abstract

Objective: To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution.

Summary background data: Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes.

Methods: Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed.

Results: Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls.

Conclusions: This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.

FIGURE 1. Fluorescent micrographs of hepatocytes after overnight culture following cryopreservation. Cells were cryopreserved in (B) HTS + 10% DMSO (HTS-cryo) or (C) culture media + 10% DMSO (Media-cryo), cultured overnight on collagen coated tissue culture ware and compared with (A) nonfrozen controls. Samples were stained with Syto13/ethidium bromide to distinguish living populations (green cells) and dead populations (red cells).

FIGURE 2. Hepatocyte viability over 14 days’ culture following cryopreservation. Long-term cell viability was assessed by determination of total intracellular LDH levels in experimental samples in comparison to controls.

FIGURE 3. Phase contrast micrographs of hepatocytes following 14 days of culture. Cells were cultured in a collagen gel 3-dimensional configuration for 14 days. Cells cryopreserved in (B) HTS + 10% DMSO exhibited morphology similar to (A) controls (intact, hexagonal cells), whereas (C) media + 10% DMSO–cryopreserved cells were morphologically different, characterized by cellular disintegration and irregular cell shape.

FIGURE 4. Levels of albumin secretion and urea synthesis of hepatocytes following cryopreservation. Figures represent typical graphical data obtained for (A) albumin secretion and (B) urea synthesis from experimental analysis. Analysis was repeated for 3 separate experiments and is reported in the text and Table 2.

FIGURE 5. a, b, Hepatocyte responsiveness to cytokine stimulation following cryopreservation. Cells were exposed to IL-6 (2.5 ng/mL) at either 2 to 5 days (a) or 7 to 10 days (b) postthaw, and fibrinogen and albumin production was monitored as a determinant of hepatocyte responsiveness. Straight lines represent cultures not exposed to IL-6, dotted lines represent cultures exposed to 2.5 ng/mL human IL-6 at days 2 to 5 or 7 to 10 (shaded box). Data revealed that cells cryopreserved in HTS + 10% DMSO responded in a similar manner to controls in both an increase in fibrinogen production and a decrease in albumin production in response to IL-6 stimulation.