Julie R. Friend

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications.

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver‐specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 × 10−9 mmol O2/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6‐day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell‐cell contacts and tight junctions between cells within spheroids. Microvilli‐lined bile canaliculus‐like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver‐specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three‐dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism.

Enhanced cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 μM β-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Development of a bioartificial liver employing xenogeneic hepatocytes.

Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations.

Formation and characterization of hepatocyte spheroids.

Several investigators have demonstrated that freshly harvested hepatocytes self-assemble into three-dimensional, compacted, freely suspended aggregates known as spheroids (1-3). These aggregates have smooth, undulating surfaces and average approx 120 µm in diameter. Hepatocyte spheroids exhibit enhanced liver-specific activities and prolonged viability, compared to cells maintained as a monolayer (4,5). Extensive cell-cell contacts, tight junctions, and microvilli-lined channels that resemble bile canaliculi have been observed between hepatocytes in spheroids (6,7). Thus, these cells appear to mimic the morphology and ultrastructure of an in vivo liver lobule. Reorganization of hepatocytes into these three-dimensional structures is hypothesized to contribute to their enhanced liver-specific functions. Because of their enhanced function and tissue-like ultrastructure, hepatocyte spheroids show great promise for use in tissue-engineering applications and drug metabolism studies.

Engineering a Bioartificial Liver Support

Publisher Summary While most extracorporeal bioartificial liver devices have been developed as a bridge to organ transplant, these devices are also often mentioned as a bridge therapy while a patients liver regenerates. The time for a human liver to regenerate to the point where it could adequately sustain function for survival may take weeks, or perhaps even months. Most bioartificial liver devices have only been tested in animals for times on the order of hours. Whether they will stand up to the longer duration of therapy needed to allow for regeneration has yet to be seen. Though there are several obstacles to be overcome, results to this point have been encouraging, and it is likely that these extracorporeal devices will serve as successful “bridge-to-transplant” therapies.

Development of a bioartificial liver device.

Liver disease continues to be a challenge clinically, with 30,000 patients dying each year from liver failure (1). Although liver transplantation can successfully treat many patients undergoing liver failure, the scarcity of donor organs severely limits this treatments application. For this reason, many investigators are pursuing alternatives to total organ transplantation, from living donors to cell transplantation. One additional approach is the development of a hybrid, bioartificial liver as an extracorporeal device for the temporary treatment of acute liver failure. This approach has demonstrated early success, and may provide an important clinical treatment in the near future. In addition, a bioartificial liver reactor is useful for prolonged in vitro studies of hepatocyte function. This chapter will provide information on the design and use of such a reactor for in vitro applications.

Polarity and Bile Canaliculi-Like Channels in Self-Assembled Rat Hepatocyte Spheroids

Rat hepatocytes, when cultured on moderately-adhesive substrates or in suspension, self-assemble into three dimensional, compacted, spherical aggregates called spheroids [1, 2]. In addition to maintaining higher liver-specific functions than monolayer cultures, spheroids bear structural similarity to native liver tissue. The surface of the spheroid is smooth, and cell-cell borders are barely distinguishable. In addition, pores are present on the surface of the spheroid and adjacent hepatocytes inside share microvilli-lined channels demarcated by tight junctions [3, 4]. Such differentiated structures are often indicative of hepatocyte polarization, the segregation of specific proteins to defined membrane domains.