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.