The liver plays an important role in a broad spectrum of physiological functions such as metabolism, storage, synthesis and release of vitamins, carbohydrates, proteins, lipids and cyclic tetrapyrroles (1). The liver also detoxifies and inactivates endogenous and exogenous substances such as toxins and metals, and activates proenzymes and coagulation factors (2). Furthermore, the liver produces bile involved in intestinal lipid absorption and secretes a majority of circulating plasma proteins. Therefore, the liver is one of important organs in our body and liver failure is associated with a high risk of mortality. Currently, the only effective way for liver failure is liver transplantation (3). Because of shortage of organ availability, it is necessary to find alternative ways to temporarily support the failing liver until a compatible donor can be found. Various bioartificial liver (BAL) systems have been used for clinical trials as a bridge for liver transplantations in patients with liver failure (4). Long term and stable liver-specific functions of hepatocytes should be considered in the development of BAL support systems.

Spheroid culture is very effective in the upregulation of liver-specific functions of hepatocytes due to the high similarity to real tissues in many aspects than monolayer cells although the diffusion limitation of oxygen and nutrients by the big size of spheroid of hepatocytes should be considered. In this review, spheroid formation methods of hepatocytes in the extracellular matrix (ECM) are covered for enhancing liver-specific functions.

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Kobayashi et al. (5) reported that galactose-derivatized polystyrene (PS), poly(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide (PVLA) is an excellent artificial ECM for hepatocytes because it guides hepatocyte adhesion through the asialoglycoprotein receptors (ASGPR)-galactose interaction between ASGPR of hepatocytes and highly concentrated galactose moieties along the polymer chains. Also, they reported that morphologies of hepatocytes adhered to PVLA at the lower galactose density (0.5 μg/ml) showed spreaded shapes whereas the morphologies of hepatocytes adhered to PVLA at the higher galactose (100 μg/ml) showed round ones and the round morphologies of hepatocytes were found to trigger the spheroid formation in the presence of epidermal growth factor (EGF) (6). Yin et al. (7) also reported that hepatocytes formed hepatocyte spheroids 1 day after cell seeding with better liver-functions at a high galactose density of 513 nmol/cm² on the PET surface as an artificial ECM, suggesting that spheroid formation of hepatocytes is dependent on the galactose density in the artificial ECM.

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Many researches have been focused on the design of ECM topology for the spheroid formation of hepatocytes because the topology of ECM affects cell morphology and this structure resembles the tight cell-cell contact in native liver and tight junctions (8). It is generally known that three-dimensional ECMs more induce spheroid formation of hepatocytes with better differentiated functions than two-dimensional ones due to the provision of better model systems for physiologic situations (9).

Chua et al. prepared highly porous nanofiber scaffolds by electrospinning of poly (epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) and introduced galactose onto the nanofiber surface (10). The results indicated that hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20∼100 microm, resulting in an integrated spheroid-nanofiber construct. Feng et al. prepared nanofibrous galactosylated chitosan (GC) scaffolds by electrospinning and evaluated liver functions (11). The results indicated that hepatocytes cultured on GC nanofibrous scaffold formed stably spheroid formation and exhibited high liver-specific functions.

Recently, Takahashi et al. prepared collagen- or Matrigel- nanopillar sheets and evaluated liver functions (12). The results indicated that the hepatocytes cultured on collagen- or Matrigel-coated nanopillar sheets formed spheroids with higher structural polarity and functional bile canaliculi compared with conventional two-dimensional culture method.

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Microfabrication is an emerging and powerful technique for spheroid formation of hepatocytes with well-controlled size and geometry. Microwells of defined size and aspect ratio can be fabricated by micromolding on cell-non-adhesive inert polymers like agarose (13) and poly (ethylene glycol) (PEG) (14) for the spheroid formation of hepatocytes. Makazawa et al. fabricated polydimethylsiloxane (PDMS) chip having microcavities by a photo-lithography technique and evaluated liver-specific functions (15). The results indicated that most hepatocytes formed a single spheroid in each cavity until 3 days of culture and the spheroid configuration was maintained for at least 14 days of culture. Also, liver-specific functions such as albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase activity of hepatocytes on the PDMS chip were higher than those of a monolayer dish. Miyamoto et al. developed a cell array technique by two combined innovations composed of micropatterning by a hydrophilic polymer and use of bovine carotid artery-derived HH cells as feeder cells for the formation of hepatocyte spheroid (16). The results showed that the rat hepatocytes formed spheroids on the cryopreserved HH cell array and the CYP3A drug metabolism activities of the hepatocytes were well maintained on the cryopre-served and cell arrays. Yoshimoto et al. prepared inverted cell patterned surface using PEG gel by a photolitho-graphic technique and seeded hepatoma cells on the constructed surface (17). The results showed that spheroids of the cells were formed on the unmodified PEG gel domains without feeder cells. Cocultures of HepG2 and Balb/3T3 (18), hepatocytes and mouse embryonic stem (mES) cells (19), and fetal mouse liver cells and nonparenchymal liver cells (20) on the micropatterned surfaces were performed to enhance more liver-specific functions than micropatterned ones with monocultures.

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Lin et al. used hanging-drop culture of hepatocytes to form spheroid formation although the method was initially developed for culture of stem cell embryoid bodies. The technique is useful for generation of spheroid formation of defined sizes, cell numbers and composition although it is labor-intensive and difficult for massive production (21).

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The spheroid formation of hepatocytes was obtained by culturing on a non-adhesive substratum such as poly (N-isopropylacrylamide) (22), alginate (23), and agarose (24) owing to enhancing the cell-cell interaction although it is not easy to control the size of hepatocyte spheroid.

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Tzanakakis et al. (25) reported that hepatocytes cultured on positively charged surfaces (Falcon Primaria) adhered to form a monolayer with spreading shapes on day 1 and the hepatocytes underwent spheroid formation after 48h through actin filament-mediated tissue reorganization. Verma et al. (26) also reported that HepG2 cells cultured on chitosan surfaces with appropriate seeding density resulted in the spheroid formation and exhibited higher amount of albumin and urea synthesis compared to uncoated dishes. However, Matsushita et al. (27) reported that primary human fetal hepatocytes did not form spheroids on a poly (L-lysine)- or poly (L-arginine)-coated dish that had a positively charged surface whereas the cells formed spheroids on a poly (L-glutamic acid)- or poly (L-aspartic acid)-coated dish that had a negatively charged surface.

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Spinner culture systems were used to form large numbers of hepatocyte.

Spheroids (28) although they can experience a strong shear force. To overcome the problem, a rotational cell culture was recently used (29).

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Brophy et al. (30) used rocked technique to induce spheroid formation of hepatocytes. The spheroid formation by rocked technique was more rapid and more efficient than rotational technique. The hepatocyte spheroids in rocked culture showed stable expression of over 80% of 242 liver-related genes.

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Medium components have been used to enhance spheroid formation of hepatocytes. The round shapes of hepatocytes were found to trigger the spheroid formation in the presence of EGF (6), HGF and sodium butylate (31), collagen (32), dexamethasone (33) and Eudragit (34).

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External forces such as dielectrophoresis (35), magnetic fields (36) and ultrasound trap (37, 38) were used to enhance cell aggregation and spheroid formation from single cells although specialized equipment is necessary in spheroid formation of the cells. Spheroid formation of HepG2 adhered on a PHEMA-coated surface and encapsulated in alginate capsules was formed within 30 and 5 min, respectively, by the ultrasound trap with good liver-specific functions (37).

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Heterotypic cell interactions between parenchymal cells and nonparenchymal neighbors are of fundamental importance in physiology (39) and tissue engineering (40) because coculture of hepatocytes with other cells enhances spheroid formation of hepatocytes and liver-specific functions. Coculture of rat hepatocytes with pancreatic islets (41) and rat hepatocytes with NIH/3T3 fibroblasts (42, 43) enhanced spheroid formation and functions of hepatocytes.

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Gwak et al. (44) developed a new method for the effective spheroid formation of hepatocytes by addition of poly (lactic-co-glycolic acid) nanospheres to hepatocyte cultures in spinner flasks although the mechanism is not clear.

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Accumulated evidence showed that spheroid formation of hepatocytes is very effective in the enhancement of liver-specific functions of hepatocytes. The spheroid formation of hepatocytes will contribute to liver tissue engineering because they induce long term and stable liver-specific functions of hepatocytes although several obstacles should be overcome. First, diffusion limitation of nutrients and oxygen to the big size of spheroid should be considered. Second, spheroid studies are needed to correlate how well the system represents real tissues because spheroid formation is an artificially reconstructed system. Spheroid formation-based 3-D culture systems continue to advance in all aspects, including new applications in combination with micro-electro-mechanical systems and new drug evaluation.

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