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

Abstract

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. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. 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 SSB 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. Continuously fed bioreactor systems combined with nutrient 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.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

“…Perfusion was technically established by the incorporation of a porous filter (20‐ to 40‐µm pore size) as a cell retention device similar to that in Weegman et al [24]. The culture volume was kept constant by applying the same flow rates for addition of fresh medium (feed) via a head plate port and for the removal of depleted medium (waste) via the outflow filter (supplemental online Fig.…”

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

“…Alternatively, choose another outflow tube based on the specific requirements of the culture. NOTE: Choose the OT pore size to remove only medium and cell debris, leaving cell aggregates in culture (as described in section 6 and publication 3 for more details).…”

“…1. Calculate the feed rate using the adjusted feed rate equation described in the literature 3 and in the provided representative results section below. 2.…”

“…In order to generate large numbers of viable and functional human cells for transplantation, regulation of the culture conditions is imperative. Depletion of nutrients, along with buildup of metabolic waste are major contributors to senescence and metabolic changes that reduce the quality of the cell product [1][2][3] . This procedure demonstrates a method to culture mammalian cells in spheroids using a stirred bioreactor combined with an adjusted rate perfusion feeding system to regulate glucose in a physiological range 4 throughout the duration of the culture.…”

“…However, this batch-feeding method still requires frequent manipulation, introduces variability in the cell environment, and increases the risk of contamination [5][6][7][8][9] . Stirred suspension bioreactors (SSB) provide better mixing and decreased handling 3,[10][11][12][13][14][15][16][17][18][19][20] , but like static cultures, require manual medium changes that contribute to potentially damaging fluctuations in nutrient and waste product levels. Perfusion feeding of SSB cultures reduces these problems by continuous infusion and removal of medium, but large changes in nutrient levels due to cell growth remain an issue.…”