A Technique for Porcine Hepatocyte Harvest and Description of Differentiated Metabolic Functions in Static Culture

Sielaff, Timothy D.; Hu, Michael Y.; Rao, Sridhar; Groehler, Kristine E.; Olson, Daidre; Mann, Henry J.; Remmel, Rory P.; Shatford, Russell A.; Amiot, Bruce; Hu, Wei Shou; Cerra, Frank B.

doi:10.1097/00007890-199505270-00017

Cited by 56 publications

(28 citation statements)

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“…Optimization of bioreactor parameters and culturing conditions is expected to further increase the levels of liver-specific functions of hepatocyte spheroids. Phase I biotransformations via the cytochrome P450 family have yet to be characterized in a spheroid-entrapment BAL, though static monolayer cultures and spheroids entrapped into collagen gel have demonstrated ability to clear lidocaine to its metabolite monoethylglycinexylidide (MEGX) (Lazar et al, 1995a;Sielaff et al, 1995b). Initial characterizations of the BAL in vitro (Nyberg et al, 1992a) and in an anhepatic rabbit model (Nyberg et al, 1993) were conducted utilizing primary rat hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

“…Although not as extensively studied as rats, pigs were chosen as an attractive alternative. Routine liver harvests have yielded approximately 20 X lo9 cells at greater than 90% viability (Sielaff et al, 1995b). Other researchers have also recently reported investigations on the effectiveness of primary porcine hepatocytes as the functionally active component of hybrid liver support systems (Rozga et al, 1994;Uchino et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…Hepatocytes were harvested from 10-kg pigs (Midwest Swine Research, Gibbon, MN) utilizing sequential in situ portal vein perfusion with oxygenated ethylene glycinetetraacetic acid and 0.1% collagenase (Boehringer Mannheim, Indianapolis, IN), as described elsewhere (Lazar et al, 1995b;Sielaff et al, 1995b). Postharvest hepatocyte viability was greater than 90% as determined by trypan blue exclusion.…”

Section: Hepatocyte Culturementioning

confidence: 99%

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Hollow fiber bioartificial liver utilizing collagen‐entrapped porcine hepatocyte spheroids

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Lazar

et al. 1996

Biotech & Bioengineering

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Hepatocyte Culturementioning

confidence: 99%

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Hollow fiber bioartificial liver utilizing collagen‐entrapped porcine hepatocyte spheroids

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Lazar

et al. 1996

Biotech & Bioengineering

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“…19 Briefly, following general anesthesia and portal vein cannulation, in vivo perfusion was performed with a calciumfree HEPES-buffered solution (143 mmol/L NaCl, 6.7 mmol/L KCl, 10 mmol/L HEPES, 100 mg/dL ethylene glycol-bis-aminoethyl ether [pH 7.4]), and then extracorporeally perfused (300 mL/min ϫ 10 minutes) with a second HEPES-buffered solution (67 mmol/L NaCl, 6.7 mmol/L KCl, 4.8 mmol/L HEPES, 1.0 g/dL fatty acid-free bovine albumin [pH 7.6]) containing 0.05% collagenase D (clostridiopeptidase A, Boehringer Mannheim Corp., Indianapolis, IN). The liver was then placed in a glass dish containing 4°C Williams' E medium (Life Technologies, Grand Island, NY), supplemented with 10% calf serum, 2 mmol/L L-glutamine, 15 mmol/L HEPES, 1.5 mg/L insulin, 10,000 U/L penicillin G, and 100 mg/L streptomycin sulfate.…”

Section: Hepatocyte Isolationmentioning

confidence: 99%

Caspase Inhibition Reduces Apoptotic Death of Cryopreserved Porcine Hepatocytes

et al. 2001

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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...

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“…However, due to a lack of human organ availability the current main source of hepatocytes for bioartificial systems is xenogeneic material. Primary porcine hepatocytes have preferably been used as the xenograft candidates regarding differentiated metabolic functions and high-yield retrieval (Sielaff et al 1995;te Velde et al 1995;Gregory et al 2000). Preparation of hepatocytes from pig liver has been shown to deliver a sufficient amount of cells for a bioartificial system (De Bartolo and Bader 2001).…”

Section: Cell Sourcementioning

confidence: 99%

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

Diekmann¹,

Bader

Schmitmeier

2006

Cytotechnology

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The liver is the most important organ for the biotransformation of xenobiotics, and the failure to treat acute or acute-on-chronic liver failure causes high mortality rates in affected patients. Due to the lack of donor livers and the limited possibility of the clinical management there has been growing interest in the development of extracorporeal liver support systems as a bridge to liver transplantation or to support recovery during hepatic failure. Earlier attempts to provide liver support comprised non-biological therapies based on the use of conventional detoxification procedures, such as filtration and dialysis. These techniques, however, failed to meet the expected efficacy in terms of the overall survival rate due to the inadequate support of several essential liver-specific functions. For this reason, several bioartificial liver support systems using isolated viable hepatocytes have been constructed to improve the outcome of treatment for patients with fulminant liver failure by delivering essential hepatic functions. However, controlled trials (phase I/II) with these systems have shown no significant survival benefits despite the systems' contribution to improvements in clinical and biochemical parameters. For the development of improved liver support systems, critical issues, such as the cell source and culture conditions for the longterm maintenance of liver-specific functions in vitro, are reviewed in this article. We also discuss aspects concerning the performance, biotolerance and logistics of the selected bioartificial liver support systems that have been or are currently being preclinically and clinically evaluated.

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A Technique for Porcine Hepatocyte Harvest and Description of Differentiated Metabolic Functions in Static Culture

Cited by 56 publications

References 0 publications

Hollow fiber bioartificial liver utilizing collagen‐entrapped porcine hepatocyte spheroids

Hollow fiber bioartificial liver utilizing collagen‐entrapped porcine hepatocyte spheroids

Caspase Inhibition Reduces Apoptotic Death of Cryopreserved Porcine Hepatocytes

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

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