Approaches for automized expansion and differentiation of human MSC in specialized bioreactors

Background and experimental approach A main challenge in cell therapies and other tissue regeneration approaches is to produce a therapeutically significant cell number. For expansion of mesenchymal stem cells (MSC) the cultivation on 2D plastic surfaces is still the conventional procedure, even though the culture conditions differ significantly from the 3D environment in vivo. Additionally, static amplification of MSC is a labour-intensive procedure. We therefore used a specialized rotating bed bioreactor in order to maximize ex vivo expansion of MSC. MSC were isolated from umbilical cord (UC) by explant method approach under xeno-free conditions. UC-MSC were thereafter expanded under dynamic conditions in a novel rotating bed bioreactor. The bioreactor system was designed to enable integration of sensors for online monitoring of various parameters (e.g. pH, pO2, pCO2) and hence, allow ensured cultivation under well controlled and reproducible conditions. Beside cell expansion, directed differentiation of MSC was also achieved in bioreactors. MSC lack the ability to grow in 3D direction and build functional tissue in vitro. Thus, it is necessary to seed and culture MSC on 3D matrices to obtain functional implants. For guided differentiation towards the osteogenic lineage, MSC were cultivated on ceramic porous matrices under dynamic conditions. Custom-made miniaturized perfusion bioreactors for parallel testing were designed and optimized for that purpose. Methods MSC isolation was achieved as described previously [1]. Briefly, umbilical cord tissue is cut into pieces (approx. 0.5 cm) and cultivated for 10 days in aMEM containing 15% human serum in cell culture flasks. Cells grow out of the tissue pieces and adhere to the cell culture plastic. Subsequently, cells are harvested and subcultivated in aMEM containing 10% human serum. UC-MSC were expanded in a rotating bed bioreactor (Figure 1A). The bioreactor chamber is a cylindrical bioreactor shell, comprising an inlet (bed) fixed to a magnet whereas the bioreactor chamber is hold by that magnet to an engine. The inlay is rotating, while the shell is fixed. The inlay comprises cell culture plastic slides with an all over surface of 2000 cm, requiring approximately 130 ml cell culture medium to be completely covered. The bioreactor is equipped with a feed circulation for fresh medium and removal of waste. An additional circulation to pH and pO2 sensor electrodes enables online monitoring. Sampling is performed through a septum in the bioreactor shell. Gas mixture of air and CO2 is supplied by an overlay stream. The whole bioreactor set up is situated in a GMP conform breeder, enabling sterile handling as well as an environmental temperature of 38°C. The system is connected to a control unit, which comprises gas regulation, pumps and software for parameter set up and monitoring. UCMSC were seeded (1,500 cells/cm) in the bioreactor for 24 h hours and expanded for 5 days under dynamic conditions. Medium feed was adjusted depending on glucose consumption. After 5 days of cultivations UCMSC were harvested by flushing the bioreactor with accutase for 20 min. MSC were counted, examined regarding their senescence (b-galactosidase), proliferation capacity (glucose/lactate) and differentiation * Correspondence: [email protected] Department for Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria Full list of author information is available at the end of the article Neumann et al. BMC Proceedings 2013, 7(Suppl 6):P47 http://www.biomedcentral.com/1753-6561/7/S6/P47