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.

Evaluation of the design and implementation of municipal ice cleat distribution programs for the prevention of ice-related fall injuries among older adults in Sweden.

INTRODUCTION: The risk for outdoor falls tends to increase during winter due to icy road conditions. Several Swedish municipalities have introduced programs that provide their senior citizens with a pair of ice cleats in an attempt to tackle this problem. In this paper, we perform a process evaluation to identify potential barriers to the success of these programs and analyze the logic of their design. METHODS: We sent a survey to all 290 Swedish municipalities to collect data on the characteristics of ice cleat distribution programs. We also performed focus-group interviews with older adults to gain insight into their thoughts about ice cleat programs. We synthesized our data with existing literature on ice cleats and behavior change theory to populate a logic model to identify and analyze hidden assumptions and potential flaws using program theory analysis. RESULTS: On average, about 40% of the eligible population living in the intervention municipalities collected a pair of ice cleats. While we identified some other, but mostly minor, barriers to implementation, the main barrier appears to be a lack of scale (i.e., insufficient procurement and distribution of ice cleats), as 90% of all purchased ice cleats were eventually distributed. While previous research suggests that ice cleats can decrease injury risks if worn, we find that there is limited evidence on the effects of distribution on ice cleat use. Our interviewees emphasized the potential utility of ice cleats for staying safe and active during winter but stressed that ice cleats need to be user-friendly and of high-quality to increase the likelihood that a distribution program encourages behavior change. CONCLUSION: Existing ice cleat distribution programs appear to have reached a meaningful share of the targeted population. Additional research is required to assess their effects on ice cleat use and injury rates.

Simulating Impacts of Ice Storms on Forest Ecosystems.

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. Current models suggest that the frequency and intensity of ice storms could increase over the coming decades in response to changes in climate, heightening interest in understanding their impacts. Because of the stochastic nature of ice storms and difficulties in predicting when and where they will occur, most past investigations of the ecological effects of ice storms have been based on case studies following major storms. Since intense ice storms are exceedingly rare events it is impractical to study them by waiting for their natural occurrence. Here we present a novel alternative experimental approach, involving the simulation of glaze ice events on forest plots under field conditions. With this method, water is pumped from a stream or lake and sprayed above the forest canopy when air temperatures are below freezing. The water rains down and freezes upon contact with cold surfaces. As the ice accumulates on trees, the boles and branches bend and break; damage that can be quantified through comparisons with untreated reference stands. The experimental approach described is advantageous because it enables control over the timing and amount of ice applied. Creating ice storms of different frequency and intensity makes it possible to identify critical ecological thresholds necessary for predicting and preparing for ice storm impacts.

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.

Extracellular Antifreeze Protein Significantly Enhances the Cryopreservation of Cell Monolayers.

The cryopreservation of cells underpins many areas of biotechnology, healthcare, and fundamental science by enabling the banking and distribution of cells. Cryoprotectants are essential to prevent cold-induced damage. Here, we demonstrate that extracellular localization of antifreeze proteins can significantly enhance post-thaw recovery of mammalian cell monolayers cryopreserved using dimethyl sulfoxide, whereas they show less benefit in suspension cryopreservation. A type III antifreeze protein (AFPIII) was used as the macromolecular ice recrystallization inhibitor and its intra/extracellular locations were controlled by using Pep-1, a cell-penetrating peptide. Flow cytometry and confocal microscopy confirmed successful delivery of AFPIII. The presence of extracellular AFPIII dramatically increased post-thaw recovery in a challenging 2-D cell monolayer system using just 0.8 mg·mL-1, from 25% to over 60%, whereas intracellularly delivered AFPIII showed less benefit. Interestingly, the antifreeze protein was less effective when used in suspension cryopreservation of the same cells, suggesting that the cryopreservation format is also crucial. These observations show that, in the discovery of macromolecular cryoprotectants, intracellular delivery of ice recrystallization inhibitors may not be a significant requirement under "slow freezing" conditions, which will help guide the design of new biomaterials, in particular, for cell storage.

Slip resistance and wearability of safety footwear used on icy surfaces for outdoor municipal workers.

BACKGROUND: Outdoor workers experience high injury rates in the winter due to slipping on ice and snow. Our testing program has demonstrated that most safety footwear does not provide adequate slip-resistance and/or comfort in icy conditions. OBJECTIVE: Our objective was to determine which of the most commonly worn safety footwear available to outdoor municipal workers in Toronto, Ontario, Canada would best prevent slips on icy surfaces and which models had good wearability. METHODS: We selected 45 of the most popular types of winter footwear worn by these workers and applied our Maximum Achievable Angle (MAA) test method to rate the slip-resistance of the footwear. A ten-point rating scale was used for recording participants' perceptions of wearability. The MAA test measured the steepest ice-covered incline that participants can walk up and down without experiencing a slip. RESULTS: Of the 45 types of footwear tested, only one model achieved an MAA score of 8 degrees that exceeded our cut-off for acceptable performance set at 7 degrees. Secondary measures of performance including thermal insulation; wearability and heaviness of footwear tested were also ranked. CONCLUSION: Our results demonstrate that footwear manufactures have the opportunity to differentiate their footwear by investing in slip-resistant outsole materials.

Vulnerability of cotton subjected to hail damage.

This paper establishes the quantitative relationships between hail fall parameters and crop damages by examining the impacts of 49 hail hazard scenarios on cotton in the bud stage and boll stage. This study utilizes simulated cotton hail hazard to analyze the following data: hail size, hail fall density, and crop damages (i.e., defoliation rate, branch breaking rate, and the fruit falling rate). The results are as follows: 1) cotton vulnerability increased via an increase in crop damages as the hail hazard magnitude increased; 2) crop damages exhibit significant logistic relationships with hail diameter and hail fall density, and the fit was better at the bud stage than at the boll stage; 3) cotton is more vulnerable to hail hazard at the bud stage than at the boll stage, and the bud stage is a critical period for cotton hail disaster prevention and mitigation; and 4) damages to cotton plant at the bud stage and boll stage were less sensitive to hail size from hail fall density. Thus, we suggest that hail diameter can be used as the priority indicator to predict hail-induced crop damages. These results provide a sound basis for developing a comprehensive evaluation of hail damage in cotton, which is crucial for promoting sustainable cotton production. We recommend that the impacts of hail-induced crop damages on yield and fiber quality need to be addressed further in future studies.

Changes in the Excitability of Corticobulbar Projections Due to Intraoral Cooling with Ice.

The aim of this study was to assess the effects of ice applied to the oral cavity on the excitability of corticobulbar projections to the swallowing muscles. The subjects were 8 healthy adult volunteers (mean age 29.0 ± 4.9 years). Motor-evoked potentials (MEPs) were recorded from the suprahyoid muscle complex using surface electrodes. Two blocks of 20 MEPs with a test stimulus intensity of 120% of the resting motor threshold were recorded at rest (baseline). Subjects then underwent 5-min thermal stimulation by either of 3 different types: (1) "ice-stick inside mouth," (2) "ice-stick on neck," and (3) "room temperature inside mouth." Blocks of 20 MEPs were then recorded immediately and at 5-min intervals for the following 15 min. There was a significant difference in the effects of the 3 interventions on the amplitude of the MEPs following stimulation (two-way ANOVA: INTERVENTION × TIME; F8,84 = 3.76, p < 0.01). One-way ANOVA was used to evaluate the changes over time for each intervention type. Only "ice-stick inside mouth" caused an increase in the MEPs (one-way ANOVA main effect of TIME: F4,28 = 4.04, p = 0.010) with significant differences between baseline and P10 (mean difference 0.050; confidence interval (CI) 95% 0.019-0.079; p = 0.004). There were no significant effects of either "ice-stick on neck" or "room temperature inside mouth" (F4,28 = 1.13, p = 0.36; F4,28 = 1.36, p = 0.27, respectively). Ice stimulation within the oral cavity increases the excitability of the cortical swallowing motor pathway.

Superchilling of muscle foods: Potential alternative for chilling and freezing.

Superchilling is an attractive technique for preservation of muscle foods which freezes part of the water and insulate the food products from temperature fluctuations thereby enhancing the shelf-life during storage, transportation and retailing. Superchilling process synergistically improves the product shelf-life when used in combination with vacuum or modified atmospheric packaging. The shelf-life of muscle foods was reported to be increased by 1.5 to 4.0 times relative to traditional chilling technique. Advantages of superchilling and its ability to maintain the freshness of muscle foods over freezing has been discussed and its potential for Industrial application is highlighted. Present review also unravel the mechanistic bases for ice-crystal formation during superchilling and measures to ameliorate the drip loss. The future challenges especially automation in superchilling process for large scale Industrial application is presented.

Cold Panniculitis After Ice Therapy for Supraventricular Tachycardia.

A late preterm infant presenting with supraventricular tachycardia (SVT) was admitted to the pediatric intensive care unit because of poor systolic function seen on echocardiogram. The hospitalization was complicated by multiple breakthrough episodes of SVT requiring ice placed on the face during each repeat episode. The infant was later diagnosed as having cold panniculitis secondary to the application of ice to the face on multiple occasions. In children who are hemodynamically stable during SVT episodes, ice is used as first-line treatment. It is important to be aware of how often ice is being applied to the face and the duration of time to limit complications such as cold panniculitis.

Ice pack-induced perniosis: a rare and underrecognized association.

Perniosis, or chilblain, is characterized by skin lesions that occur as an abnormal reaction to exposure to cold and damp conditions. It can present as an idiopathic dermatosis or in association with an underlying connective tissue or autoimmune disease. Differentiation by histopathologic examination is controversial. Herein, we report a unique case of a 73-year-old woman who developed acquired perniosis on the buttocks from the use of ice packs to treat chronic low back pain.

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms.

All modern molecular biology and microbiology is underpinned by not only the tools to handle and manipulate microorganisms but also those to store, bank, and transport them. Glycerol is the current gold-standard cryoprotectant, but it is intrinsically toxic to most microorganisms: only a fraction of cells survive freezing and the presence of glycerol can impact downstream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of microorganisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, and successful cryopreservation shown as low as 1.1 wt % of additive. The mechanism of protection is demonstrated to be linked not only to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram-negative, Gram-positive, and mycobacteria strains. This represents a step-change in how microorganisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

Hiding in Plain Sight: Contaminated Ice Machines Are a Potential Source for Dissemination of Gram-Negative Bacteria and Candida Species in Healthcare Facilities.

BACKGROUND Contaminated ice machines have been linked to transmission of pathogens in healthcare facilities. OBJECTIVE To determine the frequency and sites of contamination of ice machines in multiple healthcare facilities and to investigate potential mechanisms of microorganism dispersal from contaminated ice machines to patients. DESIGN Multicenter culture survey and simulation study. SETTING The study took place in 5 hospitals and 2 nursing homes in northeastern Ohio. METHODS We cultured multiple sites on ice machines from patient care areas. To investigate potential mechanisms of microbial dispersal from contaminated ice machines, we observed the use of ice machines and conducted simulations using a fluorescent tracer and cultures. RESULTS Samples from 64 ice machines in the 5 hospitals and 2 nursing homes (range, 3-16 per facility) were cultured. Gram-negative bacilli and/or Candida spp were recovered from 100% of drain pans, 52% of ice and/or water chutes, and 72% of drain-pan grilles. During the operation of ice machines, ice often fell through the grille, resulting in splattering, with dispersal of contaminated water from the drain pan to the drain-pan grille, cups, and the hands of those using the ice machine. Contamination of the inner surface of the ice chute resulted in contamination of ice cubes exiting the chute. CONCLUSIONS Our findings demonstrate that ice machines in healthcare facilities are often contaminated with gram-negative bacilli and Candida species, and provide a potential mechanism by which these organisms may be dispersed. Effective interventions are needed to reduce the risk of dissemination of pathogenic organisms from ice machines. Infect Control Hosp Epidemiol 2018;39:253-258.

Ice-associated norovirus outbreak predominantly caused by GII.17 in Taiwan, 2015.

BACKGROUND: On 5 March 2015, Taiwan Centers for Disease Control was notified of more than 200 students with gastroenteritis at a senior high school during excursion to Kenting. We conducted an outbreak investigation to identify the causative agent and possible vehicle of the pathogen. METHODS: We conducted a retrospective cohort study by using a structured questionnaire to interview all students for consumed food items during their stay at the resort. Students were defined as a gastroenteritis case while having vomiting or diarrhea after the breakfast on 4 March. We inspected the environment to identify possible contamination route. We collected stool or vomitus samples from ill students, food handlers and environmental specimens for bacterial culture for common enteropathogens, reverse transcription polymerase chain reaction (RT-PCR) for norovirus and enzyme-linked immunosorbent assay (ELISA) for rotavirus. Norovirus PCR-positive products were then sequenced and genotyped. RESULTS: Of 267 students enrolled, 144 (54%) met our case definition. Regression analysis revealed elevated risk associated with iced tea, which was made from tea powder mixed with hot water and self-made ice (risk ratio 1.54, 95% confidence interval 1.22-1.98). Ice used for beverages, water before and after water filter of the ice machine and 16 stool and vomitus samples from ill students were tested positive for norovirus; Multiple genotypes were identified including GI.2, GI.4 and GII.17. GII.17 was the predominant genotype and phylogenetic analyses showed that noroviruses identified in ice, water and human samples were clustered into the same genotypes. Environmental investigation revealed the ice was made by inadequate-filtered and un-boiled water. CONCLUSIONS: We identified the ice made by norovirus-contaminated un-boiled water caused the outbreak and the predominant genotype was GII.17. Adequately filtered or boiled water should be strongly recommended for making ice to avoid possible contamination.

A cold hard menace: A contaminated ice machine as a potential source for transmission of carbapenem-resistant Acinetobacter baumannii.

During an investigation of potential sources of transmission of multidrug-resistant gram-negative bacilli on a spinal cord injury unit, we recovered genetically related carbapenem-resistant Acinetobacter baumannii isolates from the stool of 3 patients, the hands of a nurse, and an ice machine water outlet spout and drain. Our findings suggest that contaminated ice machines could serve as a potential reservoir for dissemination of multidrug-resistant gram-negative bacilli.

Cryobiological parameters of multipotent stromal cells obtained from different sources.

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol-1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol-1 and 27.4 ± 0.9 kcal mol-1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol-1 and 19.5 ± 0.5 kcal mol-1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to -30 °C, followed by 3 °C/min to -80 °C.

Experimental provocation of 'ice-cream headache' by ice cubes and ice water.

Background There are various studies on experimentally provoked 'ice-cream headache' or 'headache attributed to ingestion or inhalation of a cold stimulus' (HICS) using different provocation protocols. The aim of this study was to compare two provocation protocols. Methods Ice cubes pressed to the palate and fast ingestion of ice water were used to provoke HICS and clinical features were compared. Results The ice-water stimulus provoked HICS significantly more often than the ice-cube stimulus (9/77 vs. 39/77). Ice-water-provoked HICS had a significantly shorter latency (median 15 s, range 4-97 s vs. median 68 s, range 27-96 s). There was no difference in pain localisation. Character after ice-cube stimulation was predominantly described as pressing and after ice-water stimulation as stabbing. A second HICS followed in 10/39 (26%) of the headaches provoked by ice water. Lacrimation occurred significantly more often in volunteers with than in those without HICS. Discussion HICS provoked by ice water was more frequent, had a shorter latency, different pain character and higher pain intensity than HICS provoked by ice cubes. The finding of two subsequent HICS attacks in the same volunteers supports the notion that two types of HICS exist. Lacrimation during HICS indicates involvement of the trigeminal-autonomic reflex.

Assessing health impacts of the December 2013 Ice storm in Ontario, Canada.

BACKGROUND: Ice, or freezing rain storms have the potential to affect human health and disrupt normal functioning of a community. The purpose of this study was to assess acute health impacts of an ice storm that occurred in December 2013 in Toronto, Ontario, Canada. METHODS: Data on emergency department visits were obtained from the National Ambulatory Care Reporting System. Rates of visits in Toronto during the storm period (December 21, 2013 - January 1, 2014) were compared to rates occurring on the same dates in the previous five years (historical comparison) and compared to those in a major unaffected city, Ottawa, Ontario (geographic comparison). Overall visits and rates for three categories of interest (cardiac conditions, environmental causes and injuries) were assessed. Rate ratios were calculated using Poisson regression with population counts as an offset. Absolute counts of carbon monoxide poisoning were compared descriptively in a sub-analysis. RESULTS: During the 2013 storm period, there were 34 549 visits to EDs in Toronto (12.46 per 1000 population) compared with 10 794 visits in Ottawa (11.55 per 1000 population). When considering year and geography separately, rates of several types of ED visits were higher in the storm year than in previous years in both Toronto and Ottawa. Considering year and geography together, rates in the storm year were higher for overall ED visits (RR: 1.10, 95 % CI: 1.09-1.11) and for visits due to environmental causes (RR: 2.52, 95 % CI: 2.21-2.87) compared to previous years regardless of city. For injuries, visit rates were higher in the storm year in both Toronto and Ottawa, but the increase in Toronto was significantly greater than the increase in Ottawa, indicating a significant interaction between geography and year (RR: 1.23, 95 % CI: 1.16-1.30). CONCLUSIONS: This suggests that the main health impact of the 2013 Ice Storm was an increase in ED visits for injuries, while other increases could have been due to severe weather across Ontario at that time. This study is one of the first to use a population-level database and regression modeling of emergency visit codes to identify acute impacts resulting from ice storms.