Compared to traditional 2D-cultured mesenchymal stem cells (MSCs), 3D-MSCs offer distinct advantages in disease treatment. However, large-scale culture of 3D-MSCs remains labor-intensive and time-consuming. Thus, developing cryopreservation method for 3D-MSCs is essential for clinical application. Existing cryopreservation techniques primarily focus on 2D-cultured MSCs, and vitrification methods such as Cryotop are not suitable for large-scale applications, often leading to cytotoxicity due to high concentrations of cryoprotective agents. To address these challenges, we developed an innovative vitrification method using microfluidics, which involved encapsulating 3D human umbilical cord MSCs in GelMA hydrogel to create 3D-MSCs hydrogel microspheres (3D-MSCsHM). This approach significantly enhanced the survival rates of MSCs while reducing the need for cryoprotective agents. The entire process could be completed in 30 min, yielding 96 % viability and functionality upon rewarming. Proteomic analysis further revealed that improved viability and functions post rewarming were linked to enhance mitochondrial function, increased antioxidant proteins, and elevated growth factors. Furthermore, this method showed effective therapeutic outcomes in wound healing in a mouse model, comparable to those achieved with fresh 3D-MSCs. The presented vitrification technique offers a practical solution for the cryopreservation of multicellular stem cell tissues, enhancing their therapeutic applications.Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.