Three-dimensional stroma engineered models would enable fundamental and applicative studies of human tissues interaction and remodeling in both physiological and pathological conditions. In this work, we propose a 3D vascularized stroma model to be used as in vitro platform for drug testing. A pullulan/dextran-based porous scaffold containing pre-patterned microchannels of 100 μm diameter is used for co-culturing of fibroblasts within the matrix pores and endothelial cells to form the lumen. Optical clearing of the constructs by hyperhydration allows for in-depth imaging of the model up to 1 mm by lightsheet and confocal microscopy. Our 3D vascularized stroma model allows for higher viability, metabolism and cytokines expression compared to a monocultured vascular model. Stroma-endothelium cross-talk is then investigated by exposing the system to pro and anti-angiogenic molecules. The results highlight the protective role played by fibroblasts on the vasculature, as demonstrated by decreased cytotoxicity, restoration of nitric oxide levels upon challenge, and sustained expression of endothelial markers CD31, vWF and VEGF. Our tissue model provides a 3D engineered platform for in vitro studies of stroma remodeling in angiogenesis-driven events, known to be a leading mechanism in diseased conditions, such as metastatic cancers, retinopathies and ischemia, and to investigate related potential therapies.

Liver tissue engineering approaches aim to support drug testing, assistance devices, or transplantation. However, their suitability for clinical application remains unsatisfactory. Herein, we demonstrate the beneficial and biocompatible use of porous pullulan-dextran hydrogel for the self-assembly of hepatocytes and biliary-like cells into functional 3D microtissues. Using HepaRG cells, we obtained 21 days maintenance of engineered liver polarity, functional detoxification and excretion systems, as well as glycogen storage in hydrogel. Implantation on two liver lobes in mice of hydrogels containing 3800 HepaRG 3D structures of 100 ​μm in diameter, indicated successful engraftment and no signs of liver toxicity after one month. Finally, after acetaminophen-induced liver failure, when mice were transplanted with engineered livers on left lobe and peritoneal cavity, the survival rate at 7 days significantly increased by 31.8% compared with mice without cell therapy. These findings support the clinical potential of pullulan-dextran hydrogel for liver failure management.

Freeze-drying is a process of choice to texture hydrogel scaffolds with pores formed by an ice-templating mechanism. Using state-of-the-art microscopies (cryo-EBSD, mu CT, CLSM), this work evidences and quantifies the effect of crosslinking and ice nucleation temperature on the porous structure of thin hydrogel scaffolds freeze-dried at a low cooling rate. We focused on a polysaccharide-based hydrogel and developed specific protocols to monitor or trigger ice nucleation for this study. At a fixed number of intermolecular crosslinks per primary molecule (p = 5), the mean pore size in the dry state decreases linearly from 240 to 170 mu m, when ice nucleation temperature decreases from 6-degrees C to -18 degrees C. When ice nucleation temperature is fixed at -10 degrees C, the mean pore size decreases from 250 to 150 mu m, as the crosslinking degree increases from p = 3 to p = 7. Scaffold infiltration ability was quantified with synthetic microspheres. The seeding efficiency was assessed with MC3T3-E1 individual cells and HepaRG (TM) spheroids. These data collapse into a single master curve that exhibits a sharp transition from 100 % to 0 %-efficiency as the entity diameter approaches the mean pore size in the dry state. Altogether, we can thus precisely tune the porosity of these 3D materials of interest for 3D cell culture and cGMP production for tissue engineering.

The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.

Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 µm to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4α and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.

BACKGROUND & AIMS:Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis.METHODS:In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties.RESULTS:P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation.CONCLUSION:P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases.LAY SUMMARY:During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.