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

MeSH Terms

Animals
Collagenases
Drug Evaluation, Preclinical
Fluorescent Antibody Technique
Gene Expression
Hepatocytes*
Liver
Liver, Artificial
Methods
Mice*
Printing, Three-Dimensional
Real-Time Polymerase Chain Reaction
Collagenases

Figure

  • Fig. 1 Perfusion for isolation of primary hepatocytes and 3-dimensional (3D) bio-printing. To obtain primary hepatocytes, we uses a 2-step perfusion method, in which perfusion solution is injected through the portal vein (A, B). Panel B is a magnification of panel A and the red arrow indicates portal vein. Panel C shows primary hepatocytes cultured for 1 day. The 3D bio-printer can print the alginate and hepatocyte mixture with air pressure (D). Seven layered 3D bio-hepatic structures were printed (E; 25 mm × 25 mm).

  • Fig. 2 (A-D) Morphological change with time of primary hepatocytes cultured in 3-dimensional (3D) alginate scaffold. Three-dimensional printed hepatocytes migrated and aggregated for 14 days. In addition, they aggregated 3D hepatocyte structures after 7 days (arrow in D, E). Panel E is magnified region of Panel D.

  • Fig. 3 Gene expression in primary hepatocytes cultured by 3-dimensional (3D) bio-printing. Albumin (A), HNF-4α (B), and Foxa3 (C) gradually increased with time. (D) ASGR1 expression decreased slightly on day 14. Expression continued for at least 14 days. MEF, mouse embryonic fibroblasts.

  • Fig. 4 Immunofluorescence detection of hepatic-specific proteins. (A, G, and M) The photo of hematoxilin & eosin (H&E) staining, and (B, H, N) black box is a magnified region . Panels A to R are hepatocytes morphology in 3-dimensional scaffold, and immunofluorescence photo of hepatocyte specific protein (albumin and cytokeratin 18 [CK18]) shows in panels C-F, I-L, and O-R with time.


Reference

1. Yu SJ. A concise review of updated guidelines regarding the management of hepatocellular carcinoma around the world: 2010-2016. Clin Mol Hepatol. 2016; 22:7–17.
2. Vinken M, Maes M, Oliveira AG, Cogliati B, Marques PE, Menezes GB, et al. Primary hepatocytes and their cultures in liver apoptosis research. Arch Toxicol. 2014; 88:199–212.
3. Mandrycky C, Wang Z, Kim K, Kim DH. 3D bioprinting for engineering complex tissues. Biotechnol Adv. 2016; 34:422–434.
4. Klaunig JE, Goldblatt PJ, Hinton DE, Lipsky MM, Chacko J, Trump BF. Mouse liver cell culture. I. Hepatocyte isolation. In Vitro. 1981; 17:913–925.
5. Swift B, Brouwer KL. Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes. Mol Pharm. 2010; 7:491–500.
6. Mathijs K, Kienhuis AS, Brauers KJ, Jennen DG, Lahoz A, Kleinjans JC, et al. Assessing the metabolic competence of sandwich-cultured mouse primary hepatocytes. Drug Metab Dispos. 2009; 37:1305–1311.
7. Boess F, Kamber M, Romer S, Gasser R, Muller D, Albertini S, et al. Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems. Toxicol Sci. 2003; 73:386–402.
8. Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. 2002; 32:505–520.
9. Choi HJ, Choi D. Successful mouse hepatocyte culture with sandwich collagen gel formation. J Korean Surg Soc. 2013; 84:202–208.
10. Kwon YJ, Lee KG, Choi D. Clinical implications of advances in liver regeneration. Clin Mol Hepatol. 2015; 21:7–13.
11. Smidsrod O, Skjak-Braek G. Alginate as immobilization matrix for cells. Trends Biotechnol. 1990; 8:71–78.
12. Martinsen A, Skjak-Braek G, Smidsrod O. Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnol Bioeng. 1989; 33:79–89.
13. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials. 1999; 20:45–53.
14. Arterburn LM, Zurlo J, Yager JD, Overton RM, Heifetz AH. A morphological study of differentiated hepatocytes in vitro. Hepatology. 1995; 22:175–187.
15. Bell CC, Hendriks DF, Moro SM, Ellis E, Walsh J, Renblom A, et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep. 2016; 6:25187.
Full Text Links
  • ASTR
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr