3D cultures produced exosomes with significantly increased yield and superior functional properties.
Exosomes from three-dimensional cultures (3D-Exo) enhanced cell proliferation and migration compared to those from traditional two-dimensional cultures (2D-Exo).
3D-Exo treatment accelerated wound closure and reduced inflammation in a mouse skin injury model.
Distinct microRNA expression profiles were observed in 3D-Exo, suggesting activation of regenerative signaling pathways.
Simplified
BACKGROUND: Extracellular vesicle (EV)-based cell-free therapies have emerged as a powerful alternative to stem cell transplantation in regenerative medicine, owing to their ability to promote tissue repair while avoiding safety concerns associated with live-cell therapies. However, traditional two-dimensional (2D) cell cultures used for EV production are constrained by low exosome (Exo) yields and limited biological activity.
OBJECTIVE: This study introduces a novel and scalable three-dimensional (3D) culture platform based on a (HA) and L-ornithine methyl ester (Orn) hydrogel to enhance the production and therapeutic efficacy of stem cell-derived exosomes.
METHODS: The HA-Orn hydrogel was fabricated via a simple and mild crosslinking strategy, forming a biomimetic matrix that promotes spontaneous spheroid formation. Exosomes derived from 3D cultures (3D-Exo) were compared with those from 2D cultures (2D-Exo) in terms of yield, molecular composition, and biological functions.
RESULTS: 3D-Exo exhibited significantly increased yield and superior functional properties, including enhanced stimulation of cell proliferation, migration, angiogenesis, and extracellular matrix remodeling. In vivo, 3D-Exo treatment accelerated wound closure and reduced inflammation in a mouse skin injury model, demonstrating robust therapeutic efficacy and safety. Mechanistic studies revealed distinct miRNA expression profiles and activation of regenerative signaling pathways in 3D-Exo.
CONCLUSION: This work presents a cost-effective, scalable, and bioinspired 3D culture system for high-yield and functionally enhanced Exo production. The HA-Orn hydrogel platform offers significant translational potential for advancing cell-free regenerative therapies, particularly in the context of wound healing.
Key numbers
97%
Wound Closure Rate
Wound closure rate achieved by 3D-Exo treatment by day 14.
3D-Exo significantly higher than 2D-Exo
Increase in Exosome Yield
Comparison of exosome production between 3D and 2D cultures.
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Declarations. Ethics approval and consent to participate: This study did not involve human participants. This study involving experimental mice was conducted in accordance with the ethical standards of Central South University and followed the National Act on the Use of Experimental Animals (People’s Republic of China). Ethics approval was obtained from the Experimental Animal Ethics Committee of Central South University for the project titled “Study on the Effectiveness of Spherical Cultured MSCs Exosomes in Chronic Wound Treatment and the Design of Biomimetic Exosomes” (Approval ID: CSU-2024-0133, Approval Date: May 13, 2024). This approval ensures that all procedures were performed to minimize pain and discomfort to the animals and adhered to the national guidelines for the care and use of laboratory animals. The hUC-MSCs used in this study were commercially obtained from Hunan Shengbao Biotechnology Co., Ltd. ( www.hnssbsw.org/ ) It has been confirmed by the supplier that the cells were collected with an ethical approval and the donors had provided informed consent for the use of their cells in research. Consent for publication: Not applicable. The research does not involve individual participants or personal data. Competing interests: The authors declare no competing of interests.
