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Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

ABSTRACT: Ultrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave field and studied their viability, their functions, and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a conventional 2D system. In addition, constructs created in the AFB and subsequently cultured outside of it had improved functionality including higher albumin and urea production than 2D or pellet cultures. The viability of Huh7 cells grown in the ultrasound field to 5FU anti-cancer drug was comparable to that of cells cultured in the 2D system, showing rapid diffusion into the aggregate core. We have shown that AFB formed 3D cell constructs have improved functionality over the conventional 2D monolayer and could be a promising model for anti-cancer drug testing.

SUBMITTER: Khedr MMS

PROVIDER: S-EPMC6404912 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

Khedr Mogibelrahman M S MMS Messaoudi Walid W Jonnalagadda Umesh S US Abdelmotelb Ahmed M AM Glynne-Jones Peter P Hill Martyn M Khakoo Salim I SI Abu Hilal Mohammed M Abu Hilal Mohammed M

Biomicrofluidics 20190101 1

Ultrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave field and studied their viability, their functions, and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a co ...[more]

PMID: 30867882

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Project description:The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.

Project description:Background & Aims: The increasing requirements for hepatocytes in therapeutics and pharmacy call for an efficient system that enables large-scale production of functional hepatocytes in vitro. Hepatoblast (HB) hold the ability to regenerate liver when damaged, combined with organoid (org) technology, the culture of human pluripotent stem cell (hPSCs) derived HB-orgs was established under 2D culture system, enabling long-term expansion of HB in vitro. However, the expensive cost and the limitation of existing methods impede the further scale up of the production. Methods: Under 3D suspension culture conditions with the support of Matrigel at low concentration in conjunction with employing the developed (defined) medium, we developed a novel system to efficiently and reproducibly generate expandable HB-orgs and functional polarized hepatocyte organoids (P-hep-orgs) derived from hESCs. HB-orgs were further scaled up by dynamic culture in spinner flasks, and the maturity and function of P-hep-orgs was validated by transcriptome analysis and in vitro and in vivo hepatic functional assays. scRNA-seq was used to further profile HB-orgs and P-hep-orgs. Results: Our HB-orgs exhibited biopotency that could differentiate into functional hepatocytes and cholangiocytes. They proliferated actively at least for 15 passages with defined medium through avoiding the excessive autophagy under 3D suspension culture condition. HB-orgs showed higher proliferation rate in dynamic culture conditions and could reach 10^12 cell number at the initiate number of 3 million cells in only 4 weeks. With further differentiation, P-hep-orgs displayed characteristics of hepatocytes in liver, including the polarization state which mediated by integrin-AMPK signalling pathway, molecular features and improved liver functions. Moreover, P-hep-orgs were used for predicting the toxicity of various compounds, and modeling the development of hepatic lipid accumulation, as well as rescuing acute liver failure of mice. Conclusion: Our work provided a cost-efficient method for large-scale generation of 3D HB-orgs and 3D P-hep-orgs that hold considerable potential to be a robust platform for disease research, drug screening/development and toxic study, as well as clinical applications.

Dataset Information

Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

Publications

Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

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