Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro

Abstract

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

Editor’s Highlight: Modeling Compound-Induced FibrogenesisIn VitroUsing Three-Dimensional Bioprinted Human Liver Tissues

“…The punctate patterning and distribution of mesenchymal marker vimentin is preserved in 3D bioprinted liver and facilitates key heterotypic cell-cell interactions critical for supporting phenotypic features of uninjured liver ( Fig. 1E and F), and sustained viability for at least 4 weeks post-printing (Nguyen, et al, 2016). Tissues comprising cryopreserved primary HCs, HSCs, and ECs were fabricated reproducibly on the membranes of standard 24-well culture inserts, thus enabling the use of this system to conduct routine in vitro toxicity testing ( Fig.…”

“…Three-dimensional bioprinted liver tissues were manufactured by Organovo (San Diego, California) using primary cryopreserved human HCs (Life Technologies, Carlsbad, California), HSCs (ScienCell, Carlsbad, California), and human umbilical vein ECs (Becton Dickinson, Tewksbury, Massachusetts), using patented protocols (U.S. Patents 8,241,905 B2;8,852,932;9,222,932 B2;9,222,932 B2) as described previously (Forgacs et al, 2012(Forgacs et al, , 2014Murphy et al, 2015;Nguyen et al, 2016;Shepherd et al, 2015). Each commercial cell supplier provides assurances that the cells come from tissues collected in compliance with applicable laws and provided based on informed consent by the donors.…”

“…Briefly, HSCs and ECs were cultured prior to tissue fabrication and cryopreserved HCs were thawed and prepared for use according to the manufacturer's instructions. Separate bio-inks comprising parenchymal cells (100% cellular paste, generated via compaction) or NPCs (150e 6 cells/mL formulated in NovoGel 2.0 Hydrogel) were prepared and loaded into separate heads of the NovoGen Bioprinter platform (Organovo) housed within a standard biosafety cabinet (Forgacs et al, 2012(Forgacs et al, , 2014Jakab et al, 2008;Murphy, et al, 2015;Nguyen, et al, 2016;Shepherd, et al, 2015). An automated computer script was then executed to precisely deposit the bio-inks in a two-compartment planar geometry onto the membranes of standard 24-well 0.4 mm transwell membrane inserts (Corning, Tewksbury, Massachusetts) via continuous deposition, with NPCs comprising the border regions of each compartment and HCs filling each compartment such that the cell ratios roughly approximated physiologic ratios and the final tissue thickness was approximately 500 mm (Murphy and Atala, 2014).…”

“…The recent availability of bioprinted human liver tissue models that incorporate both parenchymal (ie, HCs) and NPCs (ie, HSCs and ECs) in a 3D context has created the opportunity to examine progressive liver injury in response to known pro-fibrotic modulators and compounds (Nguyen et al, 2016). In this study, we utilized this novel model system to establish conditions for monitoring tissue responses after treatment with fibrogenic agents, including transforming growth factor-b1 (TGF-b1), and prototype fibrogenic compounds methotrexate (MTX), and thioacetamide (TAA).…”

Progress in scaffold‐free bioprinting for cardiovascular medicine

“…Then, the hydrogel is dissolved, leaving behind a “scaffold‐free” construct. By this approach, vascular,33 nerve,34 liver35 and kidney36 structures were demonstrated and now commercialized for in vitro pharmacological assays.…”