Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Vitrification induces critical subcellular damages in ram spermatozoa

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 10⁶ spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.     

Vitrification of one-cell mouse embryos in cryotubes

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.     

Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions

The application of the droplet vitrification cryopreservation technique to taro accessions from a range of Asia Pacific countries is presented. The optimum protocol involves excision of about 0.8 mm shoot-tips from in vitro plants, 20–40 min PVS2 exposure at 0°C followed by rapid plunge into liquid nitrogen. Thawing was done at room temperature (25°C) and shoot-tips inoculated on MS medium with 0.1 M sucrose regenerated into plantlets 4–6 weeks later. This new droplet vitrification protocol improved the mean post-thaw regeneration rates to 73–100% from 21–30% obtained with the previous cryo-vial vitrification protocol.     

Thermodynamic aspects of vitrification

Vitrification is a process in which a liquid begins to behave as a solid during cooling without any substantial change in molecular arrangement or thermodynamic state variables. The physical phenomenon of vitrification is relevant to both cryopreservation by freezing, in which cells survive in glass between ice crystals, and cryopreservation by vitrification in which a whole sample is vitrified. The change from liquid to solid behavior is called the glass transition. It is coincident with liquid viscosity reaching 10¹³ Poise during cooling, which corresponds to a shear stress relaxation time of several minutes. The glass transition can be understood on a molecular level as a loss of rotational and translational degrees of freedom over a particular measurement timescale, leaving only bond vibration within a fixed molecular structure. Reduced freedom of molecular movement results in decreased heat capacity and thermal expansivity in glass relative to the liquid state. In cryoprotectant solutions, the change from liquid to solid properties happens over a ∼10 °C temperature interval centered on a glass transition temperature, typically near −120 °C (±10 °C) for solutions used for vitrification. Loss of freedom to quickly rearrange molecular position causes liquids to depart from thermodynamic equilibrium as they turn into a glass during vitrification. Residual molecular mobility below the glass transition temperature allows glass to very slowly contract, release heat, and decrease entropy during relaxation toward equilibrium. Although diffusion is practically non-existent below the glass transition temperature, small local movements of molecules related to relaxation have consequences for cryobiology. In particular, ice nucleation in supercooled vitrification solutions occurs at remarkable speed until at least 15 °C below the glass transition temperature.     

Cryopreservation: A tool to conserve date palm in Saudi Arabia

The cryostoring of embryogenic tissue of the date palm (Phoenix dactylifera L. cv. Sagai) was examined through dehydrated-encapsulation, vitrification, and vitrification-encapsulation. The most extreme regeneration rate (53.33%) of epitomized, cryostored liquid nitrogen (+LN) treated embryos was observed when pre-embryonic masses were hatched with 0.5 M sucrose for 48 h pursued by 6 h air drying out. The most noteworthy survival rate (80.0%) of epitomized, cryopreserved embryonic cluster came about when calli were hatched with 0.3 or 0.7 M sucrose for 48 h pursued by four hours of lack of hydration, or with 0.5 M sucrose for 48 h without air drying out or with 2 h of air drying out. Following cryopreservation utilizing the embodiment vitrification convention, the most astounding survival (86.7%) as well as the greatest growth (46.7%) was accomplished when the typified vitrified, cryopreserved calli were treated with Vitrification Solution 2 for plants (PVS2) for 60 min at 25 °C. Cryopreservation utilizing the vitrification convention brought about the most extreme recuperation of 53.3%, when vitrified-cryopreserved calli were subjected to PVS2 solution for 30 min at 25 °C. Most extreme (40%) regeneration of vitrified, cryopreserved embryonic calli was seen when these calli were treated with PVS2 solution for 60 min at 25 °C. The outcome got amid this investigation of regrowth after cryopreservation of the cv. Sagai was over the base suitable for a cryo-germplasm bank. Recovery and regrowth were above 30% for all the techniques developed for the cv. Sagai.     

Determination of glassy state by cryo-SEM and DSC in cryopreservation of mint shoot tips by encapsulation–dehydration

Cryopreservation of mint shoot tips grown in vitro (Mentha × piperita) was performed after encapsulation in alginate beads. Encapsulated shoot tips were first precultured in sucrose enriched medium (0.75 M) and then dried under a sterile air flow (0–6 h). After cooling in liquid nitrogen and warming in a warm water bath, alginate beads were transferred to solid culture medium for 4 weeks. The effect of dehydration time of the encapsulated shoots was evaluated for water content, cooling and warming rates, ice crystal formation and cellular vitrification, by using low temperature scanning electron microscopy and differential scanning calorimetry. Viability of the recovered material showed a close relation between the dehydration time, cooling and warming rates, ice formation avoidance and tissue vitrification. At short drying periods (up to 3 h), ice crystals were formed and the viability was low or absent. After longer drying periods (5 and 6 h), both beads and specimens became vitrified.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.     

A new vitrification device that absorbs excess vitrification solution adaptable to a closed system for the cryopreservation of mouse embryos

Several closed vitrification devices that avoid contact with liquid nitrogen have been reported. Recently, based on the Kitasato Vitrification System (KVS), we developed the Closed-KVS, which is a closed vitrification device. The KVS is an open vitrification device that can absorb excess vitrification solution. In this study, we performed two experiments to evaluate the efficacy of the Closed-KVS as a vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage. In the first experiment, the blastocysts were vitrified using either the Closed-KVS or the KVS (control device). The survival, re-expansion, and hatching rates were not significantly different between embryos vitrified using the Closed-KVS and those vitrified using the KVS. In the second experiment, we evaluated the embryonic development of the two-cell stage embryos vitrified using the Closed-KVS. There were no significant differences in the survival, blastocyst formation, or hatching rates between vitrified or non-vitrified embryos. Additionally, we evaluated the cooling and warming rates of these devices using a numerical simulation method. The cooling rates of the Closed-KVS were similar regardless of whether the outer cap was pre-cooled and were lower than those of the KVS. However, the warming rates of the Closed-KVS (irrespective of cap pre-cooling) were the same as those of the KVS (612,000 °C/min). In summary, the Closed-KVS is a novel closed vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage.     

Heterologous expression of <Emphasis Type="Italic">PDH47</Emphasis> confers drought tolerance in <Emphasis Type="Italic">indica</Emphasis> rice

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L^?1 GA₃ for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.     

Cryopreservation of Musca domestica (Diptera: Muscidae) embryos

Prior studies on cryopreserving embryos of several non-drosophilid flies established that two Drosophila melanogaster embryo cryopreservation protocols were not directly suitable for use with these species. This paper describes our work on developing a protocol for cryopreservation of embryos of the housefly, Musca domestica. Significant progress was made when permeabilization of the vitelline membrane was optimized, a vitrification solution containing ethylene glycol, polyethylene glycol, and trehalose was formulated, and when cooling and recovery of the cryopreservation protocol included a step which passed the embryos through liquid nitrogen vapor. More than 70% of housefly embryos withstand treatments of dechorionation, permeabilization, loading with cryoprotectant, and dehydration in vitrification solution, but the cooling, warming, and poststorage rearing steps still cause a considerable reduction in survival. About 53% of the vitrified M. domestica embryos hatched into larvae. Relative to the percentage of the control adult emergence, about 13% of the embryos stored in liquid nitrogen developed into fertile adults. Hatching of the F(1) progeny of adults having been cryopreserved as embryos was similar to control levels.     

Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl₂). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Measurement of cooling and warming rates in vitrification‐based plant cryopreservation protocols

Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro‐viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (T_f) and the glass transition (T_G) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice‐derived damage. Vitrification and droplet‐vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between ?20°C and ?120°C, ranged from ca. 5°C s^?1 to 400°C s^?1, while warming rates spanned from ca. 2°C s^?1 to 280°C s^?1, for the different protocols and conditions studied. A wider measuring window (0°C to ?150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1177–1184, 2014     

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 m sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 m glycerol and 0.4 m sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 m sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.     

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.     

Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 μL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2–5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.     

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.