Periodic Reporting for period 2 - MATRIXASSAY (Novel Cell Migration Assay Based on Microtissue Technology and Tissue-Specific Matrices) Okres sprawozdawczy: 2017-06-01 do 2019-05-31 Podsumowanie kontekstu i ogólnych celów projektu Most migration assays operate based on a migration zone created when a stopper is removed or a scratch is made. Both of these methods can damage the underlying tissue-specific ECM, which is critical to the proliferation and migration of the cells and therefore to the reliability and accuracy of the assay. With the foregoing in mind, the overall scientific objective of our project is the development of a novel assay, where instead of using a gap, a size-specific microtissue containing a known number of cells is placed on a tissue-specific ECM. Briefly, the project aims to exchange knowledge and skills in the areas of assembly and characterisation of functional biomaterials, mathematical modelling of mechanical properties of biomaterials and layered scaffolds and their measurement, the formation of organotypic microtissues, and the migration of cells on aligned substrates in response to well-characterised tuneable mechanical and structural properties, which will lead to the development of a novel prototype cell migration and invasion assay that offers significant advantages over existing assays. Objectives:1) Determine experimentally the structure-property relationships between collagen I matrices with controlled thicknesses and fibril diameter and alignment, and their mechanical and electromechanical properties, and to understand these relationships using mathematical modelling;2) Employ simulation and experiment to develop and model functional bonding between the dissimilar PDMS and collagen I layers;3) Optimise the formation of microtissues suitable for cell migration assays;4) Effectively disseminate knowledge intersectorally between academic and industrial consortium members; and, 5) Perform cell migration assay investigations on 96 well-plate prototypes. Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty Objective 1: a significant programme of mechanical tests have been implemented at LU to elucidate mechanical behaviour and processes at various scales and in different regimes. Elastic parameters of collagens were measured using nanoindentation and compared with the data obtained with atomic force microscopy (AFM), performed at UCD. A time-dependent (viscoelastic) response of collagens was studied using Dynamic Mechanical Analysis for a range of frequencies at physiological temperatures. The measured data for storage modulus was compared with the magnitudes obtained both with nanoindentation and AFM. Another contribution of LU towards Objective 1 was development of advanced parametric multi-scale models of fibrous materials to study evolution of deformation, damage and fracture on them. As mentioned, mechanical measurements have been performed by AFM at UCD on a wide range of collagen samples (to be reported in Deliverable 1.4 under preparation and presented below). Fibralign is currently preparing aligned collagen films having different sized areas of uniform piezoelectric domain/polarization ordering to be characterized using piezoresponse force microscopy at UCD.Objective 2: the planned mechanical measurements of freestanding films at LU was postponed – together with Deliverable D3.4 – due to an unexpected total failure of the Instron MicroTester system, suitable for performing low-load (in the range 0-5 N) tests. Other systems at LU do not provide a necessary level of precision, necessary for such measurements. A contingency plan to meet this challenge was developed, and as a result of these activities LU invested funds in acquisition of a new high-precision testing system with low-load capabilities – Instron 5944. The system is in the process of ordering, with the delivery date in August 2017. This will allow implementation of the initial test programme on freestanding collagen specimens. In the meantime, activities at UT Dallas and UCD have progressed, leading to the development of molecular collagen film coatings on PDMS and other substrates (as reported in Deliverable 1.3). The thickness of the collagen film and of the PDMS layers can be controlled. The deposition of D-periodic collagen fibrils of controlled fibril size as a function of pH is currently under investigation. Separately, alignment of collagen on glass and PDMS is a topic of investigation at UCD. It is unlikely that aligned collagen deposited by Fibralign will be transferred to PDMS due to limited availability of staff to work on this project; however, the other approaches are making headway. In addition, the effect of cross-linking of collagen on mechanical and electromechanical properties has been studied at UT Dallas and UCD by UCD staff and will give some control over the modulus of the resulting cell assay prototypes.Objective 3: as mentioned in the previous year’s report, the hanging drop plates used are now a mature product and routine procedure in cell culture where 3D spheroids and microtissues are studied. We have demonstrated the ability to prepare spheroids starting from 250 – 1000 cells, and can release the spheroids onto a well plate for a migration assay. Thus, InSphero has successfully transferred this know-how to UCD and UCD can perform migration assays with spheroids as reported at 1st and 2nd year workshops. The challenge now is in transferring such spheroids to LU in such a way that they can complete mechanical testing of them. This will be a priority in the coming months.Objective 4: there are very close and strong interactions between the beneficiaries and partners that continue to evolve through secondments. In particular, a series of mutual secondments between LU and Vornia as well as LU and InSphero enhanced knowledge transfer between the academic partner and industrial partners working in different areas. LU demonstrated to its industrial collaborators importance of understanding mechanical properties and performance of collagens for reliability of their products and the ways to use quantitative data to predict their performance. Possibilities to enhance manufacturing processes based on additional mechanical excitation were also discussed. In return, the industrial partners presented the links between the manufacture and structure of their products on their properties and performances to underpin development of experimental protocols and modelling tools. Please see Deliverable 6.3 for a full accounting of Matrixassay exchange of knowledge activities.Objective 5: cell migration assays have been performed on collagen-coated 96 well plates, and progress is being made to perform such assays on aligned collagen-coated well plates. It is expected that the first prototypes will be shipped to UCD in the coming months. In the meantime, aligned nanofiber-coated well-plates have been procured for continued testing and development of the spheroid transfer and migration imaging and analysis protocols. Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu) The main exploitable result will be the development of the Matrixassay prototype combining Fibralign and InSphero products. The prototype will be used by UCD and may be developed into a joint InSphero/Fibralign product. Matrixassay Action Logo
