Industrialization and pre-clinical testing of an oscillating perfusion bioreactor (OPB) for tissue engineering strategies

Advanced therapies and in particular tissue engineering strategies have encountered an increasing interest and a rapid evolution in the last couple of decades, due to their potential to significantly impact current therapeutic modalities by providing a virtually unlimited supply of patient-specific tissues and organs. Increasing in the number of advanced therapies undergoing both early and late-stage clinical trials as well as FDA-approved commercial products that have already entered the market strongly indicates that these therapies are emerging as a distinct healthcare sector. Renewed investor interest and recent activity among major pharmaceutical companies suggest that this industry is rapidly developing the capability and capacity to be a highly competitive, sustainable and multibillion-dollar enterprise. The translation of successful research results into the clinic however still suffers of important issues, such as cost, time, lack of ease of application and difficulty in complying with regulation. In this context the field of automated cell cultivation using highly specialized bioreactor designs and stringent bioprocess controls will be crucial for the development of biomanufacturing technologies suitable for clinical-grade production of advanced therapies. Bioreactors are generally defined as devices in which biological and/or biochemical processes develop under closely monitored and tightly controlled environmental and operating conditions. Many types of bioreactors have been designed to provide different stimuli in relation to the specific tissue to be developed. Among these, perfusion bioreactors have proven to be particularly promising in engineering different type of tissues (bone, cartilage, heart). They are employed in tissue engineering procedures to perform specific and important functions such as cell seeding on porous scaffolds and confined medium perfusion through porous scaffolds seeded with cells. Moreover they: a) allow to overcome the typical drawbacks of manual procedures (high intra and inter-individual variability, low efficiency and uniformity) and improves efficiency of seeding processes and uniformity of cells distribution inside porous scaffolds, promoting the achievement of more uniform engineered constructs; b) improve efficiency of oxygen and metabolites transfer and catabolites removal c) allow automation and monitoring of culture medium exchange procedures; d) allow physical stimulation of cells seeded into porous scaffolds, through shear stress generation; e) reducing the use of manual actions, promote the transfer of Tissue Engineering procedures from research to clinical application, improving the traceability, reproducibility, efficiency and safety of processes (key requirements for these procedures to compete with traditional therapeutic alternatives in terms of cost, Quality Control and Good Manufacturing Practice). Despite a high number of bioreactors already in the marketplace, a device accounting for all the requirements needed to be successfully used into a streamlined bioreactor-based advanced therapy strategy is still a lack. In this context the present study focuses on the industrialization and the pre-clinical testing of a technological platform – based on a prototype bioreactor (OPB, Oscillating Perfusion Bioreactor) – for tissue engineering purposes. Aim of the project was to develop a scalable and robust bioreactor, enabling flexible culture strategies and monitoring and control of the culture environment, taking into account serial manufacturability and quality assurance, for the cost effective and automated manufacturing of biological tissues. The study comprised two main phases: industrialization and pre-clinical testing. During the industrialization phase the prototype version of the bioreactor has been re-designed in order to comply with regulatory requirements in term of GMP practice for cell and tissue culture. Objectives of this phase were: 1. Oscillating platform and disposable chambers design: Oscillating platform and disposable chambers have been designed complying with the requirements specifications. Pro/Engineer CAD software was used to define design features and generate three-dimensional renderings of the bioreactor. Structural simulation, through FEM analysis, has been performed in order to verify displacements and stresses and minimize weight and materials. The culture chambers have been designed to be realized by injection molding. Attention has been paid to the choice of geometries, materials and to the simplification of the operational procedures. Furthermore carters have been realized by rapid prototyping with the aim to isolate mechanical and electrical components. 2. Sensing system development: The objective of this phase was to provide the bioreactor with a sensing system able to monitor critical culture parameters (such as pH and pO2). The data collected by the sensors are logged and monitored so as to enable online, real-time and traceable observation of culture progression. Optical pH and oxygen sensors by Presens Precision Sensing GmbH have been selected and tests have been performed in order to verify their applicability. A motor driven automated system has been designed to drive the sensors with micrometric precision in the proximity of the single culture chambers arranged in parallel, in order to serially interrogate them. Validation experiments have been carried out in order to verify correspondence with the requirements. 3. Control system development: A microcontroller-based control system able to manage all functionalities has been realized. It enables the management of a) an electric actuator allowing the oscillating platform movement; b) an electric actuator allowing the sensing system movement; c) a pH sensor and d) an oxygen sensor. User interface consist of a liquid crystal display and a keypad. 4. Validation: Validation activity has been carried out in order to demonstrate the correspondence with the requirements and the robustness and the safety of the device. It comprised: a) installation qualification (IQ); b) operational qualification (OQ); c) performance qualification (PQ), with the execution of sterility, LAL and media fill tests. Pre-clinical testing has been carried out in order to validate the bioreactor and comprised: a) Experiments in order to verify the performances of the bioreactor in seeding and culturing several cell types. In particular in the first phase, a study aiming at optimizing seeding process through Design of Experiment statistical method has been carried out. Once optimized seeding parameters using Ultrafoam scaffolds and MG63 these parameters were then used in seeding other scaffold and cell types. Finally a culture experiment has been carried out in order to verify the effect of the optimized seeding process on DNA content and cell viability at long term and to assess the reliability and robustness of the industrialized bioreactor. Optimization through DoE allowed us to identify which parameters influence the seeding results. In particular we found that the flow velocity and the seeding time influence the seeding efficiency, while the seeding density influences the cell viability. As to scaffold and cell type main differences in seeding efficiency were found in relation to scaffold type. The permeability in particular seems to most affect seeding results. In performing culture the bioreactor was able to deliver constructs with higher cell content and viability and with a better distribution of cells throughout the scaffold thickness with respect to statically cultured ones. Furthermore the bioreactor proved to be reliable and robust in performing the culture experiment. b) As final step of validation, in vivo implant and evaluation of bone grafts generated by means of the developed perfusion bioreactor have been performed in order to verify the performance of the device in carrying on a complete tissue engineering process. Ovine animal model has been chosen for this purpose. Pre-implantation analysis on grafts confirmed the effectiveness of the bioreactor in carrying out automatic and safe tissue engineering processes, delivering constructs with an osteogenic leaning. Pre-implantation analysis on grafts confirmed the effectiveness of the bioreactor in carrying on automatic and safe tissue engineering processes, delivering constructs with an osteogenic leaning. The effect of the dynamic culture conditions on the cell differentiation was investigated by studying the expression of genes involved in osteogenic differentiation and characteristic of the extracellular matrix of bone tissue. The RT-PCR results showed that the perfusion is a valid stimulus in addressing cells toward the osteogenic lineage, in presence of osteogenic culture medium. Both early and late markers are more expressed in dynamically cultured constructs than in statically cultured ones for ENGIpore scaffolds.