02279nas a2200229 4500008003900000245008800039210006900127260002700196300000700223520152800230653001901758100001801777700001401795700001401809700001801823700001401841700001501855700001401870700001401884700001701898856013401915 2014 d00aAutonomous bioreactor modules for disease models and detection of systemic toxicity0 aAutonomous bioreactor modules for disease models and detection o aPrague, Czech Republic a433 a

Advanced systems based on bioreactors and scaffolds are an essential step towards the development of more predictive and ethical alternatives to animal experiments. Size, modularity, automation, monitoring and essential design are crucial because these elements will ease the transition from old technology and accelerate their acceptance into mainstream research. Based on these requirements, the interconnected transparent sensorised “lego” bioreactors designed in our labs have been used to generate physiologically relevant disease and toxicity models which recapitulate systemic responses impossible to observe in standard cell cultures. The disease model is an interconnected bioreactor circuit with i) adipose tissue in 3D in 3 different concentrations representing normo-weight, over weight and obese body mass indices, ii) human hepatocytes on porous collagen scaffolds and iii) monolayers of human endothelial cells. High adiposity and elevated glucose levels induce systemic and endothelial inflammation in the circuit, as observed in overweight and diabetic humans (Iori et al., 2012). Using similar technology a three-tissue circuit for monitoring the absorption, distribution, metabolism and toxicity of nanoparticles was developed in the context of the EU project InLiveTox (Ucciferri et al., 2014). The results were strikingly similar to those observed in animal experiments demonstrating that the dynamic 3D in-vitro models are ethical, meaningful and economically viable replacements.

10aBioengineering1 aAhluwalia, A.1 aGiusti, S1 aSbrana, T1 aWilkinson, M.1 aMisto, A.1 aLehr, C -M1 aLiley, M.1 aHorst, S.1 aJírová, D. uhttps://www.centropiaggio.unipi.it/publications/autonomous-bioreactor-modules-disease-models-and-detection-systemic-toxicity.html02641nas a2200181 4500008003900000245010200039210006900141260003600210300000800246520196600254653001902220100001402239700001402253700001902267700001802286700001802304856013702322 2014 d00aMulti-organ-on-plate system for in-vitro studies of intestinal drug absorption and hepatotoxicity0 aMultiorganonplate system for invitro studies of intestinal drug  aEgmond aan Zee, The Netherlands a1073 a

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® 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.

10aBioengineering1 aGiusti, S1 aSbrana, T1 aGiacopelli, D.1 aDi Patria, V.1 aAhluwalia, A. uhttps://www.centropiaggio.unipi.it/publications/multi-organ-plate-system-vitro-studies-intestinal-drug-absorption-and-hepatotoxicity02073nas a2200193 4500008003900000245010100039210006900140260002700209300001100236520139000247653001901637100001401656700001501670700001401685700001801699700001501717700001401732856013301746 2014 d00aThe need of innovative technologies for new 3D relevant in-vitro models and the answer of Ivtech0 aneed of innovative technologies for new 3D relevant invitro mode aPrague, Czech Republic a45–63 a

New relevant in-vitro models are priorities in pharmaco-toxicology, cosmetic and food research to reduce the animal tests. Therefore, invivo models show ethical issues, are not time and cost effective and are progressively showing scientific limitations: for instance they fail in detection of pathogens that are species specific (Mazzoleni et al., 2009). The search of more relevant pre-clinical models forced the researcher to move from 2D to 3D in-vitro models in order to maintain the phenotype of cells (Lovit et al., 2013; Mattei et al., 2014). Even if the significant progress in material science, the metabolic requirement of 3D tissues is higher than a 2D culture and the scaffold is a limitation in nutrients transport. Dynamic cell culture chambers are then required to assure the gas/nutrient supply, waste elimination, mechanical stimulation of cells, study of cross talk between different tissues and real time monitoring of cells. Nowadays the only systems that meet all these specifications are the Ivtech technologies. Ivtech is an innovative Italian start-up that grows up to solve the needs of in-vitro experts, offering and customizing several type of transparent, dynamic and modular cell culture systems, organizing workshops and training. The goal is to expand the 3D approach and permits a significant evolution towards highly relevant in-vitro models.

10aBioengineering1 aSbrana, T1 aMattei, G.1 aGiusti, S1 aAhluwalia, A.1 aDagmar, J.1 aHorst, S. uhttps://www.centropiaggio.unipi.it/publications/need-innovative-technologies-new-3d-relevant-vitro-models-and-answer-ivtech.html02045nas a2200205 4500008004100000022001400041245010800055210006900163520140900232653001901641100001401660700001401674700001601688700001801704700001901722700001501741700001701756700001801773856004801791 2014 eng d a1860-731400aA novel dual-flow bioreactor simulates increased fluorescein permeability in epithelial tissue barriers0 anovel dualflow bioreactor simulates increased fluorescein permea3 a

Permeability studies across epithelial barriers are of primary importance in drug delivery as well as in toxicology. However, traditional in vitro models do not adequately mimic the dynamic environment of physiological barriers. Here, we describe a novel two-chamber modular bioreactor for dynamic in vitro studies of epithelial cells. The fluid dynamic environment of the bioreactor was characterized using computational fluid dynamic models and measurements of pressure gradients for different combinations of flow rates in the apical and basal chambers. Cell culture experiments were then performed with fully differentiated Caco-2 cells as a model of the intestinal epithelium, comparing the effect of media flow applied in the bioreactor with traditional static transwells. The flow increases barrier integrity and tight junction expression of Caco-2 cells with respect to the static controls. Fluorescein permeability increased threefold in the dynamic system, indicating that the stimulus induced by flow increases transport across the barrier, closely mimicking the in vivo situation. The results are of interest for studying the influence of mechanical stimuli on cells, and underline the importance of developing more physiologically relevant in vitro tissue models. The bioreactor can be used to study drug delivery, chemical, or nanomaterial toxicity and to engineer barrier tissues.

10aBioengineering1 aGiusti, S1 aSbrana, T1 aLa Marca, M1 aDi Patria, V.1 aMartinucci, V.1 aTirella, A1 aDomenici, C.1 aAhluwalia, A. uhttp://www.ncbi.nlm.nih.gov/pubmed/2475686901568nas a2200229 4500008004100000245008400041210006900125260000800194300001600202490000800218520084800226653001901074100001301093700001501106700001301121700001401134700001401148700001501162700001601177700001801193856012701211 2011 eng d00aA flexible bioreactor system for constructing in vitro tissue and organ models.0 aflexible bioreactor system for constructing in vitro tissue and  cSep a2129–21400 v1083 a

To develop in vitro models of cells, tissues and organs we have designed and realized a series of cell culture chambers. Each chamber is purpose designed to simulate a particular feature of the in vivo environment. The bioreactor system is user friendly, and the chambers are easy to produce, sterilize and assemble. In addition they can be connected together to simulate inter-organ or tissue cross-talk. Here we discuss the design philosophy of the bioreactor system and then describe its construction. Preliminary results of validation tests obtained with hepatocytes and endothelial cells are also reported. The results show that endothelial cells are extremely sensitive to small levels of shear stress and that the presence of heterotypic signals from endothelial cells enhances the endogenous metabolic function of hepatocytes.

10aBioengineering1 aVozzi, F1 aMazzei, D.1 aVinci, B1 aVozzi, G.1 aSbrana, T1 aRicotti, L1 aForgione, N1 aAhluwalia, A. uhttps://www.centropiaggio.unipi.it/publications/flexible-bioreactor-system-constructing-vitro-tissue-and-organ-models.html03577nas a2200205 4500008004100000022002200041245010600063210006900169260003300238300001400271520283400285653001903119100001503138700001403153700001403167700001803181700001703199700001703216856013803233 2011 eng d a978-1-62100-164-500aMulti-Compartmental Modular Bioreactor as Innovative System for Dynamic Cell Cultures and Co-Cultures0 aMultiCompartmental Modular Bioreactor as Innovative System for D bNova Science Publishers, Inc a159 - 1783 a

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.

10aBioengineering1 aMazzei, D.1 aGiusti, S1 aSbrana, T1 aAhluwalia, A.1 aAntolli, P G1 aLiu, Zhiming uhttps://www.centropiaggio.unipi.it/publications/multi-compartmental-modular-bioreactor-innovative-system-dynamic-cell-cultures-and-co00467nas a2200145 4500008004100000022001800041245004500059210004500104260001200149653001900161100001800180700001400198700001500212856009400227 2011 eng d aMI2011A00207300aSupporto per camere di coltura cellulare0 aSupporto per camere di coltura cellulare c11/201110aBioengineering1 aAhluwalia, A.1 aSbrana, T1 aMazzei, D. uhttps://www.centropiaggio.unipi.it/publications/supporto-camere-di-coltura-cellulare.html