A novel evaluation method for determining drug-induced hepatotoxicity using 3D bio-printed human liver tissue

Toxicol Mech Methods. 2020 Mar;30(3):189-196. doi: 10.1080/15376516.2019.1686795. Epub 2019 Nov 21.


Predicting drug-induced liver injury is important in early stage drug discovery; however, an accurate prediction with existing hepatotoxicity evaluation tools is difficult. Conventional monolayer (2D) cultures have short viabilities and are therefore inappropriate for performing long-term toxicity tests. Conventionally used 200-μm spheroids also have toxicity detection limits. The goal of this study was to develop a humanized liver tissue capable of evaluating long-term toxicity with high sensitivity. Spheroids consisting of co-cultured cryopreserved primary human hepatocytes and human hepatic stellate cells were developed using a 3D bio-printer. The "3D bio-printed liver tissue", of ∼1 mm, was then used for long-term viability assessments (over 25 days) based on ATP, albumin, and urea levels. Hepatotoxicity evaluation was performed by analyzing the expression of genes involved in drug metabolism and transport over a 2-week drug exposure period. The 3D bio-printed liver tissue showed improved viability and enhanced gene expression of enzymes related to drug metabolism and transport, as compared to the controls. Additionally, the 3D bio-printed liver tissue demonstrated a high sensitivity for hepatotoxicity evaluation when combined with pathological evaluation and measurements for ATP production, and secretion of albumin and urea. In conclusion, the 3D bio-printed liver tissue was able to detect the toxicity of compounds that was, otherwise, undetected by 2D culture and conventionally used spheroids. These findings demonstrate a 3D bio-printed liver tissue with increased accuracy of hepatotoxicity prediction in the early stages of drug discovery, as compared to currently available methods.

Keywords: 3D bio-printed liver tissue; ATP production; albumin secretion; compound toxicity detection; hepatotoxicity sensitivity; hepatotoxicity testing; long-term viability; pathological evaluation; urea secretion.

MeSH terms

  • Adenosine Triphosphate / biosynthesis
  • Bioprinting*
  • Chemical and Drug Induced Liver Injury*
  • Coculture Techniques
  • Cytochrome P-450 CYP3A / metabolism
  • Hepatic Stellate Cells / drug effects
  • Hepatocytes / drug effects
  • Humans
  • Spheroids, Cellular
  • Toxicity Tests / methods*


  • Adenosine Triphosphate
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human