1,720,986 research outputs found
DEVELOPMENT OF MEASUREMENT METHODOLOGIES IN METROLOGY FOR CELL BIOLOGY AND REGENERATIVE MEDICINE
Full text linkAim 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 factorie
Optimizing resazurin-based viability assays for P-MSC/TER308 cell line to enhance results reliability
Full text linkObjective: The results of this research contribute to the LifeSaver project, which focuses on reducing neonatal and infant mortality resulting from preterm births. The project aims to create an in vitro system simulating prenatal conditions to screen and analyze chemicals and pharmaceuticals, establishing scientifically justified regulations for their use during pregnancy. Because several papers have recently identified data inconsistencies in pre-clinical studies, a key part of the project involves optimizing cellular cytotoxicity assays to enhance the reliability of pharmacological and toxicity screening for drugs and environmental contaminants. Results: The resazurin-based viability assay was chosen as the primary method due to its widespread adoption and simplicity in assessing drug cytotoxicity. This work describes the optimization of the resazurin-based viability assay on the P-MSC/TERT308 cell line, a placenta-derived mesenchymal stem cell used within the LifeSaver project. By applying our previously described and validated Standard Operating Procedure, we fine-tuned experimental parameters, consistently obtaining reliable results with measurement uncertainty of less than 10%
Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo
No full textDirect reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through microRNAs (miRNAs) is a new emerging strategy for myocardial regeneration after ischemic heart disease. Previous studies have reported that murine fibroblasts can be directly reprogrammed into iCMs by transient transfection with four miRNAs (miRs-1, 133, 208 and 499 – termed “miRcombo”). While advancement in the knowledge of direct cell reprogramming molecular mechanism is in progress, it is important to investigate if this strategy may be translated to humans. Recently, we demonstrated that miRcombo transfection is able to induce direct reprogramming of adult human cardiac fibroblasts (AHCFs) into iCMs. Although additional studies are needed to achieve iCM maturation, our early findings pave the way toward new therapeutic strategies for cardiac regeneration in humans. This chapter describes methods for inducing direct reprogramming of AHCFs into iCMs through miRcombo transient transfection, showing experiments to perform for assessing iCM generation
Influence of a calcium carbonate scaffold on human mesenchymal stem cells proliferation and differentiation
No full textCardiac Tissue-like 3D Microenvironment Enhances Route towards Human Fibroblast Direct Reprogramming into Induced Cardiomyocytes by microRNAs
Full text linkThe restoration of cardiac functionality after myocardial infarction represents a major
clinical challenge. Recently, we found that transient transfection with microRNA combination
(miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult
human cardiac fibroblasts (AHCFs) into induced cardiomyocytes (iCMs) in vitro. However, achieving
efficient direct reprogramming still remains a challenge. The aim of this study was to investigate
the influence of cardiac tissue-like biochemical and biophysical stimuli on direct reprogramming
efficiency. Biomatrix (BM), a cardiac-like extracellular matrix (ECM), was produced by in vitro
culture of AHCFs for 21 days, followed by decellularization. In a set of experiments, AHCFs were
transfected with miRcombo and then cultured for 2 weeks on the surface of uncoated and BM-coated
polystyrene (PS) dishes and fibrin hydrogels (2D hydrogel) or embedded into 3D fibrin hydrogels
(3D hydrogel). Cell culturing on BM-coated PS dishes and in 3D hydrogels significantly improved
direct reprogramming outcomes. Biochemical and biophysical cues were then combined in 3D fibrin
hydrogels containing BM (3D BM hydrogel), resulting in a synergistic effect, triggering increased
CM gene and cardiac troponin T expression in miRcombo-transfected AHCFs. Hence, biomimetic
3D culture environments may improve direct reprogramming of miRcombo-transfected AHCFs into
iCMs, deserving further study
Characterization of AFM for elasticity analysis of biological samples: Application to diagnosis
No full textCytomorphological- and immunofluorescence-based methods are currently use for the diagnosis of malignant human tumors. However, morphological overlaps between tumor and normal cell types frequently occur. Recently, a change in cell elasticity of tissues has been recognized as a marker of tissues metastatic potential. Atomic force Microscope (AFM) can quantitatively distinguish cancer and normal cells by measuring the cell elastic modulus when operating in Force Spectroscopy mode. Thus, AFM as elasticity sensor can be a novel diagnostic method for cancer detection. With the broader aim to translate this novel method into clinical application, a preliminary metrological characterization of the AFM sensor is presented in this work. The sensor is characterized in the interested elasticity range, indenting in vitro models of biological materials with elasticity of 5 MPa–50 kPa. Measurement repeatability of 10 % is obtained in a suitable fit range with an indentation speed comparable with the sample viscous relaxation time (1 um/s). Opportunities of improvement for measurement reproducibility are discussed by analyzing surfaces images and the behavior of elasticity versus indentation depths
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The total testing process harmonization: the case study of SARS-CoV-2 serological tests
Full text linkThe total testing process harmonization is central to laboratory medicine, leading to the laboratory test's effectiveness. In this opinion paper the five phases of the TTP are analyzed, describing, and summarizing the critical issues that emerged in each phase of the TTP with the SARS-CoV-2 serological tests that have affected their effectiveness. Testing and screening the population was essential for defining seropositivity and, thus, driving public health policies in the management of the COVID-19 pandemic. However, the many differences in terminology, the unit of measurement, reference ranges and parameters for interpreting results make analytical results difficult to compare, leading to the general confusion that affects or completely precludes the comparability of data. Starting from these considerations related to SARS-CoV-2 serological tests, through interdisciplinary work, the authors have highlighted the most critical points and formulated proposals to make total testing process harmonization effective, positively impacting the diagnostic effectiveness of laboratory tests
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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