Evaluation of a Hybrid Artificial Liver Using a Polyurethane Foam Packed-Bed Culture System in Dogs

doi:10.1006/jsre.1998.5540

Journal of Surgical Research

Volume 82, Issue 2, April 1999, Pages 131-136

https://doi.org/10.1006/jsre.1998.5540 Get rights and content

Abstract

Background and aims.We developed a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver (PUF-HAL), which was effective for recovery from liver failure in rat experiments. In this report, the design of a scaled-up PUF-HAL for dogs is described and evaluated using a dog acute liver failure model.

Methods.Warm ischemic liver failure was induced with a portocaval shunt in each dog. The dogs were divided into two groups: (1) a control group (N= 4), in which each dog was attached to a PUF-HAL without hepatocytes for 9 h, and (2) a HAL group (N= 5), in which each dog was attached to a PUF-HAL with hepatocytes. Blood pressure, blood ammonia, blood glucose, serum creatinine, and other parameters related to liver function were compared between the two groups.

Results.In the HAL group, blood ammonia and serum creatinine levels were significantly lower, and blood pressure and blood glucose levels significantly higher, than those in the control group.

Conclusions.The scaled-up PUF-HAL developed for large animals is useful as a liver support system in the dog acute liver failure model.

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Citation Excerpt :
The bioreactor should ideally provide necessary microenvironment for the hepatocytes to express differentiated functions with efficient mass transport between cells and medium [5]. Bioreactors used in BAL can be classified based on how it receives hepatocytes such as encapsulated suspension perfused bed [6,7], flat plate and monolayer [8] and hollow fiber systems [9]. Adherent hepatocytes easily lose their viability and fail to express liver specific functions due to deprivation of in vivo architecture, polarity and soluble cell signaling cues [4].
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The surface design used for reducing nonspecific biofouling is one of the most important issues for the fabrication of medical devices. We present here a newly synthesized a carbohydrate-immobilized phosphorylcholine polymer for surface modification of medical devices to control the interface with living cells. A random copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and 2-lactobionamidoethyl methacrylate (LAMA) was synthesized by conventional radical polymerization. The monomer feeding ratio in the copolymer was adjusted to 24/75/1 (MPC/BMA/LAMA). The copolymer (PMBL1.0) could be coated by solvent evaporation from an ethanol solution.
Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on PMBL1.0 or poly(BMA) (PBMA)-coated PET plates. On PBMA, many adherent cells were observed and were well spread with monolayer adhesion. HepG2 adhesion was observed on PMBL1.0 because the cell has ASGPRs. Furthermore, some of the cells adhering to PMBL1.0 had a spheroid formation and similarly shaped spheroids were scattered on the surface. According to confocal laser microscopic observation after 96 h cultivation, it was found that albumin production preferentially occurred in the center of the spheroid. The albumin production of the cells that adhered to PBMA was sparse. The amount of albumin production per unit cell that adhered to PMBL1.0 was determined by ELISA and was significantly higher than that which adhered to PBMA.
Long-term cultivation of HepG2 was also performed using hollow fiber mini-modules coated with PMBL1.0. The concentration of albumin produced from HepG2 increased continuously for one month. In the mini-module, the function of HepG2 was effectively preserved for that period. On the hollow fiber membrane, spheroid formation of HepG2 cells was also observed.
In conclusion, PMBL1.0 can provide a suitable surface for the cultivation of hepatocytes and has great potential for producing reliable bioartificial liver (BAL) devices.
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Several extracorporeal bioartificial liver (BAL) devices are currently being evaluated as an alternative or adjunct therapy for liver disease. While these hybrid systems show promise, in order to become a clinical reality, BAL devices must clearly demonstrate efficacy in improving patient outcomes. Here, we present aspects of BAL devices that could benefit from fundamental advances in cell and developmental biology. In particular, we examine the development of human hepatocyte cell lines, strategies to stabilize the hepatocyte phenotype in vitro, and emphasize the importance of the cellular microenvironment in bioreactor design. Consideration of these key components of BAL systems will greatly improve next generation devices.
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Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture. The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal. For the perifusion of cells, they must be immobilized to prepare a bioreactor model. The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture. Hepatocytes were isolated, immobilized and perifused with William’s E culture medium containing 1 mM NH₄Cl and CsA (20 μM). Hepatocytes in the culture were treated in the same way. CsA disappearance from the perifusion or culture media was determined by a HPLC method. Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5±0.05 mg h⁻¹ vs 0.33±0.03 mg h⁻¹, respectively). ALT leakage was lower in the bioreactor model (60 U l⁻¹) as compared to hepatocyte culture (125 U l⁻¹). The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively). The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture. Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes. In addition, the cellular model described here inherits economic and ethical potentials.
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^☆: M. J.T. Carrondo

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Regular ArticleEvaluation of a Hybrid Artificial Liver Using a Polyurethane Foam Packed-Bed Culture System in Dogs☆

Abstract

Lancet

Gastroenterology

Hepatology

Exp. Cell Res.

Biochem. Biophys. Res. Commun.

Fulminant liver failure: ralevance of extracorporeal hybrid liver support systems

Int. J. Artif. Organs

Artificial liver: Evolution and perspectives

ASAIO J.

The artificial liver

Sci. Am. Sci. Med.

Problems treating experimentally induced acute hepatic failure

Hepatology

Effect of serial resin hemoperfusion in fulminant hepatic failure

Int. J. Artif. Organs

Hepatic support with hepatocyte suspension in a permeable membrane dialyzer

Surgery

Hybrid bioartificial liver in hepatic failure: Preliminary clinical report

Surgery

Performance of a new hybrid artificial liver support system using hepatocytes entrapped within a hydrogel

Trans. Am. Soc. Artif. Intern. Organs

A hybrid bioartificial liver composed of multiplated hepatocyte monolayers

Trans. Am. Soc. Artif. Intern. Organs

Regular Article
Evaluation of a Hybrid Artificial Liver Using a Polyurethane Foam Packed-Bed Culture System in Dogs☆