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Mattei G.,University of Pisa | Magliaro C.,University of Pisa | Giusti S.,University of Pisa | Ramachandran S.D.,Medicyte GmbH | And 3 more authors.
PLoS ONE | Year: 2017

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. © 2017 Mattei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: HEALTH.2010.4.2-9-2 | Award Amount: 9.40M | Year: 2011

The goal of HeMiBio is to develop a hepatic microfluidic bioreactor from human iPSC-derived hepatocytes, hepatic sinusoidal endothelial cells (HSEC) and stellate cells (HSC), suitable for inclusion in a repeated dose toxicity testing strategy of pharmaceuticals/cosmetic ingredients. The successful creation of such a liver-device requires (a) homotypic and heterotypic interactions between the three cell types to induce and maintain their functional, differentiated state, and (b) optimisation of the matrix, oxygenation conditions, nutrient transport and physiological shear forces. The objectives are (1) to engineer the cellular components incorporated in the bioreactor to enable specific and spatially defined enrichment of the different cells from iPSC progeny, and, by gene editing, to allow non-invasive monitoring of the cellular state (differentiation and damage). (2) Aside from the molecular sensors, an array of electro-chemical sensors will be embedded in the reactors to assess liver-specific function and cellular health under repeated dose toxicity conditions, dynamically and in a high-throughput way. Cells and sensors will be built into (3) bioreactors that will be sequentially upgraded from 2D to 3D microfluidic reactors to ultimately allow full maintenance of mature functional hepatocytes, HSC and HSEC for >28 days. (4) As the ultimate goal is to use the device as a human-based alternative to rodent long-term hepatotoxicity studies, it will be of utmost importance to provide proof of concept that the 3D-devices reveal the hepatotoxicity of prototypical hepatotoxic compounds in vivo (5). -Omics and cell functionality studies will provide evidence that liver-like cells are present, exposed and affected by the selected toxic compounds. These ambitious objectives will be achieved by the excellent project team, composed of academic/industrial partners with unique and complementary biology, physiology, toxicology and technical skills from 7 EU Member States.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: HEALTH-2009-1.4-2 | Award Amount: 15.57M | Year: 2010

The goal of VascuBone is to develop a tool box for bone regeneration, which on one hand fulfils basic requirements and on the other hand is freely combinable with what is needed in the respective patients situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging based in vivo diagnostics which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of three different types of bone defects. The chosen bone diseases differ in their requirements, to ensure a successful implementation and translation. Additionally this implementation strategy is characterized by high complexity to remove bottlenecks and limitations of bone regeneration identified in the clinical setting. Therefore definite quality criteria have to be evaluated concerning an optimal stem cell source/subpopulation as well as GF concentrations and their bioactivity in vivo. Furthermore quantitative evaluation will focus on the definition of differences between stem cell populations responsible for bone regeneration in young and old people, as a prerequisite for the development of regenerative therapies for the ageing European society. Considering a successful and prompt approval of the biomaterial, for each clinical application a minimum of modification steps in daily routine must be identified. The road map of the project and clinic trials contains pre-determined milestones, to ensure efficacy, safety, and immunological acceptance of the implant. The efficacy is quantified by high innovative MRI and PET/CT technology which is able to demonstrate the regenerative effect of biomaterials and cells in vivo. Based on the clinical data the proposal as Advanced Therapeutical Medicinal Product will be submitted to the European Medicines Agency at the end of the project.


Levy G.,Hebrew University of Jerusalem | Bomze D.,Hebrew University of Jerusalem | Heinz S.,Upcyte Technologies GmbH | Ramachandran S.D.,Medicyte GmbH | And 7 more authors.
Nature Biotechnology | Year: 2015

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 1013 to 1016 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development. © 2015 Nature America, Inc.


PubMed | Medicyte GmbH, University of Mannheim, University of Würzburg, University of Tromsø and University of Heidelberg
Type: Journal Article | Journal: PloS one | Year: 2015

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.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.1.4-2 | Award Amount: 5.38M | Year: 2012

The liver, by way of its central role in both endogenous and exogenous metabolism, is one of the most well-studied organs in the human body. Hepatic tissue and its derivatives have a wide range of in-vivo and in-vitro applications from whole organ or partial lobe transplant, bioartificial ex-vivo devices, treatment of metabolic disorders to toxicology, drug metabolism and tissue regeneration. On the one hand, suitable donor livers for solid organ transplant are in short supply, while chronic liver diseases are on the increase both in Europe and world-wide. On the other hand, in-vitro and ex-vivo technologies for recapitulating liver function still fall short of reliability, consistency and predictivity, precluding many commercial applications. There is a dire need for innovative and reproducible methods for developing functional bioartificial livers or portions of liver which can be easily transplanted or reliably integrated into extracorporeal devices, essential for treating acute liver failure and other metabolic liver disorders. To avoid the risks and complications associated with animal/human matrices, yet furnish a reliable and reproducible 3D microarchitecture capable of maintaining the detoxification and metabolic functions of healthy human liver, our aim is to fabricate a novel hepatic lobuli ECM replica seeded with stable human hepatocytes and endothelial cells using the human liver as a design template through a bottom up approach. These cell-containing bioartificial constructs will be developed and characterized in vitro to assess metabolic function, protein production and angiogenic potential. They will then be implanted in animal models through minimally invasive techniques as a solid organ transplant alternative to recombinant Factor VIII-based therapy for patients with haemophilia A.


Grant
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2013-ITN | Award Amount: 3.81M | Year: 2013

BIO-INSPIRE will develop a new horizon in Orthopaedic Therapy by: 1. Development of a technology platform that consists of new bio-active and bio-mimetic materials and their therapeutic applications for bone regenerative purposes. 2. Training of the next generation of leading Tissue Engineering scientists in a multi-disciplinary way, leading them towards excellence in individual disciplines in combination with a multidisciplinary, holistic view on the (bone tissue) biological systems to be studied. The consortium consists of a unique alliance of carefull selected Partners with a high reputation in a set of complementary disciplines (as required for a multi-disciplinary program in the field of bone regeneration) consisting of Fraunhofer, Medicyte (field of Cell therapy), Fujifilm Life Science (Bio-Materials), ISTEC (Bone Mineralisation), Erasmus MC (Growth Factors), Un of Bologna, Bone Therapeutics (Orthopaedic Therapy). This combination provides a unique multidisciplinary research environment for 16 hosted Fellows. The industrial participation is high, being the coordinator and chairing the project and hosting 8 Fellows out of 16 Fellows, immersing those Fellows in both an academic and an industrial research setting. This project will provide: - A new generation of scientists, capable to participate in multi-disciplinary, pan-European project teams, mastering their individual disciplines in combination with a multi-disciplinary, holistic view. - A new class of bio-material prototypes, bio-mimetic, triggering cascades of bone regenerative processes, ready for clinical trials beyond BIO-INSPIRE for a variety of therapeutic needs. - A robust pan-European network for development of bio-mimetic materials, consisting of Academic and Industrial participants, acting as an European Network for Innovation and Education for talented scientists in the field of tissue regenerative technologies and medical therapies.

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