Real time monitoring of metabolism of cells encapsulated in 3D bioprinted hydrogels to bioengineer a liver sinusoid model
Final Report Abstract
Bioengineered or in vitro liver models are pivotal in early drug development as they serve in assessing drug induced liver toxicity (DILI). Currently, many in vitro liver models (2D monolayer cultures or organoids) do not aptly represent DILI as they lack intricate cellular organization and flow conditions of a native liver. In a liver sinusoid, hepatocytes, endothelial cells, and other cell types experience dynamic conditions such as blood flow, gradients of glucose & oxygen (O2) etc. - mimicking these conditions in an in vitro model would be essential for studying DILI. The aim of the project is to develop an in vitro liver sinusoid model using 3D bioprinting method and to develop strategies to mimic the cellular organization and directional flow conditions as in a native liver sinusoid. Directional flow conditions in 3D bioprinted constructs could lead to development of gradients of nutrients and O2, potentially aiding in establishment of zonation of liver. Real-time O2 monitoring method was used to evaluate O2 dynamics in bioprinted constructs, which was used to correlate rate of metabolism of cells in the constructs. Also, this method was used to characterize O2 diffusion properties of various bioinks. Among the studied bioinks, gelatinalginate (GA) composite was determined as a suitable bioink due to its intrinsic high biocompatibility and desirable O2 diffusion properties. Biocompatibility and functionality of HepG2 (a hepatic carcinoma cell line) and HepaRG cells was assessed in GA bioinks. O2 consumption rate in bioprinted constructs cultured in normoxic and hypoxic conditions was evaluated. A decrease in O2 consumption rate in drug treated bioprinted constructs was observed. The decrease was prominent in rifampicin treated constructs in comparision to acetaminophen treatment. Gene expression analysis of CYP3A4 and MRP2 (related to drug metabolism) and albumin genes was performed on drug bioprinted constructs. An increase & decrease in expression of MRP2 & albumin, respectively was observed in drug treated HepaRG laden bioprinted constructs. A perfusable in vitro liver sinusoid model was developed by fabricating hollow HepG2 cell laden tubes (using coaxial 3D bioprinting method). Followed by seeding the tube lumen with human umbilical vein endothelial cells (HUVEC). The tube lumen was perfused with coculture media for 5 days in a custom-made bioreactor. Complete HUVEC colonization of the tube lumen was observed. Addition of 5mM acetaminophen to the perfusion coculture media resulted in significant disruption of HUVEC morphology. Based on the results obtained in this work, a correlation between O2 dynamics in bioprinted hepatic constructs and drug toxicity (evaluated by gene expression analysis) can be potentially established. The developed perfusable in vitro liver sinusoid model could be potentially tailored to achieve zonation of liver and could potentially serve as an apt model to assess drug toxicity of new drugs.
Publications
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Oxygen-sensitive nanoparticles reveal the spatiotemporal dynamics of oxygen reduction during magnesium implant biodegradation. npj Materials Degradation, 6(1).
Zeller-Plumhoff, Berit; Akkineni, Ashwini Rahul; Helmholz, Heike; Orlov, Dmytro; Mosshammer, Maria; Kühl, Michael; Willumeit-Römer, Regine & Gelinsky, Michael
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3D bioprinting of Gelatin-Alginate bioinks for biofabrication of in vitro liver sinusoid model. Procedia CIRP, 125, 96-100.
Akkineni, Ashwini Rahul; Lode, Anja & Gelinsky, Michael
