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Electrical stimulation subsystem. The electrical stimulation subunit is composed of a programmable electrical stimulator and eight transmission lines that deliver the electrical signal to four pairs of platinum electrodes. The stimulator can be freely programmed with different patterns and then triggered by a PC. The electrode insertion in the culture chamber is obtained simply by superimposing the electromechanical lid (Fig. 5) to the chamber. A novel, versatile, GLP-compatible bioreactor for electromechanical conditioning of engineered tissues Nasser Sadr (1) , Stefania Adele Riboldi (1) , Stefano Lorenzoni (1) , Franco Maria Montevecchi (2) and Sara Mantero (1) . (1) Department of Bioengineering, Politecnico di Milano, Milano, Italy (2) Department of Mechanics, Politecnico di Torino, Torino, Italy Background . By enabling the maintenance of controlled chemical and physical environmental conditions, bioreactors revealed to be useful tools to prove that electromechanical stimulation improves tissue development in vitro, especially in the case of tissues which are subjected to such stimuli during embryogenesis and growth (i.e. skeletal and cardiac muscle tissue) [1,2]. Bioreactor general function is the control and the delivery of accurate and reproducible electromechanical stimulus patterns to promote growth and differentiation of several cell phenotypes and biological tissue development. Introduction Bioreactor Requirements Results System overview. Key constitutive elements of the bioreactor are: • A culture chamber • A mechanical stimulation subsystem • An electrical stimulation subsystem • A system controller managing whole experiment Mechanical stimulation subsystem. The mechanical stimulation subsystem is composed of a stepping motor driving two drive shafts screwed onto a crossbar that fixes four mobile grips (Fig. 4). The characteristics of both the spring and the silicon sheet (used to realize the grasping system) can be customized to adapt holding surface and pressure to the consistency of different biological constructs. Aim of this Study. With the aim to provide researchers with a yielding tool for extensive experimentation in vitro in biological laboratories regardless of specific research field, we developed an innovative device to dynamically culture cells respecting all the GLP rules. [1] Dennis, R. et al. 2001, " Excitability and Contractility of Skeletal Muscle Engineered from Primary Cultures and Cell Lines “, Am. J. Physiol., Cell Physiol., 280(2), pp. C288-C295 [2] Powell, C. A. et al. 2002, “ Mechanical Stimulation Improves Tissue-Engineered Human Skeletal Muscle “, Am. J. Physiol., Cell Physiol., 283(5), pp. C1557-C1565. Fig. 1 Schematic diagram of bioreactor elements. The biological constructs receive mechanical stimuli through a stepper motor-driver-axis control subsystem. The electrical stimuli are delivered by an electrical stimulator. All the experiment is managed by a PC based system controller. Culture Chamber. The transparent culture chamber (Fig. 2, a) represents the sterile and cytocompatible environment where cell cultures take place. Special attention is spent on: • Culture chamber design • Stimuli transmission point design in order to avoid medium stagnation points (preferred targets for microbial contamination) and to preserve sterility during the whole experiment (allowing at the meantime biological constructs stimulation). Gas exchanges take place through a couple of HEPA filter positioned on the culture chamber. Fig. 2 Culture chamber. The Plexiglas ® culture chamber (a) houses four biological constructs holding them by means of a system of symmetrical grips. In order to preserve the sterility of the chamber, each motion transmitting drive shaft is provided with two dynamic diaphragms (b - 1). To deliver the electrical stimuli in a sterile way a tight D connector is used, positioned on the lid of the culture chamber (c). Fig. 4 Grip in opened and closed position. The grip is composed of a Plexiglas ® body (1) and a stainless steel mobile part (2). The holding pressure is provided by a compression spring (3) and it is uniformly distributed using silicone sheets (4). Fig. 5 The electro-mechanical lid. It is equipped with a rank to house eight electrodes which are inserted simply by superimposing the lid to the chamber. Conclusion Our bioreactor is a forefront powerful device capable of mechanically and/or electrically conditioning 3D engineered constructs of different origins. It generates a wide range of pattern combinations permitting to adjust the dynamic culture parameters to the specific cell species, to the specific tissue but also to the specific developmental phase of cultured cells. All the system is developed considering GLP compatibility of the bioreactor and of all the protocol of use Bioreactor uses and lacks. According on these results many bioreactors have been developed in the last twenty years in order to foster: • Cell proliferation and differentiation studies • Tissue engineering studies However, most of developed bioreactors, being designed to suit specific applications (adapting preexisting laboratory equipment) , lack in versatility and Good Laboratory Practice (GLP) pertinence . General design principles. The first design principle is a high versatility both of the actuators and of the stimulation patterns, so that the device can be employed with several biological constructs and for different applications. The design has also to respect the GLPs providing tools and protocols for biological laboratory use Constrains and requirements. • Ease of sterilization and sterility maintenance • Ease of use (assembly in sterile conditions under a laminar flow hood, medium exchange, cleaning, use for non-trained staff) • Small dimensions, suitable for positioning in a cell culture incubator and to minimize medium volume • No medium stagnation during exchange operations • Housing of experimentally significant number of specimens • Visual inspection possibility System controller. A specific software is developed on LabVIEW ® (National Instruments) permitting to: • To define arbitrary mechanical stimulation patterns • To define arbitrary mechanical-electrical synchronization patterns • To overview all interesting parameters during the experiment Specimen insertion tools. A complete protocol and some specific tools (Fig. 3) are developed with the purpose of an easy, fast and GLP-compatible specimen insertion in the culture chamber under laminar flow hood. Fig. 3 Insertion tools. A (1) mounting table and a (2) couple of crossbars are used during specimen insertion.
