Final Report Summary - NANOBIO4TRANS (A new nanotechnology-based paradigm for engineering vascularised liver tissue for transplantation)
Executive Summary:
NanoBio4Trans has merged hiPSC-, perfusable hybrid scaffolds (PHS)- and biosensor technologies to develop new tools to be used in biomedical research beyond the present state-of-the-art. The outcome has been the development, optimisation and validation of a highly vascularised in vivo-like BAL as a potential extracorporeal bioartificial liver, ready to be perfused with human blood plasma. The BAL has been characterised and its metabolic capability and other essential functions (e.g. clearance of ammonia, bilirubin, toxins and bile acids) has been assessed. Precision-cut liver slices (PCLS) incubated in Liver-on-a-Chip systems has been used together with new LC-based bioassays for quality control of the newly engineered tissue.
Major steps have involved: (1) Fabrication of different types of PHS with different geometries, architectures, and porosities. (2) Exploration of in vitro cellular systems for development of novel 3D cell/tissue culture systems starting from hiPSCs, using perfusion based BAL-on-a-Chip systems for optimisation of 3D culture and perfusion conditions, and further adaptation for potential up-scaling to larger size BAL support system to form BALs in the order of cm3 to dm3. (3) Comparison and validation of the developed fluidic BAL-on-a-Chip system with fluidic Liver-on-a-chip, containing precision-cut liver slices (PCLS) obtained from surgical waste from transplant, used as the gold standard to which the BAL systems have been compared.
Novel strategies to make 3D scaffolds with microporous structured, random, and combined structured/random channel network architecture have been achieved, using a combination of 3D filament printing of sacrificial moulds, polymer casting and sugar/salt leaching procedures, realising woodpile and hexagonal scaffold architecture. Nanostructured interpenetrating networks (IPNs) of hydrogel deposits inside these 3D scaffolds have been achieved, which has made it possible to deliver a drug that specifically can activate gene expression in cells residing in the interior of the 3D scaffolds. The developed strategy allows scaling up of scaffold size in dm3 and in principle larger scale, the main challenge being the availability of enough cells for seeding inside.
Scaling up of hiPSCs production through 3D spheroid cultures has successfully been realised, and freezing procedures for easy transport of cells/spheroids to partners. Defined medium for the expansion of undifferentiated hiPSCs and differentiation in an optimised culture system with achieved mature hepatocytes with increased CYP-activities and CYP-induction function has been achieved. The developed protocols towards hepatocyte protocols are very robust, and more than 28 cell lines have been tested for their ability to grow undifferentiated as well as successful differentiation into not only hepatocytes but also other cell types (lung- and beta cell progenitors). State-of-the art maturity and functionality of hepatocytes and hepatospheres derived from hiPSCs has been achieved.
Two types of BAL support systems have been developed: (1) 16-bioreactor array intended for improved throughput optimization experiments of cell culture and differentiation conditions. (2) 8-bioreactor array incorporating needle-electrodes for bioimpedance analysis of real time cell proliferation in 3D over time. Efficient operation of the BAL support system requires precise control of their key physical, chemical and physiological parameters. To implement this, advanced optochemical- and electrochemical sensors and assays have been developed and applied for monitoring hiPSC-derived liver tissue and human precision cut liver slices (PCLS).
The human BAL was moreover assessed and compared to human PCLS. Various functions, such as Phase I and II metabolism, synthesis, as well as expression of hepatic genes responsible for liver development, metabolism, synthesis and transport, were tested. This is the first time that the function of hiPSC-derived hepatocytes has been compared to fresh human tissue, and it revealed that these hiPSC-derived human hepatocytes cultured under the applied conditions show as yet unrivalled functionality, with excellent comparability of liver functions with fresh liver tissue.
Project Context and Objectives:
Summary description of context and objectives (see also attached pdf-Project context and objectives for final report)
Context:
Organ transplantation has been a major scientific and clinical breakthrough in the 20th century, in spite of many associated problems, such as lack of organ donors and/or rejection of the transplanted organs and life-long heavy medication with side effects. The innovative therapeutic approach of the 21th century is focusing on bioartificial organs to circumvent present problems, using a multidisciplinary and complementary approach to achieve the targeted goals. Tissue engineering and stem cell biology have uncovered groundbreaking opportunities for cellular re-programming, i.e. some cell types can be changed into a pluripotent stem cell (PSC) by over-expressing key transcription factors. These, so called induced pluripotent stem cells (iPSC)[1] share the two key characteristics with embryonic stem cells (eSC): self-renewal and pluripotency (i.e. they can differentiate to form any cell type in the human body). Crucially they are generated from adult cells circumventing many ethical concerns associated with using human eSC. Tander the discovery of human iPSC (hiPSC) enables the growth of an almost unlimited supply of a patient´s own cells, potentially conferring the ability to grow and regenerate tissues and organs[2] from ‘self’, which is envisaged to resolve many organ rejection[1, 3] related issues. Similarly, recent developments in material science and nanobiotechnology have produced engineered materials and devices that can be manipulated and controlled by physical and chemical means, resulting in unique functional or analytical properties. The fusion of these fields with resulting synergistic effects will open up yet unexplored scientific possibilities. By involving SMEs with relevant and leading expertise in key scientific fields, NanoBio4Trans has taken an additional step forward, by transferring the scientific results into exploitation phase, focusing on a new paradigm for creating a bioartificial liver (BAL).
Objectives:
NanoBio4Trans set out to create a new paradigm to engineer cutting-edge scalable highly vascularised BAL on the following hypotheses:
• The use of hiPSC as the starting material, is foreseen to enable the construction of personalised artificial organs from a patient´s own cells, which is expected to result in reduced organ rejection and increased availability of lifesaving organs for transplantation.
• Growth and differentiation into in vivo-like BALs is possible if hPSC-derived liver tissue is highly vascularised, i.e. penetrated with blood-vessel like channels lined with endothelial cells, and with immediate and nearby supply of the necessary growth factors and signalling molecules that support its growth and differentiation.
• Creation of novel scalable two-component perfusable hybrid scaffolds (PHSs).
• The use of integrated optical and electrical (bio)sensing systems for monitoring real time effects and changes during tissue growth will allow control and surveillance of BAL formation, with envisaged feed-back control.
In a short-term perspective (3 years), a personalised/patient specific liver seems unrealistic since it is very likely that the patient will already be dead before any hiPSC have been produced from a potentially liver deficient patient´s own cells to result in a BAL derived from these cells. In a long-term perspective (8-10 years), however, it is realistic to build libraries and biobanks with patient derived hiPSCs, as a first crucial step towards a patient specific BAL. The main challenge has been to produce a fully functional BAL. Researchers worldwide are attempting to develop artificial livers (ALs) and BALs in different ways, some of which are already in clinical testing. So far, work is mainly focused on the creation of an extracorporeal liver for use as a liver support system (LSS) before an appropriate liver has been found for transplantation or until recovery of the patients own liver. In order to avoid immune rejection of the liver, the patient should ideally receive a transplant from somebody with an identical blood type (AB0 system). A more short-term step towards a BAL, less prone to rejection, may thus be to produce and store hiPSCs derived from individuals with different blood type from which blood type specific BALs could be produced.
By fusing hiPSC-, polymer hybrid scaffolds- and biosensor technologies, new tools can be developed to be used in transplantation and biomedical research beyond present state-of-the-art. The final goal has been to develop, optimise and validate a highly vascularised in vivo-like BAL - as an extracorporeal bioartificial liver (EBAL) - ready to be perfused with human blood plasma, to be exploited in the medical technology of the 21th century. Partner SMEs have had a leading role in this process concerning both innovation and exploitation aspects.
NanoBio4Trans integrates PHS-, sensor- and hiPSC technologies into a BAL support system in which a highly vascularised BAL will grow under real time control and surveillance. This new concept is a vital step forward in future developments of personalised/patient specific liver for transplantation.
The NanoBio4Trans strategy has been to follow a step-by-step development of an increasing order of complexity, thus diminishing risks and assuring gain of knowledge beyond the state-of-the-art. Major steps are:
• To explore different in vitro cellular systems for development of novel 3D cell/tissue culture systems starting from (a) existing endothelial cells and moving on to (b) hiPSCs, as they have become available in the project, (c) using in a first instance miniaturised fluidic sensor array systems (BAL-on-a-Chips) for optimisation of 3D culture and perfusion conditions, and (d) further adaption for up-scaling to a large size BAL support system to form BALs in the order of cm3 to dm3.
• To compare and validate the developed BAL system with fluidic tissue slice sensor array systems (Liver-on-a-chip), containing precision-cut liver slices obtained from surgical waste from transplant. This reference system has been used as the gold standard to which the BAL systems will be compared.
Project Results:
See attached document: Section 4.1-Final NanoBio4Trans report-publishable summary.pdf
Potential Impact:
See attached document: Section 4.1-Final NanoBio4Trans report-publishable summary.pdf
List of Websites:
http://www.nanobio4trans.eu/
Coordinator:
Professor Jenny Emnéus
Technical University of Denmark, Department of Micro- and Nanotechnology
Ørsteds Plads 345B, DK-2800 Kgs. Lyngby, Denmark
jenny.emneus@nanotech.dtu.dk
NanoBio4Trans has merged hiPSC-, perfusable hybrid scaffolds (PHS)- and biosensor technologies to develop new tools to be used in biomedical research beyond the present state-of-the-art. The outcome has been the development, optimisation and validation of a highly vascularised in vivo-like BAL as a potential extracorporeal bioartificial liver, ready to be perfused with human blood plasma. The BAL has been characterised and its metabolic capability and other essential functions (e.g. clearance of ammonia, bilirubin, toxins and bile acids) has been assessed. Precision-cut liver slices (PCLS) incubated in Liver-on-a-Chip systems has been used together with new LC-based bioassays for quality control of the newly engineered tissue.
Major steps have involved: (1) Fabrication of different types of PHS with different geometries, architectures, and porosities. (2) Exploration of in vitro cellular systems for development of novel 3D cell/tissue culture systems starting from hiPSCs, using perfusion based BAL-on-a-Chip systems for optimisation of 3D culture and perfusion conditions, and further adaptation for potential up-scaling to larger size BAL support system to form BALs in the order of cm3 to dm3. (3) Comparison and validation of the developed fluidic BAL-on-a-Chip system with fluidic Liver-on-a-chip, containing precision-cut liver slices (PCLS) obtained from surgical waste from transplant, used as the gold standard to which the BAL systems have been compared.
Novel strategies to make 3D scaffolds with microporous structured, random, and combined structured/random channel network architecture have been achieved, using a combination of 3D filament printing of sacrificial moulds, polymer casting and sugar/salt leaching procedures, realising woodpile and hexagonal scaffold architecture. Nanostructured interpenetrating networks (IPNs) of hydrogel deposits inside these 3D scaffolds have been achieved, which has made it possible to deliver a drug that specifically can activate gene expression in cells residing in the interior of the 3D scaffolds. The developed strategy allows scaling up of scaffold size in dm3 and in principle larger scale, the main challenge being the availability of enough cells for seeding inside.
Scaling up of hiPSCs production through 3D spheroid cultures has successfully been realised, and freezing procedures for easy transport of cells/spheroids to partners. Defined medium for the expansion of undifferentiated hiPSCs and differentiation in an optimised culture system with achieved mature hepatocytes with increased CYP-activities and CYP-induction function has been achieved. The developed protocols towards hepatocyte protocols are very robust, and more than 28 cell lines have been tested for their ability to grow undifferentiated as well as successful differentiation into not only hepatocytes but also other cell types (lung- and beta cell progenitors). State-of-the art maturity and functionality of hepatocytes and hepatospheres derived from hiPSCs has been achieved.
Two types of BAL support systems have been developed: (1) 16-bioreactor array intended for improved throughput optimization experiments of cell culture and differentiation conditions. (2) 8-bioreactor array incorporating needle-electrodes for bioimpedance analysis of real time cell proliferation in 3D over time. Efficient operation of the BAL support system requires precise control of their key physical, chemical and physiological parameters. To implement this, advanced optochemical- and electrochemical sensors and assays have been developed and applied for monitoring hiPSC-derived liver tissue and human precision cut liver slices (PCLS).
The human BAL was moreover assessed and compared to human PCLS. Various functions, such as Phase I and II metabolism, synthesis, as well as expression of hepatic genes responsible for liver development, metabolism, synthesis and transport, were tested. This is the first time that the function of hiPSC-derived hepatocytes has been compared to fresh human tissue, and it revealed that these hiPSC-derived human hepatocytes cultured under the applied conditions show as yet unrivalled functionality, with excellent comparability of liver functions with fresh liver tissue.
Project Context and Objectives:
Summary description of context and objectives (see also attached pdf-Project context and objectives for final report)
Context:
Organ transplantation has been a major scientific and clinical breakthrough in the 20th century, in spite of many associated problems, such as lack of organ donors and/or rejection of the transplanted organs and life-long heavy medication with side effects. The innovative therapeutic approach of the 21th century is focusing on bioartificial organs to circumvent present problems, using a multidisciplinary and complementary approach to achieve the targeted goals. Tissue engineering and stem cell biology have uncovered groundbreaking opportunities for cellular re-programming, i.e. some cell types can be changed into a pluripotent stem cell (PSC) by over-expressing key transcription factors. These, so called induced pluripotent stem cells (iPSC)[1] share the two key characteristics with embryonic stem cells (eSC): self-renewal and pluripotency (i.e. they can differentiate to form any cell type in the human body). Crucially they are generated from adult cells circumventing many ethical concerns associated with using human eSC. Tander the discovery of human iPSC (hiPSC) enables the growth of an almost unlimited supply of a patient´s own cells, potentially conferring the ability to grow and regenerate tissues and organs[2] from ‘self’, which is envisaged to resolve many organ rejection[1, 3] related issues. Similarly, recent developments in material science and nanobiotechnology have produced engineered materials and devices that can be manipulated and controlled by physical and chemical means, resulting in unique functional or analytical properties. The fusion of these fields with resulting synergistic effects will open up yet unexplored scientific possibilities. By involving SMEs with relevant and leading expertise in key scientific fields, NanoBio4Trans has taken an additional step forward, by transferring the scientific results into exploitation phase, focusing on a new paradigm for creating a bioartificial liver (BAL).
Objectives:
NanoBio4Trans set out to create a new paradigm to engineer cutting-edge scalable highly vascularised BAL on the following hypotheses:
• The use of hiPSC as the starting material, is foreseen to enable the construction of personalised artificial organs from a patient´s own cells, which is expected to result in reduced organ rejection and increased availability of lifesaving organs for transplantation.
• Growth and differentiation into in vivo-like BALs is possible if hPSC-derived liver tissue is highly vascularised, i.e. penetrated with blood-vessel like channels lined with endothelial cells, and with immediate and nearby supply of the necessary growth factors and signalling molecules that support its growth and differentiation.
• Creation of novel scalable two-component perfusable hybrid scaffolds (PHSs).
• The use of integrated optical and electrical (bio)sensing systems for monitoring real time effects and changes during tissue growth will allow control and surveillance of BAL formation, with envisaged feed-back control.
In a short-term perspective (3 years), a personalised/patient specific liver seems unrealistic since it is very likely that the patient will already be dead before any hiPSC have been produced from a potentially liver deficient patient´s own cells to result in a BAL derived from these cells. In a long-term perspective (8-10 years), however, it is realistic to build libraries and biobanks with patient derived hiPSCs, as a first crucial step towards a patient specific BAL. The main challenge has been to produce a fully functional BAL. Researchers worldwide are attempting to develop artificial livers (ALs) and BALs in different ways, some of which are already in clinical testing. So far, work is mainly focused on the creation of an extracorporeal liver for use as a liver support system (LSS) before an appropriate liver has been found for transplantation or until recovery of the patients own liver. In order to avoid immune rejection of the liver, the patient should ideally receive a transplant from somebody with an identical blood type (AB0 system). A more short-term step towards a BAL, less prone to rejection, may thus be to produce and store hiPSCs derived from individuals with different blood type from which blood type specific BALs could be produced.
By fusing hiPSC-, polymer hybrid scaffolds- and biosensor technologies, new tools can be developed to be used in transplantation and biomedical research beyond present state-of-the-art. The final goal has been to develop, optimise and validate a highly vascularised in vivo-like BAL - as an extracorporeal bioartificial liver (EBAL) - ready to be perfused with human blood plasma, to be exploited in the medical technology of the 21th century. Partner SMEs have had a leading role in this process concerning both innovation and exploitation aspects.
NanoBio4Trans integrates PHS-, sensor- and hiPSC technologies into a BAL support system in which a highly vascularised BAL will grow under real time control and surveillance. This new concept is a vital step forward in future developments of personalised/patient specific liver for transplantation.
The NanoBio4Trans strategy has been to follow a step-by-step development of an increasing order of complexity, thus diminishing risks and assuring gain of knowledge beyond the state-of-the-art. Major steps are:
• To explore different in vitro cellular systems for development of novel 3D cell/tissue culture systems starting from (a) existing endothelial cells and moving on to (b) hiPSCs, as they have become available in the project, (c) using in a first instance miniaturised fluidic sensor array systems (BAL-on-a-Chips) for optimisation of 3D culture and perfusion conditions, and (d) further adaption for up-scaling to a large size BAL support system to form BALs in the order of cm3 to dm3.
• To compare and validate the developed BAL system with fluidic tissue slice sensor array systems (Liver-on-a-chip), containing precision-cut liver slices obtained from surgical waste from transplant. This reference system has been used as the gold standard to which the BAL systems will be compared.
Project Results:
See attached document: Section 4.1-Final NanoBio4Trans report-publishable summary.pdf
Potential Impact:
See attached document: Section 4.1-Final NanoBio4Trans report-publishable summary.pdf
List of Websites:
http://www.nanobio4trans.eu/
Coordinator:
Professor Jenny Emnéus
Technical University of Denmark, Department of Micro- and Nanotechnology
Ørsteds Plads 345B, DK-2800 Kgs. Lyngby, Denmark
jenny.emneus@nanotech.dtu.dk
