Cardiac tissue engineering (TE) aims at fabricating in vitro cardiac tissues as substitutes of native damaged ones. To this purpose, an adequate 3D micro-environment, resembling the in-vivo extracellular matrix (ECM), and specific pysical/chemical stimuli are required. We introduce a novel method to develop micrometer-scale platforms able to electrically stimulate cell-laden hydrogels exploiting three-dimensional stainless steel electrodes. Two configurations of the electrodes were implemented, in order to obtain different directions of the resultant electric fields: parallel and orthogonal to the sample. Devices were first studied through computational analysis, so as to have a qualitative and quantitative idea of their electric behavior. Then electrical characterizations of the platforms were performed: the time constant of the system was assessed to understand if an adequate stimulation, referring to literature, could be given through the electrodes. Further, voltage and current trends were examined along the platforms culture chambers to find out if the fabrication process was repeatable and whether comparable devices responses to electric stimuli were obtainable. As regards the 3D matrix three hydrogels were tested, embedding bone marrow stromal cells (BMSCs) within them: methacrylated gelatin (GelMA), collagen and fibrin gels. Polymerization parameters and preparation protocols were optimized for each hydrogel at different scale sizes, then analyzing cellular response in terms of viability, elongation and metabolic activity. Electric stimuli (cyclic, biphasic, square voltage pulses) were applied in parallelized experiments to cell-laden fibrin gels inserted in our novel microplatforms, studying BMSCs response up to 4 days of culture.
L'ingegneria dei tessuti (TE) cardiaca mira alla fabbricazione di tessuti cardiaci in vitro come sostituti di quelli nativi danneggiati. A questo scopo, un micro-ambiente 3D adeguato, che assomigli alla matrice extracellulare (ECM) in vivo, e specifici stimoli fisici/chimici sono richiesti. Proponiamo qui un metodo innovativo per sviluppare piattaforme alla micro-scala capaci di stimolare elettricamente idrogeli seminati con cellule sfruttando elettrodi tridimensionali in acciaio inossidabile. Due configurazioni degli elettrodi sono state implementate, per ottenere diverse direzioni dei campi elettrici risultanti: parallela e ortogonale al campione. I dispositivi sono prima stati studiati attraverso analisi computazionali, così da avere un'idea qualitativa e quantitativa del loro comportamento elettrico. Caratterizzazioni elettriche sulle piattaforme sono quindi state eseguite: la costante di tempo del sistema è stata determinata per capire se una stimolazione adeguata, secondo letteratura, potesse essere fornita attraverso gli elettrodi. Inoltre, gli andamenti di voltaggio e corrente sono stati esaminati lungo le camere di coltura delle piattaforme per scoprire se il processo di fabbricazione fosse ripetibile e se risposte comparabili dei dispositivi agli stimoli elettrici fossero ottenibili. Per quanto riguarda la matrice 3D tre diversi idrogeli sono stati testati, incorporandovi cellule stromali del midollo osseo (BMSCs): gelatina metacrilata (GelMA), gel di collagene e gel di fibrina. I parametri di polimerizzazione e i protocolli di preparazione sono stati ottimizzati per ciascun gel a diverse scale di grandezza, analizzando poi la risposta cellulare in termini di vitalità, morfologia e attività metabolica. Stimoli elettrici (onde quadre di voltaggio cicliche e bifasiche) sono stati applicati in esperimenti in parallelo su gel di fibrina seminati con cellule inseriti nelle nostre innovative micro-piattaforme, studiando la risposta delle BMSCs fino a 4 giorni di coltura.
Design and development of a micro-scale platform able to perform electrical stimulation on cellular constructs cultivated in a 3D environment
MANELLI, ANDREA;STURLA, GIORGIA
2013/2014
Abstract
Cardiac tissue engineering (TE) aims at fabricating in vitro cardiac tissues as substitutes of native damaged ones. To this purpose, an adequate 3D micro-environment, resembling the in-vivo extracellular matrix (ECM), and specific pysical/chemical stimuli are required. We introduce a novel method to develop micrometer-scale platforms able to electrically stimulate cell-laden hydrogels exploiting three-dimensional stainless steel electrodes. Two configurations of the electrodes were implemented, in order to obtain different directions of the resultant electric fields: parallel and orthogonal to the sample. Devices were first studied through computational analysis, so as to have a qualitative and quantitative idea of their electric behavior. Then electrical characterizations of the platforms were performed: the time constant of the system was assessed to understand if an adequate stimulation, referring to literature, could be given through the electrodes. Further, voltage and current trends were examined along the platforms culture chambers to find out if the fabrication process was repeatable and whether comparable devices responses to electric stimuli were obtainable. As regards the 3D matrix three hydrogels were tested, embedding bone marrow stromal cells (BMSCs) within them: methacrylated gelatin (GelMA), collagen and fibrin gels. Polymerization parameters and preparation protocols were optimized for each hydrogel at different scale sizes, then analyzing cellular response in terms of viability, elongation and metabolic activity. Electric stimuli (cyclic, biphasic, square voltage pulses) were applied in parallelized experiments to cell-laden fibrin gels inserted in our novel microplatforms, studying BMSCs response up to 4 days of culture.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/106826