title: Influence of platelet lysate on the recovery and metabolic performance of cryopreserved human hepatocytes upon thawing.
abstract: BACKGROUND
  Storage of human hepatocytes is essential for their use in research and liver cell transplantation. However, cryopreservation and thawing (C/T) procedures have detrimental effects on the viability and functionality compared with fresh cells. The aim of this study was to upgrade the standard C/T methodology to obtain better quality hepatocytes for cell transplantation to improve the overall clinical outcome.
  
  
  METHODS
  Human hepatocytes isolated from donor livers were cryopreserved in University of Wisconsin solution with 10% dimethyl sulfoxide (standard medium), which was supplemented with 10% or 20% of platelet lysate. Thawing media supplemented with up to 30 mM glucose was also investigated. The effects on cell viability, adhesion proteins (e-cadherin, β-catenin, and β1-integrin) expression, attachment efficiency, apoptotic indicators, Akt signaling, ATP levels, and cytochrome P450 activities have been evaluated.
  
  
  RESULTS
  The results indicate that the hepatocytes cryopreserved in a medium supplemented with platelet lysate show better recovery than those preserved in the standard medium: higher expression of adhesion molecules, higher attachment efficiency and cell survival; decreased number of apoptotic nuclei and caspase-3 activation; maintenance of ATP levels; and drug biotransformation capability close to those in fresh hepatocytes. Supplementation of thawing media with glucose led to a significant decrease in caspase-3 activation and to increased adhesion molecules preservation and Akt signal transduction after C/T. Minor nonsignificant changes in cell viability and attachment efficiency were observed.
  
  
  CONCLUSIONS
  These promising results could lead to a new cryopreservation procedure to improve human hepatocyte cryopreservation outcome.
authors.affiliation: Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain.
authors.forename: Laia
authors.initials: L
authors.lastname: Tolosa
authors.forename: Ana
authors.initials: A
authors.lastname: Bonora-Centelles
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Donato
authors.forename: Vicente
authors.initials: V
authors.lastname: Mirabet
authors.forename: Eugenia
authors.initials: E
authors.lastname: Pareja
authors.forename: Alejandro
authors.initials: A
authors.lastname: Negro
authors.forename: Silvia
authors.initials: S
authors.lastname: López
authors.forename: José V
authors.initials: JV
authors.lastname: Castell
authors.forename: M José
authors.initials: MJ
authors.lastname: Gómez-Lechón
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000493:Allopurinol / Q000494:pharmacology; D017209:Apoptosis; D001792:Blood Platelets / Q000166:cytology*; D053148:Caspase 3 / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D004789:Enzyme Activation; D005615:Freezing; D005978:Glutathione / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D019188:Organ Preservation Solutions / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011887:Raffinose / Q000494:pharmacology; D015398:Signal Transduction; D013048:Specimen Handling / Q000379:methods*; D016896:Treatment Outcome
chemical_list: D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D000255:Adenosine Triphosphate; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose