Important Aspects for Bioreactor Design

With regard to tissue engineering, bioreactors are used for cell proliferation on a small scale (e.g., for individual patients) and on a large scale (e.g., for allogeneic therapy concepts), to generate 3D tissue constructs from isolated and proliferated cells in vitro and for direct organ-support devices [23]. These bioreactors should enable the control of environmental conditions such as oxygen tension, pH, temperature, and shear stress, as well as allowing aseptic operation (e.g., feeding and sampling). Furthermore, a bioreactor system should allow for automated processing steps. This is essential not only for controlled, reproducible, statistically relevant basic studies, but also for the future routine manufacture of tissues for clinical application or drug screening [7, 26]. In addition to these global requirements, specific key criteria for 3D tissue constructs based on cells and scaffolds must also be met, including the proliferation of cells, the seeding of cells onto macroporous scaffolds, nutrient (particularly oxygen) supply within the resulting tissue, and mechanical stimulation of the developing tissues [7].

The proliferation ofcells represents the first step in establishing a tissue culture. Usually, cells harvested from a biopsy must be expanded by several orders of magnitude. Cell proliferation is quite often accompanied by the dedifferentiation of cells [27, 28], with small culture dishes (e.g., Petri dishes, 12-well plates, and T-flasks) being mainly used for cell expansion. As these devices allow an increase in cell number by a factor of only about 10, several subcultivation steps are required. These are considered to be a major cause of dedifferentiation of cells. Recent studies have shown that microcarrier cultures performed in well-mixed bioreactor systems can significantly improve cell expansion [29-31].

A further critical aspect of a differentiated tissue is the extracellular matrix (ECM), as described by Alison Abbott (cited from [95]): "In mammalian tissues, cells connect not only to each other, but also to a support structure called the ECM.

This contains proteins, such as collagen, elastin and laminin, that give tissues their mechanical properties and help to organize communication between cells embedded within the matrix. Receptors on the surface of the cells, in particular a family of proteins called the integrins, anchor their bearers to the ECM, and also determine how the cells interpret biochemical cues from their immediate surroundings. Given this complex mechanical and biochemical interplay, it is perhaps no surprise that researchers will miss biological subtleties if the cells they are studying grow only in flat layers. But providing an appropriate environment in which to culture cells in three-dimensions is no easy matter (...). Some researchers use simple gels consisting of collagen, whereas others make their own gels by extracting ECM material from relevant tissues. Another popular option is the commercially available Matrigel, which consists of structural proteins such as laminin and collagen, plus growth factors and enzymes, all taken from mouse tumours." [94, 95]. Further 3D (mostly macroporous) scaffolds used for tissue engineering have been discussed previously. The cell seeding of scaffolds is an important step in establishing a 3D culture in a macroporous scaffold, as not only seeding at high cell densities but also a homogeneous distribution of cells within the scaffold is essential [32-34]. Several techniques for cell seeding have been discussed by Martin et al. [7].

A sufficient supply of nutrients, together with the removal of toxic or inhibitory substances, is crucial for long-term culture to control a constant and defined environment. In 2D "flat culture", the formation of a suitable microenvironment is disturbed by convection of the culture supernatant and the periodically exchanged media. Cell migration and interaction is limited on the 2D-culture surface, and is more or less defined by the initial seeding of cells or resuspension and may be suitable for single-layer epithelioid tissues based on a high proliferative capacity. Perfusable and cell migrational voluminous matrices - the so-called 3D-matrices - support the formation oflocal microgradients, cell migration, cell-cell-activation, leading to coordinated proliferation aggregation, the initialization of tissue forming, and tissue polarization.

All kinds of flows needed for long-term supply, such as transfusion, perfusion, circulation and convection, disturb the formation of guiding microgradients. For an ideal microenvironment, it is vital to ensure the correct balance of minimal, yet sufficient, perfusion and a maximum of self-conditioning. Sufficient perfusion ensures an optimum of nutrient and metabolite concentrations. Inhomogeneity in oxygen tension, and local accumulations of cytokines and chemokines, triggers chemotaxis and cell activation and differentiation [101, 102].

Furthermore, the size of most engineered tissues is limited as they do not have their own blood system and the cells are supplied only by diffusion [8, 25, 35]. Oxygen supply is particularly critical, as only cell layers of 100-200 ^m thickness can be supplied by diffusion [36]. However, as tissue constructs should have larger dimensions, mass-transfer limitations represent one of the greatest engineering challenges [25].

Various studies have shown that mechanical stimulation (e.g., mechanical compression, hydrodynamic pressure and fluid flow, which are important

2.3 Culture Systems and Bioreactors Used in Tissue Engineering | 57

modulators of cell physiology) can have a positive impact on tissue formation [37], particularly in the context of musculoskeletal tissue engineering, cartilage formation, and cardiovascular tissues [19, 38-45]. As yet, however, little is known about the specific mechanical forces or the ranges of application, such as magnitude, frequency, continuous or intermittent, and duty cycle [7, 17]. Further studies of these factors must be coupled with quantitative and computational analyses of physical forces experienced by cells and changes in mass transport induced by the method used.

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