A project website was created, disseminated on social media (Twitter, LinkedIn). Concurrently an OoC technology/market landscape was conducted (<10-years), complemented with competitor analysis and thought-leader interviews. Online content was created and published with keywords [drug response, precision oncology, live imaging microscopy, cell motility] for search engine optimization (SEO). Results and metrics were analysed, followed by iteration of the above process with best results for the keywords [microvascularization, liver, dissolved oxygen management]. End-user desire for OoC application flexibility was discerned from interviews, with competitor analysis identified 3 potential market competitors (Ibidi, CSEM, Lonza) for 24MW approaches. The toolkit design aimed to facilitate medium throughput 24MW microvascularized OoC AIMS as a possible market entry point, also addressing needs of existing collaborators (academia, biotechnology, pharma).
An overview of the results show successful demonstration for proof-of-principle microvascularized OoC liver model which correlates well with current state-of-the-art publications at reduced cost of biomaterials (e.g. collagen) as well as faster AIMS establishing time using the plug-n-play CubiX platform. The OoC liver model demonstrated physiological requirements when biochemical features were assessed, with future assessment to go beyond the 72h mark. This approach demonstrated advantages: (a) reduced collagen use; (b) directed liquid movement results in better shear stress control; (c) less cells used; (d) reduced media consumption; and (e) physiological cell-type ratios. Points (a), (c), (d) and (e) where end-user needs for OoC adoption; where (b) and (e) are technical improvement for OoC models. The toolkit allows for user flexibility at a reduced cost of commonly used biomaterials, adding ±20min to general methodologies, allowing end-user implementation of MF-principles on existing or novel platforms for establishing AIMS.A more technical description follows.
Bioinert stamps were designed (SolidWorks), manufactured (Outsourced) and technically validated for hydrogel patterning in 24MW (Fig. 1, Scheme 1). Hydrogel formulations (agarose-based) for patterning were reiteratively optimized during biological validation experiments. Technical validation estimated a 60% collagen use reduction per 24MW using this approach, considering collagen typically costs €2400/gram, this is a significant cost-benefit. The general experimental 24MW setup with the CubiX Mark I (Fig. 2), also used for biological validation using commercial cell lines: liver carcinoma cells (HepG2) and human umbilical vein endothelial cells (HUVECS). The latter was used to establish in vitro microvasculature monolayer (Fig. 3a) on top of a 3D collagen (3DC) surface (Fig. 3b). Actin fibre alignment analysis (Fig. 3b) showed good cellular polarization with liquid flow direction. Immunofluorescent staining (Fig. 3c) for vascular biomarkers indicated the desired cellular phenotype. HepG2 cells grown embedded within 3DC as mono-or cocultures (+HUVEC). Results (Fig. 4) show that HepG2 cells were cultured successfully for all conditions. Microscopy (Fig. 4a) and functional assessment (Fig. 4b) demonstrated successful 72h perfused gas managed culturing. Assessment of microtissue functional capacity showed that the HepG2-HUVEC cocultures, correlating with established and state-of-the-art literature.
The exploitation and dissemination of the technical result is currently (>August 2021) under investigation for intellectual property protection, where aspects are under discussion with collaborators for implementation within their respective workflows under confidentiality agreements. The technology/market landscape data was repurposed for a case-study publication (“The Challenges and Considerations for Emerging or Future Entrepreneurial Researchers in Microphysiological Systems.” – In Review) on the European Open Research Portal.
