Developing Defined and Scalable 3D Culture Systems for Culturing Human Pluripotent Stem Cells at High Densities

Abstract

Human pluripotent stem cells (hPSCs) – including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) – are very promising candidates for cell therapies, tissue engineering, high throughput pharmacology screens, and toxicity testing. These applications require large numbers of high quality cells; however, scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple, efficient, fully defined, scalable, and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here, we describe additional design rationale and characterization of this system. For instance, we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast, using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally, hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally, there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny.

An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells

“…The low yield and frequent cell passaging make the 2D culture systems not a good choice as personalized cellular biomanufacturing system. In 3D suspension cultures, cells frequently aggregate into large cell agglomerates434446. Due to the diffusion limits, the supply of growth factors, nutrients and oxygen to and the depletion of the metabolic waste from the cells located at the center of cell agglomerates became insufficient, leading to slow cell proliferation, apoptosis and uncontrolled differentiation.…”

“…However, it also generated hydrodynamic stress that negatively affects the cell growth and quality5556. High cell density in the culture also promotes spheroid agglomeration46. Considering all these factors, cells are generally seeded at low density (e.g.…”

“…Even under these optimized conditions, cells grow slowly and cell death is common. For instance, iPSCs typically expand 4 times per 4 days to yield around 2.0 × 10 6 cells/ml in spinner flasks46. It is good to note that cells occupy <0.4% of the medium volume with this yield.…”

“…To transfer the culture from 2D to 3D PNIPAAm-PEG hydrogels, hPSCs maintained in Matrigel-coated 6-welll plate were treated with Accutase (Life Technologies) at 37 °C for 5 minutes and dissociated into single cells4346. Dissociated cells were mixed with 10% PNIPAAm-PEG (Cosmo Bio, USA) solution dissolved in E8 medium on ice and cast on tissue culture plate, then incubated at 37 °C for 10 minutes to form hydrogels before adding warm E8 medium containing 10 μM ROCK inhibitor (Y-27632, Selleckchem).…”

“…The bioprocessing takes advantage of the discovery that human iPSCs could be expanded in 3 dimension (3D) thermoreversible Poly(N-isopropylacrylamide)-Poly(ethylene glycol) (PNIPAAm-PEG) hydrogels at high growth rate and yield4346. In this paper, we first developed a protocol that could efficiently differentiate human iPSCs into NSCs in the PNIPAAm-PEG hydrogel.…”