Recent advances in the application of cultured hepatocytes into bioartificial liver

Abstract

Key Words: Bioartificial/Liver, Liver/Cytology

INTRODUCTION

Over the past 3 decades, various experimental liver support systems have been studied. Early artificial liver support systems included hemodialysis, extracorporeal liver perfusion, human cross circulation, charcoal hemoperfusion, hepatodialysis, fresh blood, and plasma exchange transfusion. These systems were developed to remove toxic substances from the blood. Clinical trials showed that these detoxification systems could promote the recovery of consciousness in patients with deep coma, although the survival of patients was not improved. Recently, advances in biotechnology and tissue engineering have brought into focus the importance of biological components for a hybrid artificial liver support system (HALSs). Biological components may include isolated enzymes, cellular components, slices of liver, or cultured hepatocytes. Hepatocyte systems have shown the greatest promise for HALSs. The advantages of hepatocyte systems may be summarized as follows: (1) supplying crucial liver specific metabolic functions; (2) being easily scaled up; (3) having immunoisolation from the host defenses by semipermeable membrane; and (4) being cryopreserved for later use. The disadvantage of hepatocyte systems is the problem of maintaining normal hepatocyte viability and function at high cell density necessary for clinical application. With the development of cell culturing technology and cell engineering technology, great progress has been made in the research of using cultured hepatocytes as a bioreactor to provide hepatic support.

Current situation of cultured hepatocytes system research

Cultured hepatocytes must maintain good viability and function to be used in HALSs. However, when they are cultured on a plastic surface with standard cell-culture medium, they flatten and become agranular; tissue specific functions are lost in 3 to 5 d, followed by hepatocyte death within 1 to 2 wk[1]. Therefore, the techniques of cell culture should be improved. The approaches include: (1) addition of growth factors and hormones to culture medium; (2) co-culturing of hepatocytes with nonparenchymal liver cells; (3) using biologic gel or matrix; and (4) cultivation of cells in hollow fiber.

Addition of growth factors and hormones to culture medium

It has been reported that hormones and growth factors, such as insulin, glucagon, dexamethasone, epithelial growth factor (EGF), and hepatocytes growth factor (HGF), may play an important role in modulating the differentiation and proliferation of cultured hepatocytes as well as maintaining tissue-specific functions.

According to Hamad’s results, EGF can prolong the survival of cultured hepatocytes and stimulate hepatocyte proliferation[2]. The DNA contents of hepatocytes were reported to be well preserved in media supplemented with insulin and EGF by Hamad[2] and Dich et al[3] respectively.

It has also been reported that glucagon is necessary for maintaining the synthesis of urea by cultured hepatocytes for about 2 wk. Moreover, Takahashi et al[4] reported in 1993 that the growth and differentiation functions of hepatocytes cultured at different densities were modulated by EGF and insulin when hepatocytes were cultured at a low cell density of 2.5 × 10⁴ cells/cm². Conversely, cellular functions were induced by hormones at a high cell density of 12.5 × 10⁴ cells/cm², whereas the hepatocyte proliferation was suppressed.

Co-culture with nonparenchymal liver cells (NPC)

Co-culturing is a technique in which two more cell types are cultured together, resulting in cell behavior and physiological responses that would not occur if the cell types were cultured alone. It was first reported by Puck and Marcus in 1955 in studies with Hela cells. One of the most successful methods of co-culturing is using mito-mycin-C-treated “feeder layers” of another cell type (often fibroblasts), which are thought to supply the primary cells with nutrients or factors. Begue et al[5] found that the cytochrome P-450 content in adult rat hepatocytes was maintained over a 10-d period when these cells were co-cultured with another rat liver epithelial cell type.

Use of biologic gel and cell engineering techniques

Hepatocytes are anchorage dependent. Currently, biologic gel, microcarriers, micro-encapsules, and Poly-N-P-Vinybenzyl-D-Lactomide (PVLA) are used to culture hepatocytes with high density and slight differentiation.

Immobilized hepatocytes in hydrogel or microencapsule

Miura et al[6] demonstrated the long term maintenance of the capacity to synthesize urea, albumin, and glucose as well as the ability to detoxify hepatic toxins like phenols and fatty acids when isolated hepatocytes were encapsuled within calcium alginate gel. The encapsulating technique can avoid immunological hazards and maintain the cellular function of isolated hepatocytes. Micro-encapsulated hepatocytes are mainly used in hepatocytes transplantation, and HALSs produced using such cells would be large and, therefore, inconvenient for clinical use.

Microcarrier hepatocytes

Van Wezel reported the microcarrier technique in 1967. It has the advantage of increasing the adhesive area of culturing cells (the surface area of 1 g dextran is 0.6 m²), thus facilitating transplantation of large quantities of hepatocytes in very small volumes of microcarrier suspension. Being cultured on the collagen-covered microcarriers, hepatocytes can maintain long-term cellular function and growth. Kasai et al[7] found that the metabolic activity of rat hepatocytes attached to collagen-coated multiporous cellulose microcarriers can be preserved for at least 1 week. Nezuil et al[8] developed a HALSs using porcine hepatocytes attached to microcarriers and placed on the outer surface of hollow fibers. The HALSs was used to treat a 33-year-old male patient with alcohol induced cirrhosis. The patient’s mental status greatly improved 2 h after initiation of HALSs treatment. Three weeks later, the patient’s liver function improved gradually and steadily. He underwent orthotopic liver transplantation 6 months later.

PVLA hepatocytes

Recently, rapid progress has been made in research on using PVLA to culture hepatocytes. In hepatocytes cultured on the PVLA coated dishes, growth and functional activity was regulated by the simulated three-dimensional environment in vivo[9]. Hepatocytes cultured on PVLA coated dishes are able to maintain a high level of differentiation functions and longevity because PVLA has the ligand, b-galactose for asialogly protein receptors on hepatocytes[10,11]. Kobayashi et al[12] found that the regulation of differentiation function and proliferation of hepatocytes is low for cells cultured on dishes coated with high level PVLA (200 μg/mL) and is high for those with low level PVLA (1 μg/mL). Moreover, bile acid release was maintained at higher levels in hepatocytes attached on dishes coated with a high level of PVLA.

Toke et al[13] found that the hepatocytes initially attached onto PVLA substratum and then migrated together to form multilayer aggregations by the stimulation of growth factors, such as EGF and insulin. They observed many orifices of tube-like structures on the surface of the multilayer aggregation by scanning electron microscopic analysis. Transmission electron microscopic analysis of the aggregation revealed the maintenance of endoplasmic reticulum and the appearance of bile canalicular-like structure. This tissue-reconstruction of primary cultured rat hepatocytes exhibited long-term maintenance of specific cellular functions, such as the secretion of albumin and bile acid, and retained mitochondrial enzyme activity. These data showed that PVLA could potentiate the differentiation and proliferation of hepatocytes. However, hepatocytes cultured with high proliferation and high maintenance of differentiation function could not be achieved with the same coating concentration. Further refinement of the PVLA hepatocytes systems is expected to assist in the development of HALSs.

Hollow fiber hepatocytes

Hollow fiber hepatocyte bioreactors consists of two compartments: (1) an intraluminal compartment within the fibers and (2) an extraluminal compartment outside the fibers and within the rigid housing. Hepatocytes are cultured within the extraluminal compartment, and the patient’s blood is circulated within the capillaries. Through pores in the walls of the hollow fiber, communication between the compartments occurs. Toxic substance in the patient’s blood permeates through the pores and is acted on by the hepatocytes attached on the hollow fibers.

Hollow fiber systems have several advantages, such as immunological separation between the patient and the support hepatocytes and providing a large membranous area for hepatocytes to attach. Recently, several versions of hollow fiber membrane based systems have been reported in the literature[14-16]. Sussman et al[15] cultured a CAS cell line on the fibers, and Nyberg et al[16] proposed a novel hollow fiber based HALSs. The primary hepatocytes were entrapped in cylindrical gels inside the lumen of the hollow fibers.

Recent experimental studies with these devices have demonstrated their efficacy in animal models of acute liver failure. Rozga et al[17] treated seven patients with acute liver failure with a HALSs based on hollow fiber bioreactor. These patients recovered gradually and underwent liver transplantation. However, randomized clinical trials are still needed to evaluate these forms of HALSs.

Problems and future aspects of the cultured hepatocytes systems

In the last 5 years, research on cultured hepatocytes in bioartificial liver has made rapid progress with the aid of recent advances in cryobiology and tissue culture. Isolated hepatocytes or differentiated cell lines can be cultured at a density of 10⁷/mL. However, for clinically applicable HALSs, the cell density needs to be increased to 10⁸/mL. Therefore, much effort should be made to obtain the effective HALSs.

As mentioned above, only hepatocytes have been used in HALSs to date. Nonparenchymal liver cells were confined to co-culturing. Some experts proposed that the ideal HALSs may be developed with co-cultured hepatocytes systems. Thus, further studies are needed to ascertain its feasibility.

The development of HALSs is still in its infancy. Some of the HALSs designs using cultured hepatocytes have satisfied the rigors of animal testing and now are being studied in phase I clinical trials. We expect that the randomized clinical trials will establish the value of bioartificial liver therapy for patients with hepatic failure.