Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

Annals of Surgical Treatment and Research 2017³â 92±Ç 2È£ p.67 ~ p.72

±è¿äÇÑ(Kim Yo-Han) - Hanyang University College of Medicine Department of Surgery
°­±³Áø(Kang Kyo-Jin) - Hanyang University College of Medicine Department of Surgery
Á¤Àç¹Î(Jeong Jae-Min) - Hanyang University College of Medicine Department of Surgery
¹é½Â»ï(Paik Seung-Sam) - Hanyang University College of Medicine Department of Pathology
±èÁö¼÷(Kim Ji-Sook) - Hanyang University College of Medicine Department of Pathology
¹Ú¼ö¾Æ(Park Su-A) - Korea Institute of Machinery and Materials Department of Nature-Inspired Nanoconvergence Systems
(Kim Wan-Doo) - Korea Institute of Machinery and Materials Department of Nature-Inspired Nanoconvergence Systems
¹ÚÁö¼±(Park Ji-Sun) - Korea Institute of Machinery and Materials Department of Nature-Inspired Nanoconvergence Systems
ÃÖµ¿È£(Choi Dong-Ho) - Hanyang University College of Medicine Department of Surgery

Abstract

Purpose: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved.

Methods: To generate 3D bio-printed structures (25 mm ¡¿ 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6?8 weeks old mice by a 2-step collagenase method. Samples of 4 ¡¿ 107 hepatocytes with 80%?90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes.

Results: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4¥á and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time.

Conclusion: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

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Hepatocytes, Three-dimensional printing, Culture, Maintenance
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