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Hydrogels are widely used in stem cell therapy due to their extensive tunability and resemblance to the extracellular matrix (ECM), which has a three-dimensional (3D) structure. These features enable various applications that enhance stem cell maintenance and function. However, fast and simple hydrogel fabrication methods are desirable for stem cells for efficient encapsulation and to reduce adverse effects on the cells. In this study, we present a one-pot double-crosslinked hydrogel consisting of polyethylene glycol (PEG) and collagen, which can be prepared without the multi-step sequential synthesis of each network, by using bio-orthogonal chemistry. To enhance the adipogenic differentiation efficiency of adipose-derived stem cells (ADSCs), we added degradable components within the hydrogel to regulate matrix stiffness through cell-mediated degradation. Bio-orthogonal reactions used for hydrogel gelation allow rapid gel formation for efficient cell encapsulation without toxic by-products. Furthermore, the hybrid network of synthetic (PEG) and natural (collagen) components demonstrated adequate mechanical strength and higher cell adhesiveness. Therefore, ADSCs grown within this hybrid hydrogel proliferated and functioned better than those grown in the single-crosslinked hydrogel. The degradable elements further improved adipogenesis in ADSCs with dynamic changes in modulus during culture and enabled the retrieval of differentiated cells for potential future applications.

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Three-dimensional spheroid culture enhances cell-to-cell interactions among stem cells and promotes the expression of stem cell properties; however, subsequent retrieval and delivery of these cells remain a challenge. We fabricated a thermoresponsive fiber-based microwell scaffold by combining electrospinning and hydrogel micropatterning. The resultant scaffold appeared to facilitate the formation of cellular spheroids of uniform size and enabled the expression of more stem cell-secreting growth factor genes ( EGF, IGF-1, FGF1, FGF2, and HGF), pluripotent stem cell-related genes ( SOX2 and NANOG), and adult epithelial stem cell-related genes ( LGR4, LGR5, and LGR6) than salivary gland stem cells in a monolayer culture (SGSCmonolayer). The spheroids could be retrieved efficiently by decreasing temperature. SGSC-derived spheroid (SGSCspheroid) cells were then implanted into the submandibular glands of mice at 2 weeks after fractionated X-ray irradiation at a dose of 7.5 Gy/day. At 16 weeks post-irradiation, restoration of salivary function was detected only in SGSCspheroid-implanted mice. The production of submandibular acini specific mucin increased in SGSCspheroid-implanted mice, compared with PBS control. More MIST1+ mature acinar cells were preserved in the SGSCspheroid-implanted group than in the PBS control group. Intriguingly, SGSCspheroid-implanted mice exhibited greater amelioration of tissue damage and preservation of KRT7+ terminally differentiated luminal ductal cells than SGSCmonolayer-implanted mice. The SGSCspheroid-implanted mice also showed less DNA damage and apoptotic cell death than the SGSCmonolayer-implanted mice at 2 weeks post-implantation. Additionally, a significant increase in Ki67+AQP5+ proliferative acinar cells was noted only in SGSCspheroid-implanted mice. Our results suggest that a thermoresponsive fiber-based scaffold could be of use to facilitate the production of function-enhanced SGSCspheroid cells and their subsequent retrieval and delivery to damaged salivary glands to alleviate radiation-induced apoptotic cell death and promote salivary gland regeneration.