Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.     

Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.     

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.     

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.     

Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes

Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.     

Mixed-ligand modification of polyamidoamine dendrimers to develop an effective scaffold for maintenance of hepatocyte spheroids

Compared with a monolayer culture, hepatocyte spheroids are known to maintain liver function for long periods. We found that hepatocytes formed spheroids when cultured on polyamidoamine dendrimers modified with fructose. Because galactose is a ligand for the asialoglycoprotein receptor on the hepatocyte cytoplasmic membrane, it was chosen as another ligand for modification in order to maintain adhesion of spheroids for long periods. Simultaneous modification of dendrimers with fructose and galactose had a marked effect on the time length of spheroid adhesion. Suppression of apoptosis and necrosis was observed in hepatocyte spheroids cultured on a dendrimer modified with fructose and galactose (F/G dendrimer). Moreover, the hepatocyte spheroids cultured on the F/G dendrimer had higher activities of liver-specific functions, such as urea synthesis and albumin gene expression, than did those cultured on single-ligand-modified dendrimers. The expression of heat shock protein (HSP) genes was examined to evaluate the stress response of cells to scaffolds. The hepatocytes cultured on the F/G dendrimer had very low expression levels of both HSP60 and HSP70 mRNAs. Thus immobilization of mixed-ligand-modified dendrimers could generate a suitable surface for hepatocyte spheroid formation. These dendrimers could be a powerful tool for generating custom-made scaffolds for cells other than hepatocytes by selecting the ligands suitable for each cell type.     

Receding cytochrome P450 activity in disassembling hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes or suspended in stirred vessels. These spheroids exhibit prolonged viability, enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures. Upon transfer to collagen coated surface, or upon the addition of fetal bovine serum (FBS) to the culture, these spheroids began to disassemble and spread on the surface. The dynamics of cytochrome P450 CYP1A1/2 activity in the course of spheroid disassembly was examined in situ by detection of the fluorescent product, resorufin, of ethoxyresorufin O-dealkylation. Optical sectioning of the disassembling spheroids by confocal microscopy demonstrated that hepatocytes that reverted to monolayer exhibited markedly lower CYP1A1/2 activity than those that remained in a multilayered structure. This occurred whether the disassembly was caused by incubation with FBS-containing medium or by cultivation on a collagen-coated surface. When spheroids were cultured on the surface of agar, the disassembly process was retarded even in the presence of FBS. However, even in those intact spheroids, the exposure to FBS markedly decreased CYP1A1/2 activity. The decreased CYP1A1/2 activity was correlated to a diminished smooth endoplasmic reticulum as seen in the transmission electron micrograph. The results clearly demonstrate that the disassembly of hepatocyte spheroids led to decreased CYP1A1/2 activity. Furthermore, FBS contained a factor that caused CYP1A1/2 to decrease even in intact spheroids.     

3D hepatocyte monolayer on hybrid RGD/galactose substratum

Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell-cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.     

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.     

An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids

Since oxygen is one of the critical limiting factors for maintaining cell viability and function, a great deal of effort is being focused on improving the oxygen supply to three-dimensional (3D) cellular constructs. Here, we report a technique to construct spheroids utilizing 3D culture chips with a rapid and simple method for the replication of the surface structures of a polydimethylsiloxane (PDMS) mold. The resultant spheroid culture chip made it possible to rapidly yield high numbers of the spheroids at a time as well as to obtain uniform spheroids with a narrow size distribution and to collect the spheroids easily and noninvasively. The most important feature of this spheroid culture chip is that it enables direct oxygen supply to the cells because the chip is made with only gas-permeable PDMS. When human hepatoma HepG2 cells were grown on the oxygen-permeable chips as a model for liver cells, the cellular growth was remarkably enhanced, and the anaerobic glycolysis was significantly reduced compared to the non-oxygen-permeable chips. Furthermore, the oxygen-permeable chip improved the albumin secretion rates compared to the conventional spheroid culture system after 10 days. Histochemical and immunohistochemical analyses demonstrated that the oxygen-permeable chip dramatically prevented hypoxia in the core of the spheroids and subsequent central necrosis. Surprisingly, the diameters of approximately 400 and 600?μm were estimated to be the threshold of the hypoxic and survival size, respectively, for the HepG2 spheroids in the oxygenated chip. These results indicate that this chip is useful for engineering 3D cellular constructs with high viability and functionality for tissue engineering.     

Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures

Bioengineering approaches, such as co-cultures of multiple cell types, that aim to mimic the physiological microenvironment may be beneficial for optimizing cell function and for engineering tissues in vitro. This study describes a novel method for preparing a spheroid microarray on microfabricated hydrogels, alone or in co-cultures. Photocrosslinkable chitosan was synthesized and utilized for fabricating hydrogel microstructures through a micromolding process. The chitosan surface was initially cell repellent but became increasingly cell adhesive over time. By using this unique property of chitosan hydrogels, it was possible to generate patterned co-cultures of spheroids and support cells. In this scheme, cells were initially microarrayed within low shear stress regions of microwells. Human hepatoblastoma cells, Hep G2, seeded in these wells formed spheroids with controlled sizes and shapes and stably secreted albumin during the culture period. The change of cell adhesive properties in the chitosan surface facilitated the adhesion and growth of a second cell type, NIH-3T3 fibroblast, and therefore enabled co-cultures of hepatocyte spheroids and fibroblast monolayers. This co-culture system could be a useful platform for studying heterotypic cell-cell interactions, for drug screening, and for developing implantable bioartificial organs.     

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.     

Maintenance of morphology and function of mixed liver cell spheroids under collagen gel environment

Mixed liver cell spheroids from rats consisting of hepatocytes and nonparenchymal cells lose three-dimensional structure and function when cultured on dishes. In order to maintain the configuration and function of the spheroids, we cultured them with collagen gel in various conditions, such as on the surface of collagen gel (group 2), between two collagen gel layers (group 3), and within collagen gel (group 4). Spheroids cultured on a standard collagen-coated dish were used as controls (group 1). Culture was continued for 10 days. Phase-contrast microscopy revealed that the spheroids of group 1 lost their spheroidal configuration and became a monolayer within 24 h. Group 2 spheroids also spread out to a monolayer, but thus occurred at 24 to 48h. In group 3, spheroid configuration was sustained until day 10, though slightly flattened. In group 4, the spheroid configuration was well maintained throughout the culture period. Urea synthesis of the spheroids cultured with collagen gel was significantly higher than in group 1 between days 1 and 3. Albumin synthesis of three experimental groups was also significantly higher than that of group 1. Although three experimental groups showed no difference in urea synthesis, albumin production by spheroids in groups 3 and 4 was better maintained than in group 2, even toward the end of the culture period. It is concluded that mixed liver-cell spheroid culture within collagen gels showed better maintenance of their configuration and function.     

Mature human hepatocytes from ex vivo differentiation of alginate-encapsulated hepatoblasts

Alginate gel was used to provide encapsulation to support the growth and eventually the differentiation of hepatic progenitors cells derived from human fetal livers. The encapsulated cells aggregated into spheroids within a few days in culture and continued to grow for at least 4 weeks in a serum-free medium. The hepatic progenitor cells in the spheroids undergo differentiation, as indicated by the appearance of functions of mature hepatocytes such as the detoxification of ammonia, albumin secretion, expression of the adult cytochrome P450 isozyme CYP3A4 and enzymatic activity typical of CYP2C9. Along with the expression of mature hepatic markers, these progenitor cells lost features typical of immature liver cells such as epithelial cell adhesion molecule. In addition to the acquisition of mature biochemical functions, the spheroids also developed a bile ducts, suggesting that they had differentiated into tissues resembling those in an intact liver.     

Bionic 3D spheroids biosensor chips for high-throughput and dynamic drug screening

To perform the drug screening, planar cultured cell models are commonly applied to test efficacy and toxicity of drugs. However, planar cultured cells are different from the human 3D organs or tissues in vivo. To simulate the human 3D organs or tissues, 3D spheroids are developed by culturing a small aggregate of cells which reside around the extracellular matrix and interact with other cells in liquid media. Here we apply lung carcinoma cell lines to engineer the 3D lung cancer spheroid-based biosensor using the interdigitated electrodes for drug efficacy evaluation. The results show 3D spheroid had higher drug resistance than the planar cell model. The anticarcinogen inhibition on different 3D lung cancer spheroid models (A549, H1299, H460) can be quantitatively evaluated by electric impedance sensing. Besides, we delivered combination of anticarcinogens treatments to A549 spheroids which is commonly used in clinic treatment, and found the synergistic effect of cisplatin plus etoposide had higher drug response. To simultaneously test the drug efficacy and side effects on multi-organ model with circulatory system, a connected multiwell interdigitated electrode arraywas applied to culture different organoid spheroids. Overall, the organization of 3D cancer spheroids-based biosensor, which has higher predictive value for drug discovery and personalized medicine screening, is expected to be well applied in the area of pharmacy and clinical medicine.     

An injectable spheroid system with genetic modification for cell transplantation therapy

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-μm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications.     

体外肝细胞培养及在药物筛选中的应用

肝脏是人体内最复杂的器官之一,负责执行多种功能,是药物毒性检测的重要靶器官。体外培养肝细胞是进行药物毒性检测的重要途径。传统的体外培养主要是让细胞在不同成分的培养基中生长,或将细胞接种于主要由体内ECM成分如胶原或基质胶组成的基底上成层生长,但易快速丧失肝特异性功能。为解决此问题,学者们研究设计了多种能更好地模拟肝脏体内微环境特征的精加工技术,以进行肝脏细胞的体外培养。在三维支架上培养肝细胞,如球状聚集体和细胞片层,可促进细胞-细胞以及细胞-基质间的相互作用和肝细胞分化,维持肝细胞特异性功能,并形成类似体内的结构。最近,脱细胞基质已被用作支持理想的细胞-基质相互作用的细胞培养支架。本文就体外二维和应用球状聚集体、细胞片层、脱细胞基质进行三维培养肝细胞的具体技术进行了综述,并简要介绍其在药物筛选中的应用。     

Morphological and cytoskeletal changes of pancreatic cancer cells in three-dimensional spheroidal culture

Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. We used a NanoCulture plate to determine the spheroid-forming ability of pancreatic ductal adenocarcinoma (PDAC) cell lines and compared the morphology and expression of cytoskeletal proteins of PDAC cells to those in two-dimensional (2D) cultures. All examined PDAC cells grew as monolayers in 2D culture. PANC-1 and KLM-1 formed spheroids in 3D culture, but PK-45H and MIAPaCa-2 did not. Strong expression of F-actin was observed in the cells attached to the surface of the plate, which formed cell projections in 3D culture. F-actin was detected on the grids of the NanoCulture plate in PANC-1 cells but not in PK-45H. The levels of tubulin expression in cells were higher in 3D culture than in 2D culture. The expression level of E-cadherin mRNA in PANC-1 and KLM-1 was higher than that in PK-45H and MIAPaCa-2. In conclusion, PDAC cells showed morphological changes, spheroid formation, and alterations of cytoskeletal proteins in 3D culture. E-cadherin might be one of the key molecules involved in spheroid formation of PDAC cells. The 3D spheroidal culture system was a useful method for cell imaging with contrast-phase microscopy and confocal microscopy.