Optimizing Key Process Parameters for Scale-Up Production of Insulin-Synthesizing β Cells Derived from iPSCs

I. Abstract:

Type 1 diabetes is an autoimmune disease that leads to the destruction of pancreatic β-cells and requires lifelong insulin therapy. Pancreatic islet transplantation offers a promising solution, yet it faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and hold the promise of large-scale production. However, current differentiation protocols—primarily conducted in mixed or 2D environments—lack the ideal conditions for scalability and suspension culture.

We investigated a series of bioreactor-scale-up parameters that could influence the differentiation process. The study employed an optimized HD-DoE protocol, which was designed for scalability and implemented in a 0.5-L (PBS-0.5 Mini) vertical-wheel bioreactor.

We have developed a three-stage suspension culture process that transitions from adherent to suspension culture. The TB2 medium supports the growth of iPSCs during scale-up. A staged optimization approach and extended differentiation time were employed to enhance the expression of marker genes and maturation of islet-like clusters derived from iPSCs. Continuous bioreactor operation was used to investigate the effects of nutrient and growth limitations on differentiation. Comparison of the continuous bioreactor with a control bioreactor using varying culture media revealed metabolic changes and a differentiation profile more closely resembling that of beta cells. Cryopreserved aggregates collected from the experiments were thawed and showed maintained viability and insulin-secretory capacity, indicating their potential for storage and future therapeutic transplantation.

This study shows that increasing the duration of the phase or limiting medium supplementation to reduce lactate accumulation can enhance the differentiation capacity of insulin-producing cells cultured in large-scale suspension cultures.

II. Excerpt from the experimental content: Analysis of nutrient consumption and metabolites

To identify potential alternative carbon and nitrogen sources for the cells, we analyzed amino acid metabolism in both the control group and the continuous bioreactor throughout the entire culture period (Fig. S5A-B). Amino acid concentrations were determined using the Rapid Media Amino Acid and Vitamin Analyzer Rebel (908 Devices). Essential amino acids—including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine—remained stable throughout the culture period. However, several amino acids were completely depleted in both culture media, including L-aspartic acid after 5 days and L-glutamic acid after 16 days. Amino acid metabolism is critical for the normal function of pancreatic β-cells; alanine and glutamine are well known for their roles in regulating β-cell function and insulin secretion. At the end of the culture, the concentrations of glutamine and alanine were higher than those in the fresh medium, suggesting that these amino acids did not limit cell growth (Fig. S5A-B). Nevertheless, the source of their increase remains unknown; unlike previous observations that attributed their rise to the GlutaMAX™ supplement, no such increase was observed in the control bioreactor. In contrast, the control bioreactor, which experienced frequent medium changes at different stages and throughout the extended endocrine induction phase, did not show elevated levels of alanine and glutamine compared to the initial medium. No other significant differences were noted between the two bioreactors. As previously mentioned, the bioreactor with restricted medium supplementation exhibited better differentiation capacity than the bioreactor with frequently changing control medium. Regulation of amino acid concentrations and serum deprivation have been shown to promote the development of pancreatic β-cells derived from human stem cells.

In addition, FLEX2 (Nova Biomedical) was used to evaluate the results from both cultures, and the levels of Gln, Glu, NH4+, Na+, K+, Ca++, pH, PCO2, and PO2 were analyzed throughout the entire culture period (Figure S6A-B). In the continuous bioreactor, the osmolarity of the culture medium steadily increased but remained within the range of 280–320 mOsm/kg. This increase can be attributed to the accumulation of solutes generated from nutrient metabolism and other waste products. By contrast, in the control bioreactor, the osmolarity of the culture medium fluctuated as the medium was supplemented at different stages of cell differentiation. The levels of glutamine and glutamate were also assessed, and both showed a gradual depletion over time, which is consistent with measurements obtained using the Rebel analyzer. The two bioreactors exhibited comparable performance in terms of biological differentiation, except for significant differences in the continuous bioreactor, where the pH continuously declined and the expected oxygen consumption rate was higher. Although the gas concentrations measured in the reaction medium might be influenced by the time interval between sample collection and measurement, the overall impact on all samples was uniform. Overall, the data indicated that after 10 days of culture—or following the PP-induced differentiation stage—PO2 levels began to decline steadily. Despite the fact that the volume of gas in the headspace above the reaction medium was identical in both bioreactors (500 mL), the oxygen flux entering the reaction medium might have been insufficient to compensate for the increased oxygen consumption in the 0.5-L continuous vessel, compared to the control bioreactor where medium supplementation varied.

Source of literature: doi.org/10.21203

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