title: Analysis of multivariables during porcine liver digestion to improve hepatocyte yield and viability for use in bioartificial liver support systems.
abstract: In order to achieve optimal BALSS function, preparation of porcine hepatocytes with high yield, viability, and P450 activity is known to be important. To date hepatocyte yields have varied from 0.58 x 10(10) to 3.45 x 10(10) and viabilities from 75% to 95% within and between laboratories, even when using the same digestion methods and procedures, indicating that hepatocyte isolation during porcine liver digestion is not fully optimized. The aim of this work was to identify the critical parameters affecting cell recovery during porcine liver harvesting by investigating 21 variables involved in the process, including pig body and liver weight, different digestion times of perfusates, pH, a range of concentrations of sodium and chloride in EDTA, and collagenase perfusates. Univariate and multivariate analysis of a retrospective study (n = 23) revealed that low perfusate pH during the process of digestion had a positive effect on hepatocyte yield (p < 0.05), while high (relative) concentrations of sodium and chloride in the perfusates had significant negative effects on hepatocyte viability (both p < 0.05). Sodium and chloride had narrow optimal ranges for achieving a >90% viability. These findings were then tested in a prospective study (n = 10) and further verified. High hepatocyte viabilities (91.8+/-1.6% p = 0.036) and yields (2.56+/-0.48 x 10(10)) were achieved consistently, and P450IA1 activity was increased after sodium and chloride concentrations and pH in the perfusates were controlled. The physiological mechanism by which sodium and chloride affects hepatocyte viability during porcine liver digestion is discussed.
authors.affiliation: Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital and Department of Surgery, and University of Sydney, NSW.
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: K
authors.initials: K
authors.lastname: Woodman
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: C
authors.initials: C
authors.lastname: Harbour
authors.forename: B
authors.initials: B
authors.lastname: Johnston
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: M
authors.initials: M
authors.lastname: Horvat
authors.forename: N
authors.initials: N
authors.lastname: Koutalistras
authors.forename: X
authors.initials: X
authors.lastname: Luo
authors.forename: J
authors.initials: J
authors.lastname: Watson
authors.forename: A G
authors.initials: AG
authors.lastname: Sheil
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D019164:Liver, Artificial; D015999:Multivariate Analysis; D010477:Perfusion / Q000379:methods; D011446:Prospective Studies; D012189:Retrospective Studies; D013552:Swine; D020858:Tissue and Organ Harvesting
chemical_list: D004492:Edetic Acid; D019363:Cytochrome P-450 CYP1A1; D017364:Collagenases