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Abstract Extracellular vesicles (EVs) secreted by human‐induced pluripotent stem cells (hiPSCs) have great potential as cell‐free therapies in various diseases, including prevention of blood–brain barrier senescence and stroke. However, there are still challenges in pre‐clinical and clinical use of hiPSC‐EVs due to the need for large‐scale production of a large quantity. Vertical‐Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC‐EVs using a scalable aggregate or microcarrier‐based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3‐D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA‐seq, respectively. Thein vitrofunctional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3‐D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA‐seq. The microcarrier cultures had at least 17–23 fold higher EV secretion, and EV collection in mTeSR had 2.7–3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA‐seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt‐related pathways). hiPSC‐EVs demonstrated the ability of stimulating proliferation and M2 polarization of microgliain vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti‐aging study. 

Abstract Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic, while only elastic properties have been investigated in the past. How the viscoelastic matrix regulates stem cell patterning is critical for cell‐ECM mechano‐transduction. Here, this study fabricated different methacrylated hyaluronic acid (HA) hydrogels using covalent cross–linking, consisting of two gels with similar elasticity (stiffness) but different viscoelasticity, and two gels with similar viscoelasticity but different elasticity (stiffness). Meanwhile, a second set of dual network hydrogels are fabricated containing both covalent and coordinated cross–links. Human spinal cord organoid (hSCO) patterning in HA hydrogels and co‐culture with isogenic human blood vessel organoids (hBVOs) are investigated. The viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. More viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels result in higher interneuron marker expression. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects in the co‐culture of hSCOs and hBVOs. In addition, more viscoelastic hydrogels can lead to more Yes‐associated protein nuclear translocation, revealing the mechanism of cell‐ECM mechano‐transduction. This research provides insights into viscoelastic behaviors of the hydrogels during human organoid patterning with ECM‐mimicking in vitro microenvironments for applications in regenerative medicine.