Industrialization of a perfusion bioreactor: Prime example of a non-straightforward process.

Bioreactors are essential enabling technologies for the translation of advanced therapies medicinal products from the research field towards a successful clinical application. In order to speed up the translation and the spread of novel tissue engineering products into the clinical routine, tissue engineering bioreactors should evolve from laboratory prototypes towards industrialized products. In this work, we thus challenged the industrialization process of a novel technological platform, based on an established research prototype of perfusion bioreactor, following a GMP-driven approach. We describe how the combination of scientific background, intellectual property, start-up factory environment, wise industrial advice in the biomedical field, design, and regulatory consultancy allowed us to turn a previously validated prototype technology into an industrial product suitable for serial production with improved replicability and user-friendliness. The solutions implemented enhanced aesthetics, ergonomics, handling, and safety of the bioreactor, and they allowed compliance with the fundamental requirements in terms of traceability, reproducibility, efficiency, and safety of the manufacturing process of advanced therapies medicinal products. The result is an automated incubator-compatible device, housing 12 disposable independent perfusion chambers for seeding and culture of any perfusable tissue. We validated the cell seeding process of the industrialized bioreactor by means of the Design of Experiment approach, whilst the effectiveness of perfusion culture was evaluated in the context of bone tissue engineering.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion.

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell-seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non-crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Bioreactors in tissue engineering: scientific challenges and clinical perspectives.

In this Chapter we discuss the role of bioreactors in the translational paradigm of Tissue Engineering approaches from basic research to streamlined tissue manufacturing. In particular, we will highlight their functions as: (1) Pragmatic tools for tissue engineers, making up for limitations of conventional manual and static techniques, enabling automation and allowing physical conditioning of the developing tissues; (2) 3D culture model systems, enabling us to recapitulate specific aspects of the actual in vivo milieu and, when properly integrated with computational modeling efforts and sensing and control techniques, to address challenging scientific questions; (3) Tissue manufacturing devices, implementing bioprocesses so as to support safe, standardized, scaleable, traceable and possibly cost-effective production of grafts for clinical use. We will provide evidences that fundamental knowledge gained through the use of well-defined and controlled bio-reactor systems at the research level will be essential to define, optimize, and moreover, streamline the key processes required for efficient manufacturing models.

Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds.

Skeletal myogenesis is a complex process, which is known to be intimately depending on an optimal outside-in substrate-cell signaling. Current attempts to reproduce skeletal muscle tissue in vitro using traditional scaffolds mainly suffer from poor directionality of the myofibers, resulting in an ineffective vectorial power generation. In this study, we aimed at investigating skeletal myogenesis on novel biodegradable microfibrous scaffolds made of DegraPol, a block polyesterurethane previously demonstrated to be suitable for this application. DegraPol was processed by electrospinning in the form of highly orientated ("O") and nonorientated ("N/O") microfibrous meshes and by solvent-casting in the form of nonporous films ("F"). The effect of the fiber orientation at the scaffold surface was evaluated by investigating C2C12 and L6 proliferation (via SEM analysis and alamarBlue test) and differentiation (via RT-PCR analysis and MHC immunostaining). We demonstrated that highly orientated elastomeric microfibrous DegraPol scaffolds enable skeletal myogenesis in vitro by aiding in (a) myoblast adhesion, (b) myotube alignment, and (c) noncoplanar arrangement of cells, by providing the necessary directional cues along with architectural and mechanical support.

A new electro-mechanical bioreactor for soft tissue engineering.

By enabling the maintenance of controlled chemical and physical environmental conditions, bioreactors proved that electro-mechanical stimulation improves tissue development in vitro, especially in the case of tissues which are subjected to stimuli during embryogenesis and growth (i.e. skeletal and cardiac muscle tissue). However, most of the bioreactors developed in the last 20 yrs, designed to suit specific applications, lack versatility. With the aim to provide researchers with a yielding, versatile tool, we designed and realized in this study an electro-mechanical stimulator capable of dynamically culturing four biological constructs, delivering assignable stretching and electrical stimulation patterns. The device has been conceived to be easy to handle and customizable for different applications, while ensuring sterility along with stimuli delivery. The gripping equipment, modular and adaptable to scaffolds of different consistencies, is provided with dedicated tools for supporting sample insertion into the culture chamber performed under a laminar flow hood. As to performance, a wide range of electro-mechanical stimulation patterns and their relative occurrence can be accomplished, permitting the adjustment of the dynamic culture parameters both to the specific cell species and to the developmental phase of the cultured cells.