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

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

Histological characterization of 3D liver tissues.

<p>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).</p

Measurement of tissue ATP and the secreted factor albumin from3D liver tissues over 28 days.

<p>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.</p

Experimental Timeline for Trovafloxacin toxicity studies.

<p>Experimental Timeline for Trovafloxacin toxicity studies.</p

Expression of and function of CYP enzymes in 3D liver tissues.

<p>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.</p

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

<div><p>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.</p></div

CYP3A4 Activity Experimental Timeline (Rif = Rifampicin, Mid = Midazolam).

<p>CYP3A4 Activity Experimental Timeline (Rif = Rifampicin, Mid = Midazolam).</p

Histological Effects of Trovafloxacin in 3D bioprinted liver tissues.

<p>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.</p

Biochemical Effects of Trovafloxacin and Levofloxacin in 3D bioprinted liver tissues.

<p>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.</p