Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device.

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice.

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 × 10 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days.

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.

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.

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 IC50 values for each compound correctly classified them as potent inhibitors.

Long-term culture and expansion of primary human hepatocytes.

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 10 to 10 cells from a single human hepatocyte isolate.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.

Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro.

Optimization of upcyte® human hepatocytes for the in vitro micronucleus assay.

"Upcyte(®) human hepatocytes" have the unique property of combining proliferation with the expression of drug metabolising activities. In our current study, we evaluated whether these cells would be suitable for early in vitro micronucleus (MN) tests. A treatment period of 96 h without a recovery period was most reliable for detecting MN formation in upcyte(®) hepatocytes from Donor 740.

Upcyte® microvascular endothelial cells repopulate decellularized scaffold.

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality.

Generation of proliferating human hepatocytes using Upcyte® technology: characterisation and applications in induction and cytotoxicity assays.

1. We have developed a novel technique which causes primary human hepatocytes to proliferate by transducing them with genes that upregulate their proliferation. 2.