Periodic Reporting for period 1 - MImETIC INDiRECT (MIcrovascularizED Tumour-on-chip for Cellular ImagiNg of Drug Response and Cell moTility)
Periodo di rendicontazione: 2019-08-01 al 2021-07-31
MImETIC INDiRECT aimed to create a versatile microfluidic (MF)/organ-on-chip (OoC) toolkit for cost-effective, standardized methodologies creating advanced in vitro models (AIM) to study cell motility and drug response. Envisioned toolkit versatility fostered cross-platform compatibility under perfusion or static AIM approaches exploiting commercially available 24-multiwell (24MW) consumables.
The OoC field currently lacks well-defined or established regulatory guidelines, where commercial entities each provide an unique, non-standardized products for end-users in academia and industry. Regulatory uncertainty and reluctance of adoption is echoed in the OoC markets, with conservatively low predicted compound annual growth rates (CAGRs). Adoption of OoC technologies require significant financial and investment where a plethora of patents, publications and commercial products instils confusion in end-users. Comparative advantages of OoC to “golden standard” technologies do not always demonstrate cot-incentive advantages for robust scientific application. These sentiments were echoed in a 2017/18 European Research Commission Joint Research Panel report. A 40-60% cost-saving and higher success turnover is predicted with OoC adoption in the drug development pipeline, currently requiring 13-15 years and ±USD2.6 billion per compound. However, end-users recognize OoC advantages for improved physiological recapitulation in vitro, once standardization and cost-barriers are addressed.
Objectives were informed by initiating a technology- and market-landscape, with step-wise toolkit development: (i) design, fabrication and technical validation of consumables and biomaterial blends; and (ii) biological validation with proof-of-concept using commercially available cell lines to establish a microvascularized in vitro liver model. The Cherry CubiX platform was used to demonstrate proof-of-principle by providing multiparametric control of liquid perfusion to mimic blood flow; temperature regulation; and dissolved gasses (O2, CO2) management.
In conclusion, it was successfully demonstrated that a standardized, robust toolkit and methodology can be established on a multiparametric platform (Cherry CubiX) to study AIMS liver pathology. However, fostering technology adoption remains a challenge in a small market embroiled in regulatory uncertainty, with early adopters currently the target end-users.
The OoC field currently lacks well-defined or established regulatory guidelines, where commercial entities each provide an unique, non-standardized products for end-users in academia and industry. Regulatory uncertainty and reluctance of adoption is echoed in the OoC markets, with conservatively low predicted compound annual growth rates (CAGRs). Adoption of OoC technologies require significant financial and investment where a plethora of patents, publications and commercial products instils confusion in end-users. Comparative advantages of OoC to “golden standard” technologies do not always demonstrate cot-incentive advantages for robust scientific application. These sentiments were echoed in a 2017/18 European Research Commission Joint Research Panel report. A 40-60% cost-saving and higher success turnover is predicted with OoC adoption in the drug development pipeline, currently requiring 13-15 years and ±USD2.6 billion per compound. However, end-users recognize OoC advantages for improved physiological recapitulation in vitro, once standardization and cost-barriers are addressed.
Objectives were informed by initiating a technology- and market-landscape, with step-wise toolkit development: (i) design, fabrication and technical validation of consumables and biomaterial blends; and (ii) biological validation with proof-of-concept using commercially available cell lines to establish a microvascularized in vitro liver model. The Cherry CubiX platform was used to demonstrate proof-of-principle by providing multiparametric control of liquid perfusion to mimic blood flow; temperature regulation; and dissolved gasses (O2, CO2) management.
In conclusion, it was successfully demonstrated that a standardized, robust toolkit and methodology can be established on a multiparametric platform (Cherry CubiX) to study AIMS liver pathology. However, fostering technology adoption remains a challenge in a small market embroiled in regulatory uncertainty, with early adopters currently the target end-users.
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.
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.
Toolkit proof-of-concept was demonstrated using commercial 24MW consumables, CubiX Mark I prototype, peristaltic pumps, as well as golden standard static culture approaches where complementary example protocols demonstrate 2D/3D in vitro culturing as well as opensource image analysis approaches. This enabled generating results equivalent to commercial available OoC/MPS chips in a medium-throughput format without the single chip inter-OoC variability between experiments. The plug-n-play approach of the toolkit designed for this project aims to encourage end-user experimentation and subsequent adoption, allowing end-user flexibility demonstrating MF-principles for OoC/MPS without significant financial investment. The toolkit is currently under investigation for novelty and patentability, presently already adopted or integrated as part of the Cherry CubiX Mark I offering for academic and pharmaceutical collaborators.