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

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 bioprinting system.Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method.Samples of 4 × 10 7 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.