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Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes

ABSTRACT: In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

ORGANISM(S): Homo sapiens

PROVIDER: GSE166256 | GEO | 2021/11/23

REPOSITORIES: GEO

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Project description:The liver is the most common site for colorectal cancer (CRC) metastasis and new tissue culture models are needed to study hepatic metastasis from CRC as none of the current models mimic the biological, biochemical and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of cancer extracellular matrix (ECM) as decellularized scaffolds retained their tissue-specific features and biological properties. In the present study we created a 3D model of CRC and matched CRC liver metastasis (CRLM) using patient-derived decellularized ECM scaffolds seeded with HT-29 CRC cell line. Here we show increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. CRLM scaffolds also induced activation of epithelial-mesenchymal transition (EMT) with cancer cells showing loss of E-cadherin expression and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacethylation, cellular response to stress metabolic processes, response to oxygen level and to starvation. The complex 3D culture environment reduced the HT-29 cell response to anti-CRC drug 5-FU when used at standard IC50 determined in 2D cultures. In conclusion, our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of liver CRC metastasis formation and progression. Transcriptomic analysis was performed comparing the global gene expression profiles of HT29 cells grown on CRLM and HL scaffolds. The transcriptomic profile of each 3D subtype was compared with HT29 cells grown in plastic. Hierarchical clustering analysis revealed that the gene expression signature of recellularized CRLM scaffolds was more comparable to recellularized HL scaffolds, than to HT29 cells grown in 2D. Gene set enrichment analysis (GSEA) of the differently expressed genes (DEG) up-regulated in recellularized CRLM scaffolds compared to HT29 grown in 2D revealed that genes involved in demethylation, deacethylation and metabolic process belong to the most enriched biological pathways in repopulated-CRLM scaffolds. Comparing the DEG between recellularized CRLM scaffolds vs HT29 grown in 2D with a series of a-priori defined gene-set, called Hallmark, we obtained concordant results with the “HYPOXIA” and “EPITHELIAL_MESENCHYMAL_TRANSITION” pathway, enriched in recellularized CRLM, supporting the idea that 3D culture models are the ability to re-create a complex environment in terms of oxygen tension, nutrients, and metabolic gradient, similarly to the in vivo environment.

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Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes

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