技术资料

The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections,the rejected organs from organ donation programs,and the need to use cells immediately. To facilitate accessibility to these precious tissue resources,we have established an effective method for serum-free cryopreservation of the cells,allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers,some serum-free,designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80-90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective,both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors,as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules,particularly CD44,the hyaluronan receptor,E-cadherin,β4 integrin in hHpSCs,and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications. View Publication

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. View Publication