Biofabrication of 3D adipose tissue via assembly of composite stem cell spheroids containing adipo-inductive dual-signal delivery nanofibers.

Reconstruction of large 3D tissues based on assembly of micro-sized multi-cellular spheroids has gained attention in tissue engineering. However, formation of 3D adipose tissue from spheroids has been challenging due to the limited adhesion capability and restricted cell mobility of adipocytes in culture media. In this study, we addressed this problem by developing adipo-inductive nanofibers enabling dual delivery of indomethacin and insulin. These nanofibers were introduced into composite spheroids comprising human adipose-derived stem cells (hADSCs). This approach led to a significant enhancement in the formation of uniform lipid droplets, as evidenced by the significantly increased Oil red O-stained area in spheroids incorporating indomethacin and insulin dual delivery nanofibers (56.9 ± 4.6%) compared to the control (15.6 ± 3.5%) with significantly greater gene expression associated with adipogenesis (C/EBPA, PPARG, FABP4, and adiponectin) of hADSCs. Furthermore, we investigated the influence of culture media on the migration and merging of spheroids and observed significant decrease in migration and merging of spheroids in adipogenic differentiation media. Conversely, the presence of adipo-inductive nanofibers promoted spheroid fusion, allowing the formation of macroscopic 3D adipose tissue in the absence of adipogenic supplements while facilitating homogeneous adipogenesis of hADSCs. The approach described here holds promise for the generation of 3D adipose tissue constructs by scaffold-free assembly of stem cell spheroids with potential applications in clinical and organ models.

Engineering pre-vascularized 3D tissue and rapid vascular integration with host blood vessels via co-cultured spheroids-laden hydrogel.

Recent advances in regenerative medicine and tissue engineering have enabled the biofabrication of three-dimensional (3D) tissue analogues with the potential for use in transplants and disease modeling. However, the practical use of these biomimetic tissues has been hindered by the challenge posed by reconstructing anatomical-scale micro-vasculature tissues. In this study, we suggest that co-cultured spheroids within hydrogels hold promise for regenerating highly vascularized and innervated tissues, bothin vitroandin vivo. Human adipose-derived stem cells (hADSCs) and human umbilical vein cells (HUVECs) were prepared as spheroids, which were encapsulated in gelatin methacryloyl hydrogels to fabricate a 3D pre-vascularized tissue. The vasculogenic responses, extracellular matrix production, and remodeling depending on parameters like co-culture ratio, hydrogel strength, and pre-vascularization time forin vivointegration with native vessels were then delicately characterized. The co-cultured spheroids with 3:1 ratio (hADSCs/HUVECs) within the hydrogel and with a pliable storage modulus showed the greatest vasculogenic potential, and ultimately formedin vitroarteriole-scale vasculature with a longitudinal lumen structure and a complex vascular network after long-term culturing. Importantly, the pre-vascularized tissue also showed anastomotic vascular integration with host blood vessels after transplantation, and successful vascularization that was positive for both CD31 and alpha-smooth muscle actin covering 18.6 ± 3.6µm2of the luminal area. The described co-cultured spheroids-laden hydrogel can therefore serve as effective platform for engineering 3D vascularized complex tissues.

Composite Multicellular Spheroids Containing Fibers with Pores and Epigallocatechin Gallate (EGCG) Coating on the Surface for Enhanced Proliferation of Stem Cells.

Multicellular spheroids are formed by strong cell-cell and cell-extracellular matrix interactions and are widely utilized in tissue engineering for therapeutic treatments or ex vivo tissue modeling. However, diffusion of oxygen into the spheroid gradually decreases, forming a necrotic core. In this study, polycaprolactone (PCL) fibers with pores and epigallocatechin gallate (EGCG) coating on their surface to provide a structural framework within the spheroids and investigated their ability to mitigate diffusional limitation and control over the proliferation of human adipose-derived stem cells (hADSCs) is engineered. The DNA content of composite spheroids prepared from fibers and hADSCs decreased in unadjusted cells (1224 ± 134 ng), in those with fibers with a smooth surface (SF) (1447 ± 331 ng), and in those EGCG-coated with SF (E-SF) (1437 ± 289 ng). Cells with fibers with pores on the surface (PF) (2020 ± 32 ng) and those with EGCG-coated PF (E-PF) (1911 ± 80 ng) increased after 7 days of culture, with a significantly greater number of proliferating cells (29 ± 8% and 30 ± 8%, respectively). These results indicate that physical modification through the formation of pores on the fiber surface alleviates diffusion limitation of composite spheroids, playing a dominant role over chemical modification.