For most investigators the culture of mammalian cells has meant the use of traditional flasks or dishes. While this technique has proved useful for many studies, it is widely recognized that cells grown in a 2-D environment tend to dedifferentiate and lose the specialized features of the tissues from which they were derived.
Biologists are now coming to the realization that 3-D cell-to-cell and cell-to-extracellular matrix interactions are critical to the maintenance of differentiation in culture. These considerations are obviously important for basic science, but also in the context of therapeutic applications of cell culture. The emerging technology of tissue engineering will require efficient, large-scale 3-D cell culture techniques.
The advantages of 3-D cell culture have been demonstrated by culturing cells embedded in various natural or artificial substrates that mimic the effects of extracellular matrices. While this culture technique has been important to establish the benefits of 3-D culture, it is inadequate to achieve scale-up production of tissue-engineered 3-D therapeutics.
A new cell culture technology has been developed that shows promise in addressing the shortcomings of conventional cell culture apparatus for 3-D culture. This technology was created at NASA’s Johnson Space Center to simulate the effects of microgravity on cells in a ground-based culture system. The bioreactor, the Rotating Wall Vessel (RWV) from Synthecon (www.synthecon.com), is a cylindrical vessel that maintains cells in suspension by slow rotation about its horizontal axis with a coaxial tubular silicon membrane for oxygenation (Figure 1).
Several features distinguish the RWV from conventional cell culture technologies:
• Solid body rotation—when the vessel is rotated, the culture media rotates at the same angular velocity as the vessel wall with laminar fluid flow. In this environment, the damaging effects of turbulence and shear stress are minimized.
• Gentle mixing of media induced by particle sedimentation.
• Absence of a headspace: Unlike roller bottles, the vessel is completely filled with culture media, avoiding the turbulence created by a headspace.
• Delivery of oxygen is accomplished via a coaxial silicone membrane avoiding bubbles, which can create cell-damaging turbulence.
The forces acting on a cell or aggregate of cells are illustrated in Figure 2. The sedimentation velocity due to gravity, Vs, is composed of an inwardly directed component, Vsr, and a tangential component, Vst. There is an outwardly directed motion, Vcr, produced by centrifugal force, and a tangential component, Vct, from the Coriolis force. The resolution of these forces on cells or aggregates produces a slow descent through the culture media as the vessel turns. Because the net forces on the cells are substantially reduced, this culture environment is sometimes referred to as simulated or modeled microgravity.