Nutrient Regulation By Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods.  Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product, as these cells are particularly sensitive to nutrient levels in the environment.  Cells were compared in static, stirred suspension bioreactors, and continuously fed stirred suspension bioreactors for cell expansion rates and medium nutrient levels.  While stirred bioreactors cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, such as glucose, which were consumed by the cultures between feedings.  Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed.  Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and stirred bioreactor culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products.  Thus, a perfusion feeding system of continuously fed bioreactor systems combined with feed rate regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies, and will facilitate the translation of cell culture from the research lab to clinical applications.