Vapor Phase Of Nitrogen Research Articles

Sustained Hematopoietic Engraftment Potential after Prolonged Storage of Cryopreserved Hematopoietic Stem Cells Used in Salvage Autologous Stem Cell Transplantation

Salvage autologous hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with relapsed multiple myeloma (MM). Peripheral blood stem cells (PBSCs), a source of hematopoietic stem cells (HSCs), are collected before the first transplantation, and adequate quantities of PBSCs can be collected and stored potentially for years to support at least 2 transplantations for eligible patients. To ensure the safety of salvage HSCT in the treatment of patients in subsequent relapse, PBSCs must retain the potential to engraft even after several years of cryopreservation. Although PBSC viability has been studied extensively using in vitro techniques, few publications describe the most rigorous functional potency measure, of patients receiving a myeloablative conditioning regimen. This study describes a large single-institution experience evaluating the engraftment kinetics of PBSCs used in salvage transplantation after multiple years of storage compared with first transplantation for the same patients in the treatment of MM. A retrospective chart review of patients with MM undergoing HSCT between 2000 and 2021 identified 89 patients who received salvage autologous PBSCs stored for >1 year after first HSCT. PBSCs were cryopreserved and stored in vapor-phase liquid nitrogen refrigerators at ≤-150°C. All patients received a PBSC product from the same collection cycle for both transplantations. Differences in CD34+ cell doses and days to engraftment between the first and salvage transplantations were tested using the paired 2-tailed t-test and Wilcoxon signed-rank test. Univariate and multivariable linear regressions were used to determine the association between storage time and days to engraftment, adjusting for CD34+ cell dose and conditioning regimen in the multivariable model. The median duration of storage between the day of initial collection and salvage transplant was 5.4 years (range, 1.0 to 19.7 years). Engraftment kinetics demonstrated a sustained neutrophil engraftment (absolute neutrophil count >0.5×109 cells/L) at a median of 11 days after both the first and salvage transplantations (range, 8 to 15 days and 8 to 19 days, respectively; P < .05). The median time to sustained platelet engraftment (>20×109 cells/L without transfusion support) was 13.5 days after the first HSCT and 14 days after salvage HSCT (range, 9 to 27 days and 10 to 56 days, respectively; P=.616). After adjusting for CD34+ cell doses and conditioning regimens, there was no association between the duration of cryopreservation and days to neutrophil engraftment (r=0.178, P=.130) or platelet engraftment (r=0.244, P=.100). Engraftment kinetics of the salvage HSCT are comparable to those of the first HSCT even when products are stored in vapor-phase nitrogen refrigerators for a median of 5.4 years. There is no association between the duration of storage and time to engraftment when controlling for CD34+ cell dose and conditioning regimen. Prolonged storage of cryopreserved HSC products is a safe practice for MM patients undergoing salvage autologous HSCT.

Evaluation of the TMRW vapor phase cryostorage platform using reproductive specimens and in vitro extended human embryo culture

To assess the impact of shipment and storage of sperm, oocytes, and blastocysts in vapor phase nitrogen compared with static storage in liquid phase nitrogen. Prospective cohort-matched study. Multiple invitro fertilization laboratories in an invitro fertilization network. Fifty-eight human embryos, 32 human oocytes, 15 units of bovine semen. Vapor vs. liquid nitrogen. The postwarming survival of oocytes, sperm, and blastocysts, and the developmental potential of blastocysts during invitro extended culture. Custom-designed labware, for use with the TMRW platform, enables continuous temperature monitoring during shipment and/or storage in the vapor phase robotic storage system. The highest temperature recorded for specimens shipped to a domestic laboratory was -180.2 °C with a mean ± SD of -190.4 ± 0.5 °C during shipment and -181.1 ± 0.6 °C during storage. Likewise, specimens shipped internationally had a high of -180.2 °C with a mean ± SD of -193.5 ± 0.6 °C during shipment and -181.2 ± 0.7 °C during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The viability of mammalian gametes and embryos was equivalent between the vapor phase and liquid phase storage. The evaluated system did not have any deleterious effects on the postwarming survival of sperm, oocytes, and blastocysts. The postwarming developmental potential of human blastocysts during invitro extended culture was unaffected by storage and handling in the vapor phase nitrogen TMRW platform when compared with static liquid phase nitrogen storage. Our results suggest that the vapor phase cryostorage platform is a safe system to handle and store reproductive specimens for human assisted reproductive technology.

ASSESSMENT OF COMPLETE END TO END VAPOR PHASE NITROGEN SHIPPING AND STORAGE ON GAMETE AND BLASTOCYST QUALITY

ASSESSMENT OF COMPLETE END TO END VAPOR PHASE NITROGEN SHIPPING AND STORAGE ON GAMETE AND BLASTOCYST QUALITY

Key events during the cryopreservation of t cells

Background & Aim Slow, controlled cooling is essential for successful cryopreservation of t cells. In practice, samples are typically cooled at a controlled rate of around 1°C min−1 before being transferred at around -80°C to -100°C. However, other than dehydration, the physical and biological events occurring as the temperature is lowered in t cells (and mammalian cells more widely) have been under-studied. Placing samples in the liquid or vapor phase of nitrogen too early could result in rapid cooling rates, leading to intracellular ice formation and cell death. However, there is little basis in the literature for setting the controlled cooling endpoint at -80°C or below. The present work explored the critical physical and biological events during the cooling of t cells (immortalized jurkat t cell line) through a combination of biological and physical measurements (Differential Scanning Calorimetry and Fourier transformed Infra-Red Spectroscopy). Methods, Results & Conclusion During cooling in a VIA Freeze controlled rate freezer, cells were seen to undergo a membrane transition from a liquid crystal to a gel state at -1.0°C ± 0.8°C. Ice nucleation occurred at -7.7°C ± 1.4°C in 1mL cryovials and was characterized by a rapid release of heat and macroscopic solidification of the sample. Cells dehydrate on cooling after ice nucleation to remain in osmotic equilibrium with the extra-cellular channels between ice crystals. A further transition was observed using DSC at -46.9°C ± 1.3°C. This transition was only present in cryovials during cooling when cells were present and is an intracellular glass transition. The intracellular glass transition temperature corresponded to the cells becoming osmotically inactive, reaching their maximally dehydrated state. Biologically samples following controlled cooling and plunged into liquid nitrogen below this temperature showed good post-thaw outcome, whereas samples plunged into liquid nitrogen above this point had substantial reduction in post-thaw viability and function. An extracellular glass transition at -123°C was observed and below this temperature samples could be safely stored and shipped. These results suggest that controlling cooling until -47°C is reached in t cells is critical for a successful cryopreservation, and no biological advantage is conferred by further cooling. Shorter controlled-rate cooling cycles could thus safely be applied, saving time and potentially allowing more cryopreservation cycles to be completed a day.

Stability Program for Hematopoietic Progenitor Products from Apheresis (HPC-A) at an Academic Institution

We perform stability testing on HPC-A products stored in vapor phase nitrogen from 1 week to 5 years. Stability is defined as CD34 viability of 70% or higher (±40% from baseline), and negative sterility results at 14 days. Potency is defined as 21 days or less to ANC engraftment if infused. We performed a comparison on frozen products from deceased patients or soon to be transplanted products stored for more than 10 years (group 3, n=7) or less than 10 years (group 2, n=10). The objective of our analysis was to establish a method to predict stability and potency, with clinical and economic implications. Thirteen patients were consented on an IRB approved protocol, and request for exemption was presented to the IRB for the remaining. Freshly isolated samples (group 1, n=3) provided control data. The demographic characteristics of the cellular products were matched between groups, except that older samples were volume reduced using the COBE-2991 device, (p=0.007). Samples in group 2 were stored for a median time of 3.6 years (range 0.42, 7.1), and samples in group 3 for 12.31 years (range10.12-14.34), respectively. We observed a post-thaw TNC decrease of 30% in group 2 and 36% in group 3 (p=0.9). CD34 counts decreased by 6.5% in group 2, and 11.37% in group 3 (p=0.7). We observed a 26% reduction of viable CD34 cells in group 2 vs. 34% in group 3 (p=0.1). All but two patients in group 3 received the first transplant, with products stored for a median of 2 days in group 1, 9 days in group two, and 137 days in group 3 (p=0.03). Cellular composition was consistent among products. All products engrafted ANCs within 12 days (p=0.5) and PLT within 17 days (p=0.4). Four patients in group 2 and 1 patient in group 3 received a second transplant. Median storage for group 2 was 3.6 years (0.42-7.09), compared with 12.3 years (10.12-14.34) in group 3 (p=1). All second transplants achieved prompt engraftment of both ANCs and PLT (≤12 d, p=0.2; 0.046, resp.). Regarding the ability of lineage depleted CD34 stem cells to form colonies in vitro, we collected data from 13 independent hematopoietic colony-forming unit assays. A statistically significant reduction in colony formation for both groups 2 and 3 was observed when compared with group 1. Other authors have reported successful engraftment in the absence of colony formation in vitro. Therefore, we decided to perform a functional evaluation of the aldehyde dehydrogenase (ALDH) enzyme activity using a commercially available flow cytometry kit in alternative. As expected, ALDH functionality correlated with CD34 viability (Pearson's coefficient: 0.4). (Figure 1). HPC-A products stored longer than 10 years resulted in efficient engraftment after the infusion of standard doses of viable CD34 cells. Viability assessed with 7-AAD is a simple and essential measure to assess the stability of frozen products, and the ALDH assay is an efficient adjunct to assess the functionality of CD34 stem cells.

Étude prospective comparant l’efficacité de la vitrification ovocytaire en système fermé ou en système ouvert

ObjectivesOocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). MethodsForty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). ResultsThe global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). ConclusionTaking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.

A novel filtration device for point of care preparation of cellular therapies

Many biological samples are stored at cryogenic temperatures (i.e. temperatures below -1500C) to preserve their viability. Typically, these samples are stored in liquid nitrogen vapor-phase freezers. The underlying assumption is that biological samples show highly reduced degradation and metabolic activity while below Tg (the glass transition temperature). On the other hand, every time a sample is manipulated or temporarily removed from an LN2 freezer, the sample will experience thermal excursions marked by warm-up rates of several degrees per second. In these cases, the risk of harming a sample by inadvertently crossing the Tg threshold is very likely. This paper’s objective is to provide supporting data to fully characterize the thermal excursions of cryogenically frozen single vials filled with H2O during typical transient temperature events. Specifically, we focus our attention on the cold-chain steps that involve transferring a single vial from an LN2 vapor environment to either an ambient temperature or transportable dry ice (-800C) environment. In general, the warm-up rates experienced by the biological sample correlate to the thermal energy exchanged with the warmer environment via convective and conductive heat transfer. The magnitude of the heat transfer is driven by multiple factors: the warmer environment temperature and the overall time of exposure, the size and shape of the vial, its placement in a cryobox, the type of handling (manual or automated), the handling speed, etc. In this paper, we rationalize all the above factors and present experimental measurements supported by calibrated finite elements simulations showing typical expected warm-up rates. As a result, best-practice time constants for handling vials of biological samples without risking excessive thermal excursions above Tg are suggested.

Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality.

Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing ("fast-rate" or "slow-rate") and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p < 0.05) and also for 50% compared to 20% D/P (two test variations). Effect of precooling D/P or WBC-enriched product and of storage in vapor-phase or liquid nitrogen was marginal. Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary.

Successful short‐term cryopreservation of volume‐reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential

Cord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150°C on critical quality control variables of CB collections to investigate the possible use of mechanical freezers at -150°C as an alternative to storage in liquid- (or vapor-) phase nitrogen. A total of 105 CB units were thawed and washed at different time intervals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recoveries were calculated. Also, total absolute colony-forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. Significant differences (p < 0.05) were observed in certain variables (TNCs, MNC numbers, viability) when they were examined in relation with time intervals, while others (CD34+, CD133+) were relatively insensitive (p = NS) to the duration of time interval the CB units were kept in cryostorage condition. The data presented suggest that cryopreservation of CB units in a mechanical freezer at -150°C may represent an alternative cryostorage condition for CB cryopreservation.

Development of a Simplified Ice-Free Cryopreservation Method for Heart Valves Employing VS83, an 83% Cryoprotectant Formulation

We have previously demonstrated storage of ice-free cryopreserved heart valves at -80°C without the need for liquid nitrogen, with the aims of decreasing manufacturing costs and reducing employee safety hazards. The objectives of the present study were a further simplification of the ice-free cryopreservation method and characterization of tissue viability. Porcine pulmonary heart valves were permeated with an 83% cryoprotectant solution (VS83) followed by rapid cooling and storage at -80°C. The cryoprotectants were added and removed in either single or multiple steps. Fresh untreated frozen controls employing 10% dimethylsulfoxide and controlled rate freezing to -80°C, and storage in vapor phase nitrogen were also performed. After rewarming and washing, cryopreserved leaflets were compared with fresh controls using the resazurin reduction metabolism assay. Comparison of valve tissues in which the cryoprotectants were added and removed in either single or multiple steps demonstrated similar viability results for the muscle, conduit, and leaflet components. The ice-free cryopreserved conduit and leaflet components were significantly less viable than either fresh or frozen tissues. The muscle component, although less viable, was not significantly different. The changes in tissue viability were a function of cryoprotectant exposure, and resulting cytotoxicity, not temperature reduction during storage. TUNEL staining showed that ice-free cryopreservation did not induce significant amounts of apoptosis, suggesting that necrosis is the predominant cell death pathway in ice-free cryopreserved heart valves. There was very little difference in cell viability when the cryoprotectants were added and removed in a single step versus multiple steps. Ice-free cryopreserved valve tissues demonstrated very low viability compared with controls. These results support further simplification of the ice-free cryopreservation method.

Characterization of a simplified ice-free cryopreservation method for heart valves

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at --80 °C or with 10 % DMSO and control rate freezing to --80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.

Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6mol/l cryoprotectant (4.65, 4.65, and 3.31mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at -80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P<0.05). After 3 and 6months in vivo valve function was determined by two-dimensional echo-Doppler and at 7months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P=0.0403, P=0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P<0.05) and infiltrating CD3+ T-cells (P<0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at -80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.

IVF treatment using intentionally vitrified and transported blastocysts obtained from donor cryo-egg bank may provide an efficient treatment option for recipients around the world

Patients requiring donor eggs but living far from donor source may prefer to obtain treatment locally while still benefiting from the experience of a clinic with an established oocyte cryopreservation program. In the current study, we aimed to evaluate the efficiency of IVF, when frozen sperm (of the recipient's partner) was shipped to the “Egg-Bank”, used to ICSI vitrified-warmed donor eggs, and the resulting embryos were vitrified and shipped back to the recipient location thus ready to be warmed and transferred. Longitudinal observational study. Oocytes were obtained from appropriately screened and tested donors using a GnRH antagonist protocol. Eggs were vitrified at the MII stage. Selected eggs were warmed 3 hours prior to ICSI. Embryos that developed to blastocyst stage were vitrified and shipped in vapor phase nitrogen. Two hours after warming, embryos were hatched and transferred to recipients who had received standard, medicated endometrial preparation. A total of 141 eggs were warmed from 16 donors (age: 26.4 ± 2.7). Mean age of the 19 recipients was 41.1 ± 3.6.Tabled 1Number (%)Mean ± SDNo. Recipients19No. Oocytes survived (%)129 (91.4)6.8 ± 2.6No. Oocytes fertilized (%)102 (79.0)5.4 ± 1.5No. Blastocysts (%)74 (72.5)3.9 ± 1.5No.Embryos vitrified (%)72 (70.5)3.8 ± 1.5No. FET cycles12No. Embryos warmed and transferred211.75 ± 0.45No. Clinical pregnancies (%)6 (50.0)No. Implantations (%)11/21 (52.3) Open table in a new tab The study data demonstrate that recipients of donor eggs can be efficiently treated “long distance” by using the combination of 1, cryopreserved donor eggs; 2, ICSI with shipped frozen/thawed sperm; 3, vitrified embryos shipped back to recipient location and 4, warming and transferring embryos to recipients. Improvements in cryopreservation technology now seem to substantiate the condition whereby recipients can obtain an efficient and more comfortable treatment anywhere in the world without the expense and inconvenience of long distance travel.

Long-Term Cryopreservation of Autologous Stem Cell Grafts: Impact of Patient Age, Initial Collection, and Stem Cell Mobilization Regimen.

AbstractAbstract 1178 Background:Effectively cryopreserving hematopietic peripheral blood progenitor cells (HPC(A)) until time of transplant is critical for successful autologous stem cell transplant. It is important to demonstrate long-term HPC(A) viability and recovery and also to identify patient characteristics that may influence this. We evaluated HPC(A) products cryopreserved over 16 year period. We analyzed potential correlations between total nucleated cell (TNC) and CD34+ recovery and viability and patient age, initial collection, and impact of stem cell mobilization regimen. Method:Samples were obtained from expired patients, chosen based on storage duration but randomly selected in regard to patient and product characteristics; 23 samples were obtained from 18 patients. Five patients had 2 samples each; for duplicate samples, data were averaged (collection dates and thaw dates were similar). All HPC(A) products were frozen in autologous plasma and 10% DMSO, then stored in liquid phase or vapor phase nitrogen. Products were uniformly thawed and washed to remove DMSO. Analysis included 7-Amino-actinomycin D viability, TNC and CD34+ cell counts and recovery. Some samples had post-thaw CD34+ cell counts that exceeded pre-thaw count; for these, the recovery was set to 100%. Spearman rank correlations were used to analyze association between the different parameters. Wilcoxon rank sum test and analysis of covariance were used to compare patient groups. Result:Median patient age at time of stem cell collection was 54 (range 2–75) years. A majority of patients had lymphoma (61%) or multiple myeloma (22%). Most (56%) were mobilized using G-CSF+VP16, 33% with G-CSF alone, and 11% other. Samples were cryopreserved for a median of 8 years (range 1–16). Median (range) for total CD34+ × 106 collected was 547 (43-5845). Median (range) for CD34+/kg × 106 was 8 (1-59). Median (range) count/bag for TNC × 108 was 144 (37-285) and for CD34+ × 106 was 105 (8-805). Median (range) recovery and viability were 85% (32-213%), and 70% (46-85%) respectively for TNC; and 97% (16-359%) and 80% (61-98%), respectively for CD34+ cells.Pre- and post- thaw TNC and CD34+ counts were both highly correlated (r=0.95, p<.0001 in both cases – figures 1a and b). There was no significant association between TNC recovery and viability (r=-0.07, p=.79) or between CD34+ cell recovery and viability (r=-0.21, p=.41). Storage duration did not impact CD34+ recovery or viability (r=-0.01, p=.98 and -0.18, p=.47, respectively); or TNC viability (r=-0.13, p=.62). There was, however a significant positive correlation between the storage duration and TNC recovery (r=0.81, p<.0001).Age did not significantly impact TNC or CD34+ recovery (r=-0.05, p=0.86, and r=-0.26, p=0.29, respectively); however there was suggestion of negative impact on viability (r=-0.45, p=0.06 and r=-0.42, p=0.08, respectively, figures 1a and 1b). Overall, there did not appear to be a correlation between the initial TNC count/bag or total CD34+ cell dose on recovery or viability (r=0.29, r=-0.22, r=0.18, r=-0.38, respectively; all p>0.12). However, when patients were stratified according to total collection >25×106 CD34+ cells/kg or ≤25 × 106 CD34+ cells/kg, there was better %CD34+ cell count recovery between groups (p = 0.07, median recovery 81% for 13 patients with ≤25 × 106 CD34+ cells/kg and 100% for 5 patients with >25×106 CD34+ cells/kg). For mobilization regimen, there was no significant difference in TNC recovery or viability (p = 0.19 and p=0.76, respectively) or CD34+ viability (p = 0.53), however there was suggestion that CD34+ recovery was greater with G-CSF+VP16 (p=0.06, see figure 2), independent of storage time. Conclusion:Products cryopreserved for 16 years retain acceptable recovery and viability. Unexpectedly, storage duration positively correlated with TNC recovery, and CD34+ cell recovery of >100% was noted in several samples. Reasons for this are unclear, but are likely related to changes in enumeration method or use of methods validated for counting fresh cells. Patient age was suggested to negatively impact post-thaw HPC(A) viability. Our data also suggested that the mobilization regimen or the CD34+ cell collection yield may affect the CD34+ cell recovery, possibly reflecting differences in graft characteristics. The number of patients included is relatively small and these findings warrant further studies for validation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Storage of human oocytes in the vapor phase of nitrogen

To evaluate the effectiveness of long-term vapor-phase nitrogen storage of vitrified human oocytes as a strategy for preventing the risk of cross-contamination due to direct contact with the liquid nitrogen (LN). Prospective randomized study. Private infertility center, IVI, Valencia. Oocyte donors (n = 44) and recipients (n = 46). Vitrification by the Cryotop method. Storage of vitrified oocytes in a vapor-phase nitrogen storage freezer and a traditional LN storage tank. Donation of the surviving oocytes and evaluation of fertilization, embryo development, and clinical results. Survival, fertilization, and cleavage rates. Embryo quality and clinical outcome. Survival was 95.3% (vapor-phase nitrogen) and 94.5% (LN). Fertilization rates (73.1% and 71.7%) or cleavage on day 2 (95.6% and 94.7%), day 3 (84.5% and 79.9%), and blastocyst formation (54.7% and 53.9%) were similar between vapor-phase nitrogen and LN. Implantation, clinical, and ongoing pregnancy rates were similar for vapor-phase nitrogen (40.5%, 58.1%, and 48.8%, respectively) and LN groups (33.7%, 53.3%, and 46.6%, respectively). The vapor-phase nitrogen system permits the storage of oocytes vitrified, maintaining their potential to develop into competent embryos in a similar manner as those stored in a traditional LN freezer. This approach represents a practical alternative that prevents cross-contamination during the storage of vitrified samples.

Comparison of cryopreserved human sperm in vapor and liquid phases of liquid nitrogen: effect on motility parameters, morphology, and sperm function

To compare the effects of cryopreserved sperm in vapor and liquid phases of liquid nitrogen on sperm motility, morphology, and sperm function. Experimental study. Andrology laboratory at Ramathibodi Hospital, Thailand. Thirty-eight semen samples with normal motility and sperm count were collected from 38 men who were either patients of an infertility clinic or had donated sperm for research. Each semen sample was divided into two aliquots. Samples were frozen with static-phase vapor cooling. One aliquot was plunged into liquid nitrogen (-196 degrees C), and the other was stored in vapor-phase nitrogen (-179 degrees C) for 3 days. Thawing was performed at room temperature. Motility was determined by using computer-assisted semen analysis, sperm morphology was determined by using eosin-methylene blue staining, and sperm function was determined by using a hemizona binding test. Most of the motility parameters of sperm stored in the vapor phase were not significantly different from those stored in the liquid phase of liquid nitrogen, except in amplitude of lateral head displacement. The percentages of normal sperm morphology in both vapor and liquid phases also were not significantly different. There was no significant difference in the number of bound sperm in hemizona between sperm cryopreserved in both vapor and liquid phases of liquid nitrogen. Cryopreservation of human sperm in a vapor phase of liquid nitrogen was comparable to cryopreservation in a liquid phase of liquid nitrogen.

Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB

Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.

Direct nitriding of large grains of aluminum with 2 mm size

Aluminum grains with 2 mm size have been completely nitrided in one batch process. Aluminum grains with added promoter (13.8 wt.% of Y(NO3)3·6H2O) were almost completely nitrided by heating at 1300°C for 2 h in N2 atmosphere. It was deduced that aluminum was vaporized by heat of initial nitriding to react with nitrogen in vapor phase in a short period of time. Nitriding of large-sized aluminum was also attempted without additive. Electrolytically etched aluminum wires were also used as starting material. The aluminum wires without additive were also completely nitrided at 1300°C by preheating at 600°C for 2 h. It seemed that the oxide layer produced by a trace amount of oxygen in N2 gas during preheating activated the initial nitriding. Finally, the complete nitriding of aluminum grains was achieved by preheating at 600°C, without the additive. The oxygen content in the product was only 0.3%, in spite of the oxidation of the aluminum surface. This is because the surface area of the aluminum grains with 2 mm size is relatively small.