DEVELOPMENT OF MEASUREMENT METHODOLOGIES IN METROLOGY FOR CELL BIOLOGY AND REGENERATIVE MEDICINE

Aim of this thesis is the development of measurement methodologies in metrology for cell biology and regenerative medicine. Regenerative medicine is a novel branch of medicine based on the use of autologous stem cells and biocompatible medical devices to regenerate and repair damaged tissues of patients, i.e. by using three-dimensional scaffolds to implant stem cells into the tissue to be regenerated. Stakeholders of metrology for regenerative medicine are: health care providers who require safe, reliable and cost effective treatments, supported by evidence and approved by regulators; regulators who require standard materials and traceable data demonstrating the safety and efficacy of new products and treatments; medical products companies who require advanced and traceable techniques to develop new products and need methods to measure processes, such as cell growth on scaffolds, to ensure quality and efficiency of the medical products implanted into the patients. Consequently, regenerative medicine has the important requisite of a real time monitoring and not invasiveness neither destructiveness processes to measure the cell-scaffold interactions, in order to preserve the samples from any contamination or modification. Thus non-invasive measurement methodologies need to be developed for analysing the 3D cell culture on scaffolds and, in order to evaluate the uncertainty, highly reproducible measurement procedures are strongly required to minimize the type A uncertainties and to define the type B uncertainties. The non-invasive and non-destructive measurement of cell-scaffold interactions (i.e. stem cell proliferation and differentiation on scaffolds) is one of the most effective methodology to answer the need of testing the efficacy of the design, production/manufacturing, development and performances of stem cell-scaffold products. To satisfy the requirements and the needs for metrology in regenerative medicine, for this thesis it has been chosen to develop a measurement methodology for cellular activity (proliferation and differentiation) on 3D Biocoral® scaffolds and to conduce a metrological study to evaluate the uncertainty of the methodology. This thesis has been developed in the Bioscience group of the Italian National Metrological Institute (Istituto Nazionale di Ricerca Metrologica - INRIM). The main important contributes of this thesis to the metrology in biosciences have been: • to lay the foundations for a metrological approach to cell biology and particularly to regenerative medicine research and applications; • to address the filling of the lack of traceability in the metrology for cell biology metabolic methodologies used to evaluate cellular activities in living sample with non-invasive procedures. The main results and originalities achieved during this PhD work are: • a metabolic assay, the resazurin/resorufin assay, for the first time, has been metrologically characterized and the uncertainty of the measurement has been evaluated; • the resazurin/resorufin assay has been for the first time tailored for a 3D cell culture on Biocoral® scaffolds and the uncertainty of the measurement has been evaluated; • it was demonstrated that Biocoral® induces osteodifferentiation of stem cells and for the first time it was demonstrated on human mesenchymal stem cells; • it was demonstrated, for the first time, that the resazurin/resorufin metabolic assay can be a methodology to detect not only the proliferation but also the differentiation of stem cells on Biocoral® scaffolds; A description of the METREGEN regional project, which this thesis is part of, will follow in the introduction. The chapter 1 will give an overview on regenerative medicine field and its application with scaffolds, particularly referring to the Biocoral® scaffold. The resazurin/resorufin methodology will be deeply described in chapter 2 with a uncertainty budget evaluation and discussion. Chapter 3 will present in details a series of experiments made to establish and characterize a hMSCs in vitro 2D culture, establish a hMSCs in vitro 3D culture on Biocoral, tailor the resazurin/resorufin assay for 3D cell culture on Biocoral and evaluate the hMSCs osteodifferentiation induced by Biocoral scaffolds. All the results have been analysed with a metrological approach to evaluate the uncertainty. Finally, the conclusion will give a recapitulation and some interesting perspective of employment for the resazurin/resorufin methodology to final users, such as the cell factories.