«Visse tutta una lunga vita a fare professione di pessimismo»: Michelstaedter vs Schopenhauer

[English]:This paper examines the meaning of Carlo Michelstaedter’s judgement on Arthur Schopenhauer, according to which Schopenhauer does not fully accept the consequences of his own system of thought but only makes a «profession of pessimism», in the light of Michelstaedter’s categories of «Persuasion» and «Rhetoric»./ [Italiano]: Il saggio analizza, alla luce delle categorie di «persuasione» e «rettorica», il significato del giudizio espresso dal filosofo goriziano Carlo Michelstaedter su Arthur Schopenhauer secondo il quale quest’ultimo, lungi dall’accogliere pienamente le conseguenze del proprio sistema di pensiero, avrebbe fatto soltanto «professione di pessimismo»

A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes

Transfection describes the delivery of exogenous nucleic acids (NAs) to cells utilizing non-viral means. In the last few decades, scientists have been doing their utmost to design ever more effective transfection reagents. These are eventually mixed with NAs to give rise to gene delivery complexes, which must undergo characterization, testing, and further refinement through the sequential reiteration of these steps. Unfortunately, although microfluidics offers distinct advantages over the canonical approaches to preparing particles, the systems available do not address the most frequent and practical quest for the simultaneous generation of multiple polymer-to-NA ratios (N/Ps). Herein, we developed a user-friendly microfluidic cartridge to repeatably prepare non-viral gene delivery particles and screen across a range of seven N/Ps at once or significant volumes of polyplexes at a given N/P. The microchip is equipped with a chaotic serial dilution generator for the automatic linear dilution of the polymer to the downstream area, which encompasses the NA divider to dispense equal amounts of DNA to the mixing area, enabling the formation of particles at seven N/Ps eventually collected in individual built-in tanks. This is the first example of a stand-alone microfluidic cartridge for the fast and repeatable preparation of non-viral gene delivery complexes at different N/Ps and their storage

VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding

The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications

Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa

Microfabricated Physiological Models for In Vitro Drug Screening Applications

Microfluidics and microfabrication have recently been established as promising tools for developing a new generation of in vitro cell culture microdevices. The reduced amounts of reagents employed within cell culture microdevices make them particularly appealing to drug screening processes. In addition, latest advancements in recreating physiologically relevant cell culture conditions within microfabricated devices encourage the idea of using such advanced biological models in improving the screening of drug candidates prior to in vivo testing. In this review, we discuss microfluidics-based models employed for chemical/drug screening and the strategies to mimic various physiological conditions: fine control of 3D extra-cellular matrix environment, physical and chemical cues provided to cells and organization of co-cultures. We also envision future directions for achieving multi-organ microfluidic devices

In vitro mechanical stimulation to reproduce the pathological hallmarks of human cardiac fibrosis on a beating chip and predict the efficacy of drugs and advanced therapies

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients

Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy

A New Algorithm to Analyze the Video Data of Cell Contractions in Microfluidic Platforms

Проблематика. Одним из быстро развивающихся направлений в науке является тканевая инженерия с использованием технологии “лаборатория на микрочипе”. Для оценки механических сокращений клеток используются оптическая микроскопия и анализ видеоданных. Однако известные методы регистрируют только факт сокращения клеток и существенно искажают форму и амплитуду полезного сигнала. Поэтому задачи корректного количественного анализа таких видеоизображений являются актуальными. Цель. Целью работы является разработка алгоритма анализа видеоданных механических сокращений кардиомиоцитов на микрочипе для определения их функциональных и структурных свойств на тканевом уровне. Методика реализации. Разработанный алгоритм анализа видеоизображений реализован программным кодом Matlab 2016. Для апробации предложенного метода были проанализированы данные о сокращения клеток кардиомиоцитов, выращенных в микрочипе. Исследовались три группы клеток: выращенные без стимуляции, а также клетки, которые стимулировались электрическими полями 5 и 25 В/см. Форма стимулирующих импульсов – прямоугольная, частота – 1 Гц. Результаты. Предложенный алгоритм анализа видеоданных, позволяет оценить скорость сокращения клеток в микрометрах за секунду. Кроме того, он позволяет разложить механические колебания клеток на компоненты. Алгоритм использовался для оценки изменения скорости сокращения кардиомиоцитов в зависимости от интенсивности напряжения и частоты возбуждения. Выводы. Предложенный метод анализа видеоданных сокращений клеток кардиомиоцитов на микрочипе не требует каких-либо вспомогательных биомаркеров или сред. Анализ видеоизображений позволяет оценить амплитуду и скорость колебаний, форму сигнала, пространственно- неоднородное распределение механической активности клеток. Показано, что импульсное электрическое поле в диапазоне 5–25 В/см на частоте 1 Гц при культивировании клеток оказывает отрицательное влияние на сократительную способность кардиомиоцитов

Predicting human cardiac QT alterations and pro-arrhythmic effects of compounds with a 3D beating heart-on-chip platform

Determining the potential cardiotoxicity and pro-arrhythmic effects of drug candidates remains one of the most relevant issues in the drug development pipeline. New methods enabling to perform more representative pre-clinical in vitro studies by exploiting induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are under investigation to increase the translational power of the outcomes. Here we present a pharmacological campaign conducted to evaluate the drug-induced QT alterations and arrhythmic events on uHeart, a 3D miniaturized in-vitro model of human myocardium encompassing iPSC-CM and dermal fibroblasts embedded in fibrin. uHeart was mechanically trained resulting in synchronously beating cardiac microtissues in one week, characterized by a clear field potential (FP) signal that was recorded by means of an integrated electrical system. A drug screening protocol compliant with the new International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines was established and uHeart was employed for testing the effect of 11 compounds acting on single or multiple cardiac ion channels and well-known to elicit QT prolongation or arrhythmic events in clinics. The alterations of uHeart's electrophysiological parameters such as the beating period, the FP duration, the FP amplitude and the detection of arrhythmic events prior and after drug administration at incremental doses were effectively analyzed through a custom developed algorithm. Results demonstrated the ability of uHeart to successfully anticipate clinical outcome and to predict the QT prolongation with a sensitivity of 83.3%, a specificity of 100% and an accuracy of 91.6%. Cardiotoxic concentrations of drugs were notably detected in the range of the clinical highest blood drug concentration (Cmax), qualifying uHeart as a fit-to-purpose pre-clinical tool for cardiotoxicity studies