@conference {2280, title = {Multi-organ-on-plate system for in-vitro studies of intestinal drug absorption and hepatotoxicity}, booktitle = {ESTIV 2014}, year = {2014}, pages = {107}, address = {Egmond aan Zee, The Netherlands}, abstract = {

More meaningful in-vitro models which simulate the physiological conditions of native tissue are becoming essential in the pharmaceutical field, for early and rapid screening of drug candidates. Here, we describe a multi-organ-on-plate system based on single and double flow mini bioreactor modules for dynamic in-vitro studies of intestinal drug absorption, drug metabolism and more relevant toxicity studies. The double flow module for membrane culture was firstly characterized using computational fluid dynamic models and measurements of pressure gradients, in order to indentify the optimal flow rates for maximizing the passage of solutes through the membrane. Then, cell culture experiments were performed with fully differentiated Caco-2 cells seeded on the semi-permeable membrane as a dynamic model of the intestinal epithelium, connected to a single flow chamber with metabolically competent human upcyte{\^A}{\textregistered} hepatocytes (Medicyte GmbH, Germany) seeded on a 3D collagen cryogel. First we assessed the role of flow in modulating the passage of compounds across the epithelial barrier. Then toxicity tests were performed by administering different concentrations of hepatotoxic compounds (i.e. Diclofenac, Nimesulide, industrial nanoparticles) in the apical compartment of the MB, compared the data with cell cultures in transwells. Our results show: i) the presence of flow significantly increases translocation of all molecules tested across the membrane, ii) flow conditioned Caco-2 cells are more permeable to small hydrophilic compounds, despite having high TEER values iii) although they display higher levels of phenotypic markers (tight junctions, albumin expression etc), cells in the system are more susceptible to drug induced toxicity. In conclusion, the multi-organ-on-plate system predicts drug adsorption and toxicity better than traditional cell cultures and could be used to reduce, refine and eventually replace animal tests.

}, keywords = {Bioengineering}, author = {S. Giusti and Sbrana, T. and D. Giacopelli and V. Di Patria and A. Ahluwalia} } @conference {1804, title = {Monitoring cell culture 3D microenvironments: oxygen and pH nano-sensors within hydrogels}, booktitle = {National Conference of Bioengineering}, year = {2012}, address = {Rome, Italy}, abstract = {

Ratiometric pH and O2 nanosensors were fabricated independently using a fluorophore that produces a signal proportional to the concentration of the analyte of interest, and a second fluorophore that produces a reference signal, insensitive to the analyte of interest. These fluorophores emit at different wavelengths and were either entrapped or covalently bound, within an inert optically transparent polymeric matrix. In this work uniform sized 3D micro-scaled alginate hydrogel constructs of approximately 100-300 $μ$m were fabricated. Through dual incorporation of HepG2 cells and nanosensors within these alginate constructs we aim to have a real-time, non-invasive method to measure microenvironmental pH and O2 content. Ratiometric fluorescent output from the microenvironment is used to monitor O2 concentrations and pH during cell culture. Measurements show that 3D micro-scaled constructs are suitable for cell growth and proliferation. Moreover O2 and pH values within the hydrogel cellularised microspheres are shown to have physiological values that enable the maintenance of the hepatic phenotype

}, keywords = {Bioengineering}, author = {Tirella, A and Carroll, L. A. and Aylott, J and A. Ahluwalia} } @conference {1800, title = {{Monitoring microconstructs: cell and nanosensor encapsulation in alginate micro-beads}}, booktitle = {TERMIS 3rd World Congress}, year = {2012}, address = {Vienna, Austria}, abstract = {

Monitoring and controlling the microenvironment of cell cultures is an ongoing challenge for many researchers. Much research has been conducted characterising individual aspects such as 3D architecture, mechanical properties, biochemicals, etc. The biggest deficits in existing models for monitoring analytes within the cellular environment is the lack of appropriate means for non invasive, real-time and integrated monitoring of the cellular responses. Nanosensors can overcome these issues: they are porous polymeric nanoparticles that are sensitive to a range of analytes including pH and O2. Microfabrication techniques are innovative tools to obtain controlled microstructures with a defined 3D architecture. In this work uniform sized 3D micro-scaled hydrogel constructs of approximately 300-400 um diameter were fabricated. Through dual incorporation of cells and nanosensors within these constructs we aim to have a real-time, non invasive method to measure microenvironmental pH value and O2 content. Ratiometric fluorescent output from the microenvironment is used to monitor O2 and pH during cell culture. Measurements show that 3D micro-scaled constructs are suitable for cell growth and proliferation. Moreover O2 content and pH within hydrogel cellularised microspheres are shown to have physiological values which enable the maintenance of the hepatic phenotype.

}, keywords = {Bioengineering, Nanotools}, author = {Tirella, A and A. Ahluwalia and Aylott, J and Carroll, L. A.} } @conference {1803, title = {A multi-stimuli environment for cardiac tissue engineering}, booktitle = {TERMIS 3rd World Congress}, year = {2012}, address = {Vienna, Austria}, abstract = {

The identification of the ideal cell source to generate cardiac tissue able to integrate into the host myocardium and with the contractile system is crucial for cardiac engineering. Amongst different cell sources so far proposed, human adult Cardiac Progenitor Cells (hCPCs) show the ability to proliferate and differentiate toward cardiac lineages when grown in appropriate microenvironmental conditions. It is widely accepted that conventional 2D cultures may provide a physiological environment for growing cells. For this reason the need to have an engineered microenvironment, matching physiological requirements, is crucial. A 3D context with spatial and time varying distribution of regulatory factors using mechanically matched scaffolds and bioreactors could represent an in vitro cell culture model being able to more closely reflects the in vivo conditions. In the present study, the possibility of using biocompatible and biodegradable scaffolds of collagen based or derivatives hydrogels in combination with Linneg/Sca-1pos hCPCs gathered from human heart biopsies was investigated. Bio-constructs were placed in the low shear, high flow MCmB (MultiCompartment modular Bioreactor) and the combined effects of dynamic culture conditions and 3D scaffolds on cell morphology and differentiation were studied in order to investigate the possibility of fabricating stem cell-derived cardiac patches to replace infarcted tissue.

}, keywords = {Bioengineering, Bioreactors, Dynamic 3D Cultures}, author = {Tirella, A and A. Ahluwalia and P. Di Nardo and Gaudiello, E and S. Giusti and F. Pagliari} } @conference {1805, title = {Modelling and realisation of smart graded hydrogel scaffolds}, booktitle = {Euromat-FEMS}, year = {2011}, address = {Montpellier, France}, abstract = {

European Congress and Exhibition on Advanced Materials and Processes - Poster F12-P-2-08

}, keywords = {Bioengineering}, author = {Tirella, A and G. Mattei and F. Montemurro and G. Vozzi and A. Ahluwalia} } @inbook {1286, title = {Multi-Compartmental Modular Bioreactor as Innovative System for Dynamic Cell Cultures and Co-Cultures}, booktitle = {Bioreactors: Design, Properties and Applications}, number = {978-1-62100-164-5}, year = {2011}, pages = {159 - 178}, publisher = {Nova Science Publishers, Inc}, organization = {Nova Science Publishers, Inc}, chapter = {Multi-Compartimental Modular Bioreactor as Innovative System for Dynamic Cell Cultures and Co-Cultures}, abstract = {

In this chapter, the design, fabrication process and preliminary tests of a Multi- Compartmental Modular Bioreactor used as a system for dynamic cell cultures and co-cultures is described. Although the microwell (MW) plate has become a standard in cell culture, the complexity of the physiological environment is not replicated in petri dishes or microplates. All cells are exquisitely sensitive to their micro-environment which is rich with cues from other cells and from mechanical stimuli due to flow, perfusion and movement. Microwells do not offer any form of dynamic chemical or physical stimulus to cells, such as concentration gradients, flow, pressure or mechanical stress. This is a major limitation in experiments investigating cellular responses in-vitro since the complex interplay of mechanical and biochemical factors is absent. Most researchers and industry have started to accept that classical in vitro experiments offer poor predictive value or mechanistic understanding and are shifting their interests to new technologies such as bioreactors. For this reason, a large number of bioreactor systems for cell culture have been recently designed and described. With the purpose of developing cell culture models to establish a physiological-like interaction between different cell types, a novel Multi-Compartmental Modular Bioreactor (MCmB)was realized. The modular chamber was designed with shape and dimensions similar to the 24-MultiWell allowing an easy transfer of microwell protocols. The MCmB consists of a cell culture chamber made of bio-compatible silicon polymer, with excellent self-sealing proprieties, transparency and flexibility. The modular chambers can be also connected together in series or in parallel as desired, in order to allow cell-cell cross-talk or replicate in vitro models of metabolism or diseases using allometric design principles. In this chapter we describe the bioreactor design process starting from a finite-element method (FEM) model, developed in order to study the shear stress and the oxygen concentration at the cell surface. A further version of the MCmB is also described, in which a semipermeable membrane is placed into the bioreactor allowing to create a double-chamber system (MCmB-dc) for biological barriers simulation like for example lung or intestine. Allometric methods for designing in-vitro organ models using combinations of different cell types or tissues cultured in different chambers are also presented. Allometric laws mathematically correlate non linear quantities such as organ mass, blood flow, blood retention time and metabolic rate. Using these laws the modules can be assembled in various configurations enabling organ and system physiology to be recapitulated in vitro. Preliminary experiments using the modules are also described.

}, keywords = {Bioengineering}, issn = {978-1-62100-164-5}, author = {D. Mazzei and S. Giusti and Sbrana, T. and A. Ahluwalia}, editor = {Antolli, P. G. and Zhiming Liu} } @conference {1640, title = {Microfabbricazione di scaffold in alginato con propriet{\`a}~ meccaniche modulabili}, booktitle = {Congresso Nazionale Biomateriali 2008}, year = {2008}, pages = {{\textendash}}, keywords = {Bioengineering}, author = {Orsini, A and Tirella, A and A. Ahluwalia and G. Vozzi} } @conference {1794, title = {Microfluidic Gradient Maker for Pattern Generation}, booktitle = {TERMIS-EU}, year = {2008}, pages = {879}, address = {Porto, Portugal}, abstract = {

poster

}, keywords = {Bioengineering}, author = {Tirella, A and D. Mazzei and G. Vozzi and A. Ahluwalia} } @article {1489, title = {A microfluidic gradient maker for toxicity testing of bupivacaine and lidocaine}, journal = {Toxicology in vitro}, volume = {22}, year = {2008}, pages = {1957{\textendash}64}, abstract = {

A great deal of effort is being dedicated to the development of new devices able to conduct effective in vitro toxicology analyses. This paper describes the use of a microfluidic gradient maker for the toxicological analysis of two conventional local anesthetics, bupivacaine and lidocaine on cell cultures. The microfluidic device was designed and simulated using COMSOL Multiphysics and the concentration gradient in the microfluidic network was analysed through a fluidodynamic and diffusive study. Subsequently the device was fabricated with soft lithography, casting PDMS in a master to obtain channels about 250 microm deep. Both drugs were tested on C2C12 myoblasts and an analysis was performed using propidium iodide staining followed by an imaging processing routine to obtain quantitative dose-response profiles in the gradient maker. The system was critically compared with microwell-based toxicity testing. The results show that the GM is a more sensitive method for detection of cell toxicity, and compared with testing of drug toxicity using microwells with individual cell cultures, allows one shot testing with a single cell culture exposed to a large number of concentrations. However, the flow rates required to obtain a suitable concentration range across the device may damage shear sensitive cells.

}, keywords = {Bioengineering}, issn = {0887-2333}, doi = {10.1016/j.tiv.2008.09.016}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18940244}, author = {Tirella, A and Marano, M and Vozzi, F. and A. Ahluwalia} } @article {1274, title = {MULTI-CAVITY BIOREACTOR CHAMBER}, year = {2008}, keywords = {Bioengineering}, issn = {P122454GB}, author = {A. Ahluwalia and D. Mazzei and Vinci, B.} } @article {1653, title = {Microfabrication for tissue engineering: rethinking the cells-on-a scaffold approach}, journal = {JOURNAL OF MATERIALS CHEMISTRY}, year = {2007}, pages = {{\textendash}}, keywords = {Bioengineering}, issn = {0959-9428}, author = {G. Vozzi and A. Ahluwalia} } @article {1656, title = {Microfabrication of fractal polymeric structures for capillary morphogenesis: applications in therapeutic angiogenesis and in the engineering of vascularized tissue}, journal = {JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. PART B, APPLIED BIOMATERIALS}, volume = {81}, year = {2007}, pages = {462{\textendash}468}, keywords = {Bioengineering}, issn = {1552-4981}, author = {BIANCHI, F and ROSI, M. and G. Vozzi and EMANUELI, C. and MADEDDU, P. and A. Ahluwalia} }