Biopreservation is a process by which the degradation of biologics is suppressed to enable the recovery of structure, viability, and function. Cryopreservation is the most common method for long-term biopreservation, using temperatures lower than 0 °C, generally ranging between -80 °C and -196 °C. At ultra-low temperatures, biochemical activity is effectively suspended, allowing materials to be preserved for extended periods without degradation or loss of function. This technology is essential across a range of sectors, including cell and gene therapy, regenerative medicine, biomanufacturing, precision agriculture, and biodiversity conservation.
Understanding how freezing imposes physical and chemical stress on biological systems was foundational to the development of modern cryobiology. Ice formation and solute imbalance were identified as two primary drivers of cryoinjury due to their ability to induce membrane rupture, osmotic shock, and intracellular damage.
To mitigate these effects, cryoprotective agents (CPAs) were discovered and developed based on their ability to reduce ice formation, regulate osmotic stress, and stabilize cellular structures. Small molecules such as glycerol and dimethyl sulfoxide (DMSO) became widely adopted due to their ability to permeate cells and suppress intracellular ice formation. Other CPAs, including hydroxyethyl starch (HES) and disaccharides like trehalose, act extracellularly to promote vitrification, minimize osmotic shifts, and protect membrane integrity.
Nature has also offered valuable insight. Freeze-tolerant organisms such as Antarctic fish and overwintering insects produce antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) that lower the equilibrium freezing point below that of the melting point. In addition, these natural glycoproteins also inhibit ice recrystallization and help maintain small, stable ice crystals during freezing and thawing.
Despite these advances, conventional CPAs are not without limitations. Although AFPs offer a compelling biological model, their strong ice-binding activity can induce dynamic ice shaping (DIS), resulting in sharp, elongated, spicular ice crystals that intensify cryoinjury. Their large and structurally complex nature also makes their cost-effective manufacturing challenging. In contrast, the relatively cheap CPA, DMSO, remains the gold standard, although it is cytotoxic and has been shown to dramatically impair post-thaw function. When administered to patients, DMSO can be associated with adverse side effects, underscoring the need for safer, more targeted preservation strategies.
IRIs come on the scene
To overcome these limitations, researchers have explored the development of synthetic ice recrystallization inhibitors (IRIs). These small molecules are specifically designed to mitigate the cellular damage from uncontrolled ice growth during cryopreservation and, ultimately, enable more flexible storage conditions and improve post-thaw cell recovery and viability, providing increased post-thaw quality. By reducing variability in post-thaw recovery and enhancing functional outcomes, this technology is redefining cryopreservation across a broad spectrum of applications. Pioneering work out of the University of Ottawa, Department of Chemistry and Biomolecular Sciences, demonstrated that small molecules could be rationally designed to mimic the IRI activity of natural AFPs without inducing dynamic ice shaping. Through iterative structure-activity studies, chemical scaffolds that potently inhibit ice recrystallization while remaining non-toxic, synthetically accessible, and highly tunable were identified. These compounds interfere with ice crystal growth kinetics, avoiding the sharp morphologies associated with AFPs.
PanTHERA CryoSolutions has advanced this work by developing proprietary IRIs that are compatible with conventional cryopreservation protocols. These compounds can be used in combination with traditional CPAs, providing a complementary mechanism of action that directly targets recrystallization—an under-addressed but significant source of cryoinjury, especially during transient warming or thawing. In doing so, they also enable the potential reduction of CPA concentration, helping to minimize cytotoxicity without compromising protection. By mitigating ice-induced mechanical damage and reducing variability in post-thaw cell recovery, IRIs represent a promising tool for improving the safety, reproducibility, and scalability of cryopreservation across multiple cell types and applications.
Results from IRI application
Cryopreservation outcomes vary widely depending on the cell type and stress conditions encountered, and IRIs can support them in different ways. As shown in Figure 1, the addition of IRIs to induced pluripotent stem cells (iPSCs) during cryopreservation can increase post-thaw viability and recovery without affecting pluripotency.1,2 The inclusion of IRIs can also facilitate a faster reestablishment of neuronal network activity and synaptic function compared to controls for iPSC-derived neurons (iPSC-Ns) (Figure 1).1,2
A growing number of systematic studies and recent reviews now document these advantages of IRIs across multiple systems.3 Platelet cryopreservation has shown measurable preservation of key surface markers and morphology with IRI supplementation, and hematopoietic stem and progenitor cells (HSPCs) cryopreserved with IRI-containing formulations have demonstrated improved post-thaw function and potency, including superior engraftment with umbilical cord (UCB) transplant models.
Beyond enhancing viability and functional recovery, IRIs also provide resilience to unintentional transient warming events (TWEs) during storage or handling—conditions that frequently lead to recrystallization-related injury and are difficult to control with conventional CPAs alone. For RBCs, the inclusion of IRIs during cryopreservation can not only enable the reduction of glycerol concentration while significantly improving membrane integrity, but as highlighted in Figure 2, RBCs with IRI can maintain higher integrity after repeated warming cycles.2,4 The protective effects extend to complex tissue. Rat lungs and livers perfused with IRIs exhibited greater post-thaw membrane integrity compared to organs preserved with DMSO alone.3
Together, these data underscore the potential for IRIs to improve the consistency, safety, and scalability of cryopreservation across a wide range of clinically and commercially relevant cell types, including T cells, NK cells, CAR T cells, iPSCs, and more.
Why it matters
As cryopreservation becomes increasingly central to medicine, biotechnology, and conservation, the limitations of conventional cryoprotectants are becoming more apparent. Ice recrystallization is being increasingly recognized as a critical source of cryoinjury that compromises post-thaw cell quality and function. IRIs offer a targeted, next-generation solution to this challenge. By mitigating ice-induced damage, enhancing post-thaw recovery, and enabling reductions in cytotoxic agents like DMSO, IRIs improve both the safety and reliability of cryopreservation protocols. Their versatility across a wide range of cell types positions them as a foundational tool in the future of precision biopreservation.
References
1. Alasmar S, Huang J, Chopra K, et al. Improved cryopreservation of human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons using ice-recrystallization inhibitors. Stem Cells. 2023;41(11):1006-1021. doi:10.1093/stmcls/sxad059. PMID: 37622655.
2. PanTHERA CryoSolutions, part of BioLife Solutions. Introducing New Possibilities in Science & Innovation. 2025. https://pantheracryo.com/science-innovation/, Figures 2 and 6.
3. McMunn LE, Walsh EM, Ben RN. Targeted development and optimization of small-molecule ice recrystallization inhibitors (IRIs) for the cryopreservation of biological systems. Cryo Lett. 2024;45(2):69-87. doi:10.54680/fr24210110112. PMID: 38557986.
4. Briard JG, Poisson JS, Turner TR, Capicciotti CJ, Acker JP, Ben RN. Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming, and thawing. Sci Rep. 2016;6:23619. doi:10.1038/srep23619. PMID: 27021850; PMCID: PMC4817074.
Rebecca Mercier, PhD, is a cell biologist at PanTHERA CryoSolutions, now part of BioLife Solutions. Ana Clementin, PhD, is the executive director at PanTHERA CryoSolutions, now part of BioLife Solutions. Jason Acker, PhD, is a professor at the University of Alberta, Department of Laboratory Medicine and Pathology, and co-founder of PanTHERA CryoSolutions, now part of BioLife Solutions. Robert Ben, PhD, is a professor at the University of Ottawa, Department of Chemistry and Biomolecular Sciences and co-founder of PanTHERA CryoSolutions, now part of BioLife Solutions.