Viscosities encountered during the cryopreservation of dimethyl sulphoxide systems

This study determined the viscous conditions experienced by cells in the unfrozen freeze concentrated channels between ice crystals in slow cooling protocols. This was examined for both the binary Me₂SO-water and the ternary Me₂SO-NaCl-water systems.Viscosity increases from 6.9 ± 0.1 mPa s at −14.4 ± 0.3 °C to 958 ± 27 mPa s at −64.3 ± 0.4 °C in the binary system, and up to 55387 ± 1068 mPa s at −75 ± 0.5 °C in the ternary (10% Me₂SO, 0.9% NaCl by weight) solution were seen. This increase in viscosity limits molecular diffusion, reducing adsorption onto the crystal plane. These viscosities are significantly lower than observed in glycerol based systems and so cells in freeze concentrated channels cooled to between −60 °C and −75 °C will reside in a thick fluid not a near-solid state as is often assumed.In addition, the viscosities experienced during cooling of various Me₂SO based vitrification solutions is determined to below −70 °C, as is the impact which additional solutes exert on viscosity. These data show that additional solutes in a cryopreservation system cause disproportionate increases in viscosity. This in turn impacts diffusion rates and mixing abilities of high concentrations of cryoprotectants, and have applications to understanding the fundamental cooling responses of cells to Me₂SO based cryopreservation solutions.     

Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me₂SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at −10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.     

Cryopreservation by slow cooling of rat neuronal cells

Although primary neuronal cells are routinely used for neuroscience research, with potential clinical applications such as neuronal transplantation and tissue engineering, a gold standard protocol for preservation has not been yet developed. In the present work, a slow cooling methodology without ice seeding was studied and optimized for cryopreservation of rat cerebellar granular cells. Parameters such as cooling rate, plunge temperature and cryoprotective agent concentration were assessed using a custom built device based on Pye's freezer idea. Cryopreservation outcome was evaluated by post thawing cell viability/viable cell yield and in culture viability over a period of 14 days. The best outcome was achieved when 10% of Me₂SO as cryoprotective agent, a cooling rate of 3.1 ± 0.2 °C/min and a plunge temperature of −48.2 ± 1.5 °C were applied. The granular cells cryopreserved under these conditions exhibited a cell viability of 82.7 ± 2.7% and a viable cell yield of 28.6 ± 2.2%. Moreover, cell viability in culture remained above 50%, very similar to not cryopreserved cells (control). Our results also suggest that post-thaw viability (based on membrane integrity assays) not necessarily reflects the quality of the cryopreservation procedure and proper functionality tests must be carried out in order to optimize both post thaw viability/cell yield and in culture performance.     

Cryopreservation of embryos and larvae of the edible sea urchin loxechinus albus (Molina, 1782)

The natural population of the edible red sea urchin, Loxechinus albus, is decreasing due to overfishing. The embryos and larvae of the species are highly useful for monitoring marine pollution, which makes it necessary to conserve gametes, embryos and larvae to facilitate their use in diverse areas of aquaculture and environmental quality monitoring. This need can be met by cryopreserving individuals representing the different developmental stages to provide an ongoing supply of genetic material of the species. The present study establishes a reproducible protocol for cryopreserving red sea urchin blastula and larvae. Toxicity tests were conducted in the first stage of this study using two permeable cryoprotectors, dimethyl sulfoxide (Me₂SO) and propylene glycol (PG), at three concentrations (5%, 10% and 15%). The tests were repeated in the second stage, but mixing the cryoprotectors with 0.04 M of trehalose (TRE), a non-permeable cryoprotector. Cryopreservation tests were conducted in the third stage employing different freezing rates: 2 °C/min, 3 °C/min, 3.5 °C/min, 4 °C/min and 4.5 °C/min, using the cryoprotectors that yielded the highest post-incubation survival rates.The highest post-freezing survival rates for blastula (76 ± 7%) and larvae (79 ± 7%) were obtained with Me₂SO at 10% + 0.04 M of trehalose, with freezing rates of 3 °C/min and 4.5 °C/min, respectively.     

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me₂SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN₂ at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me₂SO) with VS3 (3 M PG and 3 M Me₂SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.     

Cryopreservation of swine colostrum-derived cells

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me₂SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me₂SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least −30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me₂SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.     

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation,cellular viability,and metabolism

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me₂SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me₂SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me₂SO during cryopreservation. We found that the addition of trehalose to Me₂SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me₂SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me₂SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.     

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA¹) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity.First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me₂SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me₂SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me₂SO. A mixture of Me₂SO, ethylene glycol and glucose (ratio 4:2:1– termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me₂SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures.In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.     

D1-S169A substitution of photosystem II reveals a novel S2-state structure

In photosystem II (PSII), photosynthetic water oxidation occurs at the O₂-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S₀–S₄) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S₂ to S₁ transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7–2.7× slower rate of charge recombination with Q_A⁻ relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an E_a of 5.87 ± 1.15 kcal mol⁻¹ for decay back to the S₁ state, compared to 0.80 ± 0.13 kcal mol⁻¹ for the wild-type S₂ state. In addition, we find that ammonia does not affect the S₂-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S₂ state of D1-S169A PSII. The altered S₂ state of D1-S169A PSII provides insight into the S₂➔S₃ state transition.     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Cryopreservation of sperm from seven-band grouper,Epinephelus septemfasciatus

In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me₂SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6 ± 8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min^?1); however, the post-thaw motility of sperm cooled to ?30 °C was significantly lower than that of other cooled temperatures (?40 to ?70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me₂SO with 95% FBS and cooled to at least ?40 °C before immersion in liquid nitrogen.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Hybrid cooling vest for cooling between exercise bouts in the heat: Effects and practical considerations

While continuous cooling strategies may induce some ergonomic problems to occupational workers, cooling between work bouts may be an alternative for cooling them down in hot environments. The purpose of this study was to assess the effects of wearing a newly designed hybrid cooling vest (HCV) between two bouts of exercise. Inside a climatic chamber set at an air temperature of 37 °C and a relative humidity of 60%, twelve male participants underwent two bouts of intermittent exercise interspersed with a 30 min between-bout recovery session, during which HCV or a passive rest without any cooling (PAS) was administered. The results indicated that thermoregulatory, physiological, and perceptual strains were significantly lower in HCV than those in PAS during the recovery session (p≤0.022), which were accompanied with a large effect of cooling (Cohen's d=0.84–2.11). For the second exercise bout, the exercise time following HCV (22.13±12.27 min) was significantly longer than that following PAS (11.04±3.40 min, p=0.005, d=1.23) During this period, core temperature T_c was significantly lower by 0.14±0.0.15 °C in HCV than that in PAS. The heart rate drift over time was declined by 2±2 bpm min⁻¹ (p=0.001, d=1.00) and the rise in physiological strain index was reduced by 0.11±0.12 unit min⁻¹ (p=0.010, d=0.96) following the use of HCV. These findings suggested that using HCV could accelerate between-bout recovery and improve subsequent exercise performance by the enlarged body core temperature margin and blunted cardiovascular drift.     

Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.     

Heat acclimation does not modify autonomic responses to core cooling and the skin thermal comfort zone

Exercise heat acclimation (HA) is known to magnify the sweating response by virtue of a lower threshold as well as increased gain and maximal capacity of sweating. However, HA has been shown to potentiate the shivering response in a cold-air environment. We investigated whether HA would alter heat loss and heat production responses during water immersion. Twelve healthy male participants underwent a 10-day HA protocol comprising daily 90-min controlled-hyperthermia (target rectal temperature, T_re 38.5 °C) exercise sessions. Preceding and following HA, the participants performed a maximal exercise test in thermoneutral conditions (ambient temperature 23 °C, relative humidity 50%) and were, following exercise, immersed in 28 °C water for 60 min. Thermal comfort zone (TCZ) was also assessed with participants regulating the temperature of a water-perfused suit during heating and cooling. Baseline pre-immersion T_re was similar pre- and post-HA (pre: 38.33 ± 0.33 °C vs post: 38.12 ± 0.36 °C, p = 0.092). The T_re cooling rate was identical pre-to post-HA (−0.03 ± 0.01 °C·min⁻¹, p = 0.31), as was the vasomotor response reflected in the forearm-fingertip temperature difference. Shivering thresholds (p = 0.43) and gains (p = 0.61) were not affected by HA. TCZ was established at similar temperatures, with the magnitude in regulated water temperature being 7.6 (16.3) °C pre-HA and 5.1 (24.7) °C post-HA (p = 0.65). The present findings suggest that heat production and heat loss responses during whole body cooling as well as the skin thermal comfort zone remained unaltered by a controlled-hyperthermia HA protocol.