Publicaciones científicas
Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling
Pedro Vicente 1 2, Joana I Almeida 1 2 3, Inês E Crespo 1 2, Nikolaus Virgolini 1 2, Inês A Isidro 1 2, Maria Eréndira Calleja-Cervantes 4, Juan R Rodriguez-Madoz 4 5 6, Felipe Prosper 4 7 5 6, Paula M Alves 1 2, Margarida Serra 8 9
Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates.
We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors' stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors' stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes.
This condition promoted higher HLC production (maximum cell concentration: 2 × 106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6 × 106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity.
Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.undefined.
CITA DEL ARTÍCULO 2024 Oct 19;14(1):24599. doi: 10.1038/s41598-024-75582-z
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