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Biochemical processes develop

6.0 Bioreactors

Bioreactors are generally defined as devices in which biological and/or biochemical processes develop under closely monitored and tightly controlled environmental and operating conditions (e.g. pH, temperature, pressure, nutrient supply and waste removal). A bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering. The high degree of reproducibility, control and automation introduced by bioreactors for specific experimental bioprocesses, has been key for their transfer to large-scale applications.

Bioreactor technologies intended for tissue engineering can be used to grow functional cells and tissues for transplantation, and for controlled in vitro studies on the regulation effect of biochemical and biomechanical factors on cell and tissue development. The primary objectives of these systems are to establish spatially uniform cell distributions on three dimensional scaffolds, to maintain desired concentrations of gases and nutrients in the culture medium, and to expose developing tissue to appropriate physical stimuli.

A variety of different bioreactors have been developed and used to grow cells and tissues. The different types include spinner flask, rotating wall vessel, hollow-fiber and direct perfusion bioreactors. Spinner flask bioreactors have been used for the seeding of cells into 3D scaffolds and for subsequent culture of the constructs (Vunjak-Novakovic et al., 2002). During seeding, cells are transported to and into the scaffold by convection. During culture, medium stirring enhances external mass-transfer but also generates turbulent eddies, which could be detrimental for the development of the tissue.

Rotating-wall vessels provide a dynamic culture environment to the constructs, with low shear stresses and high mass-transfer rates. The vessel walls are rotated at a rate that enables the drag force (Fd), centrifugal force (Fc) and net gravitational force (Fg) on the construct to be balanced; the construct thus remains in a state of free-fall through the culture medium (Unsworth and Lelkes, 1998).

Hollow-fiber bioreactors can be used to enhance mass transfer during the culture of highly metabolic and sensitive cell types such as hepatocytes. In one configuration, cells are embedded within a gel inside the lumen of permeable hollow fibers and medium is perfused over the exterior surface of the fibers (Jasmund and Bader, 2002).

Direct perfusion bioreactors have been shown to enhance: (i) growth, differentiation and mineralized matrix deposition by bone cells (Bancroft et al., 2002, Goldstein et al., 2001), (ii) proliferation of human oral keratinocytes (Carrier et al., 2002), (iii) albumin synthesis rates by hepatocytes (Kim et al., 2000), (iv) expression of cardiac-specific markers by cardiomyocytes (Carrier et al., 2002) and (v) GAG synthesis and accumulation by chondrocytes (Pazzano et al., 2000). When incorporated into a bioreactor design, direct perfusion can thus be used as a valuable tool for enhancing cell survival, growth and function. However, the effects of direct perfusion can be highly dependent on the medium flow-rate and the maturation stage of the constructs, as recently demonstrated for 3D cultures of chondrocytes (Davisson et al., 2002). Therefore, optimizing a perfusion bioreactor for the engineering of a 3D tissue must address a careful balance between the mass transfer of nutrients and waste products to and from cells, the retention of newly synthesized extracellular matrix components within the construct, and the fluid induced shear stresses within the scaffold pores.

High density cell seeding and uniform distribution of cells on scaffolds is the first step in establishing a 3D culture, and might play a crucial role in determining the progression of tissue formation (Vunjak-Novakovic et al., 1998). Seeding cells into scaffolds at high densities has been associated with enhanced tissue formation in 3D constructs, including higher rates of cartilage matrix production (Freed et al., 1997), increased bone mineralization (Holy et al., 2000), and enhanced cardiac tissue structure (Carrier et al., 1999).

The initial distribution of cells within the scaffold after seeding has been related to the distribution of tissue subsequently formed within engineered constructs (Freed et al., 1998) suggesting that uniform cell-seeding could establish the basis for uniform tissue generation. However, even with a small 3D scaffold it can be a significant challenge to distribute a high density of cells efficiently and uniformly throughout the scaffold volume.

Although static loading of cells onto a scaffold is by far the most commonly used seeding method, several studies reported low seeding efficiencies (Holy et al., 2000) and non-uniform cell distributions within scaffolds (Kim et al., 1998), owing, in part, to the manual- and operator-dependent nature of the process. Significantly higher efficiencies and uniformities were obtained when poly glycolic acid non-woven meshes were seeded in stirred-flask bioreactors (Bursac et al., 1996). Perfusion seeding has been reported to be a more effective way to improve both seeding efficiency and cell distribution than static seeding or the stirring-flasks bioreactor. Perfusion seeding bioreactors have been designed for engineering vascular grafts, cartilage, hepatocyte and cardiac tissues (Wendt et al., 2003).

It has long been known that the supply of oxygen and soluble nutrients becomes critically limiting for the in vitro culture of 3D tissues. The consequence of such a limitation is exemplified by early studies showing that cellular spheroids larger than 1 mm in diameter generally contain a hypoxic, necrotic center, surrounded by a rim of viable cells (Sutherland et al., 1986). Because engineered constructs should be at least a few mm in size to serve as grafts for tissue replacement, mass-transfer limitations represent one of the greatest challenges to be addressed.

The rotating wall bioreactor can generate dynamic flow to improve nutrients and wastes transfer and to provide a low stress. Research results have shown that properties of engineered tissue cultured in the rotating wall bioreactor were superior to those of static or stirring-flask bioreactor. The efficacy of rotating wall vessel (RWV) bioreactors for the generation of tissue equivalents has been demonstrated using chondrocytes (Vunjak-Novakovic et al., 1999), cardiac cells (Carrier et al., 1999) and various tumor cells (Rhee et al., 2001). After a few weeks of cultivation in the RWVs, cartilaginous constructs had biochemical and biomechanical properties superior to those of static or stirred-flask cultures and approaching those of native cartilage (Vunjak-Novakovic et al., 1999). On the basis of these studies, it was proposed that the RWV bioreactor would support the engineering of tissues and organoids as in vitro model systems of tissue development and function (Unsworth and Lelkes, 1998)

Bioreactors that perfuse medium either through or around semi-permeable hollow fibers have been used successfully to maintain the function of highly metabolic cells (e.g. hepatocytes) by increasing the mass transport of nutrients and oxygen This concept has been extended to engineered tissues by perfusing culture medium directly through the pores of the cell-seeded 3D scaffold, thereby reducing mass transfer limitations both at the construct periphery and within its internal pores

Increasing evidence suggests that mechanical forces, which are known to be important modulators of cell physiology, might increase the biosynthetic activity of cells in bioartificial matrices and, thus, possibly improve or accelerate tissue regeneration in vitro (Butler et al., 2000). Various studies have demonstrated the validity of this principle, particularly in the context of musculoskeletal tissue engineering.

For example, cyclical mechanical stretch was found to: (i) enhance proliferation and matrix organization by human heart cells seeded on gelatin-matrix scaffolds (Akhyari et al., 2002), (ii) improve the mechanical properties of tissues generated by skeletal muscle cells suspended in collagen or Matrigel (Powell et al., 2002) and (iii) increase tissue organization and expression of elastin by smooth muscle cells seeded in polymeric scaffolds (Kim et al., 1999).

Despite numerous proof-of-principle studies showing that mechanical conditioning can improve the structural and functional properties of engineered tissues, little is known about the specific mechanical forces or regimes of application (i.e. magnitude, frequency, continuous or intermittent, duty cycle) that are stimulatory for a particular tissue. In addition, engineered tissues at different stages of development might require different regimes of mechanical conditioning owing to the increasing accumulation of extracellular matrix and developing structural organization

The role of bioreactors in applying mechanical forces to 3D constructs could be broadened beyond the conventional approach of enhancing cell differentiation and/or extracellular matrix deposition in engineered tissues. For example, they could also serve as valuable in vitro models to study the pathophysiological effects of physical forces on developing tissues, and to predict the responses of an engineered tissue to physiological forces on surgical implantation. Together with biomechanical characterization, bioreactors could thus help in defining when engineered tissues have a sufficient mechanical integrity and biological responsiveness to be implanted (Demarteau et al., 2003). Moreover, quantitative analysis and computational modeling of stresses and strains experienced both by normal tissues in vivo for a variety of activities and by engineered tissues in bioreactors, could lead to more precise comparisons of in vivo and in vitro mechanical conditioning, and help to determine potential regimes of physical rehabilitation that are most appropriate for the patient receiving the tissue (Butler et al., 2000).

By providing a comprehensive level of monitoring and control over specific environmental factors in 3D cultures, bioreactors can provide the technological means to perform controlled studies aimed at understanding which specific biological, chemical or physical parameter plays which function in engineering a defined tissue. This fundamental interdisciplinary research will provide the basis for identifying environmental and operating conditions required for the generation of specific tissues. At this stage, the transition from laboratory- to industrial-scale will require a switch from highly flexible bioreactors to specialized bioreactors, implementing the defined bioprocesses in a standardized way. The resulting devices will provide an economically viable approach to the automated manufacture of functional grafts, possibly bringing cell-based tissue engineering approaches down from the ivory tower and making them clinically accessible at a larger scale.

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