EFFECTS OF DIFFERENT COMBINATIONS OF CRYOPROTECTANTS ON SPERMATOZOON VITALITY OF CHINESE SCALLOP,CHLAMYS FARRERI,DURING LOW TEMPERATURE EQUILIBRIUM

The effects of membrane penetrating anti-freezing agents(MPAAs), DMSO(dimethyl sulfoxide),glycerol,EG(ethylene glycol) and methanol in combination with different cryoprotective additives suchas carbohydrates,macromolecules and inorganic compounds on the spermatozoon vitality of Chinesescallop,Chlamys farreri,during 1 h 0℃ equilibrium were investigated.When only MPAAs existed,the detrimental effects of different MPAAs ranked in the following order:DMSO【methanol【EG【glycerol.When carbohydrates were added into MPAAs solution,5% glucose caused larger decrease ofspermatozoon vitality than 2.4% lactose.5% glucose or 2.4% lactose in 7.5% glycerol caused com-plete damage.10% yolk was best in maintaining the spermatozoon vitality except when used incombination with 10% methanol.10% milk significantly decreased spermatozoon vitality in EG andmethanol and enhanced its vitality in glycerol,but did not significantly influence it in DMSO.Glycinewas apparently detrimental to spermatozoon vitality.The average

Effect of Combinations of Sucrose and Cryoprotectants on the Survival of Catfish Embryos （Pangasidae hypophthamus）

Although cryopreservation of embryos has been used in most terrestrial animals, the application of this technique has not been succeeded for aquatic animals. In this study, the authors investigate the effect of different combinations of sucrose （SUC, C12H22OH） and cryoprotectants （CPAs） on the survival of the catfish embryos （Pangasidae hypophthalmus） at low temperatures （4, 0 and -20 ℃） for short-term storage. For this aim, embryos with somites and optic cups were exposed to different combinations of sucrose with methanol （SUC ＋ MeOH）, 1.2-propylene glycol （SUC ＋ PROH） and ethylene glycol （SUC ＋ EG） at four concentrations ratios： （1） 0.5 M SUC ＋ 0.5 M CPA; （2） 1 M SUC ＋ 0.5 M CPA; （3） 0.5 M SUC ＋ 1 M CPA; （4） 1 M SUC ＋ 1 M CPA for 40 min at 4, 0 and -20 ℃. Embryos kept in water at room temperature （RT）, 4, 0 and -20℃ were used as controls. The survival rate was expressed as a percentage of hatched embryos per total embryos treated. The results showed that the hatching rate declined significantly when embryos were stored in water at 0 ℃ and -20℃. For embryos at 0 ℃ storage, the highest survival rate （87.78%） was obtained with 1 M SUC ＋ 1 M MeOH combination while at -20 ℃, only embryos in the combined treatments of 0.5 M SUC ＋ 1 MEG and 0.5 M SUC ＋ 1 M PROH reached the hatching stage （40% and 83.33%, respectively）. In conclusion, the results showed that the catfish embryos are sensitive to sub-zero temperatures and the combined treatment of 0.5 M sucrose and 1 M propylene glycol can be used to protect catfish embryos from damages caused by low temperature （0 ℃ and -20 ℃）.

Exploring novel cell cryoprotectants based on neutral amino acids

Cryoprotectants play a key role in cell cryopreservation because they can reduce cryoinjuries to cells associated with ice formation.To meet the clinical requirements of cryopreserved cells,cryoprotectants should be biocompatible,highly efficient and easily removable from cryopreserved cells.However,integration of these properties into one cryoprotectant still remains challenging.Herein,three biocompatible neutral amino acids,includingβ-alanine,γ-aminobutyric acid andε-aminocaproic acid,are first reported to have the potential as such ideal cryoprotectants.The results demonstrate that they can inhibit ice formation and reduce osmotic stress to provide extracellular and intracellular protection,thereby ensuring high cryopreservation efficiency for both anuclear and nucleated cells.More importantly,due to the remarkable osmotic regulation ability,the neutral amino acids can be rapidly removed from cryopreserved cells via a one-step method without causing observable damage to cells,superior to the current state-of-the-art cryoprotectants—dimethyl sulfoxide and glycerol.This work provides a new perspective to develop novel cryoprotectants,which may have dramatic impacts on solvent-free cryopreservation technology to support the cell-based applications,such as cell therapy and tissue engineering,etc.

Study on Cryopreservative Effect of Three Penetrating Cryoprotectants with Three Kinds of Extenders on Rabbit Spermatozoa

Glycerol,dimethyl suffixod (DMSO) and acetamide were adopted to verify the cryoprotective ability of penetrating cryoprotectants with three kinds of extenders for rabbit sperm. Three extenders (A,B and C) and three level of the final concentrations of glycerol and DMSO (2%, 3% and 4% ),and acetamide 2%,4% and 5% were used with Extender A, 4% glycerol had better cryoprotective effect for rabbit sperm motility and 5% acetamide was better with Extender C. 4% acetamide was better in being combined with Extender A for sperm acrosome integrity ratio,5% acetamide was better with Extender B and 2% acetamide was better with Extende C. With extender A,there was not difference among three penetrating cryoprotectants for plasma membrane integrity. With Extender B, 3% glycerol was better than 4% acetamide for plasma membrane in- tegrity,and 3% glycerol with Extender C was better than 2% - 4% DMSO and 2% acetamide.

Trial productions of freeze-dried Lactobacillus plantarum culture using dairy by-products as cryoprotectants:Viability and characterization of cultures

This study is a research on the utilization of dairy industry by-products in the practical production and long-term preservation of industrial microbial cultures.Accordingly,lyophilized cultures were obtained by freeze-drying of Lactobacillus plantarum bacteria in large quantities and for a constant period of time in skimmed milk-based medium containing 5%acid casein,rennet casein,whey and demineralized whey.The cryoprotectants did not have significant protection against freeze-drying process,moreover,the use of rennet casein significantly reduced viability.The efficiency of the preservatives in preserving the viability during storage was very successful compared to the control culture.Casein-enriched cultures had high water activity and moisture,but high viability was maintained during storage.The powder morphologies of the cultures were differentiated by the use of different cryoprotectants.Particle sizes of the cultures were parallel to the moisture content.Particle size and distribution increased with storage.By thermogravimetric analysis(TGA)and differential scanning calorimetry(DSC)analysis,it was determined that the addition of casein protein increased the degradation temperature of the cultures.The production of freeze-dried cultures became difficult with the use of casein.However,it was generally concluded that the protective properties of skimmed milk used for the preservation of L.plantarum could be improved by the addition of dairy by-products as a protective agent.

The use of machine learning to predict the effects of cryoprotective agents on the GelMA-based bioinks used in extrusion cryobioprinting

Cryobioprinting has tremendous potential to solve problems to do with lack of shelf availability in traditional bioprinting by combining extrusion bioprinting and cryopreservation.In order to ensure the viability of cells in the frozen state and avoid the possible toxicity of dimethyl sulfoxide(DMSO),DMSO-free bioink design is critical for achieving successful cryobioprinting.A nontoxic gelatin methacryloyl-based bioink used in cryobioprinting is composed of cryoprotective agents(CPAs)and a buffer solution.The selection and ratio of CPAs in the bioink directly affect the survival of cells in the frozen state.However,the development of universal and efficient cryoprotective bioinks requires extensive experimentation.We first compared two commonly used CPA formulations via experiments in this study.Results show that the effect of using ethylene glycol as the permeable CPA was 6.07%better than that of glycerol.Two datasets were obtained and four machinelearning models were established to predict experimental outcomes.The predictive powers of multiple linear regression(MLR),decision tree(DT),random forest(RF),and artificial neural network(ANN)approaches were compared,suggesting an order of ANN>RF>DT>MLR.The final selected ANN model was then applied to another dataset.Results reveal that this machine-learning method can accurately predict the effects of cryoprotective bioinks composed of different CPAs.Outcomes also suggest that the formulations presented here have universality.Our findings are likely to greatly accelerate research and development on the use of bioinks for cryobioprinting.

Effects of cryoprotectant treatments on bovine sperm function and osmolyte content

The hypothesis that addition and removal of cryoprotectants to and from spermatozoa would initiate regulatory volume decrease, and lead to osmolyte loss and reduced sperm function, was tested. Common cryoprotectants, in the absence of freezing and thawing, affected bovine ejaculated spermatozoa by lowering their total and progressive motility in medium, reducing their migration through surrogate cervical mucus, damaging sperm head membranes and inducing sperm tail coiling. Sperm function was slightly better maintained after cryoprotectants were added and removed in multiple small steps rather than in a single step. The intracellular content of the polyol osmolytes, D-sorbitol and myo-inositol, exceeded that of the zwitterion osmolytes, L-carnitine and L-glutamate. Certain cryoprotectants reduced intracellular L-camitine and L-glutamate concentration but not that of myo-inositol or D-sorbitol. Multistep treatments with some cryoprotectants had advantages over one-step treatments in mucus penetration depending on the original amount of intracellular camitine and glutamate in the spermatozoa. Overall, sperm quality was best maintained by multistep treatment with glycerol and propanediols that were associated with decreased intracellular glutamate concentration. Bovine spermatozoa seem to use glutamate to regulate cryoprotectant-induced cell swelling.

Enhacement of the Viability of <i>Lactobacillus</i><i>plantarum</i>during the Preservation and Storage Process Based on the Response Surface Methodology

Objective: The Response Surface Methodology (RSM) is a commonly used system to optimize cell viability of probiotic strains when they are subjected to different preservation and storage processes. Methods and Results: To determine the optimal levels of incorporation of several cry oprotectants (skim milk, sucrose and trehalose) in the freeze-drying process of Lactobacillus plantarum, a range of experiments based on a Rotational Central Composite design (CCD) were conducted. The results were adjusted to a quadratic model, resulting in the presence of interaction between the different variables. Solving a regression equation, we obtained the optimum concentrations of cryoprotective agents: 24.06% milk powder, 6.22% sucrose, 5.63% trehalose. To visualize the interactions between the three variables involved in the study, Design Expert? software was used. Conclusions: The analysis reveals that while trehalose has a direct effect on the viability of L. plantarum, skim milk and sucrose exert quadratic effects. There are also interactions between cryoprotectants, which emphasize the synergies produced between milk and sucrose and between sucrose and trehalose, which allows maintaining the viability of L. plantarum. Significance and Impact of the Study: The addition of new oligosaccharides as trehalose in premixtures for functional feed can maintain the viability of L. plantarum during longer periods of time, ensuring the proper administration of probiotics to their destinations.

Preservation efficiency of new cryoprotectant used for Acidithiobacillus ferrooxidans in liquid nitrogen

The efficiency of a new cryoprotectant,GP,for the preservation of Acidithiobacillus ferrooxidans（A.ferrooxidans） strain DC in liquid nitrogen was investigated.The optimal concentration of this new cryoprotectant for the maximal viable cell recovery and the highest ferrous ion oxidation activity was determined.The results show that 30%（volume fraction） GP is optimal for the cryopreservation with 84.4% of cells surviving,completely oxidizing ferrous ions within 120 h,and growing to a final density of 5.8×107 cell/mL after 6 d in the culture.Furthermore,the optimal residual GP concentration for viable cell recovery after culture of thawed cells in 9K medium for 6 d is 0.6%（volume fraction）.At this concentration,strain DC completely oxidizes ferrous ions within 108 h and grows to a final cell density of 6.8×107 mL-1.Thus,GP is a simple,effective cryoprotectant for the preservation of A.ferrooxidans strain DC in liquid nitrogen.

Human Sperm Freezing: Mini Update

Sperm freezing is widely used in ART clinics around the globe. Very little has actually changed with respect to cryopreservation protocols and methodology of freezing over the last 50 years. The aim of this paper is to briefly review the basic principles that underlie freezing and ice crystal formation and also provide a brief overview of newer sperm freezing techniques like sperm vitrification and freeze drying of sperm.

Oocyte Cryopreservation in Human Assited Reproduction

Embryo cryopreservation（CP） has became a very important part of the clinical use of in vitro fertilization. Oocyte CP offers more advantages compared with embryo freezing with regard to less ethical, legal and moral problems. However, the efficiency of this procedure is still low, which prevents its clinical application in wide range. The aim of our paper is to review the basic principles, technical and safety aspects and current status of oocyte cryopreservation in human assisted reproduction.

Protective effect of exogenous adenosine triphosphate on hypothermically preserved rat liver

AIM:To clarify the protective effect of exogenous adenosine triphosphate(ATP)on hypothermically preserved rat livers. METHODS:Establishment of continuous hypothermic machine perfusion model,detection of nucleotides in hepatocytes with HPLC,measurement of activities of LDH and AST in the perfusate,observation of histopathological changes in different experiment groups,and autoradiography were carried out to reveal the underlying mechanism of the protective effect of ATP. RESULTS:The intracellular levels of ATP and EC decreased rapidly after hypothermic preservation in control group,while a higher ATP and EC level,and a slower decreasing rate were observed when ATP-MgCl_2 was added to the perfusate (P<0.01).As compared with the control group,the activities of LDH and AST in the ATP-MgCl_2 group were lower(P<0.05). Furthermore,more severe hepatocyte damage and neutrophil infiltration were observed in the control group.Radioactive [α-^(32)P]ATP entered the hypothermically preserved rat hepatocytes. CONCLUSION:Exogenous ATP has a protective effect on rat livers during hypothermical preservation.However,Mg^(2+) is indispensable,addition of ATP alone produces no protective effect.The underlying mechanism may be that exogenous ATP enters the hypothermically preserved rat liver cells.

Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.

Production and characterization of an extracellular polysaccharide of antarctic marine bacteria Pseudoalteromonas sp. S-15-13

Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.

Advances in Cryopreservation of Organs

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4–12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients＇ conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.

Effects of Cryoprotective Agents on the Bovine Articular Chondrocyte Viability

Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0 6, 0 9, 1 2 and 1 5 mol/L and at 4 ℃ and 37 ℃ and removed at 37 ℃ in one step. CPA stepwise addition and removal at 0 6 and 1 2 mol/L and at 37 ℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.

Effect of freezing on the passive mechanical properties of arterial samples

Little mechanical data is available on human arteries because of the difficulty of testing artery samples often obtained from autopsy, while arteries are still considered “fresh”. Various solutions mimicking the physiological environment have been used to preserve artery samples from harvesting to testing. Cryopreservation might provide a means to preserve the mechanical properties of arteries for days or weeks after harvesting. The objective of this study is to investigate the effect of several preservation methods, including simplified cryopreservation methods, on the passive mechanical properties of arteries. Eighteen fresh cruciform samples were mechanically tested. Samples were divided in three groups based on preservation medium and freezing method: isotonic saline solution, Krebs-Henseleit buffer solution with dimethyl sulfoxide (DMSO), and dipped in liquid nitrogen. In each group, half of the samples were stored at -20℃ and the other half at -80℃. Two months later, all the tissues were thawed at 4℃ and mechanical tests were repeated. Preservation of arteries for two months in Krebs solution with DMSO (at -20℃ or at -80℃) or in isotonic saline solution at -20℃ were the methods that least changed the mechanical properties of the arteries.

A Review on the Protection Mechanism of Trehalose on Plant Tissues and Animal Cells

Trehalose is a non-reducing disaccharide composed of glucose molecules connected byα-glycosidic bond.This soluble substance plays an important role of protecting green algae and other lower plants from stress.It can help plants cope with extreme environments such as severe cold,drought and high salinity,regulate the stomatal conductance and water utilization rate of plants,and participate in the growth and metabolism regulation of plants as a signal molecule.As an impermeable cryoprotectant,trehalose is widely used in the refrigeration protection of various animal cells and tissues due to its non-toxicity and high efficiency.According to the research results at home and abroad in recent years,the protection,regulation and mechanism of trehalose on plant tissues and animal cells were summarized,so as to provide a theoretical basis for the further development and utilization of trehalose.

Major physiological adjustments in freezing-tolerant grey tiger longicorn beetle(Xylotrechus rusticus) during overwintering period

1Xylotrechus rusticus （Linnaeus） is one of the most destruc-tive woodborers in northeast China;it damages poplar and is listed as a domestic forestry quarantine pest. Overwintering larvae were collected during October 2012 and March 2013 in Harbin, China, to quantify indi-cators related to the insect’s overwintering strategy and the major cryo-protectants. The supercooling points （SCPs）, which ranged from-14.7＆#176;C to -2.9＆#176;C, were higher than the lethal temperatures of LT50 （-33.64＆#176;C） and LT99 （-40.17＆#176;C） after 24 h exposure. , also the minimum mean daily temperature （-24.5＆#176;C） and mean monthly temperature （-18.0＆#176;C） at the sampling site in January during 2008-2012. Thus, X. rusticus is a typical freezing-tolerant insect. Glycerol serves as a major cryoprotectant for overwintering larvae , because it was the only polyol accumulated during the winter and it also had a significant negative correlation with the SCP （p=0.033, R=0.907）. The glycogen and lipid are major sources of ener-gy and their levels decreased substantially in the middle of overwintering, when glycogen had a significant correlation with the SCP （p= 0.006, R=0.971） whereas the lipid contents did not. Moreover, inter-conversions between glycerol and glycogen, as well as mannose and glycogen, were suggested by their negative correlations. The water content did not change obviously during the winter and was not correlated with the SCP. The free amino acids in the hemolymph and the total protein contents of the bodies of larvae changed significantly during winter, although both had no correlations with the SCP.

An improved loopless mounting method for cryocrystallography

Based on a recent loopless mounting method, a simplified loopless and bufferless crystal mounting method is developed for macromolecular crystallography. This simplified crystal mounting system is composed of the following components： a home-made glass capillary, a brass seat for holding the glass capillary, a flow regulator, and a vacuum pump for evacuation. Compared with the currently prevalent loop mounting method, this simplified method has almost the same mounting procedure and thus is compatible with the current automated crystal mounting system. The advantages of this method include higher signal-to-noise ratio, more accurate measurement, more rapid flash cooling, less x-ray absorption and thus less radiation damage to the crystal. This method can be extended to the flash-freeing of a crystal without or with soaking it in a lower concentration of cryoprotectant, thus it may be the best option for data collection in the absence of suitable cryoprotectant. Therefore, it is suggested that this mounting method should be further improved and extensively applied to eryocrystallographic experiments.