Caspase Inhibition Reduces Apoptotic Death of Cryopreserved Porcine Hepatocytes

Abstract

Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVADfmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% ؎ 0.9%) were cryopreserved in 60 mol/L ZVAD-fmk (؉ZVAD group) or without ZVAD-fmk (؊ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVADfmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition. (HEPATOLOGY 2001;33:1432-1440.)A number of liver-assist devices have been developed, including the extracorporeal use of isolated mammalian hepatocytes to provide metabolic function in a bioartificial liver (BAL). 1,2 Criteria for successful application of a BAL include bridging patients to liver transplantation, reduction of intracranial hypertension and cerebral edema, and avoidance of liver transplantation in cases of reversible hepatic failure. 3-5 To achieve these goals, hepatocytes in the BAL provide metabolic functions to the patient in liver failure. The extent to which these goals can be achieved is dependent on several variables, including the number of viable and metabolically active hepatocytes in the device. 6 Unfortunately, hepatocyte viability declines over time following isolation, and this decline limits the effectiveness and duration of BAL therapy. Cryopreservation, which allows cells to be used weeks and months after isolation, may also contribute to premature cell death. 7,8 Several BAL systems have been designed to maintain hepatocyte viability ex vivo. One such BAL system involves the entrapment of hepatocytes in cylindrical collagen gels located in the fibers of a hollow fiber bioreactor. 9,10 An in vitro model of this BAL system using gel-entrapped hepatocytes has been developed for static culture experimentation, such as the study of mechanisms of hepatocyte death. 11 The in vitro model avoids the expenses of largescale testing, and multiple experiments can be performed simultaneously. Potential immune interactions, present with in vivo models of BAL testing, are also avoided with in vitro testing.Our recent work has shown that cell death of freshly isolated hepatocytes occurs, in part, by apoptosis. 12...

Cryopreservation

“…8 Over the last decade, there has been the emergence of numerous studies focused on understanding the role apoptosis plays in cryopreservation failure. 7,9,11,49,50,65,66,68,70,[77][78][79][80][81][82][83][84][85][86][87][88][89] The involvement of apoptotic cell death in cryopreservation has compounds for the Targeted Control of Apoptosis (TCA) during the cryopreservation process. The mitigation of the molecular-based stress responses to low temperature exposure and storage has been shown to be attainable with cryopreservation solution formulation that addresses both physical and cellular related events.…”

Biology of the corneal endothelium in health and disease

“…Such a strategy improved the cell survival in cold-stored human livers. 62 If posttransplant inhibition of apoptosis is needed, the transduction of cell survival genes such as bcl-2 or p35 into the corneal endothelial cells could be accomplished during corneal storage. 63,64 Second, the number of endothelial cells on the donor cornea might be increased by the use of growth factors and the disruption of cellular contact inhibition.…”

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands

“…The apoptotic process of different cellular groups begins with harvesting and continues during cryopreservation and cell thawing, triggering caspase-3 (33)(34)(35). We found a greater quantity of tracheal epithelial and mixed glands and higher caspase-3 immunoreactivity in the control group (without cryopreservation) than in the cryopreserved groups, which showed fewer numbers of tracheal epithelial cells and mixed glands, with a consequent reduction of caspase-3 immunoreactivity (P < 0.03, ANOVA and Tukey test) mainly in the liquid nitrogen groups.…”