Standardisation of methods for cells and tissues preparation for mechanical testing
Duration: months 2-40.
Lead beneficiary: WWU
Objectives: The main goal of this WP is to optimise methods to immobilise and prepare cells, tissues and ECMs for mechanical testing using AFM and other techniques.
WP3 will take benefit of the results achieved within the EU Tiss.EU project.
Task 3.1. Identification of robust, stable and reproducible cell culture conditions for mechanical tests.
Different cell culture conditions using cell lines will be assessed including confluency, micro-patterning, elastic substrates, surface coating (fibronectin, polylysine...) adapted to mimic each cell type physiological and clinical conditions. (Lead CNRS, IFJPAN, JUMC, BioMeca)
Task 3.2. Definition of robust, stable and reproducible preparation procedures of tissues and ECM for mechanical tests.
This task will assess the use of microtome slicing, mild fixation, de-cellularisation protocols, etc. adapted to better preserve the mechanical properties of intact, fresh tissues. Preparation methods routinely used in the clinic for histological investigation will be adapted for mechanical tests (Lead OSR, CHUGR, IFJPAN, UMIL, UB, TMT-C2RC, BioMeca)
Task 3.3. Development of standardised protocols to prepare cells and tissues for shipping, preserving viability and mechanical properties during transport.
Shipment of samples between nodes is a prerequisite for the validation of standardisation procedures (Lead WWU, OSR, UMIL, UB, JUMC, TMT-C2RC)
Task 3.4. Definition of the robust, stable and reproducible method to immobilise tissues and ECM for mechanical tests.
This will include the assessment of various biocompatible adhesives, mechanical clamps, chemical cross-linking, incubation times, etc. to best preserve tissue morphology and mechanical properties (Lead WWU, CNRS, OSR, CHUGR, IFJPAN, UMIL, UB, TMT-C2RC)
Task 3.5. Correlative approaches based on coupling AFM to other methods.
Other methods will be correlated to AFM, such as molecular localisation using fluorescent microscopy incl. super-resolution (STED/RESOLFT, PALM/STORM), histological inspection... This task will develop technical approaches to correlate structural and mechanical information (collected according to WP2) of the bio-specimens, towards better detection and diagnosis of diseases. (Lead CNRS, INSERM, UMIL, CHUGR)
Task 3.6. Multi-sample analysis: methods to perform multi-sample measurements in an automated way.
This task will develop strategies to allow automatised mechanical tests on multiple samples with the final goal of converting AFM into a middle-throughput method (in parallel with WP5). (Lead CNRS, INSERM)