Culture Systems and Bioreactors Used in Tissue Engineering

An overview of the culture systems and bioreactors used for the engineering of 3D tissue constructs, including cell maintenance, proliferation, and tissue formation, is illustrated schematically in Figure 2.1a-c.

Culture systems developed for the monolayer culture of adherent cells (T-flasks, Petri dishes, multiwell plates) are normally used for cell maintenance and proliferation. These systems allow for sterile handling procedures and are easy to use, disposable, and inexpensive [46]. By contrast, they require individual handling, for example in stages of medium exchange and cell seeding, and their usefulness is limited when large quantities of cells are required [15], though this can be overcome to some extent by using sophisticated robotics [7]. In addition, environmental parameters such as pH, pO2, and temperature cannot be controlled. A further drawback is the limited increase in cell number (approximately 10- to 20-fold during cultivation); consequently, the generation of a large number of cells requires several enzymatic subcultivation steps, accompanied by an increased passage number and cell dedifferentiation. In recent studies small well-mixed bioreactors (e.g., shake flasks, stirred vessels and "super spinner") have been suggested for cell proliferation in which the cells are grown on microcarriers [29-31]. These systems have been used for the cultivation of encapsulated cells [27, 28] or neural stem cells in single-cell suspension culture [47].

3D tissue cultures can be performed in fixed-bed and fluidized-bed bioreactors, with the cells being immobilized in macroporous carriers or in networks of fibers arranged in a column so that they are either packed (fixed-bed) or floating (fluidized-bed). The column is permanently perfused with a conditioned medium contained in a reservoir, mostly using a circulation loop. These types of reactor are very efficient for the long-term cultivation of mammalian cells to produce biopharmaceuticals, such as monoclonal antibodies, recombinant drugs including tissue plasminogen activator (tPA) and erythropoietin (EPO), or recombinant retroviruses for gene therapy [48-50]. For tissue engineering, the reactors have been investigated for several applications, including the cultivation of "liver" cells as an extracorporeal liver device [6, 18, 51], the proliferation of stem cells [52-54], the cultivation of cardiovascular cells [19] and cartilage cells [17], or as an in-vitro human placental model [55].

Roller Bottles Gene Therapy

Fig. 2.1 An overview of cell culture systems used in tissue engineering (adapted and modified from [25]). (a) Systems used for routine cultivation within an incubator, where the cells grow mainly in monolayer (e.g., 12-well-plates, Petri dishes, T-flasks or roller bottles). (b) Culture systems developed mainly for cultivation of mammalian cells,

Fig. 2.1 An overview of cell culture systems used in tissue engineering (adapted and modified from [25]). (a) Systems used for routine cultivation within an incubator, where the cells grow mainly in monolayer (e.g., 12-well-plates, Petri dishes, T-flasks or roller bottles). (b) Culture systems developed mainly for cultivation of mammalian cells, which were adapted for cultivation of tissue cells in three-dimensional structures (e.g., spinner and shake flasks, membrane-based systems such as hollow-fiber reactors or fluidized- and fixed-bed reactors). (c) Culture systems designed especially for tissue engineering mimicking the special demands of a three-dimensional tissue.

In membrane bioreactors, including hollow-fiber reactors [56], the miniPerm system [57] or the tecnomouse [58], cells are cultivated at tissue-like densities in a compartment which contains one or several types of membrane for nutrient and oxygen supply and removal of toxic metabolites. Hollow-fiber systems are widely used in the production of biopharmaceuticals, including monoclonal antibodies. Several examples of modified membrane bioreactors exist for the 3D culture of tissue cells, including hepatocytes [6, 59-64], skin cells [65] or other human cells [58, 66].

2.4 The Operation of Bioreactors | 59

Most ofthe culture systems and bioreactors discussed so far were first developed for the cultivation of mammalian cells, and subsequently adapted to the engineering of 3D tissue constructs. However, apart from some exceptions, they cannot easily be used in the generation of implantable tissue constructs, as each type of tissue intended for implantation (e.g., skin, heart valve, blood vessel, cartilage) requires a different geometric structure and a specific bioreactor design. One of the most prominent culture systems is the rotating-wall vessel [32], in which a construct remains in a state of free-fall through the medium with a low shear stress and a high mass transfer rate. This system has a wide range of practical applications [7, 15, 67]. A multipurpose culture system was introduced by Minuth et al. [68] for perfusion cultures under organotypic conditions. In this situation, several tissue carriers can be placed inside a perfusion container and, depending on the type of tissue-specific cell required, different supports can be selected. A perfused flow-chamber bioreactor with a new concept for aeration has been recently introduced [44, 69] in which tissue-specific inserts for various types of tissue (e.g., cartilage, skin, bone) can be applied.

In addition to these examples of multipurpose bioreactors, numerous tissue-specific culture systems have been suggested and reviewed [7, 8, 16-19, 22, 23, 35, 59, 70-72]. Unfortunately, the majority of these have been custom-made, with only very few having been commercialized.

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