title: Improvement of the cold storage of isolated human hepatocytes.
abstract: Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation  results in severe cryoinjury, short-term storage is currently performed at 2-8°C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not  counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer  cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution  or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better  than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes.  The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.
authors.affiliation: Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany.
authors.forename: Gesine
authors.initials: G
authors.lastname: Pless
authors.forename: Igor M
authors.initials: IM
authors.lastname: Sauer
authors.forename: Ursula
authors.initials: U
authors.lastname: Rauen
mesh_terms: D002469:Cell Separation; D048430:Cell Shape; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides; D003080:Cold Temperature / Q000009:adverse effects; D003470:Culture Media / Q000737:chemistry; D003676:Deferoxamine / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000502:physiology; D019188:Organ Preservation Solutions / Q000737:chemistry / Q000494:pharmacology; D014021:Tissue Preservation; D014022:Tissue Survival
chemical_list: D002712:Chlorides; D003470:Culture Media; C549285:Custodiol-N solution; D019188:Organ Preservation Solutions; D005947:Glucose; D003676:Deferoxamine