Periodic Reporting for period 2 - B-BRIGHTER (Better Bioprinting by Light-sheet Lithography)
Periodo di rendicontazione: 2023-10-01 al 2025-03-31
Bioprinting is considered one of the most promising methods to produce functional engineered tissues with physiological properties. Current bioprinting methods are limited by combinations of insufficient speed, spatial resolution and cell viability. Since these technologies often suffer from poor spatial resolution and inability to control biomechanical properties, they fail to mimic the heterogeneous nature of native tissues. B-BRIGHTER aims to develop a novel bioprinting technology able to produce engineered tissues with high spatial resolution at high printing speed using an original top-down lithography approach. In contrast to current bottom up, layer-by-layer bioprinting methods, B-BRIGHTER aims at ultra high-speed digital light-sheet illumination strategy to selectively photo-crosslink cell-laden hydrogels mimicking specific tissues, in confined voxels and produce three-dimensional complex geometries. Previous advances from the BRIGHTER project were extended by initiating work on building complex bioengineered skin, cornea and gut tissue models, all of which represent pioneering examples for bioengineering. These efforts did not reach the goals of the project due to the delay in the delivery of the bioprinters to the end-user partners IBEC-CERCA and TECH. Together with the work on patterning technology, bioink and application, a business case for a bioprinting product was established. The work on exploitation activities ultimately resulted in the background information for a go-no go decision for the industrialisation of a bioprinting product and a commercial path forward. The ongoing establishment of the Modulux3D entity with the support of the existing partners in moving forward is evidence of the present ‘go’ decision. Ultimately, the goal of the B-BRIGHTER project is to provide a radically new bioprinting technology to boost the performance of various engineered tissues which in turn will promote improved healthcare opportunities, as well as business and employment advances in the European Union and beyond.
The B-BRIGHTER project set out with four main goals, to produce a cost-effective and reproducible “bioink” for bioprinting, to design and build two prototype bioprinters,to use these bioprinters to produce tissues such as skin, gut, and cornea, and finally to gather experience and data that could support the future development of a business around this technology. These overarching goals were achieved through work undertaken in all of the work packages, as described in detail below.
The chemistry for hydrogel manufacture was optimised and standardised. ensuring that the materials are stable, easy to ship, and convenient to store. Bioink formulations were developed, formulated and standardised for printing gut, skin and cornea. To make the process more reliable, the bioinks also included additives that keep the cells evenly distributed during printing.
Two LS Bioprinter prototypes are operational and installed at the project partners. Work was focussed at GUF and MYC to convert a laboratory-based device built on an open optical table to a more compact device with an acceptable footprint to be used in a laboratory environment. The devices were installed at the project partners IBEC-CERCA and TECH to enable printing of tissue samples. The device was also designed to allow for real-time monitoring of the printing process for quality assurance. The work with the bioprinters included organising training for the use of the bioprinters and also producing a comprehensive manual for the device.
The main work of printing the tissue types that have been identified for the B-BRIGHTER project, namely gut, skin and cornea, was initiated by the partners IBEC-CERCA and TECH with the two bioprinters built during the project. After receiving proper training, members from both teams become fully independent end-users or the bioprinting devices and the first experiments were performed, demonstrating the suitability of the developed bioinks for producing engineered tissues. IBEC-CERCA successfully proved the printability of complex 3D gut tissue structures, mimicking the crypt and villi microfeatures of the in-vivo tissue, in an accurate and reproducible manner. First epithelized gut tissues were cultured up to 19 days and immunostained to check the main cellular markers, that were colocalized and expressed as their in vivo counterparts; proving the functionality of the constructs. In parallel, protocols for sample handling, transferring and culture into standard cell culture systems were developed and optimized, as well as other functional tests to entirely characterize the bioprinted tissues. Advances in skin and cornea tissues by TECH team showed promising results with the developed bioinks, promoting cellular spreading, migration and function.
A business case for a valid business offering has been formulated by interacting with stakeholders, utilising business case development strategies for deep tech innovations, and carrying out an innovation workshops. A spin-out company, Modulux3D, is being formed by a team stemming from GUF in Frankfurt and consisting of three researchers and a business developer. A business case has been formulated to address the need of high-speed, high-resolution bioprinters that can produce detailed three-dimensional biostructures. The primary market initially is academic and pharmaceutical development laboratories.
The chemistry for hydrogel manufacture was optimised and standardised. ensuring that the materials are stable, easy to ship, and convenient to store. Bioink formulations were developed, formulated and standardised for printing gut, skin and cornea. To make the process more reliable, the bioinks also included additives that keep the cells evenly distributed during printing.
Two LS Bioprinter prototypes are operational and installed at the project partners. Work was focussed at GUF and MYC to convert a laboratory-based device built on an open optical table to a more compact device with an acceptable footprint to be used in a laboratory environment. The devices were installed at the project partners IBEC-CERCA and TECH to enable printing of tissue samples. The device was also designed to allow for real-time monitoring of the printing process for quality assurance. The work with the bioprinters included organising training for the use of the bioprinters and also producing a comprehensive manual for the device.
The main work of printing the tissue types that have been identified for the B-BRIGHTER project, namely gut, skin and cornea, was initiated by the partners IBEC-CERCA and TECH with the two bioprinters built during the project. After receiving proper training, members from both teams become fully independent end-users or the bioprinting devices and the first experiments were performed, demonstrating the suitability of the developed bioinks for producing engineered tissues. IBEC-CERCA successfully proved the printability of complex 3D gut tissue structures, mimicking the crypt and villi microfeatures of the in-vivo tissue, in an accurate and reproducible manner. First epithelized gut tissues were cultured up to 19 days and immunostained to check the main cellular markers, that were colocalized and expressed as their in vivo counterparts; proving the functionality of the constructs. In parallel, protocols for sample handling, transferring and culture into standard cell culture systems were developed and optimized, as well as other functional tests to entirely characterize the bioprinted tissues. Advances in skin and cornea tissues by TECH team showed promising results with the developed bioinks, promoting cellular spreading, migration and function.
A business case for a valid business offering has been formulated by interacting with stakeholders, utilising business case development strategies for deep tech innovations, and carrying out an innovation workshops. A spin-out company, Modulux3D, is being formed by a team stemming from GUF in Frankfurt and consisting of three researchers and a business developer. A business case has been formulated to address the need of high-speed, high-resolution bioprinters that can produce detailed three-dimensional biostructures. The primary market initially is academic and pharmaceutical development laboratories.
The bioinks developed in this project are based on the thiolene chemistry and are, therefore, superior to other commercially available products which are based on the acryloyl chemistry (e.g. GelMa, PEGDA). They provide superior mechanical integrity, higher printing resolution, and are less cytotoxic due to a lower generation of cytotoxic reactive oxygen species. This allows a fine structuring of features in tissue bioprinting which is expected to surpass the current state of the art achieved with current commercially available bioinks. Moreover, the bioinks developed in this project are free of animal-derived components and entails full control of biomimetic features including a wide range of stiffness and the addition of biologically active peptides. Both features can be controlled separately from each other which is not found in other commercially available hydrogels to this extend.
Work in the project enables real-time monitoring of hydrogel crosslinking using fluorescent recovery after photobleaching (FRAP) and brightfield imaging as well as in situ light sheet imaging of cells. Full-thickness skin constructs of high viability displayed characteristics of both epidermal and dermal layers and remained.
Two novel high-speed, high-resolution, light-sheet-based bioprinters were designed and built during the project. The bioprinters offer real-time monitoring of the printing process for quality control.
Work in the project enables real-time monitoring of hydrogel crosslinking using fluorescent recovery after photobleaching (FRAP) and brightfield imaging as well as in situ light sheet imaging of cells. Full-thickness skin constructs of high viability displayed characteristics of both epidermal and dermal layers and remained.
Two novel high-speed, high-resolution, light-sheet-based bioprinters were designed and built during the project. The bioprinters offer real-time monitoring of the printing process for quality control.