Ann Surg Treat Res.  2017 Feb;92(2):67-72. 10.4174/astr.2017.92.2.67.

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

Affiliations
  • 1Department of Surgery, Hanyang University College of Medicine, Seoul, Korea. crane87@hanyang.ac.kr
  • 2Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.
  • 3HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul, Korea.
  • 4Department of Pathology, Hanyang University College of Medicine, Seoul, Korea.
  • 5Department of Nature-Inspired Nanoconvergence Systems, Korea Institute of Machinery and Materials, Daejeon, Korea.

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-4α 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.

Keyword

Hepatocytes; Three-dimensional printing; Culture; Maintenance
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