Frequently Asked Questions
1. How does the RCCS bioreactor work?
The Rotary Cell Culture System bioreactor consists of a horizontally rotated culture vessel with a co-axial oxygenator in the center. When the vessel is filled with culture media and rotated, the fluid rotates as a solid body around a horizontal axis. The oxygenator rotates at the same angular velocity as the outer wall. These conditions produce laminar flow and minimal shear force inside the culture vessel. The cells are maintained in suspension by the resolution of the centrifugal, gravitational and Coriolis forces, so that cells placed in the RCCS bioreactor experience minimal mechanical stresses and high mass transport (of nutrients, oxygen etc.) and are thus able to assemble into tissue-like aggregates. Gas transfer is by means of diffusion through the silicone oxygenator, thereby avoiding bubble formation and turbulence.
2. Why would I make this shift to 3D cell culture when I get reasonably good results with the culture flask?
2D cell culture has contributed greatly to our understanding of Cell biology, but it has its limitations in terms of the amount of information that can be gleaned from it. Scientists have had to content themselves with traditional cell culture techniques for the past 100 years, but this is no longer necessary. Tissue culture, by definition, strives to mimic the conditions inside the body, and obviously, living organisms are 3-D, not 2-D. So, in order to appropriately model in vivo biology, in vitro culture systems must become 3-dimensional.
3. What are the advantages of the RCCS bioreactor over other 3-D cell culture systems?
3D extracellular matrices with embedded cells in a dish or multi-well plate have been most commonly used recently for 3-D cell culture. While this model produces reasonably good 3D tissue models, it is limited by restricted mass transfer (due to the static nature of the culture and also because the matrix presents an additional barrier to mass transfer) and lack of scalability.
Dynamic culture systems such as spinner flasks, or, on a larger scale, stirred tanks provide very good mass transfer, but these systems use mechanical force which will not only damage the cells, but also prevent their aggregation.
As described earlier, RCCS bioreactors provide excellent mass transfer and low mechanical- stress conditions that allow the formation of 3D aggregates. Several studies have been conducted with these bioreactors that have shown the superiority of these bioreactors for 3D cell culture. Click on the following link for a brief look at the various studies that have successfully utilized RCCS bioreactors for 3D cell culture. http://www.synthecon.com/bibliography/index.php
4. Do the cells stay in one place in the RCCS bioreactor?
No, the cells and cell aggregates fall through media; this is one the factors that is responsible for the high mass transfer observed in these bioreactors.
5. How do I maintain the temperature in the RCCS bioreactors?
The bioreactors must be placed inside the incubator, and the temperature of the incubator can be set and maintained at 37° C.
6. How do I maintain the oxygen supply to the culture?
As mentioned earlier (question 5), the bioreactor is placed in the incubator. The air in the incubator diffuses into the culture through the silicone oxygenator according to the demands of the culture. In the case of the larger culture vessels, there are pumps that make the incubator air available to the culture. Since all the gas transfer is by means of diffusion, it does not produce bubbles and thus, turbulence.
7. At what speed should the culture vessel be rotated?
This depends on the diameter of the cell aggregate. As the vessel rotates, the cell aggregates accelerate until they reach sedimentation velocity, which is determined by the size of the cell aggregate.
ccording to the Stokes equation, the sedimentation velocity increases as the square of the radius of the cell aggregate. So, as the aggregates grow in size, they will sediment more rapidly, and it will be necessary to increase the speed of rotation in order to prevent the cell aggregates from colliding with the wall. Thus, one can begin the culture with a slow speed of rotation, such as 7rpm, and then, as the cell aggregates grow in size and become visible, increase the speed of rotation.
8. Will I see bubbles in the reactor?
The main purpose of the RCCS bioreactor is to minimize mechanical stresses on the cells. The presence of even a few bubbles will increase turbulence, and thus mechanical stresses; hence the RCCS bioreactor is operated with zero head-headspace and gas transfer to the culture is by means of diffusion in order to prevent bubble formation. Small bubbles may appear over time due to cellular respiration, and these should be removed as necessary (a syringe can be used to remove bubbles effectively).
9. What are the different applications of the RCCS bioreactors?
The original purpose of the RCCS bioreactor was to simulate microgravity. During ground experiments using this reactor, it was noticed that cells suspended in these reactors tended to form 3D aggregates. Since then, these RCCS bioreactors have been used in several fields of cell and tissue culture. Applications of the RCCS bioreactor range from basic cell biology to space biology, culturing stem cells for regenerative medicine and drug development and possibly, in the future, the development of therapies for disease and injury. For a detailed account of the various applications of the RCCS bioreactor, click here.
10. Is this a roller bottle culture?
A roller bottle consists of a closed bottle around a longitudinal axis with flexible plastic walls. Gas transport in this case is through the neck of the bottle. Also, the purpose of the roller bottle is not 3-D culture, but 2D culture. The culture grows on the walls of the bottle, and as the bottle rotates, the culture is bathed with medium. Thus, there is no similarity between these two systems (refer to question 1 for the working principle of the RCCS bioreactor).
11. When I wish to culture cells on microcarrier beads, do the cells need to be on the beads before I load them into the reactor?
The beads and the cells can be loaded into the reactor at the same time, independent of each other. The conditions inside the reactor are such that the cells automatically attach to the beads after they are both loaded into the reactor.
12. Do I need an incubator to use the RCCS bioreactor?
Yes, an incubator is necessary in order to maintain the temperature, pH and oxygenation of the culture. Also, it should be noted that water evaporates through the silicone oxygenator into the incubator. This will lead to bubbles in the culture vessel. So, humidification is needed in the incubator in order to maintain the concentration of water vapor and prevent evaporation in the culture vessel.
13. Which reactor will best suit my needs?
This depends on the application for which you need our bioreactor. Another factor that obviously plays in is the budget. If cost is a major consideration, you might want to try the RCCS D. This system allows you to run a single experiment at a time. Usually, the 4D or the 4H systems are good to begin with because most often, you will need to run a few experiments at the same time and this system is a better long term investment. We also have reactors for specific applications, such as the stem cell culture systems. We can also custom-make reactors depending on the needs of the research study. The best way to get this question answered though, would be to call us on 800-853-0740. We have research scientists as well as technical sales personnel who could recommend reactors based on your specific applications.
