November 1, 2016 (Vol. 36, No. 19)

Using a Well-Established Technology for the Cultivation of Stem Cells

Mammalian cell cultivation is routinely used to produce recombinant proteins or monoclonal antibodies (mAbs) for diagnostic and clinical applications. Since 1980 the productivity of mammalian cells cultivated in different types of bioreactors has reached the gram per liter range.

For over 20 years, hollow fiber based bioreactors have been used for protein production, including mAbs. Researchers have shown that cell densities of 1.87 x 108 cells/mL can be achieved for hybridoma cells. Other cells like Vero cells have reached a cell number of 3.81 x 107 cells/mL using a depth filter perfusion system. CHO cells have been logged at 3.35 x 107 cells/mL.

Hollow fiber technology, which was originally developed for dialysis, is still used today. Cells are separated from the circulated medium by a porous hollow fiber membrane. Utilizing the diffusion process over the hollow fiber membrane, metabolites are continuously removed from the cell compartment and nutrients are supplied to the cells from the medium compartment (Figures 1A, 1B, 1C, and 1D).

As a result of the optimized nutritional status of the cells, hollow fiber technology is now also frequently used for the expansion of stem cells like mesenchymal stromal cells (MSCs).

Figures 1A and 1B. Example set-up of hollow fiber bioreactor system, cells in suspension on the outside of the membrane. Figures 1C and 1D: Example set-up of hollow fiber bioreactor system, adherent cells on the inside of the membrane.

Stem Cell Cultivation

The number of clinical trials underway for cell-based therapies continues to rise. The cell source for nearly 400 clinical trials is MSCs. Cell numbers of 106 to 109 MSCs are needed for a single dose depending on the clinical application.

Production of this level of cells in T-flasks is inefficient due to the low volume to culture surface ratio. Additionally, there is a need to switch the manual cell culturing process in open systems to a controlled process in closed systems. This switch is required to increase the quality and efficiency of the cell culture process. This step also reduces operator-caused inconsistencies and the risk of contamination.

Unfortunately, existing large-scale bioprocessing production technologies, cannot be applied directly for this cell culture process as the manufacturing process is quite complex when these MSCs are the final product.

Additionally, MSCs are grown adherently while all proteins are grown in suspension cells (e.g. mAb-producing cells). This leads to challenges for the upstream process and the commercial demand. 

Open vs. Closed Cell-Culture System

Reproducible and controlled cell proliferation, GMP-conforming processes are essential for the clinical application of cell-based therapies. Hollow fiber bioreactor systems have been used for the clinical-scale production of human periosteum-derived stem cells (hPDCs). A number of other experiments demonstrated the comparability of hollow fiber and T-flask production.

Comparing yield and culture conditions, the hollow fiber system provides a reproducible cell proliferation process for multiple donors.

For preclinical or Phase I trials, scientists often still use standard cell culture methods multilayer flasks. This open system method is appropriate for preclinical trial production but not Phase I production. This limitation needs to be addressed early as ability to change cell therapy processing steps is restricted as clinical phases progress.

The best time to consider single-use closed systems is before Phase I trials are initiated. According to D. Clarke, writing in Bioprocess International in 2012, “A validated, closed, and presterilized process is considered to be a beneficial feature before initiating clinical trials.”

The challenge is to keep the cost of production low. Switching to a closed system requires a significant investment in a fully automated cell production bioreactor. A good compromise is the usage of semi-automated and functionality closed systems like the Cellab bioreactor system (Figures 2 and 3) to avoid switching the production method between preclinical and clinical trials.   

Figure 2. Cellab Docking stations with Cellab disposable sets in a standard incubator

Disposable Hollow Fiber Bioreactors

Disposable hollow fiber bioreactors are touted for the production of proteins like mAbs and viruses using this two-compartment technology. The benefit of this technology is obvious—a membrane separates the cell compartment from the medium compartment and increasing cell numbers in the cell compartment will not change the total volume. Stem cells, however, will require an adaptation of the cell expansion process.

Since the MSCs have to be harvested at the end of the cell culture process, the cells are growing inside of the hollow fibers on pre-coated (e.g., human fibronectin) membranes. It has been shown that human mesenchymal stem cells from bone marrow can be grown in a hollow fiber module. Staining with Alizarin Red and van Kossa showed no increased calcium concentration. Importantly, it was also shown that hMSC did not differentiate during cultivation in the hollow fiber bioreactor.

The medium supply is assured from outside of the porous hollow fiber membranes. The Cellab® bioreactor system, which is manufactured under medical device regulations, allows parallel test runs with a maximum of five hollow fiber bioreactors. Those parallel test runs can be used for process optimization. After successful process optimization the system can be used with a single hollow fiber bioreactor with a total extracellular membrane surface of 2,500 cm2.

In summary, traditional stem cell culture is done with 2D surfaces under static conditions using standard tissue culture plates and T-flasks, with a cell culture surface of several hundreds of square centimeters. During clinical trials a large number of cells has to be generated and there is a resultant need for several hundred T-flasks, which creates a high risk of contamination.

If for one treatment, 1012 MSCs will be required, 220,000 T150 flasks would need to be handled, which is inefficient for large-scale cell production. Even a change to roller bottles, multiple stack culture flasks, or cell factory systems can become unrealistic to achieve that level of cells.

Larger cell culture bioreactor systems need special equipment and occupy a lot of incubator or production space. Hollow fiber technology could be a good start to establish preclinical experiments and process optimization. It also expedites scaleup to large-scale production with fully automated equipment. Using scalable technologies from the beginning will finally lead to total time and process cost reduction as well as to efficient processes.

Figure 3. Cellab® bioreactor system—Inoculation of cells

Hans Nagels, Ph.D., is CSO and Tanja Seiler is vp business development at Cellab. For more information contact Susanne Große ([email protected]).

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