Efficient Induction of Hepatocyte Spheroids in a Suspension Culture Using a Water-Soluble Synthetic Polymer as an Artificial Matrix

Yamada, Keisuke; Kamihira, Masamichi; Hamamoto, Ryuji; Iijima, Shinji

doi:10.1093/oxfordjournals.jbchem.a022037

Cited by 50 publications

(38 citation statements)

References 28 publications

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“…No spheroids had formed after 2 weeks of culture when 30-mL spinner-flask cultures were inoculated at 5 × 10 5 cells/mL in DMEM-10 or Hepatozyme-SFM. Inclusion of a synthetic polyelectrolyte, Eudragit (Yamada et al, 1998), at 0.1% (w/v), did not resolve the problem. Liquid overlay cultures of 5 × 10 5 cells/mL of HepG2 on agarose in DMEM-10 produced numerous aggregates by day 5.…”

Section: Hepg2 Spheroidsmentioning

confidence: 98%

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Kelm¹,

Timmins²,

Brown³

et al. 2003

Biotech & Bioengineering

799

691

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Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.

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Section: Hepg2 Spheroidsmentioning

confidence: 98%

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Kelm¹,

Timmins²,

Brown³

et al. 2003

Biotech & Bioengineering

799

691

View full text Add to dashboard Cite

show abstract

“…Similar systems involve the use of poly-(2-hydroxyethylmethacrylate) (Landry et al, 1985;Tong et al, 1992) (Tobe et al, 1992), galactosylated materials (Lin et al, 1995) or poly-N-isopropyl acrylamide (Takezawa et al, 1992), and 3D scaffolds (Yamashita et al, 2001;Fukuda et al, 2003). Other systems designed to create spheroid cultures include the hanging drop method, the rotating culture system, the agitating culture system, microfabricated chips, and the microspace cell culture system (Sakai et al, 1996;Yamada et al, 1998;Kelm et al, 2003;Sakai and Nakazawa, 2007;Nakamura et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with microfabricated scaffold

Nakamura¹,

Kato²,

Aizawa³

et al. 2011

J. Toxicol. Sci.

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-The Nanoculture plate (NCP) is a recently developed plate which essentially consists of a textured surface with specific characteristics that induce spheroid formation: microfabrications with a micro-square pattern on the culture surface. The NCP can be used to generate uniform adhesive spheroids of cancer cell lines using conventional techniques without the need of any animal compounds. In this study, we assessed the performance of human hepatoma cell line HepG2 cells cultured with an NCP to evaluate the effects of the NCP on their hepatocyte-specific functions. The NCP facilitated the formation of three-dimensional (3D) HepG2 cell architecture. HepG2 cells cultured with an NCP exhibited enhanced mRNA expression levels of albumin and cytochrome P450 (CYP) enzymes compared to those cultured with a two-dimensional (2D) conventional plate. The expression levels of two specific liver-enriched transcription factors, hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer binding protein α (C/EBPα), were higher in HepG2 cells grown with the NCP than those in HepG2 cells grown with conventional plates before albumin and CYP enzymes expression levels were increased. The inducibility of CYP1A2 and CYP3A4 mRNA following exposure to inducers in HepG2 cells cultured with an NCP was comparable to that in HepG2 cells cultured with conventional plates, while the expression levels of CYP1A2 and CYP3A4 mRNA following exposure to inducers were higher when using an NCP than when using conventional plates. These results suggest that the use of an NCP enhances the hepatocyte-specific functions of HepG2 cells, such as drug-metabolizing enzyme expression, making the NCP/ HepG2 system a useful tool for evaluating drug metabolism in vitro.

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“…In static culture conditions it took 3-7 days for the hepatocytes to form spheroids [Shinji et al, 1988;Peshwa et al, 1994]. In rotation culture conditions in 1-2 days spheroid formation was observed Yamada et al, 1998]. The most recent strategies to stimulate hepatocyte spheroid formation is the use of physiological pulsatile flow [Vacanti and Langer, 1999] in special bioreactors with three-dimensional culture conditions [Gerlach et al, 1995;Lazar et al, 1995;Kim et al, 1998].…”

Section: Discussionmentioning

confidence: 99%

Optimization of Hepatocyte Spheroid Formation for Hepatic Tissue Engineering on Three-Dimensional Biodegradable Polymer within a Flow Bioreactor prior to Implantation

Török

Pollok²,

et al. 2001

Cells Tissues Organs

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We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 × 10⁶ cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams’ Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggregates (SphA) of 50–260 µm in diameter with a liver-like morphology and active metabolic function. The number of SphA was examined by phase contrast microscopy and the reductive enzyme function of the hepatocytes was tested using MTT. Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 µm in diameter but central necrosis in the SphA exceeding this size. Immunohistochemical staining confirmed albumin production of hepatocytes within the SphA. The optimal cell seeding concentration was 10 × 10⁶ cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineering of an in vivo liver substitute.

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Efficient Induction of Hepatocyte Spheroids in a Suspension Culture Using a Water-Soluble Synthetic Polymer as an Artificial Matrix

Cited by 50 publications

References 28 publications

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with microfabricated scaffold

Optimization of Hepatocyte Spheroid Formation for Hepatic Tissue Engineering on Three-Dimensional Biodegradable Polymer within a Flow Bioreactor prior to Implantation

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