Speaker
Description
"Introduction
Organ-on-a-chip (OoC) technology shows great potential to accelerate drug discovery and advance personalized medicine. Induced pluripotent stem cells (iPSCs) may enhance the predictivity of the OoC in the assessment of patient’s response to a pharmacological treatment and related toxicities. Nowadays, there are no body-on-a-chip fully based on iPSCs used for drug screening. In this context, our PEGASO project aims to develop the first iPSC-based multiorgan-on-chip for Alzheimer’s disease drug development. The full platform will be composed of six OoC devices connected and loaded with iPSCs-derived models representing the main body systems involved in dementia drug pharmacokinetics (microbiota/gut, immune system, liver, blood-brain-barrier and brain tissue). Here, we present preliminary characterization of an in vitro 3D iPSCs-based liver model to be hosted into our innovative PEGASO OoC device.
Methodology
An in vitro 3D model with commercially available human iPSC-derived hepatocytes and endothelial cells has been chosen as a liver tissue model for the dynamic culture inside the PEGASO OoC, where hepatocytes encapsulated into a collagen-poly(ethylene)glycol hydrogel and endothelial cells have been cultured interconnected once seeded on each side of a porous membrane hosted into the OoC.
A 2D model with seeded membrane was used as control. Cell viability was assessed with MTS assay while albumin and urea produced by hepatocytes with ELISA and urea colorimetric assay respectively.
The mRNA expression level of CYP3A4 in hepatocytes was evaluated with RT-PCR while the protein expression of albumin and HNF4α with Western Blot and immunofluorescence.
The shear stress and oxygen concentration to which the cells are exposed under dynamic conditions have been assessed by a computational model developed with COMSOL Multiphysics ®.
Results
Hepatocytes grown inside the hydrogel 7 days after plating exhibited comparable metabolic activity with cells in 2D control condition. Differently, the liver-specific functions, referred to albumin and urea synthesis, resulted significantly higher in the 3D model with respect to the control.
The protein expression of albumin and HNF4α, that are key hepatocytes markers, analysed with Western Blot and immunofluorescence was increased in the 3D condition.
The expression of CYP3A4 indicated a higher detoxification ability by hepatocytes in 3D condition with respect to the control.
To select the optimal flow rate for the dynamic culture of the iPSC-derived liver in our organ-on-a-chip, a computational simulation was performed with the software COMSOL Multiphysics ®, tailoring the model for hepatocytes specific requirements, that are shear stresses <0.2 Pa and oxygen consumption of 0.3 nmol/s*m3[1,2].
The numerical simulation indicated 30 µl/min as the proper medium flow rate, leading to adequate shear stresses (range 0.01-0.03 mPa) and oxygen concentrations (range 0.18-0.2 mol/m3).
Conclusions
Taken together, the biological and computational results suggest that our 3D liver model is a suitable iPSC-derived model to be hosted and cultured under perfusion in the PEGASO OoC.
Acknowledgments
PEGASO received funding from the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) under the FARE 2019 program (project code R18WWPCXLY).
References
1) MA, L. D, et al. Lab Chip, 18, 2547-2562 (2018).
2) Kang, Y. B. Biotechnol Bioeng, 112, 2571–2582 (2015)."
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