In vitro 3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes

• Published in: Animal cells and systems Vol. 21; no. 5; pp. 332 - 340
• Main Authors: Ullah, Imran, Kim, Yeongji, Lim, Malgum, Oh, Keon Bong, Hwang, Seongsoo, Shin, Yurianna, Kim, Youngim, Im, Gi-Sun, Hur, Tai-Young, Ock, Sun A
• Format: Journal Article
• Language: English
• Published: Daejeon Taylor & Francis 03.09.2017; Taylor & Francis Ltd; 한국통합생물학회
• Subjects: 3-Methylcholanthrene; Apoptosis; Culture; CYP1A; CYP1A protein; CYP3A; Cytochrome P450; Dexamethasone; Drug screening; Elongation; Gene expression; Hepatocytes; Liver; Monolayers; Morphology; mRNA; primary hepatocytes; Rat; Ribonucleic acid; RNA; Rodents; spheroid (3-D) culture; Spheroids; 생물학
• ISSN: 1976-8354; 2151-2485
• DOI: 10.1080/19768354.2017.1381151

Primary hepatocytes (PHs) are considered the 'gold standard' in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48 h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems.