Final Report Summary - SPECCC (Specialised Cell Carrier Components)
Executive Summary:
The aim of the SpeCCC project was to address the limitations in the current 3D cell culture systems that are preventing their widespread use. These limitations include: 1) Homogeneous scalable bioreactor systems have generally not been developed specifically for 3D microtissue cultures and the systems that are available (e.g. hollowfibre systems) are not ideal for 3D microtissue culture due to diffusion limitations; 2) Hydrogel carriers have been used for 3D microtissues in scalable bioreactors, but hydrogel carriers can have issues with inefficient diffusion of nutrients and waste metabolites, and lack of mechanical stability of the carrier; 3) Many of the liver cell lines currently used for 3D microtissues demonstrate poor retention of key liver specific functions. These limitations will be investigated in the SpeCCC project
The core objective of the SpeCCC project was to develop specialised functional liver cell carrier components for use in fluidised bed bioreactors, and 96 well plate formats. The other objectives were 1) to develop a homogeneous fluidised bed bioreactor which has the potential to be scaled up for use in a bioartificial liver device (BAL) and scaled down for use in a multi micro fluidised bioreactor system (for drug testing and regenerative medicine); and 2) to develop a microwell plate toxicology/drug test kit based on the functional cell carrier component.
The cell-carrier component was the central element of all these applications and therefore it was a prerequisite for their development. The two parts that make up the cell carrier component are 1) the true functional immortalised liver cell lines (human hepatocyte and human endothelial cells), and 2) the second-generation porous silicone carrier, which has a defined and optimised geometry, diameter (i.e. defined, and optimised diffusion distance), pore size, pore volume, surface (for cell attachment), specific gravity and mechanical stability.
The results obtained in the project show that the novel liver cell lines developed by ISX were not sufficiently functional to be used in a liver cell carrier component, and as a result the focus of the project was directed towards the development of platform technologies for 3D culture. Improved second generation carriers have been developed in terms of improved geometry, porosity and surface properties. Successful growth of the ISX immortalised cells in coculture and HEP G2 liver cells in single culture has been demonstrated in these improved carriers. Furthermore, the growth of HEP G2 liver cells in a micro fluidised bed and fluidised bed bioreactors has also been demonstrated. Therefore, proof of concept has been demonstrated for micro-fluidised and fluidised bioreactor systems for 3D culture of adherent cells. This represents a significant technology development in the area of adherent and 3D cell culture, which could have a disruptive impact in the domain of 3D and adherent cell culture and their associated cell and viral vector therapeutics.
Project Context and Objectives:
• To establish cell systems that are optimized for cultivation and maintenance under 3D culture conditions. These cell systems will be further designed to display properties that mimic the biological relevant cell physiology. To fully reconstitute the in vivo properties of the liver a hepatocyte cell system and an endothelial cell system (liver sinusoidal endothelial cells) will be established, authenticated, and characterized for their functions.
• To provide a baseline for the native silicone material for comparison to tissue grade plastic and modified carriers. To achieved comparability in growth and differentiated marker expression (albumin) between tissue culture grade plastic and surface charge and surface chemistry modified silicone material in 2 D cultures.
• Synthesis and characterization of porous silicone polymers with various geometries, porosities, specific gravity and pore sizes and modification of the surface of the silicones for improved cell adhesion
• Evaluation of the trial materials generated in work package 4 and based on these evaluations recommendations for improved carrier design will be provided.
• Optimisation of the porous structure for liver cell co-culture in porous silicone material. Specifically, 1) to design optimum porous carrier material for co-culture of liver cells and expression and retention of key liver specific functions.
• Design, develop and test culture systems for 1) fluidised bed perfusion bioreactor and; 2) Stirred tank suspension bioreactor.
• The main tasks for WP 7 are to test if the developed cell-carrier-systems can be used for the intended applications.1) Fluidised bed perfusion bioreactor, 2) Microfluidised bed
All the objectives have been fully achieved with the following exceptions; 1) the hepatocyte cell line was not functional and the endothelial cell line was only partially functional. As a result of this the last part of the project was focused on the development of the platform bioreactor technologies; and 2) HEP G2 cell proliferation on the second generation carriers in the suspension culture system was not sufficient to demonstrate proof of concept.
Project Results:
• Hepatocyte and endothelial continuous cell lines with full retention of the liver specific functionality of the parental primary liver cells.
• Liver cell proliferation and liver specific functionality in native silicone material compared to tissue culture plastic.
• Surface modified silicone providing equivalent liver cell proliferation and liver specific functionality to tissue culture plastics.
• Trial batches of porous silicone carriers with various surface modifications geometries, densities, porosities, and pore sizes.
• Trial batches of designed, optimised and characterised porous silicone carriers for use in stirred tank bioreactors, fluidised bed bioreactors, and microfluidised bed bioreactors.
• Documented data on the performance of the chosen second generation carriers (weighted and unweighted) for culture and level of liver specific functions of hepatocyte cell in co culture with endothelial cells from (WP 5).
• Documented data on the performance of HEP G2 cells cultured in the chosen second generation carrier (from WP 4) in suspension culture and in the fluidised perfusion bioreactor developed in WP 6.
• Developed protocol on the cultivation Hep G2 cells in the chosen second generation carrier in a microfluidised bed bioreactor system
Potential Impact:
Impact. The potential impact through the development, dissemination and use of project results
Proprietary Products from SpeCCC
The foreground intellectual property (IP) has the potential to generate substantial income for the SME participants despite the delay of 5 years. The income streams from the proprietary products derived from the IP (Table 1) can be divided into short/medium term and long-term. This largely depends on the markets they are developed for; i.e. research, toxicity testing, drug metabolism, cGMP bio-therapeutics (cell and gene therapy products), or bio-artificial organ markets. SpeCCC - IP is expected to form the technological basis for the proprietary products listed in (Table 1).
Table 1 Proprietary products developed from SpeCCC IP
Product number 1 is based on the fluidised bed scaffold based micro-bioreactor systems, and has significant advantages over the existing technologies, and therefore is well placed to establish a significant presence in the growing market for; drug metabolism studies, regenerative medicine research tools, and tissue based chemical safety testing (replacing animal-based testing). Product 2 - There an urgent need for technological innovation in cell and gene therapy manufacturing, which is not being met with the current manufacturing technologies - Product 2 has the potential to provide this much needed technological innovation.
The immobilised porous silicone scaffold and bioreactor system, developed within SpeCCC, is a platform technology with multiple high value commercial applications. The key features of the SpeCCC fluidised bed porous scaffold technology are listed in Box 1 These features will form the unique selling points (USPs) of the proprietary products described, which will give them a competitive technical advantage in their target markets.
The project was promoted at relevant scientific conferences as listed below, via a poster and leaflets.
Box 1. key features of SpeCCC immobilised bioreactor systems
The potential and achievable societal benefits of the SpeCCC technology are the ability to provide badly needed improved and potentially disruptive manufacturing technology to speed up the delivery of ground-breaking health care treatments in regenerative medicine, cell and gene therapy and vaccine technology. Scalable manufacturing technologies are currently a key bottle neck in the delivery of these treatments.
List of Websites:
www.speccc.eu
The aim of the SpeCCC project was to address the limitations in the current 3D cell culture systems that are preventing their widespread use. These limitations include: 1) Homogeneous scalable bioreactor systems have generally not been developed specifically for 3D microtissue cultures and the systems that are available (e.g. hollowfibre systems) are not ideal for 3D microtissue culture due to diffusion limitations; 2) Hydrogel carriers have been used for 3D microtissues in scalable bioreactors, but hydrogel carriers can have issues with inefficient diffusion of nutrients and waste metabolites, and lack of mechanical stability of the carrier; 3) Many of the liver cell lines currently used for 3D microtissues demonstrate poor retention of key liver specific functions. These limitations will be investigated in the SpeCCC project
The core objective of the SpeCCC project was to develop specialised functional liver cell carrier components for use in fluidised bed bioreactors, and 96 well plate formats. The other objectives were 1) to develop a homogeneous fluidised bed bioreactor which has the potential to be scaled up for use in a bioartificial liver device (BAL) and scaled down for use in a multi micro fluidised bioreactor system (for drug testing and regenerative medicine); and 2) to develop a microwell plate toxicology/drug test kit based on the functional cell carrier component.
The cell-carrier component was the central element of all these applications and therefore it was a prerequisite for their development. The two parts that make up the cell carrier component are 1) the true functional immortalised liver cell lines (human hepatocyte and human endothelial cells), and 2) the second-generation porous silicone carrier, which has a defined and optimised geometry, diameter (i.e. defined, and optimised diffusion distance), pore size, pore volume, surface (for cell attachment), specific gravity and mechanical stability.
The results obtained in the project show that the novel liver cell lines developed by ISX were not sufficiently functional to be used in a liver cell carrier component, and as a result the focus of the project was directed towards the development of platform technologies for 3D culture. Improved second generation carriers have been developed in terms of improved geometry, porosity and surface properties. Successful growth of the ISX immortalised cells in coculture and HEP G2 liver cells in single culture has been demonstrated in these improved carriers. Furthermore, the growth of HEP G2 liver cells in a micro fluidised bed and fluidised bed bioreactors has also been demonstrated. Therefore, proof of concept has been demonstrated for micro-fluidised and fluidised bioreactor systems for 3D culture of adherent cells. This represents a significant technology development in the area of adherent and 3D cell culture, which could have a disruptive impact in the domain of 3D and adherent cell culture and their associated cell and viral vector therapeutics.
Project Context and Objectives:
• To establish cell systems that are optimized for cultivation and maintenance under 3D culture conditions. These cell systems will be further designed to display properties that mimic the biological relevant cell physiology. To fully reconstitute the in vivo properties of the liver a hepatocyte cell system and an endothelial cell system (liver sinusoidal endothelial cells) will be established, authenticated, and characterized for their functions.
• To provide a baseline for the native silicone material for comparison to tissue grade plastic and modified carriers. To achieved comparability in growth and differentiated marker expression (albumin) between tissue culture grade plastic and surface charge and surface chemistry modified silicone material in 2 D cultures.
• Synthesis and characterization of porous silicone polymers with various geometries, porosities, specific gravity and pore sizes and modification of the surface of the silicones for improved cell adhesion
• Evaluation of the trial materials generated in work package 4 and based on these evaluations recommendations for improved carrier design will be provided.
• Optimisation of the porous structure for liver cell co-culture in porous silicone material. Specifically, 1) to design optimum porous carrier material for co-culture of liver cells and expression and retention of key liver specific functions.
• Design, develop and test culture systems for 1) fluidised bed perfusion bioreactor and; 2) Stirred tank suspension bioreactor.
• The main tasks for WP 7 are to test if the developed cell-carrier-systems can be used for the intended applications.1) Fluidised bed perfusion bioreactor, 2) Microfluidised bed
All the objectives have been fully achieved with the following exceptions; 1) the hepatocyte cell line was not functional and the endothelial cell line was only partially functional. As a result of this the last part of the project was focused on the development of the platform bioreactor technologies; and 2) HEP G2 cell proliferation on the second generation carriers in the suspension culture system was not sufficient to demonstrate proof of concept.
Project Results:
• Hepatocyte and endothelial continuous cell lines with full retention of the liver specific functionality of the parental primary liver cells.
• Liver cell proliferation and liver specific functionality in native silicone material compared to tissue culture plastic.
• Surface modified silicone providing equivalent liver cell proliferation and liver specific functionality to tissue culture plastics.
• Trial batches of porous silicone carriers with various surface modifications geometries, densities, porosities, and pore sizes.
• Trial batches of designed, optimised and characterised porous silicone carriers for use in stirred tank bioreactors, fluidised bed bioreactors, and microfluidised bed bioreactors.
• Documented data on the performance of the chosen second generation carriers (weighted and unweighted) for culture and level of liver specific functions of hepatocyte cell in co culture with endothelial cells from (WP 5).
• Documented data on the performance of HEP G2 cells cultured in the chosen second generation carrier (from WP 4) in suspension culture and in the fluidised perfusion bioreactor developed in WP 6.
• Developed protocol on the cultivation Hep G2 cells in the chosen second generation carrier in a microfluidised bed bioreactor system
Potential Impact:
Impact. The potential impact through the development, dissemination and use of project results
Proprietary Products from SpeCCC
The foreground intellectual property (IP) has the potential to generate substantial income for the SME participants despite the delay of 5 years. The income streams from the proprietary products derived from the IP (Table 1) can be divided into short/medium term and long-term. This largely depends on the markets they are developed for; i.e. research, toxicity testing, drug metabolism, cGMP bio-therapeutics (cell and gene therapy products), or bio-artificial organ markets. SpeCCC - IP is expected to form the technological basis for the proprietary products listed in (Table 1).
Table 1 Proprietary products developed from SpeCCC IP
Product number 1 is based on the fluidised bed scaffold based micro-bioreactor systems, and has significant advantages over the existing technologies, and therefore is well placed to establish a significant presence in the growing market for; drug metabolism studies, regenerative medicine research tools, and tissue based chemical safety testing (replacing animal-based testing). Product 2 - There an urgent need for technological innovation in cell and gene therapy manufacturing, which is not being met with the current manufacturing technologies - Product 2 has the potential to provide this much needed technological innovation.
The immobilised porous silicone scaffold and bioreactor system, developed within SpeCCC, is a platform technology with multiple high value commercial applications. The key features of the SpeCCC fluidised bed porous scaffold technology are listed in Box 1 These features will form the unique selling points (USPs) of the proprietary products described, which will give them a competitive technical advantage in their target markets.
The project was promoted at relevant scientific conferences as listed below, via a poster and leaflets.
Box 1. key features of SpeCCC immobilised bioreactor systems
The potential and achievable societal benefits of the SpeCCC technology are the ability to provide badly needed improved and potentially disruptive manufacturing technology to speed up the delivery of ground-breaking health care treatments in regenerative medicine, cell and gene therapy and vaccine technology. Scalable manufacturing technologies are currently a key bottle neck in the delivery of these treatments.
List of Websites:
www.speccc.eu