Efficient cell culturing for mesenchymal stem cell applications
A growing field of interest in medicine is cell therapy with mesenchymal stem cells (MSC). The current experiment demonstrates a successful expansion of MSC in a hollow fiber module without unspecific spontaneous differentiation.
Cultivation of human mesenchymal stem cells (hMSC) with Cellab® Bioreactor System in hollow fiber module
Simon Kordowich1, Sebastian Dressler2, Simone Valley2, Jeanette Witte2, Dierk Wittig1
1 Life Science Inkubator Betriebs GmbH & Co. KG, Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany
2 ALPHA PLAN GmbH, Juri-Gagarin-Str. 13A, 01454 Radeberg, Germany
A growing field of interest in medicine is cell therapy with mesenchymal stem cells (MSC). Standardized production processes in bioreactors need to be established. ALPHA PLAN has designed a semi-automated and easy-to-use Cellab® Bioreactor System for cell culture. The current experiment demonstrates a successful expansion of MSC in a hollow fiber module without unspecific spontaneous differentiation.
Cell culture system with hollow fiber bioreactor Cellab® is a Standardized Supply System for cell maintenance after cell seeding. The system consists of a Cellab® Docking Station in conjunction with Cellab® Disposable Sets, which will
be run in a standard incubator (Fig. 1). Cell culture medium is enriched with oxygen by a gas transfer module and circulates from medium bag through a closed tube system into bioreactors. The system feeds cells and eliminates waste
Figure 1: Cellab® Docking Stations with Cellab® Disposable Sets in a standard incubator.
The used Cellab® Disposable Set is equipped with a small hollow fiber bioreactor. This type of reactor is a twocompartment-system (Fig. 2A): Hundreds of hollow fibers are potted in a cylindrical cartridge (length x diameter: 60x12 mm). The fiber wall is a semipermeable membrane with a molecular weight cut-off (MWCO) of 30,000 Dalton. Cells grow in a volume of 1 mL inside the fibers, called intra capillary space (ICS). Cell culture medium flows through the space surrounding the fibers, called extra capillary space (ECS). Nutrient, waste and gases can diffuse through the membrane (Fig. 2B).
Figure 2A: Hollow fiber bioreactor design shows medium flow through ECS.
Figure 2B: Cross section of hollow fiber (ID 200 μm) illustrates situation during cell culture. Scale bar: 50 μm
Cell culture Human mesenchymal stem cells from bone marrow (2.7x106 cells) were inoculated into the ICS of the Cellab® Bioreactor. Medium bag was filled with 400 mL DMEM plus 10% human serum and penicillin. Cell cultivation was performed under standard conditions (37°C, 8% CO2) for 21 days. Cellab® Docking Station was adjusted at lowest level of medium flow for 10 sec every 15 min. and at a low gas flow (level 2).
Cell metabolism In continuous intervals 500 μL medium samples were taken from bioreactor and fibronectin has been analyzed with ELISA method. After cultivation of the hMSC DAPI staining was performed and cells were visualized by fluorescence microscopy. A staining with van Kossa and Alizarin Red determined calcium concentrations to indicate cell differentiation.
Maintenance of hMSC in hollow fiber bioreactors with Cellab® Bioreactor System proceeded successfully. The DAPI staining showed that cells grow inside the hollow fiber and adhere at the wall (Fig. 4A). Almost none pyknotic nucleoluses were detected. Staining with Alizarin Red and van Kossa showed no increased calcium concentration (Fig. 4B). This result indicated that hMSC did not differentiate spontaneously during cultivation in the bioreactor. Fibronectin did not rise above the basal concentration of 0.9 μg/mL in any fraction (Fig. 3). The reason is perhaps that the redundant fibronectin is rinsed away when the media flushes every 15 min. In conclusion, hMSC can be cultivated for weeks in Cellab® Bioreactor System without loss of multipotency.
Figure 4A: DAPI staining showed adherent cells inside the hollow fibers at wall of the membrane.
Figure 4B: Longitudinal sections of hollow fibers (ID 200 μm) and positive control in van Kossa and Alizarin Red staining.