Formation Of Hepatocyte Spheroids Research Articles

Hepatocyte growth factor and epidermal growth factor promote spheroid formation in polyurethane foam/hepatocyte culture and improve expression and maintenance of albumin production

In our previous pre-clinical study with pig hepatic failure, an artificial liver with polyurethane foam (PUF)/primary porcine hepatocyte spheroids had superior curative effect. We examined the effect of hepatocyte growth factor (HGF), also known as scatter factor, on the quick formation of hepatocyte spheroids and albumin production. Spheroids were formed in the pores of PUF within 3 days regardless of addition of growth factors. In particular, spheroids were formed within 1 day in medium containing 100 ng/ml HGF and 50 ng/ml epidermal growth factor (EGF). 10,000 ng/ml HGF was effective for albumin production, but the activity dramatically decreased after 6 days in EGF-free medium. On the other hand, 100 ng/ml HGF was effective for albumin production in EGF-containing medium. Albumin production rate with ≥1000 ng/ml HGF was about 1.5 times higher than that with 100 ng/ml HGF. Furthermore, albumin production rate at 3 weeks was about 1.5 times higher than that at 2 days with 1000 ng/ml HGF. The maintenance of albumin production rate depended on the activity of the individual cell and not cell growth. In other words, we were able to show the effectiveness of HGF for functional hepatocyte organoid formation in PUF pores.

Primary rat hepatocytes form spheroids on hepatocyte growth factor/heparin-immobilized collagen film and maintain high albumin production

We developed hepatocyte growth factor (HGF)/heparin-immobilized collagen film by adding HGF solution to collagen film, which immobilized heparin by chemical cross-linking. Primary rat hepatocytes maintained neither their specific morphology nor albumin synthesis on collagen film that directly immobilized HGF by cross-linking. By using HGF-supplemented medium, hepatocytes on collagen film formed typical monolayers and maintained albumin synthesis. Hepatocytes on HGF/heparin-immobilized collagen film formed spheroids after 3 days. They maintained albumin production during the culture period, the same as in HGF-supplemented medium. However, spheroids were not formed although the adhesion of hepatocytes was disturbed on heparin-immobilized collagen film, and albumin synthesis rapidly decreased during 5 days of culture. Because the mitochondrial activities of hepatocytes were not increased in all conditions, it was supposed that the cell proliferation did not occur. Therefore, it was shown that HGF/heparin-immobilized collagen film was effective for promotion of spheroid formation of hepatocytes and expression of their albumin production.

Preconditioned Cell Array Optimized for a Three-Dimensional Culture of Hepatocytes

Three-dimensional culture procedures have attracted attention in various fields of cell biology. A newly developed cell array assisted in the formation of hepatocyte spheroids by two innovations: 1) micropatterning by a hydrophilic polymer, and 2) the use of bovine carotid artery-derived HH cells as feeder cells. The former contributes to the standardization of the spheroid size and the latter to the maintenance of the spheroids. We created a way to provide a ready-to-use cell array by cryopreservation of an HH feeder cell cultured array. After inoculation of HH cells on the cell array, the culture medium was replaced by freezing medium containing dimethyl sulfoxide. Thereafter, the array was frozen and stored in a -80 degrees C deep freezer. At the start of the hepatocyte culture, the cryopreserved HH cell array was thawed by adding warmed (37 degrees C) culture medium. The morphology and biological activities of the cryopreserved HH cells were intact, as confirmed by phase contrast microscopy and functional staining with calcein and formazan. The rat hepatocytes formed perfect spheroids on the cryopreserved HH cell array without any differences from those on the freshly prepared HH cell array. The CYP3A drug metabolism activities of the hepatocytes were well maintained on the cryopreserved and fresh cell arrays. The present protocol greatly shortened the time and labor required to prepare a cell array for culturing hepatocytes.

Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function #

The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker-formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct.

ラクトース修飾絹フィブロイン基材上における初代培養ラット肝細胞のスフェロイド形成と維持

We examined the formation and maintenance of spherical multicellular aggregates (spheroids) of rat primary hepatocytes on dishes coated with lactose-silk fibroin conjugates bearing galactose residues (Lac-CY-SF) as substrate materials. Rat hepatocytes that had attached onto the conjugate-coated dishes were subsequently cultured in a medium supplemented with epidermal growth factor (EGF) and insulin. After the rat hepatocytes extended flat on the conjugate-coated dishes in 2 days of culture, these hepatocytes formed spheroids about 100 to 300 μm in diameter at day 4. After 6 days of culture the spheroids were maintained without any obvious change in size on the conjugate-coated dishes. However, the detachment of the spheroids from the conjugate-coated dishes began from day 8, and the spheroids clearly shrank at day 10. These results suggested that hepatocyte spheroid formation was induced on the Lac-CY-SF conjugate-coated dishes in the presence of EGF and insulin, but the conjugate-coated dishes were not capable of keeping the spheroids for more than 5 days.

Identification and Characterization of a Novel Prespheroid 3-Dimensional Hepatocyte Monolayer on Galactosylated Substratum

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

A method for the effective formation of hepatocyte spheroids using a biodegradable polymer nanosphere

Cultures of hepatocytes in spheroid form are known to maintain higher cell viability and exhibit better hepatocyte functions than those in monolayer cultures. In this study, a method for the formation of hepatocyte spheroids was developed using biodegradable polymer nanospheres. The addition of poly(lactic-co-glycolic acid) nanospheres to hepatocyte cultures in spinner flasks increased the efficiency of hepatocyte spheroid formation (the number of cells in spheroids divided by the total cell number) as compared with hepatocyte cultures without nanospheres (control). The viability and mitochondrial activity of the hepatocyte spheroids in the nanosphere-added cultures were significantly higher than those in the control. In addition, the mRNA expression levels of albumin and phenylalanine hydroxylase, both of which are hepatocyte-specific proteins, were significantly higher in the nanosphere-added cultures than in the control. This new culture method improves upon the conventional method of forming hepatocyte spheroids in terms of spheroid formation efficiency, cell viability, and hepatocyte function.

Hepatocyte spheroid formation on a titanium dioxide gel surface and hepatocyte long-term culture

The cell morphology and expression of differentiated functions of primary rat hepatocytes on a titanium dioxide (TiO(2)) gel surface were investigated. Polystyrene culture dishes were coated with TiO(2) gel by spin-coating an ethanol solution of titanium n-butoxide, hydrolyzing in a humidity chamber and drying with nitrogen gas. The TiO(2) gel layer formed on the polystyrene dishes was transparent and robust, and its surface was quite flat. Rat hepatocytes inoculated on the TiO(2) gel-coated polystyrene dishes gradually accumulated with increasing culture time, and then spontaneously formed many hepatocyte spheroids at 90 +/- 21 microm diameter from about 3 days of culture. The diameter of the spheroids increased during the culture, and was 151 +/- 43 microm at 14 days of culture. Ammonia removal and albumin secretion by hepatocytes on the TiO(2) gel-coated polystyrene dishes were maintained at a high level for at least 14 days of culture compared with on a type I collagen-coated dish and a non-coated polystyrene dish. These results indicate that TiO(2) gel is an adequate material for hepatocyte spheroid formation and long-term culture of spheroids.

Alginate/Galactosylated Chitosan/Heparin Scaffold As a New Synthetic Extracellular Matrix for Hepatocytes

Formation of multicellular hepatocyte spheroids in the three-dimensional culture is a potential approach for enhancing liver-specific functions in bioartificial liver (BAL) devices. In this study, as a synthetic extracellular matrix (ECM) for hepatocytes, a highly porous hydrogel (sponge-like) scaffold, 150-200 microm pore size in diameter, was fabricated with alginate (AL), galactosylated chitosan (GC), and heparin through electrostatic interaction. We attempt to select the best condition of AL/GC/heparin sponges for coculture with NIH3T3, as well as compare the liver-specific functions with monoculture. Cell adhesion to GC based on AL film was significantly increased with increasing GC concentration, but not to chitosan regardless of its concentration. The optimal concentration of GC and heparin in AL/GC/heparin sponges to perform the best liver-specific function was 1 and 6 wt% to AL contents, respectively, where albumin secretion were maintained with maximal rates. The mechanical properties in tensile strength of three types of sponges were very slightly different from one another. Cell viabilities performed on AL, AL/GC, and AL/GC/heparin sponges were 68.5, 83.3, and 90.4 % of control, respectively, after 15 days of incubation. Hepatocyte spheroids were more rapidly formed in the AL/GC and AL/GC/heparin sponges, with diameter enlarged to about 100 microm, than in AL sponges. Connexin32 and E-cadherin genes correlated with cell-to-cell adhesion were expressed in hepatocytes within AL/GC and AL/GC/heparin sponges at 36 h after incubation, but not in AL sponges. Treatment of a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, indicates that cell aggregation without GJIC does not perform the liver-specific functions for long periods. In the presence of HGF, the level of albumin secretion in AL/GC/heparin sponges was markedly elevated compared to that in AL/GC sponges. Coculture of hepatocytes in AL/GC/heparin sponges with NIH3T3 in a transwell insert resulted in significant increase of liver-specific functions, such as improved albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1 compared to those in hepatocyte monoculture. The results suggest that hepatocytes as stable spheroids enhance liver-specific functions in AL/GC/heparin sponges, providing a new synthetic ECM to design BAL devices.

Enhanced liver functions of hepatocytes cocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold

Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150–200 μm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2±3.6 and 81.3±3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 μm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell–cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18 β-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices.

Role of E-cadherin Molecules in Spheroid Formation of Hepatocytes Adhered on Galactose-Carrying Polymer as an Artificial Asialoglycoprotein Model

The role of E-cadherin in the spheroid formation of hepatocytes adhered on the poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) as a model ligand for asialoglycoprotein receptors (ASGP-R) of hepatocytes was studied. Expression of E-cadherin was increased in round hepatocytes adhered on a high-coating density of PVLA (100 microg/ml), and also in flat ones adhered on a low-coating density of PVLA (1 microg/ml) in the presence of epidermal growth factor (EGF). Hepatocyte spheroids formed on the high-coating density of PVLA in the presence of EGF after 48 h were inhibited by an anti-E-cadherin monoclonal antibody (ECCD-1). From immunofluorescence and confocal laser microscopy, E-cadherin was localized in the intercellular boundaries and concentrated at the inside surface of aggregated cells. As a result, E-cadherin could play an important role in hepatocyte assembly.

Formation of rat hepatocyte spheroids on agarose

The hepatocyte spheroid formation on the Primariatrade mark dish (PM) occurred as a result of cell migration, adhesion, and shape changes. On the other hand, hepatocytes plated on 1.5% agarose-coated dishes did not attach to the dishes. The formation of spheroids on the agarose-coated dishes was significantly more rapid than that on the PM ones. The PA/plasmin system plays an important role in the process of hepatocyte spheroid formation on PM dishes, and thus the spheroid formation on PM is inhibited by plasmin inhibitors. Nevertheless, these inhibitors did not show any effect on the spheroid formation on agarose-coated dishes. PA activities could be detected in the conditioned medium harvested from the cells cultured on PM dishes, but not in that from those cultured on the agarose-coated dishes. In summary, we have established a novel method to form hepatocyte spheroids by using agarose-coated dishes, in which some mechanism(s) independent of the PA/plasmin system is involved in their formation. The differentiated hepatocyte-specific functions of the spheroids formed on the agarose-coated dishes were expressed to a quite lesser degree than those formed on the PM ones, suggesting that the processes involved in spheroid formation are important for the expression of spheroid functions.

Dexamethasone Effects on Rat Hepatocyte Spheroid Formation and Function

Hepatocytes cultured on moderately adhesive surfaces or in spinner flasks spontaneously self-assemble into spherical tissue-like aggregates (spheroids). These spheroids have smooth surfaces and tissue-like polarized cell morphology, including bile canalicular-like channels, and maintain high viability and liver-specific functions for extended culture periods. Dexamethasone (DEX), a synthetic glucocorticoid, is known to elicit various responses in gene expression, and is often added to hepatocyte culture medium. The morphology and liver-specific protein production of hepatocyte spheroids were assessed under DEX concentrations ranging from 50 nM to 10 microM. DEX altered the kinetics of spheroid formation in a concentration-dependent fashion, with increasing concentrations inhibiting aggregation and promoting aggregate disassembly on culture dishes. DEX addition to spinner cultures resulted in smaller, more irregularly shaped spheroids and a higher incidence of aggregate clumping. Albumin and urea production were also higher in DEX cultures, but this effect was not as sensitive to concentration and occurred irrespective of the state of aggregation. RTPCR was utilized to assess the mRNA levels of extracellular matrix proteins, E-cadherin, and cytochrome P-450 enzymes. Results indicated a slight increase in fibronectin and collagen III mRNA early in the cultures, possibly contributing to the changes in morphology observed.

Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver

We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1-10 x 10(6) cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 +/- 1.0 vs. 3.1 +/- 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 +/- 3.3 vs. 4.6 +/- 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 +/- 63 vs. 92 +/- 13 micromol/L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale-up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure.

Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment

In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 °C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 m m galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 °C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 m m galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase–polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.

Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold

Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760nm were prepared by electrospinning of poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66nmol/cm2 was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50–300μm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20–100μm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.

Influence of pancreatic islets on spheroid formation and functions of hepatocytes in hepatocyte-pancreatic islet spheroid culture.

Hepatotrophic stimulation of hepatocytes is necessary to preserve long-term function of hepatocytes in hepatocyte transplantation and bioartificial liver system. The main source of hepatotrophic factors in portal venous blood seems to be the pancreatic islets. It was also reported that hepatocyte spheroids, tightly packed multicellular aggregates, showed enhanced liver-specific activities and a prolonged differentiated state compared with cells that were maintained as a monolayer. On the basis of these two facts, the authors tried to form hepatocyte-pancreatic islet spheroids and to evaluate the influence of pancreatic islets on spheroid formation and functions of hepatocytes in spheroid culture. Hepatocytes and pancreatic islet cells were harvested from adult male Sprague-Dawley rats weighing 200-250 g. Hepatocytes were cultured in spinner flasks with either basic nonstimulated medium (hepatocytes only [group BH] and cocultures with islet cells [group BI]) or hormone-stimulated medium (hepatocytes only [group HH] and cocultures with islet cells [group HI]). The size and morphology of spheroids, as determined by phase-contrast microscopy, and liver-specific functions, such as albumin secretion, urea synthesis, and ammonia removal, were compared among groups. The results were as follows: the size of spheroids, 66 +/- 53.4 microm, in group BH on day 2 was smaller than in group BI (179 +/- 66.2 microm on day 2, p < 0.05). In group BI, group HH, and group HI, smooth spheroids were observed on culture day 2. However, in group BH rugged incomplete aggregates were observed on the same day. In groups with basal medium, group BI showed better results in terms of hepatocyte-specific function such as albumin secretion, urea synthesis, and ammonia removal compared with group BH on days 2 and 3 (p < 0.05). In groups with hormone-defined medium, cocultures had no impact on albumin secretion rate, urea synthetic rate, and ammonia removal rate. In conclusion, we made a new type of hepatocyte-pancreatic islet spheroid, using a rotational culture method. Pancreatic islets in a spheroid culture system stimulated hepatocyte spheroid formation and some hepatocyte-specific functions in vitro.

Influence of Pancreatic Islets on Spheroid Formation and Functions of Hepatocytes in Hepatocyte–Pancreatic Islet Spheroid Culture

Influence of Pancreatic Islets on Spheroid Formation and Functions of Hepatocytes in Hepatocyte–Pancreatic Islet Spheroid Culture

Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.

A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.

Effect of carbohydrates attached to polystyrene on hepatocyte morphology on sugar-derivatized polystyrene matrices.

Sugar-carrying polymers have been utilized as artificial matrices for cell adhesion in tissue engineering. We have developed sugar-derivatized polystyrenes (PV-sugars) as artificial matrices, which control hepatocyte adhesion and hepatic function. Hepatocytes adhere to PV-sugar matrices in a receptor-mediated manner. In this study, we designed a new galactose-derivatized PV-sugar, poly-(6-O-p-vinylbenzyl-alpha-D-galactose) (PV6Gal) and evaluated the role of carbohydrate attached to polystyrene (PS) backbone in the morphological difference of hepatocyte cultured on PV-sugar matrices. Hepatocytes spread on monosaccharide-derivatized PV-sugars but not on disaccharide-derivatized PV-sugars. The actin filament remained aggregated in the central area of the cell body on disaccharide-derivatized PV-sugars. Hepatocyte cell bodies fully were spread on collagen, and the actin filament was almost completely reorganized. Hepatocyte spreading on monosaccharide-derivatized PV-sugars, however, was caused by protrusive cell-matrix contact like lamellipodia and the actin filament was not completely reorganized. This indicated that hepatocyte spreading on PV-sugar matrices was restricted compared with ECM-mediated cell spreading. In addition, typical spheroid formation of hepatocytes was promoted on disaccharide-derivatized PV-sugars compared with monosaccharide-derivatized PV-sugars. Although hepatocytes adhered with different affinities to PV-sugar matrices, hepatocyte morphology was not affected by the adhesion affinity. We suggest that the type of carbohydrate attached to the PS backbone governs the morphology of hepatocyte cultured on PV-sugar matrices.