Evolution in the cryopreservation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are essential for the reconstitution of the hematopoietic and immune systems and are widely used in transplantation and emerging cell-based therapies. Cryopreservation is a critical technology enabling long-term storage, banking, and distribution of HSC grafts and immuno-oncology cell products. However, the cryopreservation process exposes stem cells and progenitors to multiple sources of cryoinjury, including intracellular ice formation, osmotic shock, solute effects, and ice recrystallization, which can compromise post-thaw viability, recovery, and functional potency. For decades, dimethyl sulfoxide (DMSO) has remained the gold-standard cryoprotective agent (CPA) due to its ability to reduce osmotic stress, limit intracellular ice formation, and stabilize cellular structures. Despite its widespread use, it is associated with dose- and time-dependent cytotoxicity and adverse infusion-related reactions, motivating efforts to reduce or replace its use. This review summarizes key cryobiological principles underlying HSC preservation, including the importance of optimized CPA exposure, controlled cooling, rapid thawing, and storage below glass transition temperature. We also discuss established and emerging permeating and nonpermeating CPAs, including sugars, polymers, and carbohydrate-based ice recrystallization inhibitors (IRI), several of which have shown to improve post-thaw outcomes and engraftment in preclinical models. Finally, we review strategies to mitigate DMSO toxicity, including reduced-DMSO formulations and newly developed "all-in-one" DMSO-free cryosolutions. Collectively, these advances are driving the evolution of safer and more effective cryopreservation strategies for HSC transplantation and next-generation cellular therapeutics.