Eppendorf: For a prospective use in advanced therapies, hiPSCs need to be differentiated into the desired cell type. How straightforward was it to translate differentiation protocols which have been designed for monolayer cultures to cell aggregates in bioreactors?
RZ: Since we initiated the development of lineage-specific differentiation strategies in suspension several years ago, shortly after the first successful hiPSC culture in 3D6 , we have established a substantial degree of competency in that area as well. The most significant challenges regarding directed differentiation in suspension culture include the impact of cell aggregates size, its heterogeneity, overall cell density and defining mechanical and hydrodynamic parameters7 .
However, we also noted that the standard culture media components and differentiation-directing molecules that we are applying, as for example the WNT pathway modulators, used for mesendoderm-induction and cardiac differentiation, have equivalent effects in 2D and in 3D8 . Therefore, process transition from 2D, which is often applied for cell differentiation basic research, to 3D suspension culture is typically straightforward. However, we are convinced that in the future many differentiation strategies will benefit from advance process control abilities enabled by the bioreactor technologies, still in the early stages of development9 .
Notably, we demonstrated that stirred-tank bioreactor-based hiPSC differentiation is efficiently applicable not only for cardiac diffraction (as highlighted by references above) but also for the differentiation and production of numerous other functional hiPSC progenies, including endothelial cells10 macrophages11 and endodermal derivatives12 .
Eppendorf: In upstream bioprocessing, the feeding strategy strongly impacts cell growth and viability. Repeated batch and perfusion are two options for removing byproducts and replenishing nutrients. What do you consider the pros and cons of these two strategies?
RZ: As mentioned above, our experience suggests that perfusion feeding, despite its complexity, is the optimal tactic for advanced hPSC cultivation13 . This is due to the highly glycolytic metabolism of the rapidly growing hPSC, which, on the one hand, requires an enormous supply of extra glucose to avoid growth-limiting starvation. Moreover, on the other hand, high glucose supplementation results into a massive accumulation of secreted lactate, which may become toxic and which induces a proliferation-inhibiting acidification of the culture. These issues increase exponentially in parallel to the exponential increase in cell density5 . For these reasons, we feel that perfusion feeding is the most successfully strategy to control growth-limiting parameters, if the goal of the protocol is to optimize high density cultivation of hiPSC. Notably, in parallel to the 10-fold increase in cell density, the amount of medium required to generate a given number of cells, was reduced by 70 % in consequence to the process optimization steps.