Vitrification of 3D-MSCs encapsulated in GelMA hydrogel: Improved cryosurvival, reduced cryoprotectant concentration, and enhanced wound healing

Zixi Pu; Lei Zhang; Hongju Yang; Tianao Shao; Dan Wang; Junfeng Wang; Yaping Yan; Wei Si

doi:10.1016/j.ijbiomac.2025.139716

Vitrification of 3D-MSCs encapsulated in GelMA hydrogel: Improved cryosurvival, reduced cryoprotectant concentration, and enhanced wound healing

Int J Biol Macromol. 2025 Jan 12:139716. doi: 10.1016/j.ijbiomac.2025.139716. Online ahead of print.

Authors

Zixi Pu¹, Lei Zhang², Hongju Yang³, Tianao Shao⁴, Dan Wang⁴, Junfeng Wang⁵, Yaping Yan⁶, Wei Si⁷

Affiliations

¹ State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650000, China; Southwest United Graduate School, Kunming, Yunnan 650092, China.
² State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650000, China; Yunnan Key Laboratory of Primate Biomedical Research, Kunming, Yunnan 650500, China.
³ Division of geriatric Gastroenterology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China.
⁴ State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650000, China.
⁵ Department of Hepatic and Bile Duct Surgery, The First People's Hospital of Yunnan Province, Kunming 650032, China.
⁶ State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650000, China. Electronic address: [email protected].
⁷ State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650000, China; Southwest United Graduate School, Kunming, Yunnan 650092, China; Yunnan Key Laboratory of Primate Biomedical Research, Kunming, Yunnan 650500, China. Electronic address: [email protected].

PMID: 39809395
DOI: 10.1016/j.ijbiomac.2025.139716

Abstract

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.

Keywords: 3D culture; GelMA hydrogel; Mesenchymal stem cells; Microfluidics; Proteomic analysis; Vitrification.