title: Machine perfusion enhances hepatocyte isolation yields from ischemic livers.
abstract: BACKGROUND
  High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation.
  
  
  METHODS
  Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3h. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O(2)/5%CO(2)), as well as (3) Lifor passively oxygenated with ambient air (21%O(2)/0.04%CO(2)), all at ambient temperatures (20 ± 2°C). At hypothermic temperatures (5 ± 1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively.
  
  
  RESULTS
  Fresh livers yielded 32 ± 9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6 ± 0.6 million cells/g liver. Oxygenated Williams Medium E and Lifor recovered yields of 39 ± 11 and 31 ± 2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15 ± 7 (Lifor), 13 ± 7 (Vasosol), and 10 ± 6 (UW)million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion.
  
  
  CONCLUSIONS
  Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low viscosity.
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, United States. Electronic address: mizamis@mgh.harvard.edu.
authors.forename: Maria-Louisa
authors.initials: ML
authors.lastname: Izamis
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, United States. Electronic address: perksin@gmail.com.
authors.forename: Sinem
authors.initials: S
authors.lastname: Perk
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, United States. Electronic address: Calhoun.ck@gmail.com.
authors.forename: Candice
authors.initials: C
authors.lastname: Calhoun
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, United States. Electronic address: uygun.korkut@mgh.harvard.edu.
authors.forename: Korkut
authors.initials: K
authors.lastname: Uygun
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, United States; Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, United States. Electronic address: ireis@sbi.org.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Yarmush
authors.affiliation: Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, United States. Electronic address: fberthia@rci.rutgers.edu.
authors.forename: François
authors.initials: F
authors.lastname: Berthiaume
mesh_terms: D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D015687:Cell Hypoxia; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005978:Glutathione; D022781:Hepatocytes / Q000166:cytology / Q000637:transplantation*; D007328:Insulin; D008099:Liver / Q000166:cytology / Q000187:drug effects; D019188:Organ Preservation Solutions; D010477:Perfusion / Q000295:instrumentation* / Q000379:methods; D011887:Raffinose; D051381:Rats; D011917:Rats, Inbred Lew; D014019:Tissue Donors; D052096:Warm Ischemia / Q000379:methods*
chemical_list: D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose
keywords: Hepatocytes; Isolation; Lifor; Perfusion; Temperature; UW; Vasosol; Viscosity; Williams E