Cryopreservation is an essential tool in biomedical science, enabling the long-term storage and transport of viable cells for clinical and research applications. Dimethyl sulfoxide (DMSO) remains the most widely used cryoprotective agent (CPA), used for its ability to prevent intracellular ice formation and maintain postthaw viability. This review explores the use of DMSO-based cryopreservation across a wide spectrum of applications, including cell-based therapies, tissue and organ transplantation, reproductive medicine, and stem cell banking. Additionally, the emerging field of three-dimensional cell culture preservation is examined, emphasizing DMSO’s role in maintaining the structural and functional integrity of organoids and tissue models. While DMSO’s efficacy is well documented, concerns regarding cytotoxicity, gene expression changes, and variable cell-type sensitivities persist. Studies suggest that reduced DMSO concentrations, often in combination with alternative CPAs, may offer enhanced preservation outcomes with lower biological disruption. Continued optimization of DMSO-based cryopreservation protocols will be critical for advancing regenerative medicine, cellular therapies, and reproducible biomedical research.