To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells / endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from 6 independent donors, we focused on donor variability and cell crosstalk. Coculture was performed on calcium phosphate granules in a specific chamber (one month). MSCs were seeded first then ECs were added after two weeks, with control monocultures. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralization stage of maturation. The delayed ECs addition ECs allowed for their attachment on developed MSCs. The main impact of donor variability could be the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier’s permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using paracetamol, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of paracetamol by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.Keywords: Organ-on-chip, Liver, LSECs barrier, HepG2/C3a, coculture, microfluidic

We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self-assembling into spheroids. They adequately differentiate into hepatocyte-like cells, with hepatic features observed at day 14 post-encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a Poly-L-Lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly-designed perfused dynamic bioreactor (PDB), in the presence of the medium model for pathological plasma for 6 hours. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro-encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.