In vitro liver models allow investigation of the cell behavior in disease conditions or in response to changes in the microenvironment and are therefore valuable tools for basic research, drug screenings or toxicological analyses. Mimicking the tissue-level complexity of liver to achieve functional constructs is a major challenge, however, 3D bioprinting technologies open novel options to recreate the liver microarchitecture. Previously, we have demonstrated the high potential of coaxial extrusion-based 3D bioprinting to establish patterned co-cultures of hepatocytes and fibroblasts in core-shell fashion . The aim of the present study was to develop a liver sinusoid-like model consisting of a vascularized core compartment which is surrounded by a hepatocyte-laden shell compartment. A suitable core bioink was developed, bioprinting of the triple culture model was established and cell-cell interactions were observed.
The shell bioink consisted of 3 wt% alginate and 9 wt% methylcellulose dissolved in fresh frozen plasma (plasma-algMC) as described ; HepG2 were added immediately prior to printing. The core bioink was prepared by mixing collagen, fibrinogen and gelatin to achieve a CFG blend with final concentrations of 1.33 mg/ml C, 5 mg/ml F and 4 wt% G. Human endothelial cells (HUVEC) and fibroblasts (NHDF) were mixed into the ink immediately prior to printing. Core-shell bioprinting was conducted using a Bioscaffolder 3.1 (GeSiM) equipped with a coaxial needle as described ; crosslinking of the bioprinted constructs was done in 100 mM CaCl2 supplemented with 0.3 U/ml thrombin solution. Cell viability was examined by live/dead staining, cell proliferation was investigated by cell number quantification and EdU staining. Immunostaining was conducted to visualize endothelial tube formation in the core and to prove hepatocytes biomarker expression in the shell. Albumin secretion was analysed by ELISA.
Core-shell constructs consisting of the CFG bioink as core and the plasma-algMC bioink as shell were nicely printable and, after dual crosslinking, stable during further cultivation over at least 21 days. Plasma enhanced viability and supported proliferation, cluster formation and biomarker expression of HepG2 in the alginate-based shell compartment. Using NHDF as supportive cells, HUVEC formed a pre-vascular network in the CFG core. This network formation was also visible in the presence of HepG2 in the shell compartment, however, a competition of the HepG2 for the matrix-forming fibroblasts in the adjacent compartment was observed if a high number of HepG2 was used. The presence of HUVEC in the core compartment resulted in an increased albumin secretion of HepG2 in the shell compartment.
Based on core-shell bioprinting, a patterned triple culture model has been established which can be further developed towards a more physiological liver sinusoid model.
1 Taymour, R. et al., Sci Rep, 2021, 11, 5130
2 Ahlfeld, T. et al., ACS Appl Mater Interfaces, 2020, 12, 12557
The authors thank the European Social Fund ESF and the Free State of Saxony for financial support in the course of TU Dresden based Young Researchers Group IndivImp and the microscopy facility CFCI of TU Dresden for providing equipment and support in cell imaging.