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.
Viscosity, Density and Size
Several other culture parameters, such as the viscosity and density of the media and the size of cell aggregates, can influence the behavior of particles in the RWV. According to the Stokes equation, the sedimentation velocity of a particle in a fluid is inversely proportional to viscosity and directly proportional to the difference in density between the particles (cells) and the fluid. In practice, most investigators use standard culture medias without altering the density or viscosity.
The size of the particles, however, may change during the culture due to cell proliferation and/or recruitment of additional cells into an aggregate, causing an increase in sedimentation velocity by the square of the radius. To counteract the increase in sedimentation velocity, the speed of rotation may be increased to prevent the aggregates from hitting the vessel wall. At some point the size of the aggregates may become so large that it is impossible to maintain them in suspension without striking the vessel wall. Even with this limitation, tissue-like aggregates approaching 1 cm are possible.
Within the normal operating parameters of the vessel, cells experience minimal mechanical forces from shear stress and turbulence. These conditions are conducive to the formation of aggregates or adherence of cells to substrates such as microcarriers and extracellular matrix. As cells proliferate and reorganize, they can form 3-D structures that resemble native tissue (Figure 3). The sedimentation of 3-D cell aggregates in the vessel confers additional benefits in terms of mass transfer. The constant movement of aggregates in the media insures that nutrient, oxygen, and waste transfer will not be limited by diffusion as they are in static culture systems.
Since its beginnings at NASA, the RWV has been produced commercially as a tool for basic research as the Rotary Cell Culture System (RCCS). After more than a decade of research by investigators working in a variety of disciplines, a number of potential therapeutic applications of 3-D cell culture have emerged.
Isolated Human Pancreatic Islets
The transplantation of human islets from cadavers has recently shown promise for a long-term cure of type 1 diabetes. It is known that many of the transplanted islets do not survive, and thus it has been necessary to transplant islets from 2 to 3 pancreata to achieve insulin independence. Culturing islets in the Rotary Cell Culture System prior to transplantation produced significantly better long-term graft performance in a diabetic animal model than freshly isolated or static cultured islets. Subsequently, human islets have been cultured in a scaled-up version of the Rotary bioreactor in preparation for a human trial.
The use of 3-D models of human tissues for the study of infectious disease has considerable utility for drug development. Compared to 2-D monolayer culture, 3-D cultures more closely resemble native tissues and respond to infection by pathogens in a manner that mimics the normal infectious process. For example, 3-D cultures of lung cells in the RCCS produced structures with characteristic tight junctions, extracellular matrix, and mucins compared to 2-D cultures. When these 3-D lung models were infected with Pseudomonas aeruginosa they responded in a more physiologically relevant way. Thus, 3-D tissue models may provide a better screening tool for antimicrobial drugs than conventional 2-D culture methods.
Stem cells, both embryonic and adult, promise to revolutionize the practice of medicine in the future. However, in order to realize this potential, a number of hurdles must be overcome. Most importantly, the signaling mechanisms necessary to control the differentiation of stem cells into tissues of interest remain to be elucidated and much of the present research on stem cells is focused on this goal. Nevertheless, it will also be essential to achieve large-scale expansion and in many cases, assemble cells in 3-D as transplantable tissues. To this end, the RCCS can play a significant role.
Recently, investigators have demonstrated the capability of the RCCS to expand bone marrow mesenchymal and hematopoietic stem cells compared to static culture and facilitate the differentiation of umbilical cord stem cells into 3-D liver aggregates.
The RWV was originally conceived as a tool to study the cellular responses to microgravity. However, investigators who first used the technology quickly recognized that it could address some of the shortcomings of conventional cell culture systems, namely the deficiency of mass transport in static culture and high mechanical shear forces in stirred systems. Unexpectedly, the conditions created in the vessel were ideal for 3-D cell culture.