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February 01, 2009 (Vol. 29, No. 3)

Three-Dimensional Cultivation of Cells

Rotating Bed Bioreactor Design of System Allows for 3-D Growth

  • Cell cultivation is one of the most common techniques used in modern life science and medical research. The predominant method grows cells as mono-layers in plastic dishes. Much of the knowledge about contemporary cell biology has been generated from such 2-D cell culture models.

    These models are used to shed light on the basic principles of life at the cellular level and on the nature and origin of many diseases. They can also be applied to the study of the influence of drugs on various organisms and to the investigation of the consequences of gene manipulation. Ex vivo culturing mainly takes place in a 2-D world (including research on human stem cells and primary cells for therapeutic applications and tissue engineering).

    This kind of cell cultivation is not always suitable, particularly when growing adherent mammalian, human, and stem cells. It is increasingly recognized that cells expanded under 2-D conditions show artificial properties compared to cells derived from natural tissue. Viability, morphology, metabolism, the pattern of activated genes, and other cell properties generally differ significantly from wild-type cells.

    It is also well known that three-dimensionally organized cells in in vitro cultures better mimic biologically functional cells from natural tissues than 2-D equivalents.

  • Tissue Cell In Vivo

    Compared to in vitro 2-D cultures, natural tissue-derived cells always exist in a 3-D environment. In addition to neighboring cells they are surrounded by an extra-cellular matrix (ECM). Each cell type generates its own kind of ECM consisting of manifold bio-polymers. Collagen, proteoglucanes, hyaluronates, and elastin are main constituents (in vertebrates anorganic hydroxyapatite is also present). Muscles, cartilage, tendons, membranes, blood vessels, and bones represent examples of tissues characterized by their specific ECM.

    Cell-to-matrix as well as cell-to-cell junctions have an important influence on the quality and behavior of cells arranged in a 3-D world. Moreover, growth factors are bound to matrix constituents, and nutrient and oxygen transport to cells depends on permeability and porosity of cell-typical ECM. The action of signal molecules is modulated by ECM structures.

  • In Vitro Culturing

    As a result of the inadequacy of 2-D dish culture, there is growing interest in culturing adherent mammalian and other eukaryotic cells as tissue-like 3-D networks embedded in ECM. Most mammalian cell types only grow on suitable surfaces in the natural adherent fashion. For 3-D cultivation, many artificial matrices have been developed and explored. Among them are scaffolds consisting of (or coated with) polygluconate, polylactate, porous gels of alginate, chitosan, hyaluronate, technical fabricated matrices like dextran particles, woven cellulose-, silk- or polyamid-derived gauzes, chemically processed cartilage, and bone-replacing materials (tri-calciumphosphate/hydroxyapatite). Some of these materials are commercially available.

    The scientific literature contains many examples of cells seeded in or on these materials, grown to some extent into the third dimension. Under static culturing conditions, however, the 3-D expansion of cells remains limited in extent and time.

    Further prerequisites have to be fulfilled in vitro to better mimic natural cell and tissue formation and functionality. For example, dynamic media- and gas-flow are needed to enable exchange of nutrients and metabolites. Interruption of cell expansion by passaging must be avoided. The surface attributes of cell carriers or scaffolds (roughness, porosity, chemical, and physical features) should match with the cell type to be adhered.

    In addition, the culturing process has to consider the specific demands of a given cell/tissue-type (optimal pH-value, low or high O2-pressure, specific temperature, adequate perfusion, cocultivation with separated feeder cells, change of certain parameters during cultivation, etc.).

    All of these specifications require a much more sophisticated cell-cultivation technology enabling growth of cells as real 3-D arrangements with cells embedded in self-generated ECM. For the therapeutic preparation of human stem cells and in vitro established implants, these preparations have to be produced under strictly controlled, standardized, repeatable, and GMP-conforming conditions. Until recently, suitable instrumentation for culturing cells under these conditions was rare.

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