Introduction: Developing large vascularized tissue constructs that can maintain long-term is an ongoing challenge. A strategy that has been gaining much attention is to bioengineer prevascularize tissue constructs that allow for immediate perfusion. We present the development of a large vascularized functional human liver construct that can be maintained for 30 days with high viability Methods: Vascularized human liver tissue constructs of 1.5 cm in diameter and 6 cm in length were fabricated using an extrusion-based 3D printing system, seeded with hepatocytes (HepG2) in bulk hydrogel (alginate) and human umbilical vein endothelial cells (HUVECs) within the vascular channels. The alginate/HepG2 mixture was placed into a mold. A sacrificial gel (calcium chloride/HUVECs) was used to create vessels. Post printing, the samples were cured using calcium chloride and incubated at 37°C to liquefy the sacrificial gel. The liver tissue constructs were maintained in a media bath for 4 days, followed by media perfusion for 30 days using a peristaltic pump. The tissue samples were removed every 10 days and analyzed for cellular viability and biochemical functionality (albumin/bilirubin production). Immunohistochemistry was used to confirm the location of hepatocytes and endothelial cells. Results: Alginate and calcium chloride was successfully used to fabricate constructs patterned with internal vascular channels running the entire length of the construct. The final constructs were cylindrical and maintained their structural integrity. The liver tissue samples showed high viability following fabrication and maintained a greater than 94 percent viability throughout the 30-day time point. The liver tissue constructs produced albumin and bilirubin at levels comparable to human liver tissue (assayed at 10, 20, and 30 days). In the retrieved constructs endothelial cells were identified on the vascular channel walls surrounded by hepatocytes in the hydrogel. Conclusions: We have successfully created large vascularized liver tissue constructs patterned with hollow channels using 3D bioprinting. The combination of perfusion and internal channels maintained high viability for 30 days. The liver tissue constructs produced albumin and bilirubin levels comparable to human liver tissue. This is a major step towards creating large vascularized functional liver tissue constructs that could be used for translational applications. SOURCE OF Funding: This work is a winner of NASA’s Vascular Tissue Challenge contest. The authors thank NASA for supporting this work in a zero-gravity environment on the International Space Station.
