On the adhesion-cohesion balance and oxygen consumption characteristics of liver organoids

Liver organoids (LOs) are of interest in tissue replacement, hepatotoxicity and pathophysiological studies. However, it is still unclear what triggers LO self-Assembly and what the optimal environment is for their culture. Hypothesizing that LO formation occurs as a result of a fine balance between cell-substrate adhesion and cell-cell cohesion, we used 3 cell types (hepatocytes, liver sinusoidal endothelial cells and mesenchymal stem cells) to investigate LO self-Assembly on different substrates keeping the culture parameters (e.g. culture media, cell types/number) and substrate stiffness constant. As cellular spheroids may suffer from oxygen depletion in the core, we also sought to identify the optimal culture conditions for LOs in order to guarantee an adequate supply of oxygen during proliferation and differentiation. The oxygen consumption characteristics of LOs were measured using an O2 sensor and used to model the O2 concentration gradient in the organoids. We show that no LO formation occurs on highly adhesive hepatic extra-cellular matrix-based substrates, suggesting that cellular aggregation requires an optimal trade-off between the adhesiveness of a substrate and the cohesive forces between cells and that this balance is modulated by substrate mechanics. Thus, in addition to substrate stiffness, physicochemical properties, which are also critical for cell adhesion, play a role in LO self-Assembly

Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A [Abstract]

Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A [Abstract

Applicability of second-generation upcyte®^{®} human hepatocytes for use in CYP inhibition and induction studies

Human upcyte$^{®}$ hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte$^{®}$ hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC$_{50}$ values for each compound correctly classified them as potent inhibitors. Upcyte$^{®}$ hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte$^{®}$ hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUC$_{u}$/F$_{2}$, and C$_{max,u}$/Ind$_{50}$. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte$^{®}$ hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte$^{®}$ hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations

A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits

Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p–green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export

Evaluation of a miniaturized biologically vascularized scaffold in vitro and in vivo

In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine

Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 x 10$^6$ intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 x 10$^3$ to 1:1 x 10$^6$ human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies

Mean Pixel Intensity (MPI) for green (Integrin-β1) and red (Connexin-32) channels.

<p>The MPI was calculated from n = 3 independent experiments per substrate investigated, imaging and analysing n = 3 different DAPI-positive areas in each sample (randomly selected). Different letters indicate significant differences between groups (one-way ANOVA, <i>p</i> < 0.05): lowercase letters are referred to ANOVA results of green MPI, while capital letters denote ANOVA results of red MPI. Note that the green and red MPI levels cannot be compared due to differences in the laser power used and in the fluorophore excitation/emission efficiencies.</p