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Tutorials : Feb 1, 2009 (Vol. 29, No. 3)

Three-Dimensional Cultivation of Cells

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

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.

Ex Vivo

Zellwerk provides a novel technology for 3-D cultivation of cells that differs fundamentally from conventional 2-D methods. In ZĀ®-RP bioreactors, adherent mammalian cells, as well as other eukaryotic cells, grow three-dimensionally. This capability is a result of the rotating bed bioreactor design combined with cell type specific carriers (e.g., highly porous SponceramĀ® carriers for expansion of mesenchymal stem cells). The bioreactor operates in perfusion mode, which is essential to maintain cell viability in thick, tissue-like layers of cells embedded in ECM.

Adhered cells are gently moved alternately through medium and overlay atmosphere. This “mud-flat” environment ensures that cells receive a supply of nutrients and gases and allows the removal of waste. Bioreactor vessels with different volumes are available, enabling scale up from 50 mL to 5,000 mL media volume (Figure 1).

cGMP Cell Processing

Cell-therapy and tissue-engineering approaches, as well as biopharmaceutical production, require closely controlled environments for cell processing. Validatable seeding, expansion, and harvesting procedures must be established for safe and reproducible cell products. A 3-D cell culture system for advanced therapies and drug production must address these issues.

The Z-RP cell cultivation system (rotating bed bioreactor, GMP Breeder, control unit and control software) complies with GMP standards and allows fast transfer of research results to therapeutic applications (Figure 2).

Documented and fully automated 3-D cultivation processes can be established with the Z-RP. The Breeder provides a sterile cleanroom environment with temperature control for the bioreactor. Double UV-sterilization and other supply functions are also integrated.

The Z-RP system is a fully functional cell culture lab concentrated into 3m2. It can be used as a tool for GMP-compliant production of autologous and allogenous cell formulations. The platform is also suitable for production of complex recombinant biopharmaceuticals.

Three-dimensional cell culturing in a Z-RP cell culturing system can be used to expand stem cells and other primary cells. Mesenchymal human stem cells from different sources (e.g., bone marrow, umbilical cord vessels, adipose tissue) can be expanded to several mm thick layers of viable cells being embedded in self-generated ECM (Figure 3).

Application-specific carriers (e.g., ceramic Sponceram discs with special surface characteristics due to coated ceramic materials, macroporous carrier possessing micro- and nano-roughness and porosity, respectively) allow expansion of MSCs either without further differentiation or differentiation into specialized cells, e.g., osteocytes, myocytes, or chondrocytes.

Many other primary cells were grown in 3-D culture to functional tissue-like constructs (e.g., chondrocytes, ceratinocytes, hepatocytes, fibroblasts). In Figure 3C and 3D, results achieved with 3-D expanded hepatocytes are shown. Harvesting of cell amounts from 107 to 1010 cells can be realized using the Z-RP technology and suitable carrier. Releasing and detaching cells from ECM and the carrier surface is feasible with this system.