With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for various applications in regenerative medicine and cell-based therapies given their unique capability to differentiate into any cell type of the human body. However, human iPSCs are highly vulnerable to cryopreservation with post-thaw survival rates of 40-60%; this is due to cryoinjury resulting from ice recrystallization when using conventional slow cooling protocols. Ice recrystallization is a process where the growth of large ice crystals occurs at the expense of small ice crystals. Ice recrystallization inhibitors (IRIs) are designed to inhibit the growth of intracellular ice crystals, increasing post-thaw viability. In this study, we tested a panel of four IRIs to determine if the inhibition of ice recrystallization can decrease cellular damage during freezing and improve viability post-thaw of iPSC colonies. We supplemented commercially available and serum-free cryopreservation medium mFreSR, routinely used for the cryopreservation of iPSCs, with a class of N-aryl-D-ß-gluconamide IRIs. A 2-fold increase in post-thaw viability was observed, in a dose dependent response, for N-(4-methoxyphenyl)-D-gluconamide (PMA) at 15 mM, N-(2-fluorophenyl)-D-gluconamide (2FA) at 10 mM, and N-(4-chlorophenyl)-D-gluconamide (4ClA) at 0.5 mM over mFreSR controls. After testing the panel of four IRIs, 2FA frozen iPSCs showed an increase in cell viability, proliferation, and recovery. The addition of ROCK inhibitor (RI), commonly used to increase iPSC viability post thaw, further enhanced the survival of the iPSCs frozen in the presence of 2FA and is used routinely in research. This additive effect increased cell recovery and colony formation post thaw, resulting in increased proliferation with no adverse effects on iPSC pluripotency or differentiation capabilities. The development of improved cryopreservation strategies for iPSCs is key to establishing master clonal cell banks and limiting cell selection pressures, all while maintaining high post-thaw viability and function. This will help ensure sufficient supplies of high-quality iPSC required to meet the cell demands for cell and regenerative based therapies. Since iPSCs hold promise as a potentially unlimited cell source for a plethora of cell-based therapies, improving cryopreservation is essential to the successful deployment of iPSC-derived therapeutic cell products in the future.

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.