Bioreactors for Tissue Engineering: An Update

One of the greatest puzzles in wound healing is how to substitute or replace the defect caused by loss and damaged tissue or organs. In regenerative medicine, Tissue Engineering has been proposed to supply this demand by generating tissues in vitro. Bioreactors are the key to translate these cells and tissue-based constructs into large-scale biological products that are clinically effective, safe and financially pliable. In this review, we summarise the different up-to-date bioreactor designs being used for different cell types and special design scaffolds, and highlight advantages of different bioreactors, current challenges and the future trends. It is our belief that with efforts combined from multiple disciplinary participants, a novel bioreactor system that is capable of fast, large scale tissue culture would come about in near future

Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial

Development of a Perfusion Based System for Cardiac Decellularization

Heart failure is one of the leading causes of death worldwide. Currently, heart transplantation is the treatment of choice for end-stage heart failure, but lack of donor hearts and risk of rejection remain a challenge. Research has shown that decellularized hearts, in which cells are removed from the cardiac extracellular matrix (ECM), can provide a scaffold for use in engineering patient-specific heart transplants. This project presents a low-cost, semi-automated, small-organ (ie. rat heart) decellularization bioreactor designed for cardiac research, Testing has shown that this device can successfully decellularize rat hearts, and offers functionality similar to commercial devices

Training-to-Beat bioreactor for investigating Engineered Cardiac Tissues: design, development & validation

In last the decades, advances relevant to the generation of 3D Engineered Cardiac Tissues (ECTs) have been made. In reason of this, ECTs are now considered a great promise for in vitro studies of cardiac development, disease and, eventually, for strategies for the repair of the structure and function of the injured myocardium. Among the several physical stimuli which have been exploited to improve the functionality and maturation of ECTs, a preeminent role has been ascribed to mechanical stimulation. Appropriate mechanical stimulation can be recreated and maintained within bioreactors, which are devices/platforms devoted to mimic the physiological milieu in a monitored/controlled culture environment, where the engineered constructs can be properly stimulated. One main limitation of the bioreactor-based strategy for cardiac tissue engineering applications is that the devices which are currently used are meant to passively apply to ECTs a stimulus predefined by the user, regardless of their level of maturation along the duration of the in vitro culture. In this scenario, and trying to overcome current limitations, a novel bioreactor design has been conceived for the investigation of 3D ECTs with a biomimetic approach. Technically, the here proposed bioreactor is capable (1) to apply native-like or pathologic mechanical stimuli (cyclic strain) by means of a reliable linear actuator operating in a wide range of strains and frequencies, and (2) to monitor in real-time both chemo-physical parameters (e.g. oxygen tension, pH) of the milieu and the mechanical stiffness of ECTs by means of dedicated sensors, eventually adapting the stimulation to the actual stage of maturation of the constructs. As a proof of concept, a first experimental campaign has been carried out with a double aim: (1) to verify the bioreactor feasibility in delivering mechanical cyclical stimulation to 3D fibrin-based, ring shaped Engineered Cardiac Tissues (ECTs); (2) to assess the effect of cyclic strain on tissue maturation, contractility and modification on its mechanical properties. In detail, the bioreactor platform has been preliminarily tested to verify protocols for hold on, sterilization, and control of the delivered mechanical stimuli. Firstly, the suitability of the bioreactor platform in culturing ad-hoc designed constructs, in terms of ease of use and capability in setting the stimulation parameters, has been tested. Then, the observed maturation of ring shaped ECTs subject to sinusoidal cyclic strain within the bioreactor has confirmed the potency of the proposed approach and the instrumental role of mechanical stimulation in ECTs maturation and in the development of an adult-like cardiac phenotype responsive to electrical excitation. Even if further validation steps are required before the implementation of culture strategy fully adaptive in terms of mechanical stimuli applied to the engineered cardiac constructs, the developed bioreactor represents a valuable proof of concept for, in its most advanced operational mode, biomimetic culturing of engineered cardiac constructs

Perfusion bioreactor for liver bioengineering

End-stage organ failure has grown to become one of the key challenges for the medical community because of the high number of patients in waiting list for a transplant and the severe shortage of suitable organ donors. These, together with population ageing, have created an accumulation phenomenon of patients which increases the severity of the problem. New techniques for organ preservation, organ recovery from organs not suitable for transplant, and organ recellularization attempt to tackle this problem, appearing as some of the most promising solutions. The aim of this bachelor thesis is to continue the development of a complex liver perfusion bioreactor in order to design and develop an efficient and repeatable method for organ perfusion, decellularization and recellularization, with the final objective being the creation of a perfusion bioreactor for liver bioengineering able to be used for organ perfusion and preservation, organ decellularization and organ recellularization, able to preserve cell structure, functionality, growth and control differentiation for up to 4 weeks, while avoiding contamination and automating as much as possible the process. In order to do this, the bioreactor will include many sensors and data acquisition systems as well as control systems for pressure, flow rate, and temperature among others.Ingeniería Biomédic

Apparatus and methods for manipulation and optimization of biological systems

The invention provides systems and methods for manipulating, e.g., optimizing and controlling, biological systems, e.g., for eliciting a more desired biological response of biological sample, such as a tissue, organ, and/or a cell. In one aspect, systems and methods of the invention operate by efficiently searching through a large parametric space of stimuli and system parameters to manipulate, control, and optimize the response of biological samples sustained in the system, e.g., a bioreactor. In alternative aspects, systems include a device for sustaining cells or tissue samples, one or more actuators for stimulating the samples via biochemical, electromagnetic, thermal, mechanical, and/or optical stimulation, one or more sensors for measuring a biological response signal of the samples resulting from the stimulation of the sample. In one aspect, the systems and methods of the invention use at least one optimization algorithm to modify the actuator's control inputs for stimulation, responsive to the sensor's output of response signals. The compositions and methods of the invention can be used, e.g., to for systems optimization of any biological manufacturing or experimental system, e.g., bioreactors for proteins, e.g., therapeutic proteins, polypeptides or peptides for vaccines, and the like, small molecules (e.g., antibiotics), polysaccharides, lipids, and the like. Another use of the apparatus and methods includes combination drug therapy, e.g. optimal drug cocktail, directed cell proliferations and differentiations, e.g. in tissue engineering, e.g. neural progenitor cells differentiation, and discovery of key parameters in complex biological systems

Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology

Caractérisation et optimisation d'un réacteur pour l'ingénierie tissulaire 3D

Toward the objective to create or to replace impaired tissues, it is essential to establish a culture process allowing tissue growth in vitro . The Petri dish culture or the traditional 2D culture has only a limited potential, mainly caused by a poor oxygen mass transfer to feed larger tissue constructs. In this project, an autonomous and complete bioprocess has been built to fullfill these needs. The developed reactor is versatile because many cell culture chamber designs can be connected to it. Two proportional-integral algorithms (PI) can control the dissolved oxygen concentration and the pH. The pressure, the temperature and mass flow rate are recorded in real time. An actuator allows mimicking the cardiac output flow. In this mémoire , it will be demonstrated how the reactor has been optimized to reduce the risk of bioburden. Also, the procedures to develop the control tools of the PI algorithm will be detailed. To characterize the reactor, a RTD study will be presented. To characterize the cell culture chamber, a permeability analysis using fluorescent microscopy and magnetic resonance imaging (MRI) will be exposed. Finally, two cell culture chamber designs to orient microvessel formation have been tested and these results will be presented