Liver microtissue maintenance and disease induction
hLiMTs were comprised of a pool of PHH from 10 donors, single-donor HSC and single-donor KC and LEC (MT-02–302-05, InSphero AG). The informed consent of the human liver cells for all the subjects was obtained from the vendor. hLiMTs were generated by self-assembly of monodispersed primary cells, as described previously18,59,60. hLiMTs were maintained at 37 °C, 5% CO2 and 95% humidity. To mimic MASH disease induction and progression, hLiMTs were exposed for 10 days to either LEAN or MASH conditions (Fig. 1B). LEAN condition hLiMTs were cultured in basal hepatocyte maintenance medium (CS-07–302-01, InSphero AG). Under disease conditions, the MASH induction medium was supplemented with elevated insulin amount, glucose and fructose levels (total of 22.5 mM) (CS-07–301-01, InSphero AG), FFAs (167 µM, CP-02–302, CP-02–303, InSphero AG), and LPS (5 µg/ml LPS, Sigma-Aldrich). LPS was applied as a pulsing on day 3 of treatment. Medium was exchanged on days 0, 3, 5, and 7.
Compound treatment
The anti-fibrotic effects of reference compounds, 0.5 µM ALK5i (SB525334, Selleckchem) and 0.001 and 0.1 µM anti-TGF-β Ab (MAB1835, R&D Systems), were investigated by incubating hLiMTs in MASH induction medium containing high levels of sugars, insulin, FFAs and LPS pulse in the presence or absence of the drug (Fig. 1B). Compound treatments were performed on days 0, 3, 5 and 7, using a TECAN D300e Digital Dispenser or manually for the anti-TGF-β Ab. All conditions were normalized to vehicle controls 0.2% DMSO or PBS (only for anti-TGF-β Ab).
For the efficacy test of anti-MASH clinical compounds an optimized 3D MASH model was used. This optimized 3D MASH model was generated using the improved production protocol of the microtissues to achieve reduced levels of secretion of pro-collagen type I and tissue triglyceride levels in the LEAN conditions. The clinical test compounds were Firsocostat (HY-16901, MedChemExpress) and Selonsertib (HY-18938, MedChemExpress). These and the reference compound, ALK5i and anti-TGF-β Ab, were applied during the 10-day MASH induction protocol. The final concentrations were 0.5 µM ALK5i, 0.001 µM and 0.1 µM anti-TGF-β Ab, 0.5 µM and 10 µM Firsocostat, 2 µM and 10 µM Selonsertib, and a combination of 10 μM Selonsertib and 0.5 μM Firsocostat.
All stock solutions were prepared in DMSO except for anti-TGF-β Ab stock, which was prepared in PBS. The anti-fibrotic effects of the tested compounds were assessed according to several endpoint measurements described below.
Endpoint assays
All assays were performed with 4 to 6 biological replicates per treatment group and were repeated at least twice. For all experiments the same lots of PHH, KC, LEC and HSC were used.
Pro-collagen I secretion measurement
The levels of cleaved and secreted C-terminal human collagen type I pro-peptide in hLiMT supernatants collected from day 10 were measured with the human pro-collagen I homogeneous time-resolved fluorescence (HTRF®) assay (Cisbio). PBS (Sigma-Aldrich) diluted samples were processed according to the supplier’s instructions. Fluorescence was measured with a Tecan Spark 10M plate reader. Pro-collagen I secretion rate per hLiMT was calculated by linear fit to a standard curve. Values normalized to incubation time (days) were plotted as mean ± SD, including individual datapoints. A Brown-Forsythe version of one-way ANOVA (analysis of variance) with Welch’s correction in combination with Dunnett’s T3 multiple comparisons test vs MASH was used to calculate significant changes.
Pro-collagen III secretion measurement
The levels of cleaved and secreted N-terminal human collagen type III pro-peptide in hLiMT supernatants collected from day 10 were measured with the human N-Terminal Pro-collagen III immuno-assay (P3NP-ELISA, Cisbio). PBS diluted samples were processed according to the supplier’s instructions. Optical density (OD) at 450nm was measured with a Tecan Spark 10M plate reader. Pro-collagen III pro-peptide secretion per hLiMT was calculated by 4-parameters logistic (4-PL) mathematical fit curve. Interpolated data points below the lowest standard value were excluded from the data set. Values normalized to treatment time were plotted as mean ± SD, including individual datapoints. A Brown-Forsythe version of one-way ANOVA with Welch’s correction in combination with Dunnett’s T3 multiple comparisons test vs MASH was used to calculate significant changes.
Pro-fibrotic markers secretion
Pro-fibrotic markers TIMP-1, TIMP-2, MMP-1, MMP-2, MMP-3 and MMP-9 in cell supernatants were determined on day 10 using the Magnetic Luminex® Performance Assay: Human TIMP Multiplex Kit, LKTM003, TIMP-1 and -2 (premixed, Bio-Techne), Human MMP Multiplex Kit, LXSAHM-07 (Bio-Techne) and customized ProcartaPlex™, PPX-06 (MMP-1, -2, -3, -9, TIMP-1, eBioscience). The multiplexed assay was performed according to supplier’s instructions with minor adaptation of microparticle and Ab concentrations to analyte levels present in supernatants. Measurements were performed with a Luminex® MAGPIX® analyzer. Protein concentrations were calculated by 5 parameter logistic fit (5-PL) to a standard curve using Luminex XPonent software. Interpolated data points below the lowest standard value were excluded from the data set. Data are represented as mean values ± SD, including individual datapoints. A Brown-Forsythe version of one-way ANOVA with Welch’s correction vs MASH in combination with Dunnett’s T3 multiple comparisons test vs MASH was used to calculate significant changes. Outliers were detected based on Grubbs’ test (α = 0.05).
RNA sequencing (RNA-Seq) and analysis
RNA-Seq in the study was performed by using whole transcriptome TempO-Seq® assay consisting of 22,537 probes targeting 19,701 genes (BioSpyder Technologies, Inc.)61. TempO-Seq samples were generated as follows: upon experiment completion on day 10 of treatment, single hLiMTs were washed in PBS without Ca2 + /Mg2 + and lysed in 15 µl of 1 × Enhanced Lysis Buffer (BioSpyder Technologies, Inc.). Libraries generation from crude lysates and single-end 50 bp sequencing on Illumina HiSeq 2500 platform were performed by BioSpyder Technologies, Inc. After sample demultiplexing and obtaining sample-wise FASTQ files, reads alignment and counting were performed using TempO-SeqR data analysis tool (BioSpyder Technologies, Inc.). The gene expression data were then analyzed with the InSphero RNA-Seq analysis pipeline. In brief, the probe-wise raw count table was collapsed toward the gene-wise count table by summating counts for probes associated with the same gene. Next, the gene-wise count table was cleaned from genes that were not detected or very lowly expressed. Subsequently, data normalization, principal component analysis (PCA) and differential expression analysis (DEA) were performed as implemented in the DESeq2 R package62. Importantly, surrogate variable analysis (SVA)63 was applied to remove unintended batch effects by using the sva R package. Furthermore, the pre-ranked type of GSEA64 was performed using the clusterProfiler R library60,65. In GSEA, genes were pre-ranked by log2FC values derived from DEA and normalized enrichment score (NES) was used as an enrichment metric. The following resources from Molecular Signatures Database (MSigDB)66 version 7.5 were used to build a repertoire of gene sets queried in GSEA: BioCarta [BioCarta67], Hallmark66, PID68, Reactome69 and WikiPathways70, in total 2817 gene sets. For both, DEA and GSEA, the Benjamini–Hochberg procedure for false discovery rate (FDR)71 was used to correct p values across contrasts, and the FDR cut-offs were set to 0.01 and 0.05, respectively.
Formalin-fixation and paraffin-embedding (FFPE) of hLiMTs
On day 10 of treatment, > 18 hLiMTs per treatment group were pooled, washed with PBS, and fixed with 4% paraformaldehyde (PFA, Alfa Aesar) for 1 h at RT. The fixed hLiMTs were washed in PBS, pelleted in 1.7% agarose, and further processed with a tissue processor (Logos Microwave Hybrid Tissue Processor; Milestone Medical S.r.I) for paraffin embedding. The paraffin blocks were sectioned to 4 µm thickness using a microtome (Histocore Multicut, semimotorized Rotation Microtome; Leica Biosystems). The sections were mounted on poly-L lysin treated glass slides (HistoBond® + S with grounded edge; Marienfeld) and dried for 60 min at 65°C.
Immunohistological (IHC) and SR staining
Hematoxylin (Mayers Hematoxylin, Leica Biosystems) and eosin (eosin 2% aqueous, Chroma Waldeck) staining was performed as described previously72. For visualization of fibrillated collagen structures, a SR staining procedure73 with slight modifications, was applied. After rehydration, the sections were stained with SR solution (SR solution, Chroma Waldeck) for 5 min and stopped with 0.5% acidic acid solution (without hematoxylin counterstaining).
Quantification of fibrosis using SR-stained tissues
Digital image analysis and fibrosis quantification
SR-stained sections images of the treated tissues with ALK5i and anti-TGF-β Ab were captured using a Leica DMi8 microscope at 10x (HC PL FLUOTAR 10x/0.32 PH1) or 20x (HC PL FL L 20x/0.40 CORR PH1) magnification. Automated white balance correction was applied, and images were captured with a DMC4500 digital camera. For the clinical compounds treatments, the whole slides with SR stained sections were digitally scanned in an Aperio GT 450 DX system (Leica Biosystems Inc.) at 40 × (0.221 μm per pixel). Automated white balance correction was applied, and images were captured with a DMC4500 digital camera. For the PL images, a motorized, polarized filter was used. Images were inverted using ImageJ invert function for better visualization. The images were saved as svs or TIFF files. FibroNest™ image analysis platform was used to quantify the fibrosis in these MASH hLiMT slices. This analysis had several steps, outlined here.
Microtissue adequacy
Each microtissue was evaluated for quality by size. Microtissues which were too small (< 50% of mean microtissue area) were excluded from the study.
Fiber detection
FibroNest™ was used for the quantification of SR- positive stained collagen fibers from each image. After deconvolution (an algorithm-based image processing to extract collagens stained with SR) and selection of adequate microtissues for analysis, FibroNest™ was used to detect each collagen fiber in a stained digital histology image (Fig. 1A). The architecture (i.e., the organization and the complex buildup of multiple fibers) was measured by dividing the image into “computational windows” of 25 μm × 25 μm and then the fibrosis phenotype quantified in each window.
Analysis of fibers
FibroNest™ II image analysis software was used previously for the quantification of fibrosis in clinical and preclinical MASH tissues generating more than 300 phenotypic qFTs, which are organized into 3 different sub-phenotypes including collagen content, morphometry, and architecture21,22,23,24,25,28. Ph-FCS is a multifactorial and continuous score to quantify the phenotype of fibrosis, including collagen content, fibers morphometrics, and fibrosis architecture. Collagen-FCS is a multifactorial and continuous score for tissue collagen content with a focus on its general properties including fine and assembled collagen, collagen density, and collagen reticulation. Morphometric-FCS describes the collective morphometric traits of each individual fiber including fiber length, width, area, perimeter, and area to perimeter ratio. The morphometric score can further be segregated into fine and assembled fibers. Architecture-FCS includes entropy, inertia, correlation, and homogeneity to describe collagen fiber disorganization, compactness, patterns presence, distribution uniformity, and distortion of fibers.
The algorithm for the quantification of fibrosis in the clinical samples was adapted to MASH and LEAN hLiMTs. Only parameters and traits which were significantly changed in the MASH hLiMTs compared to the healthy LEAN control were selected for further analysis. Each collagen fiber was evaluated for histological traits such as fiber length, number of branches, and homogeneity, all of which were quantified. Additionally, collagen fibers were classified as “fine” or “assembled” based on the complexity of the collagen network, and the morphometric phenotypes which can be quantified for each subgroup. Assembled collagens appear as more reticulated collagen fiber network containing longer and thicker fibrils with many branches and nodes, whereas fine collagens are small and narrow collagen fibrils with minimal branches24. There are 32 histological phenotypic traits: collagen content (12 traits), collagen fiber morphometry (13 traits) and fibrosis architecture (7 traits). The histological traits were then evaluated further to determine a variety of statistical features, such as the mean, median and standard deviation. These are output as continuous variables defined as qFTs accounting for severity, progression, distortion, and variance for both fine and assembled collagens. A total of 316 qFTs were measured in MASH hLiMTs using FibroNest™ II platform, but only 43 qFTs were selected to generate the Ph-FCS. These 43 qFTs were preselected based on a calibration study that showed a significant changed in MASH hLiMTs vs LEAN or reference anti-fibrotic compound treatment. The 43 principal qFTs were distributed in collagen content (9), morphometric (18) and architectural (16) sub-phenotype parameters used for the assessment of severity of the disease (Supplementary Tables 1 and 2). The relative changes of each qFT between the microtissues and groups were visualized in the form of a heatmap. FibroNest™ analysis was conducted blindly to the SR-stained histological slides. The quantification of fibrosis upon treatment with ALK5i was conducted in three independent experiments to validate the algorithm. The quantification of fibrosis with reference anti-fibrotic compounds such as ALK5i and anti-TGF-β Ab was performed using FibroNest™ II software, where 43 qFTs were used and 7 to 10 microtissues per treatment group were analyzed. The optimized and next generation high sensitivity FibroNest™ III software was used for the quantification of fibrosis of clinical compounds Firsocostat and Selonsertib. FibroNest™ III software was able to detect even finer fibers of collagens than the FibroNest™ II platform. The quantification of fibrosis in optimized MASH hLiMTs using the highly sensitive and improved FibroNest™ III software enabled an increase of the number of significantly changed in MASH LiMTs vs LEAN or compound treatment qFTs from 43 to 206, covering many parameters defining the fine and assembled collagen. Out of 336 qFTs measured by FibroNest™ III software, 206 qFTs were changed in MASH vs LEAN hLiMTs and distributed among the various sub-phenotypes such as collagen content (10), morphometry (160) and architectural parameters (36) (Supplementary Table 3). For the quantification of fibrosis of MASH hLiMTs treated with clinical compounds 11 to 20 microtissues were used.
Creation of composite scores
Principal qFTs were automatically detected and combined into a normalized Ph-FCS. The Ph-FCS is a continuous fibrosis severity score (Ph-FCS, 1 to 10). The Ph-FCS is an aggregate of 3 sub-composite scores: the collagen-FCS, the morphometric-FCS and the architecture-FCS. Furthermore, each qFT is described individually for relative severity from least to most (green to red, respectively) in phenotypic heatmaps. A Brown-Forsythe version of one-way ANOVA with Welch’s correction in combination with Dunnett’s T3 multiple comparisons test vs MASH was used to calculate significant changes.
Ethical compliance information
InSphero is working in compliance with the Swiss Federal Act on Research involving Human Beings (810.30) and does not require itself to receive ethical approval in using the sourced human biological post-mortem samples in the development and production of its biological products, because those samples were received anonymized. All the methods were carried out in accordance with relevant guidelines and regulations.
Additional information
The contents of MSigDB are protected by copyright © 2004-2020 Broad Institute, Inc., Massachusetts Institute of Technology, and Regents of the University of California, subject to the terms and conditions of the Creative Commons Attribution 4.0 International License. MsigDB gene sets derived from KEGG pathways are protected by copyright, © 1995-2017 Kanehisa Laboratories, all rights reserved. EMA implementation of 3Rs principles: https://www.ema.europa.eu/en/homepage. FDA Technology Modernization Act 2.0: https://www.fda.gov/about-fda/reports/modernization-action-2022.
