A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

Abstract

SummaryUtilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

Generation of cardiac spheres from primate pluripotent stem cells in a small molecule-based 3D system

“…To enable such the purpose, we used a simple 3D sphere culture system, similar to a recent report [19]. Monolayer culture BG02 or H9 hESC colonies were gently detached from MEF-feeder after dispase treatment, resuspended in culture media and passed through the strainer with 50µm opening mesh by pipette pressure (Fig.1A-1B).…”

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

“…2a–c). Large size of hESC colonies would induce differentiation 30 and the addition of 1% methylcellulose in the culture medium can prevent clump formation 19 . The number of hESCs collected from a 1.5-L cell suspension medium reached 1.5 × 10 9 on D5 post culture, and spheres were maintained in good shape and condition, which was similar to the ones cultured with small scale (Fig.…”

“…1A). When cultured in ultra-low dishes, spheres rarely formed big clumps, and, after being transferred to culture bags, 1% methylcellulose in the medium could prevent clump formation by increasing viscosity 19,24 . Subculturing is also an essential step for robust hESC expansion.…”

“…Afterwards, human pluripotent stem cells (hPSCs) are cultured with single-cell inoculation in spinner flasks for more than 10 passages to maintain pluripotency 18 . Another strategy is that of passage in a mechanical way and supplementing functional polymers to the suspension system, which produced a yield of up to 1.4 × 10 8 hPSCs in a 200-mL cell culture bag 19 . Although some progress has been made in hESC suspension culture, mass production of good manufacturing practices (GMP)-grade hESCs for clinical application remains challenging because of clump formation in static culture systems, shear force damage in dynamic bioreactors, and the low viability caused by suboptimal passage methods 19–21 .…”

“…By utilizing ultra-low attachement dish, which have low attachment for cells 23 , we obtained optimized seeding density and culture medium, established a 3D culture system with single-cell hESCs for initial seeding, and produced cells in aggregates for proliferation. Then we progressively scaled up the system to cell culture bags while employing methylcellulose to prevent cell conglobation 19,24 , and finally reached a yield of 1.5 × 10 9 cells per 1.5-L culture system. Importantly, hESCs maintained normal morphology and pluripotency for more than 30 passages in the 3D culture system.…”