# Multicellular Organotypic Mouse Model of Alcoholic Liver Disease

> **NIH NIH R21** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2023 · $242,942

## Abstract

ABSTRACT
Alcohol-associated liver disease (ALD) is the major cause of alcohol-related mortality and encompasses
steatosis, steatohepatitis with or without progressive fibrosis, hepatocellular injury, and loss of liver function.
Despite considerable research, the specific mechanisms underlying ALD development and progression have not
been fully elucidated, partly due to the lack of in vitro and in vivo model systems that recapitulate human ALD.
While live mice are used to model features of human ALD, current models are labor-intensive, induce significant
mortality (unlike humans), include diets that induce confounding non-alcoholic steatohepatitis, and/or cannot
reproduce all features of human ALD. In contrast, we developed a simple-to-administer 16-week western diet
alcohol (WDA) model that recapitulates the inflammatory, fibrotic, and gene expression aspects of human
alcohol-associated steatohepatitis (ASH). However, it is difficult to investigate the direct effects of alcohol on
multiple liver cell types and elucidate the cell-cell interactions important for ALD pathogenesis in vivo. In contrast,
while primary liver cells can be isolated with high purity from livers to build in vitro models, they rapidly lose
phenotypic functions in 2D monocultures. To mitigate this limitation, we utilized high-throughput droplet
microfluidics to fabricate highly monodisperse extracellular matrix (ECM)-based engineered 3D liver microtissues
containing hepatocytes and liver non-parenchymal cells (NPCs) that functionally outperform conventional self-
assembled cell spheroids and cells embedded within bulk gels. Here, we will leverage the above advances to
test the novel hypothesis that microtissues containing multiple primary mouse liver cells can recapitulate the
critical features of ASH as in the WDA mouse. In aim 1, we will fabricate and optimize long-term (4+ weeks) 3D
liver microtissues containing primary mouse hepatocytes, liver endothelial cells, hepatic stellate cells, and
Kupffer cells; the role of ECM and tissue size will be investigated towards inducing high (physiologic) and stable
cell functions. We will further assess the effects of in vitro ethanol exposure on each cell type within microtissues.
In aim 2, we will investigate cell-cell interactions in liver microtissues derived from cells isolated from the WDA
mice. Microtissues will be cultured with or without ethanol and cellular functions as well as single cell RNA
sequencing data will be compared to control microtissues and existing RNA sequencing data from freshly
isolated cells from WDA mice. We will further examine the role of each of the alcohol-specific NPC type in
maintaining hepatic function and regulating inflammatory responses and fibrogenesis in microtissues. Our efforts
will yield a first-of-its-kind in vitro organotypic mouse liver model with long-term functions, which will be utilized
to elucidate the direct effects of alcohol on multiple liver cell types, the extent to which the AL...

## Key facts

- **NIH application ID:** 10667672
- **Project number:** 1R21AA030397-01A1
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** Salman R Khetani
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $242,942
- **Award type:** 1
- **Project period:** 2023-06-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10667672

## Citation

> US National Institutes of Health, RePORTER application 10667672, Multicellular Organotypic Mouse Model of Alcoholic Liver Disease (1R21AA030397-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10667672. Licensed CC0.

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