Final Report Summary - LIVEBIOMAT (Development of new polymeric biomaterials for in vitro and in vivo liver reconstruction)
The main results of the Livebiomat project are:
New HF membranes
Institute on Membrane Technology (ITM) in Italy designed and synthesised new membranes in hollow fibre configuration according to the well-known dry-wet spinning method from a polymeric blend of modified polyetheretherketone (PEEK-WC). Investigations in polymeric membrane preparation and in the understanding of their transport properties have made possible the realisation of this novel high frequency (HF) membranes for biomedical applications to be used for hepatocytes cultures.
The new PEEK-WC HF membranes yield a useful tool for the reconstruction of liver tissue model in vitro and for the reconstruction of liver tissue in vivo which allow, on the one side, to understand the mechanisms involved and regulating growth and differentiation, and on the other to study their role in the physiology and pathology of the liver.
Plasma process optimisation and immobilisation procedures
It is a class of coating that can be plasma deposited by using the lactide conventional monomer used for polymerisation of polilactic acid. By tuning plasma parameters (thus chemical structure and thickness of the coating) it is possible to tune the degradation time in water media.
Bioreactor prototype
In order to evaluate the performance of the developed bioactive polymeric materials different bioreactor prototype will be developed as dynamic tissue culture environment in which tissue specific mechanical forces (pressure, shear stress, interstitial flow), homogeneous and constant conditions will be applied and in which human hepatocytes were cultured in a optimal microenvironment allowing the simulation of the in vivo hepatic organisation.
Morphological and functional behaviours of human hepatocytes were investigated in a gas permeable membrane bioreactor and in a small scale bioreactor. In this both bio-hybrid system cells were cultured on the novel bioactive membranes which were located in tightly contact with a gaspermeable membrane foil that ensures the optimal transfer of CO2, O2 and H2O vapour to the cells adhered on the membranes and to the medium overlaying the cells.
Morphological and functional behaviours of human hepatocytes were investigated furthermore in a new crossed HF membrane bioreactor developed by ITM and based on the use of PEEK-WC and polyethersulfone (PES) HF membranes with different molecular weight (MW) cut-off and physicochemical properties and cross assembled in alternating perpendicular manner. Hepatocytes were cultured in the extraluminal compartment among the PEEK-WC HFs, devoted to provide the cells oxygenated medium containing nutrients and metabolites, and the PES HFs devoted to remove from cell compartment catabolites and cell specific products.
Surface coatings with functional groups and immobilisation of biomolecules
It is a class of deposition and/or treatment plasma processes that allow to provide biomedical surfaces with proper functional groups to be utilised for the covalent (direct or through a spacer molecule) or non covalent (e.g. through self-assembly) immobilisation of RGD and other peptides. By tuning plasma parameters (thus the distribution of chemical groups and the thickness of the modified layer) and the variables (pH, temperature, time, etc.) of the immobilisation reaction it is possible to tune the density of the biomolecules immobilised at the surface of the material. In certain cases a network of peptide is obtained, mimicking the extracellular matrix to some extent.
Design and preparation of tailor-made self-assembling peptides
During this project Instituto Químico de Sarriá (IQS) in Spain designed and synthesised three new self-assembling peptide scaffolds containing peptide motifs for proper hepatocyte instruction. The material was highly soluble in water and produced solutions at pH 3.5 when dissolved at a concentration of 1% (w/v). After buffering the solutions at pH 7.0 with PBS they turned into gels. The peptides were used to study their nanostructure characteristics by atomic force microscopy (AFM). In all cases, the modified peptides formed fibers as observed by AFM and were capable of forming hydrogels.
Design and realisation of the bioreactor using membrane and biodegradable scaffolds
A bioreactor with a gas-permeable membrane (PTFE) at the bottom has been constructed which guaranteed the maintenance of liver-specific functions in primary hepatocyte cultures This novel device was shown to be well suited for studying a variety of drugs. This small-scale bioreactor with an oxygen-permeable PTFE-membrane has been suitable for simulating the hyperoxic situation with the aim of in vitro screening of new compounds for potential antioxidant capacity.
Standardisation of isolation and freezing protocols of human liver cells
Due to the limited availability of human livers University of Leipzig in Germany established first a protocol for freezing primary animal hepatocytes immobilised in the mini bioreactor. Comparison of the parameters for functional levels of cryopreserved and unfrozen (control) hepatocytes revealed that the cryopreservation process has no major impact on the expression of liver-specific functions with respect to protein synthesis, detoxification and biotransformation. A protocol for the cryopreservation of primary animal hepatocytes, as well as primary human hepatocytes (provided by Admet Technologies) immobilised in the small-scale bioreactor has been established providing the maintenance of hepatocyte functionality following cell thawing.
New HF membranes
Institute on Membrane Technology (ITM) in Italy designed and synthesised new membranes in hollow fibre configuration according to the well-known dry-wet spinning method from a polymeric blend of modified polyetheretherketone (PEEK-WC). Investigations in polymeric membrane preparation and in the understanding of their transport properties have made possible the realisation of this novel high frequency (HF) membranes for biomedical applications to be used for hepatocytes cultures.
The new PEEK-WC HF membranes yield a useful tool for the reconstruction of liver tissue model in vitro and for the reconstruction of liver tissue in vivo which allow, on the one side, to understand the mechanisms involved and regulating growth and differentiation, and on the other to study their role in the physiology and pathology of the liver.
Plasma process optimisation and immobilisation procedures
It is a class of coating that can be plasma deposited by using the lactide conventional monomer used for polymerisation of polilactic acid. By tuning plasma parameters (thus chemical structure and thickness of the coating) it is possible to tune the degradation time in water media.
Bioreactor prototype
In order to evaluate the performance of the developed bioactive polymeric materials different bioreactor prototype will be developed as dynamic tissue culture environment in which tissue specific mechanical forces (pressure, shear stress, interstitial flow), homogeneous and constant conditions will be applied and in which human hepatocytes were cultured in a optimal microenvironment allowing the simulation of the in vivo hepatic organisation.
Morphological and functional behaviours of human hepatocytes were investigated in a gas permeable membrane bioreactor and in a small scale bioreactor. In this both bio-hybrid system cells were cultured on the novel bioactive membranes which were located in tightly contact with a gaspermeable membrane foil that ensures the optimal transfer of CO2, O2 and H2O vapour to the cells adhered on the membranes and to the medium overlaying the cells.
Morphological and functional behaviours of human hepatocytes were investigated furthermore in a new crossed HF membrane bioreactor developed by ITM and based on the use of PEEK-WC and polyethersulfone (PES) HF membranes with different molecular weight (MW) cut-off and physicochemical properties and cross assembled in alternating perpendicular manner. Hepatocytes were cultured in the extraluminal compartment among the PEEK-WC HFs, devoted to provide the cells oxygenated medium containing nutrients and metabolites, and the PES HFs devoted to remove from cell compartment catabolites and cell specific products.
Surface coatings with functional groups and immobilisation of biomolecules
It is a class of deposition and/or treatment plasma processes that allow to provide biomedical surfaces with proper functional groups to be utilised for the covalent (direct or through a spacer molecule) or non covalent (e.g. through self-assembly) immobilisation of RGD and other peptides. By tuning plasma parameters (thus the distribution of chemical groups and the thickness of the modified layer) and the variables (pH, temperature, time, etc.) of the immobilisation reaction it is possible to tune the density of the biomolecules immobilised at the surface of the material. In certain cases a network of peptide is obtained, mimicking the extracellular matrix to some extent.
Design and preparation of tailor-made self-assembling peptides
During this project Instituto Químico de Sarriá (IQS) in Spain designed and synthesised three new self-assembling peptide scaffolds containing peptide motifs for proper hepatocyte instruction. The material was highly soluble in water and produced solutions at pH 3.5 when dissolved at a concentration of 1% (w/v). After buffering the solutions at pH 7.0 with PBS they turned into gels. The peptides were used to study their nanostructure characteristics by atomic force microscopy (AFM). In all cases, the modified peptides formed fibers as observed by AFM and were capable of forming hydrogels.
Design and realisation of the bioreactor using membrane and biodegradable scaffolds
A bioreactor with a gas-permeable membrane (PTFE) at the bottom has been constructed which guaranteed the maintenance of liver-specific functions in primary hepatocyte cultures This novel device was shown to be well suited for studying a variety of drugs. This small-scale bioreactor with an oxygen-permeable PTFE-membrane has been suitable for simulating the hyperoxic situation with the aim of in vitro screening of new compounds for potential antioxidant capacity.
Standardisation of isolation and freezing protocols of human liver cells
Due to the limited availability of human livers University of Leipzig in Germany established first a protocol for freezing primary animal hepatocytes immobilised in the mini bioreactor. Comparison of the parameters for functional levels of cryopreserved and unfrozen (control) hepatocytes revealed that the cryopreservation process has no major impact on the expression of liver-specific functions with respect to protein synthesis, detoxification and biotransformation. A protocol for the cryopreservation of primary animal hepatocytes, as well as primary human hepatocytes (provided by Admet Technologies) immobilised in the small-scale bioreactor has been established providing the maintenance of hepatocyte functionality following cell thawing.
