Thermoresponsive fiber-based microwells capable of formation and retrieval of salivary gland stem cell spheroids for the regeneration of irradiation-damaged salivary glands

Abstract

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 (SGSC^monolayer). The spheroids could be retrieved efficiently by decreasing temperature. SGSC-derived spheroid (SGSC^spheroid) 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 SGSC^spheroid-implanted mice. The production of submandibular acini specific mucin increased in SGSC^spheroid-implanted mice, compared with PBS control. More MIST1⁺ mature acinar cells were preserved in the SGSC^spheroid-implanted group than in the PBS control group. Intriguingly, SGSC^spheroid-implanted mice exhibited greater amelioration of tissue damage and preservation of KRT7⁺ terminally differentiated luminal ductal cells than SGSC^monolayer-implanted mice. The SGSC^spheroid-implanted mice also showed less DNA damage and apoptotic cell death than the SGSC^monolayer-implanted mice at 2 weeks post-implantation. Additionally, a significant increase in Ki67⁺AQP5⁺ proliferative acinar cells was noted only in SGSC^spheroid-implanted mice. Our results suggest that a thermoresponsive fiber-based scaffold could be of use to facilitate the production of function-enhanced SGSC^spheroid cells and their subsequent retrieval and delivery to damaged salivary glands to alleviate radiation-induced apoptotic cell death and promote salivary gland regeneration.

Keywords: Salivary glands; fiber-based microwell; spheroid retrieval; stem cell spheroid; thermoresponsive fiber.