Alternatives to Animal Testing and Experimentation
Online ISSN : 2185-4726
Print ISSN : 1344-0411
ISSN-L : 1344-0411
Volume 21 , Issue 2
Showing 1-4 articles out of 4 articles from the selected issue
Preface
  • Taku Matsushita
    Type: Preface
    2016 Volume 21 Issue 2 Pages 50-51
    Published: 2016
    Released: March 05, 2018
    JOURNALS FREE ACCESS

    In the opening lecture of the 26th Annual Meeting of JSAAE in 2013 (Imai, 2013), Prof. K. Imai stated that three-dimensional (3D) culture was first introduced in 1952 by Prof. A. Moscona, using the "watch-glass method" or "hanging drop culture" (Moscona and Moscona, 1952) and was further advanced by Prof. Y. Kuroda, using "aggregation culture" in 1963 (Kuroda, 1963). Prof. Y. Kuroda received an award from the Japanese Society for Alternatives to Animal Experiments in 2004 for his studies. In 1986, Prof. N. Takahashi reported a technique for the isolation and long-term culture of rat hepatocytes by 3D culture (Takahashi, 1986). Subsequently, Prof. N. Koide reported the "spheroid culture" technique for primary rat hepatocytes (Koide et al., 1990).

    When we reported our study on 3D culture in 1991 (Matsushita et al., 1991), the total number of papers retrieved in PubMed, using "three dimensional culture" as the key words, was only 85 per year (Fig. 1). In 2015, the number of papers retrieved per year increased to 927 and on December 17, 2016, the total cumulative number reached 10729, as shown in Fig. 1. When the key word "cancer" was added to the search, the number of papers retrieved was 2337, and when the key word "primary" was added to the search, the number became 1314. Additionally, when "screening" or "toxicity" was added to the search, the number of papers retrieved was 838 or 397, respectively. Furthermore, when the key word "human" was added to the search, the number of papers retrieved was 6313 (Ishii et al., 2015; Ishii et al., 2016). These retrieval results suggest that the 3D culture of human cancer cells appears to be applied to drug screening, and the 3D culture of human primary cells appears to be applied to toxicity test and metabolite analysis of chemical compounds, including drug candidates.

    In this special issue on the "Application of 3D culture for human cells as alternatives to animal testing and experimentation," T. Iwasa et al. report a 3D culture of rat hepatocytes, using a new 3D culture scaffold called "Cellbed" for liver toxicity testing. Y. Kuroda et al. reported a suspended aggregation culture of cryopreserved human hepatocytes and hepatoma cell line, HepG2, using a newly developed low-acyl gellan gum, FP001, and T. Kubo et al. reported a 3D culture of a hepatic progenitor cell line, HepaRG, using VECELL Inserts, which consist of type I collagen-coated expanded polytetrafluoroethylene (ePTFE) mesh. These studies describe the new 3D culture technologies for hepatocytes or hepatic progenitor cells and include promising and informative results for the application of 3D culture for toxicity tests and metabolite analysis of chemical compounds as alternatives to animal testing and experimentation in the near future.

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ORIGINAL ARTICLES
  • Takuya Iwasa, Rie Watanabe, Kouhei Sasaki, Taku Matsushita, Akihiro Ya ...
    Type: ORIGINAL ARTICLE
    2016 Volume 21 Issue 2 Pages 52-62
    Published: 2016
    Released: March 05, 2018
    JOURNALS FREE ACCESS

    There are some cases that the results of in vitro liver toxicity testing can't correlate with the results of clinical studies or animal experiments because of difficulty in maintaining viability and activity of normal hepatocytes under in vitro culture conditions. In addition, based on the three Rs of animal experiments, the development of a culture system that can replace animal experiments is desired. In this study, we aimed to optimize a culture method using the three-dimensional cell culture scaffold "Cellbed®" that maintains the liver functions of rat hepatocytes longer than traditional monolayer culture on type I collagen-coated dishes, by coating Cellbed with various types of extracellular matrix (type I collagen, fibronectin, and laminin). As a result, culture conditions using laminin-coated Cellbed presented the most favorable effect in terms of maintenance of liver functions (based on albumin and CYP3A expression), and this was superior to monolayer culture using any other coating. As such, it was indicated that a culture method using laminin-coated Cellbed contributes to the long-term maintenance and improvement of rat hepatocyte functions. This is expected for the application of Cellbed to hepatic metabolism studies and long-term in vitro liver toxicity testing.

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  • Yukie Kuroda, Su-ryang Kim, Tatsuro Kanaki, Masato Horikawa, Yuko Seki ...
    Type: ORIGINAL ARTICLE
    2016 Volume 21 Issue 2 Pages 63-70
    Published: 2016
    Released: March 05, 2018
    JOURNALS FREE ACCESS

    The biotransformation activity of the liver is a major concern of medical and chemical production processes. Human cryopreserved hepatocytes are widely used for in vitro analysis of metabolites identification and toxicity evaluation of drug candidates or chemical compounds. However, cryopreservation causes cell damage and leads to decreased cell functionality and viability after thawing. To overcome such disadvantages, a variety of cryopreservation and culture methods have been developed. In this study, cryopreserved human hepatocytes and HepG2 cells were cultured under suspension conditions with FP001, a newly developed gellan gum-based cell culture material, after thawing. Under suspension conditions, cell damage was decreased compared with that under monolayer conditions. Combined with gene expression analysis of cell adhesion-related genes, our results suggested that FP001 contributed to decreased damage and increased viability of cryopreserved cells by altering the expression of genes involved in cell-cell adhesion.

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  • Takashi Kubo, Yukie Kuroda, Maki Hojyo, Su-ryang Kim, Shinichiro Horiu ...
    Type: ORIGINAL ARTICLE
    2016 Volume 21 Issue 2 Pages 71-79
    Published: 2016
    Released: March 05, 2018
    JOURNALS FREE ACCESS

    Cell shape influences cell functionality. We investigated the possibility that the differentiation state of HepaRG, a bipotential hepatic progenitor cell line, was changed by cultivation on VECELL Inserts, which consist of type I collagen-coated expanded polytetrafluoroethylene (ePTFE) mesh. HepaRG cells plated on VECELL Inserts possessed a round shape. Gene expression patterns obtained from HepaRG cells cultured on VECELL Inserts suggested that the cells maintained their progenitor cell-like characteristics, while not losing the capacity to differentiate into hepatocytes. Retaining cell stemness is important for the expansion of progenitor cells to maintain their differentiation potency. VECELL Inserts are a novel culture apparatus that easily retains the characteristics of hepatic progenitor cells. It may be useful for the culture and expansion of hepatic progenitor cells, and thus, provide fundamental cell source for in vitro toxicity and biotransformation assays.

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