Successful cryopreservation of human biliary tree stem/progenitor cells (hbtscs) isolated from adult liver based on Good Manufacturing Practice (GMP)

BACKGROUND AND AIM : hBTSCs isolated from adult and fetal livers are candidate sources for regenerative medicine of liver and pancreas. Freshly isolated hBTSCs w ere transplanted in patients w ith advanced liver cirrhosis. The aim of our study w as to identify the best strategy for the cryopreservation of hBTSCs. MATERIAL AND METHODS : Epithelial Cell Adhesion Molecule positive hBTSCs w ere immunoselected from liver donors, and transferred into Kubota’s Medium (KM). Thereafter, the cells w ere subjected to a gradual controlled cryopreservation (1°C/min dow n to -196 °C in liquid nitrogen) in serum-free media containing 10% dimethyl-sulfoxide (DMSO) and different concentrations of human albumin and/or synthetic human hyaluronic acid. Thaw ed cells w ere assessed for viability, expression of different genes (CD44, ITGB1, ITGB4, CDH1, PDX1, SOX17, OCT4 by RT-PCR), platting efficiency, engraftment and differentiation potential. RESULTS : Solutions containing 15% albumin ± 0.1% hyaluronic acid determined the greatest cell viability (77.8±6.5%;N=9,p<0.01) compared to 0.1%-0.05% hyaluronic acid (50.6±5.3%;N=9) or low er concentration (1.5%) of albumin (50.4±4.3%;N=9). RT-PCR show ed no difference in the expression of tested genes among different media or betw een cryopreserved and freshly isolated cells. The number of colonies in culture w as markedly higher in the medium containing 0.1% hyaluronic acid/15% albumin (31.5±8.5;N=18,p<0.01) w ith respect to the medium containing 15% albumin alone (9.0±3.4;N=18). The differentiation potential w as preserved w hen cells w ere cryopreserved in solutions containing 15% albumin ± 0.1% hyaluronic acid, since thaw ed cells show ed a higher expression of albumin/cytokeratin(CK) 18 w hen transferred in medium tailored for hepatocytes (N=5,p<0.01), higher expression of CK7/secretin receptor in medium tailored for cholangiocytes (N=5,p<0.01), and higher expression of insulin/c-peptide in medium tailored for beta-pancreatic islets (N=5,p<0.01), in comparison to hBTSCs mantained constantly in KM. CONCLUSIONS : We identified strategy and conditions for successful cryopreservation of hBTSCs. This could help in clinical trials of cell therapy for liver diseases and poses the basis for banking hBTSCs.