Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells

Abstract

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.

Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem/progenitor cells

“…Cell viability, colony formation capacity, and PD of HA-coated hBTSCs were better than uncoated cells, as verified in long-term cultures corroborating prior reports of the effects of HA on both normal cells [7, 11, 17, 28], in protecting cells under cryopreservation conditions [16], and in support of transformed cancer stem cells in vitro and in their survival and proliferation in vivo [15]. …”

Regulation and roles of the hyaluronan system in mammalian reproduction

“…Small fragments of HA induce heat shock protein and suppress apoptosis in vitro (Xu et al 2002). Similar effects promoting cryosurvival have been reported in stem cells where both post-thaw viability and phenotypic characteristics are improved by HA (Turner et al 2012).…”

Cryopreservation protocol for human biliary tree stem/progenitors, hepatic and pancreatic precursors

“…20 engraftment after cell transplantation well correlates with the level of expression of adhesion molecules. Therefore, we analysed by RT-qPCR the gene expression of different genes encoding adhesion molecules including CD44 (the hyaluronan receptor), ITGB1 (integrin beta1), ITGB4 (integrin beta 4), and CDH1 (cadherin 1).…”

“…It is distinctive in having no cytokines, growth factors or hormones other than insulin and transferrin. Even though it is not isotonic, its ability as a cryopreservation buffer has proved similar to that of the Good Manufacturing Practice (GMP) approved Cryostor-10 20 . We utilized serum-free KM that was supplemented with different concentrations of human serum-derived albumin (HSA) and/or hyaluronic acid (HA) to generate conditions effective in cryopreservation of hBTSCs from adult donors under GMP like conditions.…”