title: Effect of spheroid aggregation on susceptibility of primary pig hepatocytes to cryopreservation.
abstract: Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.
authors.affiliation: Samsung Biomedical Research Institute, Seoul, Korea.
authors.forename: J-H
authors.initials: JH
authors.lastname: Lee
authors.forename: D-H
authors.initials: DH
authors.lastname: Jung
authors.forename: D-H
authors.initials: DH
authors.lastname: Lee
authors.forename: J-K
authors.initials: JK
authors.lastname: Park
authors.forename: S-K
authors.initials: SK
authors.lastname: Lee
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000418:Albumins / Q000378:metabolism; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D002448:Cell Adhesion; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D007328:Insulin / Q000494:pharmacology; D019164:Liver, Artificial; D008297:Male; D019188:Organ Preservation Solutions / Q000737:chemistry / Q000494:pharmacology*; D011887:Raffinose / Q000494:pharmacology; D044967:Serum; D018874:Spheroids, Cellular; D013552:Swine; D013997:Time Factors; D014508:Urea / Q000378:metabolism
chemical_list: D000418:Albumins; D003451:Cryoprotective Agents; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D014508:Urea; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose; D004121:Dimethyl Sulfoxide