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.

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.

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.

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.

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.

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.

Growth and Freeze-Drying Optimization of Bifidobacterium crudilactis

Bifidobacterium crudilactis FR62/b/3 belongs to a new population of bifidobacteria isolated from raw milk and raw milk cheese. The objective of this work was to study the large scale culture of the strain and its stability in a dry formulation. Growth rate of Bifidobacterium crudilactis FR62/b/3 was optimal at a pH of 5.0 and a temperature of 37°C. At a temperature growth of 33°C and a pH of 5.0, the stationary phase was reached after 22 h, the viable cell number and the mean dry biomass concentration were respectively of 8.3 × 109 CFU/mL and of 2.1 g/L. Resistance of Bifidobacterium crudilactis FR62/b/3 to freeze-drying and effect of a variety of cryoprotectants to maintain the viability were also evaluated. Sorbitol was the most suitable cryoprotectant for freeze-drying as well as storage whereas sucrose and monosodium glutamate were only efficient during storage.

Improving native human sperm freezing protection by using a modified vitrification method

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice.However,it has been shown to have a negative impact on sperm function and structure.Vitrification as a successful alternative method has been proved to have better protective effects on human embryos,but vitrification of spermatozoa is still subject to low recovery rates.In this study,a modified vitrification method for native spermatozoa was developed.A total of 28 semen samples were included;each sample was divided into three equal parts and assigned to fresh,slow freezing,and vitrification groups.Sperm vitality,motility,morphology,DNA integrity,and acrosome reaction were assessed for each of the groups.The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing;vitrification achieves a higher recovery rate(P<0.05),motility(P<0.05),morphology(P<0.05),and curve line velocity(P<0.05)than slow freezing.Furthermore,DNA fragmentation was decreased(P<0.05)and better acrosome protection(P<0.05)was exhibited in the spermatozoa after vitrification.Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster,indicating that spermatozoa are better preserved through vitrification.In conclusion,while both slow freezing and vitrification have negative effects on sperm function and structure,the vitrification protocol described here had a relatively better recovery rate(65.8%)and showed improved preservation of several sperm quality parameters compared with slow freezing.

Influencing factors on the preservation of lytic bacteriophage VP3

Long-term and stable preservation of bacteriophages is of crucial importance.Although many efforts have been made in the past decades to explore the influence of external factors on bacteriophage preservation,there is still little understanding,and a systematic description is lacking.In this study,we explored the influence of different factors on the preservation of lytic bacteriophage VP3,one of the typing bacteriophages of Vibrio cholerae O1 biotype El Tor,and attempted to optimize its preservation.We examined external factors,including temperature,solution,and cryoprotectant,in stable cooling/freezing conditions or alternate cooling/freezing and thawing.We found that whether in Luria-Bertani(LB)medium or SM buffer,in terms of 20-week stable cooling or freezing,−20℃ was the most damaging while 4℃,−80℃,and−196℃ were protective.Thirteen cycles of alternate cooling/freezing and thawing caused a loss in the survival rates of bacteriophages.The addition of cryoprotectant,glycerol(30%,w/v)or dimethyl sulfoxide(DMSO,10%,w/v)significantly improved the survival rates of bacteriophages preserved at−20℃.However,at 4℃,−80℃,and−196℃,the cryoprotectant effect was only slightly positive or even harmful.In summary,for bacteriophage VP3,the best preservation method is to directly preserve the bacteriophage stocks in LB medium at−80℃ or−196℃ instead of storing them in SM buffer or adding cryoprotectant.Our results provided insights into the external influencing factors on bacteriophage VP3 during preservation at low temperature and can be applied to the optimization of bacteriophage preservation in the future.

Update on techniques for cryopreservation of human spermatozoa

In the 1960s,sperm cryopreservation was developed as a method to preserve fertility.Currently,techniques for the cryopreservation of human spermatozoa have been widely used in assisted reproduction.However,although sperm cryobiology has made notable achievements,the optimal method for the recovery of viable spermatozoa after cryopreservation remains elusive.Postthawing sperm quality can be affected by cryoprotectants,ice formation,storage conditions,and osmotic stress during the freezing process.This review discusses recent advances in different cryopreservation techniques,cryoprotectants,and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.