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Biomedical subjects

Kohji Nakazawa

Publications and source records attributed to Kohji Nakazawa.

8 recordsLinked to original sources
PubMed2006

Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities.

A two-dimensional microarray technique of spherical multicellular aggregates (spheroids) using a microfabricated polydimethylsiloxane (PDMS) chip and the expression of liver-specific functions of primary rat hepatocytes on the chip were investigated. The PDMS chip, which was fabricated by a photolithography-based technique, consisted of approximately 2500 cylindrical microcavities (approximately 1100 cavities/cm2) in a triangular arrangement of 330 microm pitch on a PDMS plate (20 x 20 mm); each cavity measured 300 microm in diameter and 100 microm in depth. Most hepatocytes on the PDMS chip gradually gathered and subsequently formed a single spheroid in each cavity until 3 days of culture. A part of the spheroid was attached to the bottom or wall surface of the microcavity, and the spheroid configuration was maintained for at least 14 days of culture. Albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase (EROD) activity, which is a cytochrome P-450-dependent reaction, of hepatocytes on the PDMS chip were higher than those of a monolayer dish or a flat PDMS dish without microcavities, and were maintained for at least 10 days of culture. The spheroid microarray technique appears to be promising in the development of cell chips and microbioreactors.

Animals↗
PubMed2005

Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery.

Biocompatible Materials↗
PubMed2005

Novel hepatocyte culture system developed using microfabrication and collagen/polyethylene glycol microcontact printing.

The better understanding of cell biology and cell communication requires novel culture systems that better represent the natural cell environment in tissues and organs. We developed a spherical organoid (spheroid) microarray culture system using a combination of microfabrication and microcontact printing. The system consisted of a chip that had cylindrical cavities of 300 microm diameter at a density of 700 cavities/cm2. The bottom faces of these cavities were defined as two different regions that either supported or inhibited cell adhesion. In the cell adhesion region, the center of the bottom face of a 100 microm diameter in a cavity was modified with collagen (Col), and in the non-adhesion region, the entire region around the cavity, except the Col spots, was modified with polyethylene glycol. Primary hepatocytes spontaneously formed spheroids with a uniform diameter at the center of each cavity on the chip. Hepatocytes forming spheroids had a cuboidal cell shape, similar to hepatocytes in vivo, and stably maintained liver-specific phenotypes, such as liver-enriched transcriptional factors, albumin secretion, urea cycle enzymes, and intercellular adhesion molecules. This novel culture system may be applicable as a cellular platform for fundamental studies in cell biology and tissue engineering applications.

Animals↗
PubMed2004

cDNA microarray analysis in hepatocyte differentiation in Huh 7 cells.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of a hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we previously demonstrated the upregulation of hepatocyte-specific function by spheroid formation in polyurethane foam and by culturing with the histone deacetylase inhibitor, trichostatin A (TSA), in a human hepatoma cell line (Huh 7). In this article we analyze the gene expression profile using cDNA microarray (1281 genes) in spheroid formation or culturing with TSA in Huh 7 to determine the target genes in hepatocyte differentiation. In both the spheroid formation and in the culture with TSA, the Oct-3/4 transcription factor was upregulated more thantwofold, while the early growth response-1 (EGR-1) transactivator was downregulated less than 0.5-fold. These results indicate that expressions of Oct-3/4 and EGR-1 may be key factors in the induction of hepatocyte differentiation in Huh 7.

Cell Culture Techniques↗
PubMed2003

Efficacy of a larger version of the hybrid artificial liver support system using a polyurethane foam/spheroid packed-bed module in a warm ischemic liver failure pig model for preclinical experiments.

We have reported the usefulness of a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver support system (PUF-HALSS). The aim of this study was to evaluate in detail the efficacy in serum parameters regarding the liver function of a larger version of the PUF-HALSS containing 2 x 10(10) porcine hepatocytes for clinical use in warm ischemic liver failure pigs. Warm ischemic liver failure pigs weighing 25 kg were divided into two groups: (1) a control group (n = 3), in which each pig was attached to a PUF-HALSS without hepatocytes, and (2) a HALSS group (n = 3), in which each pig was attached to a PUF-HALSS. In the HALSS group, the increase of blood ammonia was completely suppressed and blood lactate levels were significantly suppressed. The Fisher's ratio was better maintained, and the increase of total bile acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid was significantly suppressed in the HALSS group. Serum creatinine levels were significantly lower, and blood glucose levels were significantly higher in the HALSS group. Serum levels of tumor necrosis factor- a were not elevated in either group. In conclusion, the larger version of the PUF-HALSS demonstrated many advantages as a liver support system in warm ischemic liver failure pigs.

Amino Acids↗
PubMed2002

Hybrid-artificial liver support system.

We originally developed a multi-capillary polyurethane foam packed-bed module as a hybrid-artificial liver support system (HALSS) and have applied for the permission of the clinical application to our institutional ethical committee. We summarized here the history, recent obstacles in clinical applications, and future prospects of HALSS, including our own.

Animals↗
PubMed2002

High metabolic function of primary human and porcine hepatocytes in a polyurethane foam/spheroid culture system in plasma from patients with fulminant hepatic failure.

It has been demonstrated that plasma from patients with fulminant hepatic failure (FHF) interferes extensively with cellular function. We placed primary human and primary porcine hepatocytes in a polyurethane foam (PUF)/spheroid culture system and compared the metabolic functions in the plasma of patients with FHF in a 24-h stationary culture to those in a monolayer culture. The PUF/spheroid culture system using primary human and primary porcine hepatocytes significantly decreased ammonia content during 28-day culture. Fisher's ratio significantly increased at culture days 3 and 7. Tauroursodeoxycholic acid significantly increased and glycochenodeoxycholic acid and taurochenodeoxycholic acid decreased in the FHF patients' plasma at culture day 3. During at least a 24-h culture in the FHF patients' plasma, metabolic functions of primary human and primary porcine hepatocytes were almost identical. The present results indicate that the PUF/spheroid culture system using primary human or primary porcine hepatocytes demonstrated more advantageous metabolic functions in the plasma from patients with FHF than the monolayer culture.

Ammonia↗