Spheroids, as three-dimensional (3D) cell aggregates, can be prepared using various methods, including hanging drops, microwells, microfluidics, magnetic manipulation, and bioreactors. However, current spheroid manufacturing techniques face challenges such as complex workflows, the need for specialized personnel, and poor batch reproducibility. In this study, we designed a support-free, 3D-printed microwell chip and developed a compatible low-cell-adhesion process. Through simulation and experimental validation, we rapidly optimized microwell size and the coating process. We successfully formed three types of spheroids—human immortalized epidermal cells (HaCaTs), umbilical cord mesenchymal stem cells (UC-MSCs), and human osteosarcoma cells (MG63s)—on the chip. Fluorescent viability staining confirmed the biocompatibility and reliability of the chip. Finally, drug response experiments were conducted using the chip. Compared to traditional methods, our proposed strategy enables high-throughput production of size-controlled spheroids with excellent shape retention, while enhanced gas exchange during culture improves differentiation marker expression. This platform provides an efficient and cost-effective solution for biosensing applications, such as drug screening, disease modeling, and personalized therapy monitoring. Furthermore, the chip shows significant potential for real-time in vitro monitoring of cellular viability, reaction kinetics, and drug sensitivity, offering valuable advancements in biosensor technology for life sciences and medical applications.

The dermal–epidermal junction (DEJ), located between the dermal–epidermal layers in human skin tissue, plays a significant role in its function. However, the limitations of biomaterial properties and microstructure fabrication methods mean that most current tissue engineered skin models do not consider the existence of DEJ. In this study, a nanofiber membrane that simulates the fluctuating structure of skin DEJ was prepared by the composite molding process. Electrospinning is a technique for the production of nanofibers, which can customize the physical and biological properties of biomaterials. At present, electrospinning technology is widely used in the simulation of customized natural skin DEJ. In this study, four different concentration ratios of poly (lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) nanofiber membranes were prepared based on electrospinning technology. We selected a 15%PLGA + 5%PCL nanofiber membrane with mechanical properties, dimensional stability, hydrophilicity, and biocompatibility after physical properties and biological characterization. Then, the array-based microstructure model was prepared by three-dimensional (3D) printing. Subsequently, the microstructure was created on a 15%PLGA + 5%PCL membrane by the micro-imprinting process. Finally, the cell proliferation and live/dead tests of keratinocytes (HaCaTs) and fibroblasts (HSFs) were measured on the microstructural membrane and flat membrane. The results showed that 15%PLGA + 5%PCL microstructure membrane was more beneficial to promote the adhesion and proliferation of HaCaTs and HSFs than a flat membrane.

Severe skin damage poses a significant clinical challenge, as limited availability of skin donors, postoperative skin defects, and scarring often impair skin function. Traditional two-dimensional (2D) nanofibers exhibit small pore sizes that hinder cellular infiltration, unable to simulate the three-dimensional (3D) structure of the skin. To address these issues, we developed 3D porous nanofiber scaffolds composed of polycaprolactone–polylactic acid–mussel adhesive protein (PLGA-PCL-MAP) using low-temperature electrospinning combined with nano-spray technology. Meanwhile, this 3D scaffold features high porosity, enhanced water absorption, and improved air permeability. The incorporation of mussel adhesive protein (MAP) further increased the scaffold’s adhesive properties and biocompatibility. In vitro experiments demonstrated that the 3D nanofiber scaffolds significantly promoted the adhesion, proliferation, and migration of epidermal keratinocytes (HaCaTs) and human fibroblasts (HFBs), while providing ample space for inward cellular growth. Successful co-culture of HaCaT and HFBs within the scaffold revealed key functional outcomes: HaCaTs expressed keratinocyte differentiation markers CK10 and CK14, while HFBs actively secreted extracellular matrix components critical for wound healing, including collagen I, collagen III, and fibronectin. This skin substitute with a composite structure of epidermis and dermis based on three-dimensional nanofiber scaffolds can be used as an ideal skin replacement and is expected to be applied in wound repair in the future.

For healing full-thickness skin defects caused by severe injury, skin substitute grafts with structural and compositional biomimicry are critical. Vascularized spheroids, as highly active biological building blocks, can be applied in biomanufacturing to fabricate functional repair constructs. However, current methods of biofabricating endothelialized spheroids still face multiple technical limitations, including low throughput, poor uniformity, and insufficient controllability and observability. Furthermore, there is inadequate control over the spatial arrangement of the spheroids during vascularized construct biomanufacturing. We proposed a synergistic, shape-controlled spheroid–hydrogel biomanufacturing method to address these issues, and developed a highly integrated, one-stop bioprinting platform for spheroids (OBPS) with real-time monitoring. We focused on key biomanufacturing stages, spheroid fabrication, bioprinting, and in vivo validation, to establish an integrated technical framework providing for ‘fabrication–culture–assembly–application.’ Using the OBPS, we fabricated endothelialized spheroids by co-culturing human fibroblasts and human umbilical vein endothelial cells, printed fibrinogen-supplemented gelatin methacryloyl hydrogel, and bioprinted spheroids and hydrogels in situ onto full-thickness skin defects in nude mice. Animal experiments show that shape-controlled endothelialized spheroids significantly accelerated wound closure, suppressed inflammation, promoted neovascularization and collagen remodeling, and exhibited excellent tissue integration and repair potential. The OBPS system provides a novel and effective treatment for wound healing.