Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Inhibition of ice recrystallization during cryopreservation of cord blood grafts improves platelet engraftment.

BACKGROUND: Platelet engraftment following cord blood (CB) transplantation remains a significant hurdle to this day. The uncontrolled growth of ice, a process referred to as ice recrystallization, is one of several mechanisms that lead to cell loss and decreased potency during freezing and thawing. We hypothesized that reducing cell damage induced by ice recrystallization in CB units (CBUs) would reduce losses of stem and progenitor cells and therefore improve engraftment. We previously demonstrated that the ice recrystallization inhibitor (IRI) N-(2-fluorophenyl)-D-gluconamide (IRI 2) increases the postthaw recovery of CB progenitors. Herein, we set out to ascertain whether IRI 2 can enhance platelet and bone marrow engraftment activity of hematopoietic stem cells (HSCs) in cryopreserved CBUs using a serial transplantation model. STUDY DESIGN AND METHODS: CBUs were processed following standard volume/red blood cell reduction procedure and portions frozen with dimethyl sulfoxide (DMSO) supplemented or not with IRI 2. Thawed CB samples were serially transplanted into immunodeficient mice. RESULTS: Our results show that supplementation of DMSO with IRI 2 had several beneficial effects. Specifically, higher levels of human platelets were observed in the peripheral blood (p < 0.05; n = 4) upon transplant of CBUs preserved with the IRIs. In addition, human BM chimerism and the number of human CFU progenitors in the bone marrow were superior in IRI 2 recipients compared to DMSO recipients. Moreover, IRI 2 had no negative impact on the multilineage differentiation and self-renewal activities of HSCs. DISCUSSION: Taken together, these results demonstrate that supplementation of a hematopoietic graft with IRI can improve the postthaw engraftment activities of HSCs.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Modulating Intracellular Ice Growth with Cell-Permeating Small-Molecule Ice Recrystallization Inhibitors.

Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-ß-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ±â€¯0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 µM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics.

Modulation of antifreeze activity and the effect upon post-thaw HepG2 cell viability after cryopreservation.

Most antifreeze proteins (AFPs) exhibit two types of "antifreeze activity" - thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity. The mechanism of TH activity has been studied in depth and is the result of an adsorption of AFPs to the surface of ice with an ice-binding face (IBF). In contrast, the mechanism of ice recrystallization and its inhibition is considerably less understood. In this paper, we examine several different antifreeze proteins, glycoproteins and mutants of the Lolium perenne AFP (LpAFP) to understand how IRI activity is modulated independently of TH activity. This study also examines the ability of the various AF(G)Ps to protect HepG2 cells from cryoinjury. Post-thaw cell viabilities are correlated to TH, IRI activity as well as dynamic ice shaping ability and single ice crystal growth progressions. While these results demonstrate that AF(G)Ps are ineffective as cryoprotectants, they emphasize how ice crystal habit and most importantly, ice growth progression affect HepG2 cell survival during cryopreservation.

It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules.

A library of peptides and glycopeptides containing (4R)-hydroxy-L-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-ß-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.

Probing the Mechanism of Action of Small-Molecule Ice Recrystallization Inhibitors Using Proton Nuclear Magnetic Resonance Relaxation.

N-2-Fluorophenyl-d-gluconamide (2FA) improves the recovery and function of cryopreserved biological materials by inhibiting ice recrystallization. However, as for many small-molecule ice recrystallization inhibitors, the mechanism of action of 2FA is not well-understood. In this study, the IC50 of 2FA for ice recrystallization was determined to be 3.5 mM (95% CI [3.41-3.52]). 1H transverse and longitudinal relaxations were then characterized by NMR at 2FA concentrations from 0 to 10 mM and at temperatures between -15 °C and +30 °C. Corresponding activation energy of water molecule motion (EAH2O) was calculated, showing that at each concentration 2FA did not affect EAH2O in the solid state, whereas in the liquid state EAH2O was significantly higher with 2FA than for pure water. Therefore, 2FA is excluded from the ice lattice upon freezing and concentrated in the interstitial liquid phase. This restricts the migration of water molecules between ice crystals via the liquid phase, inhibiting ice recrystallization.

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization.

The ß-d-galactosyl-(1,3)-α-N-acetyl-d-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the ß-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the ß-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the ß-linked-(1,4) disaccharide exhibits more potent IRI activity than the native ß-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.

Synthesis of C-linked triazole-containing AFGP analogues and their ability to inhibit ice recrystallization.

C-Linked antifreeze glycoprotein (C-AFGP) analogues have been shown to have potent ice recrystallization inhibition (IRI) activity. However, the lengthy synthesis of these compounds is not amenable to large-scale preparation for the many commercial, industrial, and medical applications that exist. This paper describes the synthesis of triazole-containing AFGPs using a convergent solid-phase synthesis (SPS) approach in which multiple carbohydrate derivatives are coupled to a resin-bound synthetic peptide in a single step. Modified "Click" conditions using dry DMF as solvent with catalytic Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9-12 in 16-54% isolated yield. Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an essential structural feature necessary for potent IRI activity.

C-linked antifreeze glycoprotein (C-AFGP) analogues as novel cryoprotectants.

Significant cell damage occurs during cryopreservation resulting in a decreased number of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystallization during freeze-thawing is one cause of decreased viability after cryopreservation. We have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystallization to increase cell viability after cryopreservation. Our results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells.

Use of Ice Recrystallization Inhibition Assays to Screen for Compounds That Inhibit Ice Recrystallization.

Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay to measure IRI activity.

Synthesis of natural/13C-enriched d-tagatose from natural/13C-enriched d-fructose.

A concise, easily scalable synthesis of a rare ketohexose, d-tagatose, was developed, that is compatible with the preparation of d-[UL-13C6]tagatose. Epimerization of the widely available and inexpensive ketohexose d-fructose at the C-4 position via an oxidation/reduction (Dess-Martin periodinane/NaBH4) was a key step in the synthesis. Overall, fully protected natural d-tagatose (3.21 g) was prepared from d-fructose (9 g) on a 50 mmol scale in 23% overall yield, after five steps and two chromatographic purifications. d-[UL-13C6]Tagatose (92 mg) was prepared from d-[UL-13C6]fructose (465 mg, 2.5 mmol) in 16% overall yield after six steps and four chromatographic purifications.

Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization.

A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.

Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization.

Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hysteresis. The current study explores how changes in the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue 5 (n = 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization, while 4, 6, and 7 (n = 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD) spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution conformation. Variable-temperature (1)H NMR studies on truncated analogues 26-28 failed to elucidate the presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone. Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n = 3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone, orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains, with the carbohydrate moiety being exposed to bulk solvent. These results illustrate how subtle changes in conformation and carbohydrate orientation dramatically influence IRI activity in C-linked AFGP analogues.