FREQUENTLY ASKED QUESTIONS

A. Bioreactor Vessels

1. I get good results with a 2-D or transwell culture flask. Why shift to a 3-D cell culture?
   Two prime reasons to consider the 3-D culture system over the traditional 2-D cell culture: cellular de-differentiation and loss of functionality. Additionally, the 2-D system is a static system and metabolically supports a finite number of cells. Because the 3-D cell culture system produces an microenvironment similar to intact parental tissue, this system is consider the next generational step in cell-tissue technology.
    
2. What are the advantages of the RCCS bioreactor over other 3-D cell culture systems?
   3-D extracellular matrices with embedded cells in a dish or multi-well plate have been most commonly used recently for 3-D cell culture. This model can produce reasonably good 3-D tissue models, it is limited because it remains a static system and will support a lesser overall cell population than the 3-D RCCS bioreactor.
   
   RCCS bioreactors provide excellent mass transfer and low mechanical- stress conditions which allow the formation of 3-D aggregates. Several studies have been conducted with these bioreactors that have shown the superiority of these bioreactors for 3-D cell culture. Click on the bibliography page for annotated references to the various studies that have successfully utilized RCCS bioreactors for 3-D cell culture.
    
3. Do the cells/organoids stay in one place in the RCCS bioreactor?  How do I maintain the rotational speed and why?
   No, cells and cell aggregates fall through media; this is one the factors that is responsible for the high mass transfer observed in these bioreactors.
   
   The rotation sped must be adjusted to compensate for the organoids' increasing diameter due to cell proliferation and continued aggregation with surrounding organoids. So, as the organoids grow in size, they will sediment more rapidly, and it will be necessary to increase the rotational speed of the vessel to prevent the cell organoids from colliding with the vessel wall.
    
4. How do I maintain the temperature and oxygen supply in the RCCS bioreactors?
   The bioreactors must be placed inside a regulated O₂/CO₂ humidified incubator.  The temperature of the incubator can be set and maintained at the optimal temperature for that particular cell line, i.e. mammalian cells 37⁰ C; some insect cell lines at 26⁰ C.
   
   Both O₂ and CO₂ diffuse into the culture through the silicone oxygenator according to the demands of the culture. With some 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.
    
5. Do the cells need to be on microcarrier beads before infusing them into the reactor?
   No, we recommend following the manufacture's guidelines to hydrolyzing and sterilizing the beads prior to use. In our system we suggest using the Cultispher G beads (Sigma Aldrich) although the Sephadex bead is also used by a number of labs. Beads and cells can be loaded into the reactor at the same time or independent of each other. The conditions inside the bioreactors are such that the cells automatically attach to the beads after they are both loaded into the reactor. We utilize in our lab a proprietary variation of this protocol.

We suggest that you link to the Synthecon, Inc website for additional answers to commonly asked questions or contact Synthecon directly.

B. 3-D Cell Culture

1. What do you mean "systems - orientated" 3-D cell-tissue culture?
   Cell lines originate from parental tissue target selected by the investigator. If an investigator is looking at an intestinal model, we try to match cells which are peri-intestinal or originate from the intestine. Because the overall goal is to mimic the parental tissue and lessen the potential of adverse signal transduction between the cells, we avoid using cells which originate from other organ systems.
    
2. What is the timeline from cryopreserved cells to a functional 3-D model?
   We suggest a minimum of two months to engineer a functional 3-D cell-tissue model. Cryopreserved cells are initially propagated in 2-D cell culture flasks. When a  target cell density is achieved, the cells are infused into the bioreactor. In multiple cell models, this process is repeated until all of the cellular constituents have been successful infused.  The model is allowed to develop for a defined time period.
    
3. How are subsequent models developed to insure cellular and 3-D model continuity?
   We routinely freeze back cells while in the 2-D cell culture phase at low passage numbers to insure subsequent 3-D models will originate from low passage cells. We limit the passage number between 12-15 passages before "retiring" a particular cell line aliquot. If we anticipate heavy demand on a particular cell line, we will acquire several seed cell lines and maintain them in our cryo bank for future propagation.
    
4. Can an investigator use a biomechanical scaffold or hydrogels rather than the macrogelantinous bead?
   We will approach several commercial bioengineering firms which design prototype scaffolds and scaffolds that can be used in the RCCS system on behalf of the investigator. We expect the investigator to be active in the selection of the alternative scaffold. We will not commit to a particular scaffold without the direct approval of the investigator.
    
5. What are the intellectual property issues involved with the 3-d model development and usage?
   Because we currently limit our service and product development to basic research, we are covered under the intellectual property license attached to the RCCS system. If the 3-D models are designated for commercial production, then we will approach the source of the cell lines and negotiate commercial licenses.
   
   Prior to entering into a consulting arraignment with an investigator, we require signed Confidentiality Statements as a course of normal business practices. If Cell Systems 3-D is currently working with a client on a particular 3-D model/application, we will not enter into a "like or competing" agreement with another investigator unless the original investigator signs a release statement.
    
6. Is it necessary for an investigator to initially purchase the RCCS system prior to entering into an agreement to develop a 3-D prototype model with Cell Systems 3-D?
   No, as part of our consulting or developing contract with an investigator we will make a RCCS system available for 90 days post delivery of the prototype model. We will ship the RCCS system to the  investigator's laboratory, set the system up and train laboratory personal to maintain the 3-D models. Media, biochemical addidatives, tissue fixation materials and infectious agents are the responsibility of the investigator once the system arrives in their laboratory.
   
   We have existing partnerships with local histological and electron microscopy service providers which can assist the provide (see our links page) if such services are not available at their respective institutions.
    
7. If Cell System 3-D provides these services on a contract basis, then what is business objective for Cell System 3-D?
   We are looking for collaborations and partnerships with investigators in the biomedical field, governmental labs and pharmaceutical companies. Many investigators do not want to invest in the time, expense or expertise to maintain cell culture facilities. If a prototype model meets the expectations of an investigator we hope to participate in future grant applications. Likewise, we look to enter into long - term arraignments to provide additional models for research into that pathogen or other selected pathogens.