Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Stainless steel tube-based cell cryopreservation containers

This study focused on increasing the freezing rate in cell vitrification cryopreservation by using a cryopreservation container possessing rigid mechanical properties and high heat-transfer efficiency. Applying a fast freezing rate in vitrification cryopreservation causes a rapid temperature change in the cryopreservation container and has a substantial impact on mechanical properties; therefore, a highly rigid cryopreservation container that possesses a fast freezing rate must be developed. To produce a highly rigid cryopreservation container possessing superior heat transfer efficiency, this study applies an electrochemical machining (ECM) method to an ANSI 316L stainless steel tube to treat the surface material by polishing and roughening, thereby increasing the freezing rate and reducing the probability of ice crystal formation. The results indicated that the ECM method provided high-quality surface treatment of the stainless steel tube. This method can reduce internal surface roughness in the stainless steel tube, thereby reducing the probability of ice crystal formation, and increase external surface roughness, consequently raising convection heat-transfer efficiency. In addition, by thinning the stainless steel tube, this method reduces heat capacity and thermal resistance, thereby increasing the freezing rate. The freezing rate (3399 ± 197 °C/min) of a stainless steel tube after interior and exterior polishing and exterior etching by applying ECM compared with the freezing rate (1818 ± 54 °C/min) of an original stainless steel tube was increased by 87%, which also exceeds the freezing rate (2015 ± 49 °C/min) of an original quartz tube that has a 20% lower heat capacity. However, the results indicated that increasing heat-transferring surface areas and reducing heat capacities cannot effectively increase the freezing rate of a stainless steel tube if only one method is applied; instead, both techniques must be implemented concurrently to improve the freezing rate.     

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from −126 °C to −121 °C, and devitrification was initiated at −109 °C. Interestingly, samples entered rubbery state at −121 °C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (−196 °C), and two temperatures of liquid nitrogen vapors within the dewar (−50 °C and −70 °C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30 seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.     

Protective effects of iodixanol during bovine sperm cryopreservation

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep™ (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed^® with 0 (control), 1.25%, 2.5%, and 5% OptiPrep™, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3 h incubation at 37 °C for frozen-thawed samples and after 3 h and 6 h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep™ showed inferior viability (P = 0.047) and 3 h motility (P = 0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P = 0.042), acrosomal integrity (P = 0.045), and 0 h motility (P = 0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3 h motility (control, P = 0.007; 5% OptiPrep, P = 0.005; 1.25% OptiPrep, P = 0.004) and to 1.25% OptiPrep for acrosomal integrity (P = 0.001). In a search for a protection mechanism, we measured glass transition temperature (T_g) of Andromed^® and of Andromed^® with 1.25%, 2.5%, and 5% OptiPrep™. Andromed^® (-58.78 °C) and 1.25% OptiPrep™ (-58.75 °C) groups had lower mean T_g than that of the 2.5% (-57.67 °C) and the 5% (-57.10 °C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.     

Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions

Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.     

A Low Temperature Limit for Life on Earth

There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.     

Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse

Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below −100 °C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000 °C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

Slow molecular mobility in the crystalline and amorphous solid states of glucose as studied by Thermally Stimulated Depolarization Currents (TSDC)

Thermally Stimulated Depolarization Currents (TSDC) measurements on α-d-glucose have been carried out in the temperature region from −165 °C (108 K) to 120 °C (393 K). The slow molecular mobility was characterized in the crystalline and in the glassy states, as well as in the glass transition region. The influence of aging on the measured TSDC peaks of the secondary relaxation has been discussed and it was concluded that there are motional modes that are aging independent while others are affected by aging. Important discrepancies were reported in the value of the steepness index or fragility (T_g—normalized temperature dependence of the relaxation time) obtained by different, and well-established, experimental techniques. A careful discussion of the possible origins of these discrepancies is presented.     

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at − 5 °C, and then cooled slowly to − 30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.     

Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification

In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by droplet-vitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L⁻¹ sucrose for 2 days, osmoprotected in loading solution for 20-40 min at room temperature and then soaked in PVS2 solution for 90-120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method.     

Glassy State and Cryopreservation of Mint Shoot Tips

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze‐avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet‐vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low‐temperature scanning electron microscopy, as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/?1°C than in plants cultivated at 25°C. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:707–717, 2013     

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation–drying

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 m old cultures, (2) the incubation overnight in trehalose (0.5 M), (3) the drying during 48 h at 27 °C, (4) the cryopreservation in the freezer at −130 °C following a two-step decrease in temperature: a fast decrease (∼12 °C min⁻¹) from room temperature (+20 °C) to −110 °C followed by a slow decrease in temperature (∼1 °C min⁻¹) from −110 °C to −130 °C, and (5) the direct thawing in a water bath (+35 °C). The % PIB was above 70 % for all the isolates and even above 95 % for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2 m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.     

Novel Microwave Technology for Cryopreservation of Biomaterials by Suppression of Apparent Ice Formation

Ice formation inside or outside cells has been proposed to be a factor causing cryoinjury to cells/tissues during cryopreservation. How to control, reduce, or eliminate the ice formation has been an important research topic in fundamental cryobiology. The objective of this study was to test a hypothesis that the coupled interaction of microwave radiation and cryoprotectant concentration could significantly influence ice formation and enhance potential vitrification in cryopreservation media at a relative slow cooling rate. Test samples consisted of a series of solutions with ethylene glycol (a cryoprotectant) concentration ranging from 3 to 5.5M.A specific microwave resonant cavity was built and utilized to provide an intense oscillating electric field. Solutions were simultaneously exposed to this electric field and cooled to −196°C by rapid immersion in liquid nitrogen. Control samples were similarly submerged in liquid nitrogen but without the microwave field. The amount of ice formation was determined by analysis of digital images of the samples. The morphology of the solidified samples was observed by cryomicroscopy. It was found that ice formation was greatly influenced by microwave irradiation. For example, ice formation could be reduced by roughly 56% in 3.5Methylene glycol solutions. An average reduction of 66% was observed in 4.5Msolutions. Statistical analysis indicated that the main effects of microwave and ethylene glycol concentration as well as the interaction between these two factors significantly (P< 0.01) influenced ice formation amount, confirming the hypothesis. This preliminary study suggests that a combined use of microwave irradiation and cryoprotectant might be a potential approach to control ice formation in cells/tissues during the cooling process and to enhance vitrification of these biomaterials for long-term cryopreservation.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: correlation of freezing visualization with functional recovery

Cryopreservation is a well-established technique for long-term storage of viable cells and tissues. However, in recent years, application of established cryobiological principles to the preservation of multicellular tissues and organs has demanded considerable attention to ways of circumventing the deleterious effects of ice and thermal stresses in bulky tissues. As part of a multidisciplinary research program designed to study the interactions of thermo-physical events with tissue preservation, we report here on the implementation of a slow cooling (3 °C/min) and slow warming (62 °C/min) regimen towards scale-up of vitreous preservation of large tissue samples. Specifically, the correlation of thermo-physical events during vitrification of carotid artery segments with function recovery is reported using marginal thermal conditions for achieving vitrification in bulky samples. Moreover, the outcome is compared with a similar study reported previously using a 3-fold higher rate of rewarming (186 ± 13 °C/min). Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1 ml samples by imposing a low (2.6 ± 0.1 °C/min) cooling rate, between −40 and −100 °C, and a low rewarming rate (62 ± 4 °C/min) between −100 and −40 °C. Following cryoprotectant removal, the artery segments were cut into 3-4 mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore and sodium nitroprusside). In addition, non-specific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function, normalized to untreated control samples, in response to the agonists norepinephrine and phenylepinephrine was significantly better in the slowly rewarmed group of carotid segments (74 ± 9% and 62 ± 11%, respectively) than for the more rapidly warmed group 31 ± 7% and 45 ± 15%, respectively). However, EC₅₀ sensitivities were not significantly different between the groups. Thermo-physical events such as ice formation and fractures were monitored throughout the cooling and warming phases using cryomacroscopy with the aid of a purpose-built borescope device. This technique allowed a direct observation of the visual impact of ice formation on specific zones along the blood vessel segment where, in most cases, no ice formation or fractures were observed in the vicinity of the artery segments. However, in specific instances when some ice crystallization was observed to impact the artery segment, the subsequent testing of function revealed a total loss of contractility. The successful vitrification of blood vessel segments using marginal conditions of slow cooling and rewarming, provide essential information for the development of scale-up protocols that is necessary when clinically relevant size samples need to be cryopreserved in an essentially ice-free state. This information can further be integrated into computer simulations of heat transfer and thermo-mechanical stress, where the slowest cooling rate anywhere in the simulated domain must exceed the critical values identified in the current study.     

Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027), its biodegradability and potential application

Characterisation of polyhydroxyalkanoate (PHA) film produced by haloalkalitolerant Halomonascampisalis (MCM B-1027) in 14 L SS fermenter revealed it to have composition of monomer units, HB:HV as 96:4 as analysed by ¹H NMR indicating the PHA as a co-polymer of PHB-co-PHV, molecular weight by gel permeation chromatography as 2.08 × 10⁶, melting temperature 166.51 °C, tensile strength 18.8 MPa; two relaxations namely beta transition corresponding to the glass rubber transition and alpha transition corresponding to crystalline relaxation by Dynamic Mechanical Thermal analysis and only one relaxation corresponding to MWS interfacial polarisation with activation energy of 129 kJ/mol by broadband dielectric spectroscopy. Optical microscopic studies showed typical Maltese-cross pattern of spherulites. The PHA film was found to be biodegradable by standard ASTM method as well as by soil burial method. The leak proof polymer bags prepared from the film could be used as a packaging material.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.