Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation.

Lmar Babrak,Gaia Titolo,Michaela Caj,Catherine Jane Messner,Laura Suter-Dick,Enkelejda Miho

doi:10.3390/ijms22094372

Abstract

3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-β1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-β1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).

Highlights

The generation of 3D-cell culture systems encompassing multicellular interactions has enabled the generation of in vitro liver models that retain in vivo like properties and many physiological functions
We set out to isolate cells from 3D-liver MTs consisting of three cell types and to identify their individual responses to the pro-fibrotic cytokine TGF-β1
We successfully established a reproducible protocol for the dissociation of multicellular hepatic MTs that did not cause cellular stress or affect the maintenance of important cellular characteristics, such as attachment, production of albumin or responsiveness to TGF-β1

Summary

Introduction

The generation of 3D-cell culture systems encompassing multicellular interactions has enabled the generation of in vitro liver models that retain in vivo like properties and many physiological functions. Existing 3D-liver models vary regarding cell types, media, 3Darchitecture and flow conditions. These systems have conclusively demonstrated tremendous improvement in the ability to mimic and predict liver disease, hepatic metabolism, hepatotoxicity of drugs as compared to 2D, monocellular in vitro models [1]. Spheroids are a one of the most ubiquitous 3D-systems applied to the culture of liver cells and may be composed of primary or established cell lines. Either way, they are often utilized as multicellular systems of varying complexity, ranging from hepatocyte-only systems; to combination of hepatocytes with one or several types of hepatic non-parenchymal cells. Whole genome analysis of primary human hepatocytes (PHHs) and two commonly used cell lines

Methods

Results

Conclusion