{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T13:40:40Z","timestamp":1793108440368,"version":"4.5.0"},"reference-count":36,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2017,7,20]],"date-time":"2017-07-20T00:00:00Z","timestamp":1500508800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2017,7,20]],"date-time":"2017-07-20T00:00:00Z","timestamp":1500508800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Human biliary tree stem\/progenitor cells (hBTSCs) are being used for cell therapies of patients with liver cirrhosis. A cryopreservation method was established to optimize sourcing of hBTSCs for these clinical programs and that comprises serum-free Kubota\u2019s Medium (KM) supplemented with 10% dimethyl sulfoxide (DMSO), 15% human serum albumin (HSA) and 0.1% hyaluronans. Cryopreserved versus freshly isolated hBTSCs were similar <jats:italic>in vitro<\/jats:italic> with respect to self-replication, stemness traits, and multipotency. They were able to differentiate to functional hepatocytes,cholangiocytes or pancreatic islets, yielding similar levels of secretion of albumin or of glucose-inducible levels of insulin. Cryopreserved versus freshly isolated hBTSCs were equally able to engraft into immunocompromised mice yielding cells with human-specific gene expression and human albumin levels in murine serum that were higher for cryopreserved than for freshly isolated hBTSCs. The successful cryopreservation of hBTSCs facilitates establishment of hBTSCs cell banking offering logistical advantages for clinical programs for treatment of liver diseases.<\/jats:p>","DOI":"10.1038\/s41598-017-05858-0","type":"journal-article","created":{"date-parts":[[2017,7,14]],"date-time":"2017-07-14T14:55:55Z","timestamp":1500044155000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["Cryopreservation protocol for human biliary tree stem\/progenitors, hepatic and pancreatic precursors"],"prefix":"10.1038","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0924-2551","authenticated-orcid":false,"given":"Lorenzo","family":"Nevi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincenzo","family":"Cardinale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guido","family":"Carpino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniele","family":"Costantini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabina","family":"Di Matteo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfredo","family":"Cantafora","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabio","family":"Melandro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberto","family":"Brunelli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3141-4022","authenticated-orcid":false,"given":"Carlo","family":"Bastianelli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Camilla","family":"Aliberti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Monti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Bosco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pasquale Bartolomeo","family":"Berloco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pierluigi Benedetti","family":"Panici","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lola","family":"Reid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenio","family":"Gaudio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Domenico","family":"Alvaro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2017,7,20]]},"reference":[{"key":"5858_CR1","doi-asserted-by":"publisher","first-page":"2159","DOI":"10.1002\/hep.24590","volume":"54","author":"V Cardinale","year":"2011","unstructured":"Cardinale, V. et al. Multipotent stem\/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets. Hepatology \n                           54, 2159\u20132172, doi:10.1002\/hep.24590 (2011).","journal-title":"Hepatology"},{"key":"5858_CR2","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1038\/nrgastro.2012.23","volume":"9","author":"V Cardinale","year":"2012","unstructured":"Cardinale, V. et al. The biliary tree\u2013a reservoir of multipotent stem cells. Nature reviews. Gastroenterology & hepatology \n                           9, 231\u2013240, doi:10.1038\/nrgastro.2012.23 (2012).","journal-title":"Gastroenterology & hepatology"},{"key":"5858_CR3","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1111\/j.1469-7580.2011.01462.x","volume":"220","author":"G Carpino","year":"2012","unstructured":"Carpino, G. et al. Biliary tree stem\/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages. Journal of anatomy \n                           220, 186\u2013199, doi:10.1111\/j.1469-7580.2011.01462.x (2012).","journal-title":"Journal of anatomy"},{"key":"5858_CR4","doi-asserted-by":"publisher","first-page":"13","DOI":"10.3978\/j.issn.2305-5839.2012.10.02","volume":"1","author":"R Semeraro","year":"2013","unstructured":"Semeraro, R. et al. The fetal liver as cell source for the regenerative medicine of liver and pancreas. Annals of translational medicine \n                           1, 13, doi:10.3978\/j.issn.2305-5839.2012.10.02 (2013).","journal-title":"Annals of translational medicine"},{"key":"5858_CR5","doi-asserted-by":"publisher","first-page":"1194","DOI":"10.1016\/j.jhep.2014.01.026","volume":"60","author":"G Carpino","year":"2014","unstructured":"Carpino, G. et al. Evidence for multipotent endodermal stem\/progenitor cell populations in human gallbladder. Journal of hepatology \n                           60, 1194\u20131202, doi:10.1016\/j.jhep.2014.01.026 (2014).","journal-title":"Journal of hepatology"},{"key":"5858_CR6","doi-asserted-by":"publisher","first-page":"1097","DOI":"10.1016\/j.jhep.2014.06.016","volume":"61","author":"M Riccio","year":"2014","unstructured":"Riccio, M. et al. The Fas\/Fas ligand apoptosis pathway underlies immunomodulatory properties of human biliary tree stem\/progenitor cells. Journal of hepatology \n                           61, 1097\u20131105, doi:10.1016\/j.jhep.2014.06.016 (2014).","journal-title":"Journal of hepatology"},{"key":"5858_CR7","doi-asserted-by":"publisher","first-page":"2047","DOI":"10.1002\/stem.1457","volume":"31","author":"G Lanzoni","year":"2013","unstructured":"Lanzoni, G. et al. Concise review: clinical programs of stem cell therapies for liver and pancreas. Stem cells \n                           31, 2047\u20132060, doi:10.1002\/stem.1457 (2013).","journal-title":"Stem cells"},{"key":"5858_CR8","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1186\/s12876-014-0204-z","volume":"14","author":"V Cardinale","year":"2014","unstructured":"Cardinale, V. et al. Transplantation of human fetal biliary tree stem\/progenitor cells into two patients with advanced liver cirrhosis. BMC gastroenterology \n                           14, 204, doi:10.1186\/s12876-014-0204-z (2014).","journal-title":"BMC gastroenterology"},{"key":"5858_CR9","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1007\/BF02796340","volume":"18","author":"DL Novicki","year":"1982","unstructured":"Novicki, D. L., Irons, G. P., Strom, S. C., Jirtle, R. & Michalopoulos, G. Cryopreservation of isolated rat hepatocytes. In vitro \n                           18, 393\u2013399 (1982).","journal-title":"In vitro"},{"key":"5858_CR10","doi-asserted-by":"publisher","first-page":"7389","DOI":"10.1016\/j.biomaterials.2011.06.042","volume":"32","author":"OA Lozoya","year":"2011","unstructured":"Lozoya, O. A. et al. Regulation of hepatic stem\/progenitor phenotype by microenvironment stiffness in hydrogel models of the human liver stem cell niche. Biomaterials \n                           32, 7389\u20137402, doi:10.1016\/j.biomaterials.2011.06.042 (2011).","journal-title":"Biomaterials"},{"key":"5858_CR11","doi-asserted-by":"publisher","first-page":"616","DOI":"10.1097\/00007890-199303000-00028","volume":"55","author":"V Dixit","year":"1993","unstructured":"Dixit, V., Darvasi, R., Arthur, M., Lewin, K. & Gitnick, G. Cryopreserved microencapsulated hepatocytes\u2013transplantation studies in Gunn rats. Transplantation \n                           55, 616\u2013622 (1993).","journal-title":"Transplantation"},{"key":"5858_CR12","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1006\/taap.1996.0299","volume":"141","author":"C Guyomard","year":"1996","unstructured":"Guyomard, C., Rialland, L., Fremond, B., Chesne, C. & Guillouzo, A. Influence of alginate gel entrapment and cryopreservation on survival and xenobiotic metabolism capacity of rat hepatocytes. Toxicology and applied pharmacology \n                           141, 349\u2013356, doi:10.1006\/taap.1996.0299 (1996).","journal-title":"Toxicology and applied pharmacology"},{"key":"5858_CR13","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/S0168-8278(00)00086-6","volume":"34","author":"L Canaple","year":"2001","unstructured":"Canaple, L. et al. Maintenance of primary murine hepatocyte functions in multicomponent polymer capsules\u2013in vitro cryopreservation studies. Journal of hepatology \n                           34, 11\u201318 (2001).","journal-title":"Journal of hepatology"},{"key":"5858_CR14","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1016\/j.cryobiol.2006.05.004","volume":"53","author":"C Terry","year":"2006","unstructured":"Terry, C., Dhawan, A., Mitry, R. R. & Hughes, R. D. Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryobiology \n                           53, 149\u2013159, doi:10.1016\/j.cryobiol.2006.05.004 (2006).","journal-title":"Cryobiology"},{"key":"5858_CR15","doi-asserted-by":"publisher","first-page":"826","DOI":"10.1007\/BF02619627","volume":"20","author":"MJ Gomez-Lechon","year":"1984","unstructured":"Gomez-Lechon, M. J., Lopez, P. & Castell, J. V. Biochemical functionality and recovery of hepatocytes after deep freezing storage. In vitro \n                           20, 826\u2013832 (1984).","journal-title":"In vitro"},{"key":"5858_CR16","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/S0168-8278(86)80140-4","volume":"3","author":"PJ Rijntjes","year":"1986","unstructured":"Rijntjes, P. J., Moshage, H. J., Van Gemert, P. J., De Waal, R. & Yap, S. H. Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures. Journal of hepatology \n                           3, 7\u201318 (1986).","journal-title":"Journal of hepatology"},{"key":"5858_CR17","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1016\/S0011-2240(02)00011-1","volume":"44","author":"E Alexandre","year":"2002","unstructured":"Alexandre, E. et al. Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiology \n                           44, 103\u2013113 (2002).","journal-title":"Cryobiology"},{"key":"5858_CR18","doi-asserted-by":"publisher","first-page":"579","DOI":"10.1177\/096368979500400607","volume":"4","author":"RM Adams","year":"1995","unstructured":"Adams, R. M. et al. Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential. Cell transplantation \n                           4, 579\u2013586 (1995).","journal-title":"Cell transplantation"},{"key":"5858_CR19","doi-asserted-by":"publisher","first-page":"807","DOI":"10.1097\/TP.0b013e3181f24ea2","volume":"90","author":"R Turner","year":"2010","unstructured":"Turner, R., Gerber, D. & Reid, L. The future of cell transplant therapies: a need for tissue grafting. Transplantation \n                           90, 807\u2013810, doi:10.1097\/TP.0b013e3181f24ea2 (2010).","journal-title":"Transplantation"},{"key":"5858_CR20","doi-asserted-by":"publisher","first-page":"2257","DOI":"10.3727\/096368912X637000","volume":"21","author":"RA Turner","year":"2012","unstructured":"Turner, R. A., Mendel, G., Wauthier, E., Barbier, C. & Reid, L. M. Hyaluronan-supplemented buffers preserve adhesion mechanisms facilitating cryopreservation of human hepatic stem\/progenitor cells. Cell transplantation \n                           21, 2257\u20132266, doi:10.3727\/096368912X637000 (2012).","journal-title":"Cell transplantation"},{"key":"5858_CR21","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1111\/hepr.12228","volume":"44","author":"Z Wang","year":"2014","unstructured":"Wang, Z., Hisatake, G. & Yang, L. Liver-specific deceased donor risk indices. Hepatology research: the official journal of the Japan Society of Hepatology \n                           44, 159\u2013164, doi:10.1111\/hepr.12228 (2014).","journal-title":"Hepatology research: the official journal of the Japan Society of Hepatology"},{"key":"5858_CR22","doi-asserted-by":"publisher","first-page":"1191","DOI":"10.1097\/MEG.0b013e32832973fc","volume":"21","author":"E Schmelzer","year":"2009","unstructured":"Schmelzer, E. & Reid, L. M. Human telomerase activity, telomerase and telomeric template expression in hepatic stem cells and in livers from fetal and postnatal donors. European journal of gastroenterology & hepatology \n                           21, 1191\u20131198, doi:10.1097\/MEG.0b013e32832973fc (2009).","journal-title":"European journal of gastroenterology & hepatology"},{"key":"5858_CR23","first-page":"85","volume":"115","author":"V Cardinale","year":"2010","unstructured":"Cardinale, V. et al. Multipotent stem cells in the biliary tree. Italian journal of anatomy and embryology Archivio italiano di anatomia ed embriologia \n                           115, 85\u201390 (2010).","journal-title":"Italian journal of anatomy and embryology Archivio italiano di anatomia ed embriologia"},{"key":"5858_CR24","doi-asserted-by":"publisher","first-page":"1443","DOI":"10.1002\/hep.23829","volume":"52","author":"Y Wang","year":"2010","unstructured":"Wang, Y. et al. Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates. Hepatology \n                           52, 1443\u20131454, doi:10.1002\/hep.23829 (2010).","journal-title":"Hepatology"},{"key":"5858_CR25","doi-asserted-by":"publisher","first-page":"1966","DOI":"10.1002\/stem.1460","volume":"31","author":"Y Wang","year":"2013","unstructured":"Wang, Y. et al. Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis. Stem cells \n                           31, 1966\u20131979, doi:10.1002\/stem.1460 (2013).","journal-title":"Stem cells"},{"key":"5858_CR26","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1186\/s13287-017-0492-7","volume":"8","author":"L Nevi","year":"2017","unstructured":"Nevi, L. et al. Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem\/progenitor cells. Stem cell research & therapy \n                           8, 68, doi:10.1186\/s13287-017-0492-7 (2017).","journal-title":"Stem cell research & therapy"},{"key":"5858_CR27","doi-asserted-by":"publisher","first-page":"e0134677","DOI":"10.1371\/journal.pone.0134677","volume":"10","author":"V Cardinale","year":"2015","unstructured":"Cardinale, V. et al. Adult Human Biliary Tree Stem Cells Differentiate to beta-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide. PloS one \n                           10, e0134677, doi:10.1371\/journal.pone.0134677 (2015).","journal-title":"PloS one"},{"key":"5858_CR28","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1016\/S0091-679X(08)00008-3","volume":"86","author":"E Wauthier","year":"2008","unstructured":"Wauthier, E. et al. Hepatic stem cells and hepatoblasts: identification, isolation, and ex vivo maintenance. Methods in cell biology \n                           86, 137\u2013225, doi:10.1016\/S0091-679X(08)00008-3 (2008).","journal-title":"Methods in cell biology"},{"key":"5858_CR29","doi-asserted-by":"publisher","first-page":"1973","DOI":"10.1084\/jem.20061603","volume":"204","author":"E Schmelzer","year":"2007","unstructured":"Schmelzer, E. et al. Human hepatic stem cells from fetal and postnatal donors. The Journal of experimental medicine \n                           204, 1973\u20131987, doi:10.1084\/jem.20061603 (2007).","journal-title":"The Journal of experimental medicine"},{"key":"5858_CR30","doi-asserted-by":"publisher","first-page":"987","DOI":"10.1016\/j.jhep.2012.07.013","volume":"57","author":"R Semeraro","year":"2012","unstructured":"Semeraro, R. et al. Multipotent stem\/progenitor cells in the human foetal biliary tree. Journal of hepatology \n                           57, 987\u2013994, doi:10.1016\/j.jhep.2012.07.013 (2012).","journal-title":"Journal of hepatology"},{"key":"5858_CR31","doi-asserted-by":"publisher","first-page":"12132","DOI":"10.1073\/pnas.97.22.12132","volume":"97","author":"H Kubota","year":"2000","unstructured":"Kubota, H. & Reid, L. M. Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proceedings of the National Academy of Sciences of the United States of America \n                           97, 12132\u201312137, doi:10.1073\/pnas.97.22.12132 (2000).","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"5858_CR32","doi-asserted-by":"publisher","first-page":"548","DOI":"10.1002\/hep.27547","volume":"61","author":"JA Harrill","year":"2015","unstructured":"Harrill, J. A. et al. Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to liver tumorigenesis. Hepatology \n                           61, 548\u2013560, doi:10.1002\/hep.27547 (2015).","journal-title":"Hepatology"},{"key":"5858_CR33","doi-asserted-by":"publisher","first-page":"1795","DOI":"10.1056\/NEJMoa1101324","volume":"364","author":"J Kajstura","year":"2011","unstructured":"Kajstura, J. et al. Evidence for human lung stem cells. The New England journal of medicine \n                           364, 1795\u20131806, doi:10.1056\/NEJMoa1101324 (2011).","journal-title":"The New England journal of medicine"},{"key":"5858_CR34","doi-asserted-by":"publisher","first-page":"344","DOI":"10.1016\/j.biomaterials.2008.09.037","volume":"30","author":"NE Fedorovich","year":"2009","unstructured":"Fedorovich, N. E. et al. The effect of photopolymerization on stem cells embedded in hydrogels. Biomaterials \n                           30, 344\u2013353, doi:10.1016\/j.biomaterials.2008.09.037 (2009).","journal-title":"Biomaterials"},{"key":"5858_CR35","doi-asserted-by":"publisher","first-page":"e50542","DOI":"10.1371\/journal.pone.0050542","volume":"7","author":"A Pisciotta","year":"2012","unstructured":"Pisciotta, A. et al. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo. PloS one \n                           7, e50542, doi:10.1371\/journal.pone.0050542 (2012).","journal-title":"PloS one"},{"key":"5858_CR36","doi-asserted-by":"publisher","unstructured":"Ribaudo, R., Gilman, M., Kingston, R. E., Chomczynski, P. & Sacchi, N. Preparation of RNA from tissues and cells. Current protocols in neuroscience\/editorial board, Jacqueline N. Crawley\u2026 [et al.]Appendix 1, Appendix 1I, doi:10.1002\/0471142301.nsa01is07 (2001).","DOI":"10.1002\/0471142301.nsa01is07"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-05858-0","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-05858-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-05858-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,23]],"date-time":"2022-12-23T07:15:54Z","timestamp":1671779754000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-05858-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,7,20]]},"references-count":36,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2017,12]]}},"alternative-id":["5858"],"URL":"https:\/\/doi.org\/10.1038\/s41598-017-05858-0","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,7,20]]},"assertion":[{"value":"18 January 2017","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 June 2017","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"20 July 2017","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare that they have no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"6080"}}