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High Throughput Screening of Polymeric Biomaterials For eCM Periodical, 2020, Collection 1; 2020 TERMIS EU Abstracts (page 1) High Throughput Screening of Polymeric Biomaterials for Personalised 3D Printed Treatments Adja TOURé1, Lewis HART2, Laura Ruiz CANTU1, Derek IRVINE1, Wayne HAYES2, Ricky WILDMAN1 1Centre for Additive Manufacturing, University of Nottingham, Nottingham NG7 2RD 2Chemistry Department, University of Reading, Reading INTRODUCTION:Three-Dimensional (3D) printing is an established manufacturing method(1). However, the use of 3D printing is limited in industry due to the few materials available and the long development and optimisation process. A high throughput methodology has been developed allowing to quickly asses the printability and biocompatibility of a large library of materials(2,3). This method is adapted and used here, to screen a library of 64 PCL-based hyper-branched polymers and determine their suitability for a use as a gradient osteochondral interface. METHODS:To screen the synthesised polymer library, their printability (Z parameter) for inkjet printing is first assessed using a liquid handler, able to measure viscosity and surface tension (in DMF). The high-throughput assessment of mechanical and biocompatible properties is enabled by the use of a microarray strategy. Surface chemical characterisation is carried out using time-of-flight secondary ion mass spectrometry (ToF-SIMS), while localised mechanical properties (elastic moduli) are determined using atomic force microscopy (AFM). Combination of these techniques with cytotoxicity screening leads to the selection of three suitable polymers for the targeted application. RESULTS:Solubility in DMF and printability determination led to the selection of around 60% of the tested library. Subsequently, the polymer arrays were fabricated by contact printing using polymer solutions in DMF and glass slides with a super hydrophilic/hydrophobic coating as substrate. ToF-Sims surface analysis ruled out any chemical contaminations and confirmed the deposition of the desired polymers. Assessment of the mechanical properties and cytotoxicity screening led to the selection of three materials used for preliminary printing essays. DISCUSSION & CONCLUSIONS:The polymer library showed interesting mechanical properties with elastic moduli matching that of human bones and demonstrated the feasibility of manufacturing such scaffolds. Three selected materials for the library are currently being printed to create gradient scaffolds. The use of this high-throughput strategy allowed the rapid and accurate assessment of the polymers. ACKNOWLEDGEMENTS:The author would like to thank Laurence Burroughs, Xinyong Chen, Nichola Starr and Gustavo Ferraz Trindade for their scientific and technical help. REFERENCES:1. Goole, J. et al.. 3D printing in pharmaceutics: A new tool for designing customized drug delivery systems. International Journal of Pharmaceutics 499, 376–394 (2016). 2. Zhou, Z. et al. High-throughput characterization of fluid properties to predict droplet ejection for three-dimensional inkjet printing formulations. Addit. Manuf. 29, 100792 (2019). 3. Louzao, I. et al. Identification of Novel ‘inks’ for 3D Printing Using High-Throughput Screening: Bioresorbable Photocurable Polymers for Controlled Drug Delivery. ACS Appl. Mater. Interfaces 10, 6841–6848 (2018). Keywords: Additive manufacturing, Personalised medicine. www.ecmconferences.org eCM Periodical, 2020, Collection 1; 2020 TERMIS EU Abstracts (page 2) Silk-fibroin based scaffold 3D bioprinting deposition: mathematical modeling and experimental characterization Diego TRUCCO1, Cristina MANFERDINI2, Elena GABUSI2, Mauro PETRETTA3, Giovanna DESANDO3, Leonardo RICOTTI4, Shikha CHAWLA5, Sourabh GHOSH5, Gina LISIGNOLI2 1The BioRobotics Institute, Scuola Superiore Sant’Anna, Pisa, Italy; IRCSS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy 2IRCSS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy 3IRCSS Istituto Ortopedico Rizzoli, Laboratorio RAMSES, Bologna, Italy 4The BioRobotics Institute, Scuola Superiore Sant’Anna, Pisa, Italy 5Regenerative Engineering Laboratory, Department of Textile Technology, Indian Institute of Technology, New Delhi, India INTRODUCTION:Robotic dispensing-based 3D bioprinting strategies are widely used to print cell- laden hydrogel constructs. This enables the fabrication of neo-engineered constructs able to maintain cells alive and to regenerate tissues. Nowadays, efforts in this field are mainly driven by experiments and trial-and-error approaches. In order to predict the deposited filament width through extrusion-based 3D bioprinting, a mathematical model[1] was applied considering different printing parameters such as pressure and printing speed. The printing parameters were chosen to print silk fibroin(SF)-gelatin(G) based scaffolds laden with human-derived mesenchymal stromal cells (hMSCs). METHODS:The rheological characterization of cell-friendly SF-G bioink used in our previous study[2] was used to define the viscosity and the power law index. A mathematical model of deposition was formulated including the following parameters: printing pressure (0-3 bar), speed (0.5-3 mm/s) and cylindrical stainless-steel needle features (diameters: 0.2, 0.41 and 0.5 mm and lengths: 6.35, 12.7 and 25.4 mm). Then, an experimental characterization was carried out to validate the model monitoring filament width as the output. Shear stress values were also estimated to ensure cell viability during the process. Printing parameters were chosen to print 3D cell-embedded construct with open and interconnected pores to guarantee nutrients permeability. Cell viability and cartilage genes expression were investigated at different time points (days 14 and 28). RESULTS:The mathematical model was successfully validated by experimental data: the smallest discrepancy (1.5%) was found with needle diameter of 0.2 mm and length of 6.35 mm, whereas, the largest discrepancy (65.8%) with needle diameter of 0.5 mm and length of 25.4 mm. The model helped to define the best printing parameters to achieve a filament width of 200 µm using a pressure of 1.2 bar and a printing speed of 1.5 mm/s. The selected printing parameters and SF-G bioink ensured open pores formation and good cell viability until day 28. Moreover, we confirmed an increase expression of typical chondrogenic markers (SOX9, COLL2), at day 28 by both gene expression and immunofluorescence analyses. DISCUSSION & CONCLUSIONS:This study evidenced that the proposed model can be used to predict the features of the silk-based hydrogels permitting to save time and resources by avoiding trial-and-error approaches. Moreover, we confirmed that SF-G bioink enhanced cartilaginous matrix formation. ACKNOWLEDGEMENTS:This work was founded by Italian Ministry of Foreign Affairs and International Cooperation, Rizzoli fund “5x1000” and by EU Project Horizon 2020, ADMAIORA, grant N.814413. REFERENCES:[1] R Suntornnond (2016), Materials 9:756 [2] A Sharma (2019) ACS Biomater. Sci. Eng. 1518:1533 Keywords: 3D printing and bioprinting, Biomaterials www.ecmconferences.org eCM Periodical, 2020, Collection 1; 2020 TERMIS EU Abstracts (page 3) Robust Human Organoid Printing and Culture in an Integrated Plate System for Predictive Compound Screening Sangjoon LEE, Soo Yeon KANG, Sunil SHRESTHA, Pranav JOSHI, Prabha ACHARYA, Moo Yeal LEE Department of Chemical and Biomedical Engineering, Cleveland State University, Cleveland, USA INTRODUCTION:There have been several three-dimensional (3D) cell culture platforms developed, including ultra-low attachment well plates, Transwell inserts, hanging droplet plates, and microfluidic plates, but these platforms are relatively low throughput and/or unsuitable for high-throughput organoid culture and analysis in situ. METHODS:To facilitate robust organoid culture in a high-throughput screening (HTS) system, we have developed miniature “3D bioprinting” technology and an integrated plate system consisting of a pillar plate and a complementary perfusion well plate, which is highly flexible and easily combined with conventional 384-well plates to support organotypic cell cultures and multiplexed high-content imaging assays. RESULTS:We have demonstrated that our miniature 3D bioprinting platform can be used for optimizing organoid culture conditions that are critical to successfully create human tissue replicas. Several human organoids including brain, liver, intestine, and pancreas have been successfully printed, encapsulated in biomimetic hydrogels including Matrigel, differentiated, and imaged on the pillar plate platform for high-throughput compound screening. The optically clear pillar/perfusion well plates allowed direct visualization of organoids on the pillars for predictive cell-based assays. The entire organoids on the pillar plate were permeabilized, fixed, stained with primary and secondary antibodies, and cleared with tissue clearing solutions simultaneously for in situ whole organoid imaging without the need for cryosectioning. The flexible pillar and perfusion well format connected by microchannels and reservoirs made it easy to change growth media for organoid culture without the use of bulky pumps and tubes. It is compatible with standard 384-well plates and existing HTS equipment including fluorescent cell imagers and microtiter well plate readers, which is an important feature for developing HTS assays. It is easy to connect different types of organoids cultured
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