David Wendt

Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts

Three‐dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor‐based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two‐dimensional [2D]) expansion. The system, based on the perfusion of bone marrow–nucleated cells through porous 3D scaffolds, supported the formation of stromal‐like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D‐expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.

Bioreactor-based roadmap for the translation of tissue engineering strategies into clinical products

Despite the compelling clinical need to regenerate damaged tissues/organs, impressive advances in the field of tissue engineering have yet to result in viable engineered tissue products with widespread therapeutic adoption. Although bioreactor systems have been proposed as a key factor in the manufacture of standardized and cost-effective engineered products, this concept appears slow to be embraced and implemented. Here we address scientific, regulatory and commercial challenges intrinsic to the bioreactor-based translation of tissue engineering models into clinical products, proposing a roadmap for the implementation of a new paradigm. The roadmap highlights that bioreactors must be implemented throughout product development, allowing scientific, medical, industrial and regulatory parties to address basic research questions, conduct sound pre-clinical studies and ultimately facilitating effective commercialization of engineered clinical products.

Toward modeling the bone marrow niche using scaffold-based 3D culture systems

In the bone marrow, specialized microenvironments, called niches, regulate hematopoietic stem cell (HSC) maintenance and function through a complex crosstalk between different cell types. Although in vivo studies have been instrumental to elucidate some of the mechanisms by which niches exert their function, the establishment of an in vitro model that recapitulates the fundamental interactions of the niche components in a controlled setting would be of great benefit. We have previously shown that freshly harvested bone marrow- or adipose tissue-derived cells can be cultured under perfusion within porous scaffolds, allowing the formation of an organized 3D stromal tissue, composed by mesenchymal and endothelial progenitors and able to support hematopoiesis. Here we describe 3D scaffold-based perfusion systems as potential models to reconstruct ex vivo the bone marrow stem cell niche. We discuss how several culture parameters, including scaffold properties, cellular makeup and molecular signals, can be varied and controlled to investigate the role of specific cues in affecting HSC fate. We then provide a perspective of how the system could be exploited to improve stem cell-based therapies and how the model can be extended toward the engineering of other specialized stromal niches.

The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 μm) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 μm) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s(-1)) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s(-1)) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.

Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.

Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.

Over the last decade, we have witnessed an increased recognition of the importance of 3D culture models to study various aspects of cell physiology and pathology, as well as to engineer implantable tissues. As compared to well-established 2D cell-culture systems, cell/tissue culture within 3D porous biomaterials has introduced new scientific and technical challenges associated with complex transport phenomena, physical forces, and cell-microenvironment interactions. While bioreactor-based 3D model systems have begun to play a crucial role in addressing fundamental scientific questions, numerous hurdles currently impede the most efficient utilization of these systems. We describe how computational modeling and innovative sensor technologies, in conjunction with well-defined and controlled bioreactor-based 3D culture systems, will be key to gain further insight into cell behavior and the complexity of tissue development. These model systems will lay a solid foundation to further develop, optimize, and effectively streamline the essential bioprocesses to safely and reproducibly produce appropriately scaled tissue grafts for clinical studies.

Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages

IntroductionOxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture.MethodsHAC expanded in monolayer were cultured in pellets for two weeks (Phase I) or up to an additional two weeks (Phase II). In each Phase, cells were exposed to 19% or 5% oxygen. Resulting tissues and culture media were assessed to determine amounts of produced/released proteoglycans and collagens, metalloproteinases (MMPs), collagen degradation products and collagen fibril organization using biochemical, (immuno)-histochemical, gene expression and scanning electron microscopy analyses. In specific experiments, the hypoxia-inducible factor-1α (HIF-1α) inhibitor cadmium chloride was supplemented in the culture medium to assess the involvement of this pathway.ResultsIndependent from the oxygen percentage during expansion, HAC cultured at 5% O2 (vs 19% O2) during Phase I accumulated higher amounts of glycosaminoglycans and type II collagen and expressed reduced levels of MMP-1 and MMP-13 mRNA and protein. Switching to 19% oxygen during Phase II resulted in reduced synthesis of proteoglycan and collagen, increased release of MMPs, accumulation of type II collagen fragments and higher branching of collagen fibrils. In contrast, reducing O2 during Phase II resulted in increased proteoglycan and type II collagen synthesis and reduced expression and release of MMP-13 mRNA and protein. Supplementation of cadmium chloride during differentiation culture at 5% O2 drastically reduced the up-regulation of type II collagen and the down-regulation of MMP-1 mRNA.ConclusionsThe application of more physiologic oxygen percentage during specific phases of differentiation culture enhanced the biosynthetic activity and reduced the activity of catabolic enzymes implicated in cartilage breakdown. Modulation of the oxygen percentage during HAC culture may be used to study pathophysiological events occurring in osteoarthritis and to enhance properties of in vitro engineered cartilaginous tissues.

Computational evaluation of oxygen and shear stress distributions in 3D perfusion culture systems : Macro-scale and micro-structured models

We present a combined macro-scale/micro-scale computational approach to quantify oxygen transport and flow-mediated shear stress to human chondrocytes cultured in three-dimensional scaffolds in a perfusion bioreactor system. A macro-scale model was developed to assess the influence of the bioreactor design and to identify the proper boundary conditions for the micro-scale model. The micro-scale model based on a micro-computed tomography (microCT) reconstruction of a poly(ethylene glycol terephthalate)/poly(butylene terephthalate) (PEGT/PBT) foam scaffold, was developed to assess the influence of the scaffold micro-architecture on local shear stress and oxygen levels within the scaffold pores. Experiments were performed to derive specific oxygen consumption rates for constructs perfused under flow rates of 0.3 and 0.03 ml min(-1). While macro-scale and micro-scale models predicted similar average oxygen levels at different depths within the scaffold, microCT models revealed small local oxygen variations within the scaffold micro-architecture. The combined macro-scale/micro-scale approach indicated that 0.3 ml min(-1), which subjected 95% of the cells to less than 6.3 mPa shear, would maintain the oxygen supply throughout the scaffold above anoxic levels (>1%), with 99.5% of the scaffold supplied with 8-2% O(2). Alternatively, at 0.03 ml min(-1), the macro-scale model predicted 6% of the cells would be supplied with 0.5-1% O(2), although this region of cells was confined to the periphery of the scaffold. Together with local variations predicted by the micro-scale model, the simulations underline that in the current model system, reducing the flow below 0.03 ml min(-1) would likely have dire consequences on cell viability to pronounced regions within the engineered construct. The presented approach provides a sensitive tool to aid efficient bioreactor optimization and scaffold design.

Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing.

Apart from partial or total joint replacement, no surgical procedure is currently available to treat large and deep cartilage defects associated with advanced diseases such as osteoarthritis. In this work, we developed a perfusion bioreactor system to engineer human cartilage grafts in a size with clinical relevance for unicompartmental resurfacing of human knee joints (50 mm diameter × 3 mm thick). Computational fluid dynamics models were developed to optimize the flow profile when designing the perfusion chamber. Using the developed system, human chondrocytes could be seeded throughout large 50 mm diameter scaffolds with a uniform distribution. Following two weeks culture, tissues grown in the bioreactor were viable and homogeneously cartilaginous, with biomechanical properties approaching those of native cartilage. In contrast, tissues generated by conventional manual production procedures were highly inhomogeneous and contained large necrotic regions. The unprecedented engineering of human cartilage tissues in this large-scale opens the practical perspective of grafting functional biological substitutes for the clinical treatment for extensive cartilage defects, possibly in combination with surgical or pharmacological therapies to support durability of the implant. Ongoing efforts are aimed at integrating the up-scaled bioreactor based processes within a fully automated and closed manufacturing system for safe, standardized, and GMP compliant production of large-scale cartilage grafts.

Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.

Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7–8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion- as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.