背景:糖尿病是一种由于胰岛素不足或不敏感而影响全球超过5亿人的疾病。对于1型糖尿病患者,胰岛移植可以帮助调节他们的血糖水平。然而,尸体供体胰岛的稀缺限制了可以接受这种治疗的人数。为了解决这个问题,人多能干细胞为通过定向分化产生胰岛素产生细胞提供了潜在的无限来源.已经开发了几种方案来制备干细胞衍生的胰岛素产生细胞。然而,缺乏关于与这些分化方案相关的生物过程参数以及如何利用它们来增加细胞产量的知识。
方法:我们研究了各种生物工艺参数和质量目标产品概况,这些参数和质量目标产品概况可能会影响分化管道,使用CellSTACKs和垂直轮式生物反应器(PBS-Minis)以可扩展的方式使用七阶段方案。
结果:细胞在分化的所有阶段和适当表达的阶段特异性标志物中保持>80%的生存力。在胰腺祖细胞发育的最初四个阶段,细胞数量有所增加。在胰腺祖细胞阶段之后,增殖细胞的百分比逐渐下降,由Ki67阳性确定,以及在内分泌分化期间细胞的显著损失。通过最小化聚集体融合的发生,我们能够在分化的后期提高细胞产量。我们建议,葡萄糖利用和乳酸产生是细胞质量属性,在表征源自干细胞的胰岛素产生细胞时应考虑。我们的发现还揭示了糖酵解的逐渐代谢转变,在胰腺祖细胞形成的最初四个阶段,后来在内分泌分化过程中氧化磷酸化。此外,产生胰岛素的细胞对几种促分泌素有反应,包括高葡萄糖.
结论:这项研究证明了过程参数,例如葡萄糖消耗和乳酸生产率,这些参数可用于促进干细胞衍生的胰岛素生产细胞的可扩展制造。
Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield.
We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis).
Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose.
This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.