Engineered muscle tissues for disease modeling and drug screening applications

Animal models have been the main resources for drug discovery and prediction of drugs’ pharmacokinetic responses in the body. However, noticeable drawbacks associated with animal models include high cost, low reproducibility, low physiological similarity to humans, and ethical problems. Engineered tissue models have recently emerged as an alternative or substitute for animal models in drug discovery and testing and disease modeling. In this review, we focus on skeletal muscle and cardiac muscle tissues by first describing their characterization and physiology. Major fabrication technologies (i.e., electrospinning, bioprinting, dielectrophoresis, textile technology, and microfluidics) to make functional muscle tissues are then described. Finally, currently used muscle tissue models in drug screening are reviewed and discusse

In Vitro Microfluidic Models for Neurodegenerative Disorders

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease- on-a-chip models focusing on Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps

Development of micro electro mechanical devices for the study of mechanosensitive ion channels and mechanical cell properties

2010/2011The objectives of this doctoral research involve the development of tools, in particular micro-nano devices for the study of mechanical properties of single living cells and for the analyses of mechanosensitive ionic channels (MSCs). BioMEMS (Biological Micro Electro Mechanical Systems) have been devised and used to investigate MSCs and the cell mechanics in a completely innovative way. Living cells in adhesion can be studied in a physiological condition; the mechanical stretch can be controlled and measured; the MSCs activity can be evaluate using different techniques from patch clamp to AFM (atomic force microscope) or fluorescence essays. Silicon BioMEMS have been designed and tested to evaluate morphological modifications of the stretched cells, and hysteretic behavior has been assessed. However, since they are not transparent, the use of this devices has been limited. Also completely transparent devices have been designed and microfabricated. These BioMEMS will allow testing cells and combining measurements of the mechanical properties, the cell’s morphology (with optical systems and atomic force microscopy), and MSCs activity (with patch clamp and/or conductive AFM). In this doctoral research, BioMEMS have been devised and realized, the measurement set-up optimized and a surface treatment protocol developed.XXIV Ciclo198

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Development of advanced 3D tissue models and O2 imaging methodologies

This thesis presents the development and evaluation of new luminescent sensors and probes, optimisation of the tumour spheroid model and the various applications for these in vitro tissues combined with novel O2 and temperature nanoparticle probes. Here a new method for O2 sensing was introduced, measuring extracellular oxygenation using solid state polystyrene scaffold (AlvetexTM) impregnated with the phosphorescent dye PtTFPP. We accessed the toxicity, sensitivity and photostability of the O2-sensitive scaffolds and monitored cell oxygenation following seeding with both PC12 and HCT116 cells. Using TCSPC and PLIM multiplexed with fluorescent markers and fluorescent staining we could non-invasively correlate lifetime values with toxicity and drug stimulation. Several novel O2 and temperature-sensitive nanoparticle probes were evaluated. These probes displayed sufficient brightness, photostability and sensitivity. Furthermore, they showed minimal toxicity and could penetrate 3D tumour spheroids in depth, showing efficient staining and even distribution. We applied the imaging methodology to the 3D spheroid model, investigating which method of formation from "free floating", "hanging drop" and LipidureTM, produced the most uniform, viable and metabolically active in vitro tissue. Numerous applications were improved and aided by the new O2 measuring platforms. Combining the novel probes with our new 3D models we could monitor the effects of chemotherapeutic drugs in both seeded O2 scaffolds and 3D spheroids. We demonstrate the application of FLIM method for multi-parametric analysis of O2 simultaneously with temperature and confirm the existence of temperature gradients in the 3D cell-based model. Finally, we applied the O2 probe and its measurement via 2 PLIM method to elucidate the function of SPCA2 in human colon cancer HCT116 cells, grown in ambient and decreased O2 levels. We could correlate SPCA2 upregulation with hypoxia in both monolayer and in spheroids. Furthermore, we discovered that SPCA2 is up-regulated by cell density, playing a role in Mn2+ transport and cell cycle progression in cancer cells. Results show that the developed probes and techniques provide a useful tool for the highly sensitive imaging of intracellular and extracellular O2, temperature and other important parameters

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin