Fig 1.
CYP3A4 Activity Experimental Timeline (Rif = Rifampicin, Mid = Midazolam).
Fig 2.
Experimental Timeline for Trovafloxacin toxicity studies.
Fig 3.
Histological characterization of 3D liver tissues.
A) A macroscopic image of a 3D liver tissue housed in a 24 well transwell. B) H&E staining of a tissue cross-section; compartmentalization between the parenchymal and non-parenchymal fractions can be readily visualized (dashed line). C) ECM deposition assessed by Masson’s trichrome staining. D) IHC staining of the parenchymal compartment for E-cadherin (Green) and Albumin (red). E) IHC staining for CD31 (red) and desmin (green) to assess organization of the endothelial cells and the presence of quiescent hepatic stellates in the non-parenchymal compartment. F) IHC staining for desmin (green) and α-SMA (red) to assess stellate cell activation. White arrows indicate quiescent stellates in the tissue interior that stain positive for desmin and negative for α-SMA. Cells at the tissue periphery stain positive for α-SMA (white arrowhead), suggesting they have a more activated phenotype. G) Oil-red O staining of 3D liver tissue cryosections to measure lipid storage. H) PAS staining to identify glycogen granules. DAPI was utilized to stain the nuclei of the cells in all of the IHC staining samples (Blue). Scale bars in the lower right hand corner of images are 25μm (B-D, G-H) or 50μm (E, F).
Fig 4.
Immunohistochemical assessment of vimentin in bioprinted 3D liver tissues and native human liver.
A) IHC staining for vimentin (brown) to assess the non-parenchymal (mesenchymal) compartment in human liver tissue and B) 3D liver tissues. Parenchymal cells are intermingled with vimentin-positive mesenchymal cells.
Fig 5.
Measurement of tissue ATP and the secreted factor albumin from3D liver tissues over 28 days.
A) Cell Titer Glo (Promega) was used to assess the levels of ATP in 3D liver tissues over time. Data shown is the average of 3 replicates +/- standard deviation. B) Albumin was measured in the supernatant of 3D liver tissues over 28 days in culture by ELISA. Data shown is the average of 5 replicates +/- standard deviation. C) Levels of ATP in 3D liver tissues (A) were compared to those from standard 2D hepatocyte culture over 28 days by normalizing to day 3 levels. Data shown is the average of 3 replicates +/- standard deviation. D) Levels of albumin (ALB) in 3D liver tissues (B) were compared to those from standard 2D hepatocyte culture over 28 days by normalizing to day 3 levels. Data shown is the average of at least 5 replicates +/- standard deviation. All statistics (One way ANOVA) and outliers (Grubbs’ test) were calculated using GraphPad Prism software; * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.
Fig 6.
Expression of and function of CYP enzymes in 3D liver tissues.
A) Quantitative RT-PCR was used to assess levels of CYPs 1A2, 2B6, 2C9, 2D6, and 3A4 in bioprinted liver tissues at the time points indicated. Data is expressed relative quantity (RQ) * 10000 after normalization to GAPDH, and is the average of 3 tissues +/- standard deviation. B) Basal and Rifampicin-induced CYP3A4 activity, measured by the formation of 4-hydroxymidazolam at the timepoints indicated, was determined using mass spectrometry. Data shown is the average of 3 replicates +/- standard deviation. C) Quantitative RT-PCR was used to assess levels of basal and Rifampicin-induced CYP3A4 in bioprinted liver tissues at the time points indicated. Data is expressed relative quantity (RQ) after normalization to GAPDH, and is the average of 3 tissues +/- standard deviation. All statistics (One way ANOVA) and outliers (Grubbs’ test) were calculated using GraphPad Prism software; * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.
Fig 7.
Biochemical Effects of Trovafloxacin and Levofloxacin in 3D bioprinted liver tissues.
A) Measurement of secreted factor albumin in the supernatant of 3D liver tissues treated with Trovafloxacin or Levofloxacin daily for 7 days. Data is expressed as the percentage relative to the vehicle average and is the average of 10 replicates across two independent experiments +/-standard deviation. B) Measurement of ATP levels in 3D liver tissues treated with Trovafloxacin or Levofloxacin daily for 7 days. Data is expressed as the percentage relative to the vehicle average and is the average of 6 replicates across two independent experiments +/-standard deviation. C) Levels of albumin (ALB) in 3D liver tissues treated with Trovafloxacin (A) were compared to those from standard 2D hepatocyte cultures treated with Trovafloxacin daily for 7 days by normalizing to vehicle control. Data shown for 2D cultures is the average of 4 replicates +/- standard deviation. D) Levels of ATP in 3D liver tissues treated with Trovafloxacin (B) were compared to those from standard 2D hepatocyte cultures treated with Trovafloxacin daily for 7 days by normalizing to vehicle control. Data shown for 2D cultures is the average of 4 replicates +/- standard deviation. All statistics (One way ANOVA for 7A, B; Two way ANOVA for 7C, D) and outliers (Grubbs’ test) were calculated using GraphPad Prism software; * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.
Fig 8.
Histological Effects of Trovafloxacin in 3D bioprinted liver tissues.
H&E staining of an untreated (Media) or 100 μM Trovafloxacin-treated 3D liver tissue, with cross-sections captured at either 10X or 50X magnification to visualize salient features. Histological analysis shows loss of cellular adhesion (arrow) and increased hepatocyte necrosis (arrowheads) in Trovafloxacin treated tissues.