Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Generation of a Perfusable 3D Lung Cancer Model by Digital Light Processing.

Lung cancer still has one of the highest morbidity and mortality rates among all types of cancer. Its incidence continues to increase, especially in developing countries. Although the medical field has witnessed the development of targeted therapies, new treatment options need to be developed urgently. For the discovery of new drugs, human cancer models are required to study drug efficiency in a relevant setting. Here, we report the generation of a non-small cell lung cancer model with a perfusion system. The bioprinted model was produced by digital light processing (DLP). This technique has the advantage of including simulated human blood vessels, and its simple assembly and maintenance allow for easy testing of drug candidates. In a proof-of-concept study, we applied gemcitabine and determined the IC50 values in the 3D models and 2D monolayer cultures and compared the response of the model under static and dynamic cultivation by perfusion. As the drug must penetrate the hydrogel to reach the cells, the IC50 value was three orders of magnitude higher for bioprinted constructs than for 2D cell cultures. Compared to static cultivation, the viability of cells in the bioprinted 3D model was significantly increased by approximately 60% in the perfusion system. Dynamic cultivation also enhanced the cytotoxicity of the tested drug, and the drug-mediated apoptosis was increased with a fourfold higher fraction of cells with a signal for the apoptosis marker caspase-3 and a sixfold higher fraction of cells positive for PARP-1. Altogether, this easily reproducible cancer model can be used for initial testing of the cytotoxicity of new anticancer substances. For subsequent in-depth characterization of candidate drugs, further improvements will be necessary, such as the generation of a multi-cell type lung cancer model and the lining of vascular structures with endothelial cells.

3D-bioprinted HepaRG cultures as a model for testing long term aflatoxin B1 toxicity in vitro.

In recent years 3D-bioprinting technology has been developed as an alternative to animal testing. It possesses a great potential for in vitro testing as it aims to mimic human organs and physiology. In the present study, an alginate-gelatin-Matrigel based hydrogel was used to prepare 3D-bioprinted HepaRG cultures using a pneumatic extrusion printer. These 3D models were tested for viability and metabolic functions. Using 3D-bioprinted HepaRG cultures, we tested the toxicity of aflatoxin B1 (10 or 20 µM) in vitro and compared the results with 2D HepaRG cultures. There was a dose-dependent toxicity effect on cell viability, reduction of metabolic activity and albumin production. We found that 3D-bioprinted HepaRG cultures are more resistant to aflatoxin B1 treatment than 2D cultures. Although the metabolic activities were reduced upon treatment with aflatoxin B1, the 3D models were still viable and survived longer, up to 3 weeks, than the 2D culture, as visualized by fluorescence microscopy. Furthermore, albumin production recovered slightly in 3D models after one and two weeks of treatment. Taken together, we consider using 3D-bioprinting technology to generate 3D tissue models as an alternative way to study toxicity in vitro and this could also provide a suitable alternative for chronic hepatotoxicity studies in vitro.