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

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.

Keywords: Culture; Hepatocytes; Maintenance; Three-dimensional printing.

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.