A fully defined static suspension culture system for large-scale human embryonic stem cell production

Abstract

Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.

Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

“…Previous studies with mouse ESCs reported that the differential upregulation of pluripotent markers in 3-D culture was dependent on scaffold composition and stiffness [34,58]. A recent study using single cell inoculation and the expansion of human ESCs in large-scale bioreactors resulted in the maintenance of OCT4 levels but the upregulation of SOX2 in 3-D grown ESC aggregates [59]. In another study, a 3-D culture of H9 clumps in VS functionalized PEG scaffolds resulted in upregulated gene expression of SOX2 and KLF4 but not OCT4 and NANOG when compared to 2-D cultured cells [52].…”

Transcriptomic analysis of naïve human embryonic stem cells cultured in three-dimensional PEG scaffolds

“…OCT4 expression, which ranged from a slight decrease reported in thermoresponsive synthetic hydrogels [52], to no significant change in natural scaffolds composed of hyaluronic acid [30], chitosan or alginate [53]. However, upregulation of select core pluripotent markers has been observed in 3-D culture of mouse [36,54] as well as human ESCs [55,56], suggesting a dependence on scaffold composition and culture conditions. We have reported upregulation of select naïve pluripotent markers in primed [37] and naïve human ESCs In both mouse and human ESCs, focal adhesion and integrin signaling serve pleiotropic roles in anchorage, contractility, survival, proliferation as well as support or inhibition of self-renewal [63].…”

The Chinese National Stem Cell Resource Center

“… Gu et al, 2017 , Li et al, 2018 , Liu et al, 2018 , ISO 20387, 2018 , Tan et al, 2018 , Wang et al, 2015 , Wang, et al, 2019 , Wang et al, 2018 .…”