Hydrogel-Based Bioprocess for Scalable Manufacturing of Human Pluripotent Stem Cell-Derived Neural Stem Cells

Abstract

Neural stem cells derived from human pluripotent stem cells (hPSC-NSCs) are of great value for modeling diseases, developing drugs, and treating neurological disorders. However, manufacturing high-quantity and -quality hPSC-NSCs, especially for clinical applications, remains a challenge. Here, we report a chemically defined, high-yield, and scalable bioprocess for manufacturing hPSC-NSCs. hPSCs are expanded and differentiated into NSCs in microscale tubes made with alginate hydrogels. The tubes are used to isolate cells from the hydrodynamic stresses in the culture vessel and limit the radial diameter of the cell mass to less than 400 μm to ensure efficient mass transport during the culture. The hydrogel tubes provide uniform, reproducible, and cell-friendly microspaces and microenvironments for cells. With this new technology, we showed that hPSC-NSCs could be produced in 12 days with high viability (∼95%), high purity (>90%), and high yield (∼5 × 10 cells/mL of microspace). The volumetric yield is about 250 times more than the current state-of-the-art. Whole transcriptome analysis and quantitative real-time polymerase chain reaction showed that hPSC-NSCs made by this process had a similar gene expression to hPSC-NSCs made by the conventional culture technology. The produced hPSC-NSCs could mature into both neurons and glial cells in vitro and in vivo. The process developed in this paper can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future.

Engineered Microenvironment for Manufacturing Human Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells

“…To address the challenge, we previously developed a scalable and high-yield cell culture method for expanding hPSCs and other human cells (Li et al., 2018a, Li et al., 2018b, Lin et al., 2018a, Lin et al., 2018b). With this method, cells are processed into microscale alginate hydrogel tubes (AlgTubes) that are suspended in the cell culture medium.…”

Engineered human brown adipocyte microtissues improved glucose and insulin homeostasis in high fat diet-induced obese and diabetic mice

“…However, the matrix is expensive. Our group recently developed a novel microbioreactor termed AlgTubes, in which cells are cultured in alginate hydrogel microtubes [36,37,[67][68][69][70][71][72]. AlgTubes are scalable and have low cost.…”

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges

“…Hydrogels have several uses for tissue engineering. They can be used as a soft 3D ECM‐like environment, [ 115 , 119 , 226 , 227 , 228 , 229 , 230 , 231 , 232 , 233 , 234 , 235 , 236 , 237 , 238 , 239 ] as a 3D matrix filler inside porous scaffolds, [ 240 ] as components of bioinks, [ 34 , 121 , 187 , 222 , 241 , 242 , 243 , 244 , 245 , 246 ] as thin membranes which may be microstructured to produce alignment of cells, [ 126 , 247 ] or as source material to develop porous scaffolds. [ 23 , 248 , 249 ] For the first three uses, cytocompatible gelation is essential, as the cells are introduced into the hydrogel liquid solution before the hydrogel solidifies.…”