Development of a novel bio-ink for 3D bioprinting liver tissue models

Abstract

Given the essential biological role the liver plays, reliable and reproducible in vitro models are vital for furthering research in many fields. The current in vitro liver models have limitations, most often that they are two-dimensional and lack critical architectural features of the endogenous liver. The goal of this research project was to utilise stem cell-derived hepatocytes, and the cancer cell-line HepaRG, to develop a 3D tissue model that can be bioprinted in a high throughput manner. Initial experiments with human embryonic stem cells (hESCs) resulted in high levels of cell death. Robust viability of encapsulated hESCs was achieved by first generating hESCs before encapsulation. Preliminary differentiation of hESCs within the alginate was inefficient soit was necessary to adapt the differentiation protocol for stem cell-derived hepatocytes. This was achieved by preloading the alginate with growth factors andelongating the initial differentiation step. Identification of porous hydrogels was essential for permeation of crucial growth factors throughout the bioprinted structures to control differentiation. Carboxymethyl cellulose was identified as producing the most porous hydrogels in combination with alginate, determined through the leaching of fluorescent proteins from the gel. The previous results were translated to the liver cell line, HepaRG cells. The carboxymethyl cellulose and alginate composite supported the highest function from the HepaRG cells. This bioink of encapsulated HepaRGs was then bioprinted in a high throughput manner into 96 well plates using a custom designed 3D model. These HepaRG-laden structures were subsequently used for a hepatotoxicity drug assay. The structures showed expected levels of hepatotoxicity in response to these molecules. This work demonstrated the feasibility of generating reproducible and reliable liver tissue in vitro, which could be used for mid to high throughput research.