AccScience Publishing / IJB / Volume 12 / Issue 2 / DOI: 10.36922/IJB025500515
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RESEARCH ARTICLE

Development and assessment of dimethyl sulfoxide-free antifreeze gelatin methacryloyl hydrogels for integrated three-dimensional bioprinting and cryopreservation

Xin Li1,2,3,4 ,  Yukun Cao2,3,4 ,  Chengyuan Li1 ,  Chenxi Liu2 ,  Jia Tan2,5 ,  Xinli Zhou2,3,4* ,  Yang Yu6* ,  Xi Xia1*
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1 Department of Reproductive Medicine, Peking University Shenzhen Hospital, Shenzhen Peking University_the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, China
2 Department of Biomedical Engineering, University of Shanghai for Science and Technology, Shanghai, China
3 Tumor Energy Therapy Laboratory, Shanghai Co-innovation Center for Energy Therapy of Tumors, Shanghai, China
4 Cryobiology Laboratory, Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai, China
5 Key Laboratory for Tissue Engineering of Jiangxi Province, School of Medical Information Engineering, Gannan Medical University, Ganzhou, Jiangxi, China
6 Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
IJB 2026, 12(2), 025500515 https://doi.org/10.36922/IJB025500515
Received: 10 December 2025 | Accepted: 9 February 2026 | Published online: 20 February 2026
© 2026 by the Author(s).. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Three-dimensional (3D) bioprinting enables the fabrication of engineered tissues, but cell damage during printing and limitations in long-term preservation hinder practical applications. Traditional cryoprotectants, such as dimethyl sulfoxide (DMSO), introduce cytotoxicity and require complex removal, restricting immediate tissue usability. Here, we present an integrated extrusion-based bioprinting and DMSO-free antifreeze hydrogel strategy to produce cell-laden constructs with high post-thaw viability and proliferative capacity. Systematic optimization of bioink composition (6% L-proline with varying gelatin methacryloyl concentrations), extrusion parameters, and crosslinking conditions enabled high-fidelity scaffold fabrication while preserving cell viability and proliferation. Numerical simulations guided the maximum printable heights for fibers of different diameters, supporting construct scalability. Storing cell-laden 3D-printed scaffolds in cryovials at −80 °C effectively maintained high cell viability compared with alternative cooling protocols. Cells in 3D scaffolds exhibited superior post-thaw proliferation compared with two-dimensional culture, and the platform was validated using C2C12 myoblasts, achieving high survival and robust recovery of proliferative capacity. This study establishes a practical and versatile framework for integrating bioprinting and cryopreservation to support the generation of cell-laden constructs with preserved viability and structural integrity for regenerative medicine applications.

Graphical abstract
Keywords
Three-dimensional bioprinting
Cryopreservation
Gelatin methacryloyl
Antifreeze hydrogel
Dimethyl sulfoxide-free hydrogels
Printability optimization
Funding
This research was supported by the Sanming Project of Medicine in Shenzhen (No. SZSM202211043).
Conflict of interest
The authors declare no conflicts of interest.
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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing
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