Exploring Continuous And Integrated Strategies For The Up- And Downstream Processing Of Human Mesenchymal Stem Cells

Paula Alves


The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. The aim of this work is to perform a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. In particular, we have evaluated the impact of i) different operation modes (continuous and discontinuous) and ii) several TFF’s process parameters (e.g. membrane material and pore size, shear rate, permeate flux) have on cells’ expansion, recovery yield and quality (i.e. cell morphology, viability, identity and potency).

Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells’ metabolism. Regarding TFF, the combination of polysulfone membranes with pore sizes higher than 0.45 μm with lower residence and processing times (translated by higher shear rates and permeate fluxes, respectively), led to the successful concentration of hMSC up to a factor of 20. The operation of TFF either in continuous or discontinuous allowed to concentrate cells with high cell recovery yields (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration allowed higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration.

Overall, the integration of the optimized unit operations of concentration and washing led to a decrease in the bioprocess time in 40%, allowing for the recovery of over 70% of hMSC with high viability (>95%), while maintaining cells’ morphology, immunophenotype, proliferation capacity and multipotent differentiation potential.

This work shows that the integration of upstream and downstream unit operations is beneficial for cell therapy applications, where TFF showed to be an efficient methodology for the concentration and diafiltration of hMSC. The described process will have applicability to other stem cell types (e.g. human pluripotent stem cells) relevant for the cell therapy industry.