RGDS-fuctionalized alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation

Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 percent, w/v), allowed to attach to the substratum and then cryopreserved in 10 percent (v/v) dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1 C per min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93 ± 2 percent, mean and standard error) than those in suspension (52 ± 2 percent) or in unmodified alginates (62 ± 3 percent). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide per g alginate, 30 minutes loading time and 1 C per min cooling rate.     

New determinants for tolerance of coffee (Coffea arabica L.) seeds to liquid nitrogen exposure

The present work establishes for the first time that tolerance of coffee seeds to liquid nitrogen (LN) exposure depends on the initial quality of the seedlot and on the rewarming regime employed. Seedlot quality was estimated by the parameters of a quantal response model of desiccation sensitivity developed previously. The percentage of seedlings recovered from cryopreserved seeds was very well correlated with the relative humidity (RH) at which 90 percent of the initial viability was retained, RH90, as estimated by the model. Whatever the cooling regime employed, rewarming the seeds slowly by exposing them to ambient air was highly detrimental. Slow rewarming-induced viability loss was not due to imbibitional damage since seeds pre-heated at 37 degree C after slow rewarming to 0 degree C exhibited a survival percentage lower than seeds thawed rapidly to 0 degree C before sowing. The optimal hydration status for coffee seed cryopreservation was also re-examined. Drying seeds in 81 percent RH provided survival percentages considerably higher than those obtained using the drying RH always employed until now, i.e. 78 percent. A new procedure for slowly precooling the seeds prior to immersion in LN was also established. It consisted of placing the vials containing the seeds in a dry ice-bath for 25 min. Using this procedure in combination with seed drying in 81 percent RH and rapid rewarming in a 37 degree C water-bath for 30 min ensured the highest survival percentages ever obtained with coffee seeds, i.e. 89 percent, a value which was not significantly different from the initial viability percentage.     

Characteristics of Atropa belladonna hairy roots cryopreserved by vitrification method

Atropa belladonna hairy roots (clone M8) were successfully cryopreserved by using the vitrification method. A. belladonna hairy root tips were precultured on a half strength of Murashige and Skoog (MS) solid medium with 0.1 mg per L 2,4-D or without phytohormone for 1 day, and then dehydrated with PVS2 solution for 15 minutes prior to immersion into liquid nitrogen for 1 day, 1 week, 1 month and 3 months. Hairy root tips kept in liquid nitrogen were rapidly thawed at 36 degree C in a water bath. The root tips were recultured on half strength MS medium. The hairy root tips, precultured with 2,4-D before cryopreservation, showed a higher survival rate than those precultured without phytohormone. The hairy root tips, precultured with 2,4-D, showed an average survival rate of 83 percent. There was no significant difference in the viability of the hairy roots cryopreserved for different periods. The regrowth of cryopreserved hairy roots was similar to that of untreated hairy roots and tropane alkaloid productivity became stable after 4th subculture. PCR analysis of hairy roots demonstrated the conservation of the T-DNA in cryopreserved hairy roots. These results indicate that cryopreservation by vitrification method is useful to preserve A.belladonna hairy root clone M8.     

The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifoliata (L.)

The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 g water per g dry mass is believed to increase cytoplasmic viscosity. Cooling to -196 degree C was done at rates averaging between 0.17 and 1300 degree C per second, and warming at 600 or 1.35 degree C per second. Survival was assessed after 4 weeks in vitro. Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70 percent normal development ranged between 0.17 and about 1300 degree C per second for axes at a water content of 0.26 g water per g dry mass narrowing with increasing hydration to an apparent optimum at about 686 degree C per second in axes at 0.8 g water per g dry mass At 1.7 g water per g dry mass, axes cooled at 0.17 degree C per second yielded nearly 40 percent normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.     

A parametric study of freezing injury in BPH1CAFTD-2 human prostate tumor cells

Freeze injury in BPH1(CAFTD)-2 cells frozen/thawed in suspension was studied through a two-level four-parameter (2(4)) experimental design and analysis. The four parameters considered were end temperature, hold time, TNFalpha concentration, and thawing rate. Thermal parameter values chosen were based on the approximate thermal history cells would experience in the peripheral region of a cryosurgical iceball. Cell suspensions were frozen at a constant 10 degree C/min on a directional solidification stage and viability was assessed within 1 hr post-thaw using a dye exclusion assay. The parameters affecting cell survival were determined through calculation of the individual parameter effects (E) and interactions (I) according to factorial design guidelines; data set curvature (C) was also determined. Cell viability ranged from a maximum of 87.6 percent to a minimum of 17.6 percent indicating trends in cell survival were sensitive to the parameters chosen. Survival was affected by the following parameters in order: lowering the end temperature, increasing the hold time, adding TNFalpha, and reducing the thawing rate. In addition, all 2-way parameter interactions except TNFalpha hold time were statistically significant. Curvature analysis showed that cell viability at the midpoint of the data was nearly 20 percent lower than predicted based on linear interpolation. These results were verified and extended using analysis of variance (ANOVA). We conclude that cryoinjury in this tumor line can be influenced by multiple interacting thermal parameters, most importantly end-temperature and hold time, as well as the presence of the cytokine TNFalpha. Finally, although the cell type is tumorigenic results suggest the possibility of using freezing to control benign prostatic hyperplasia (BPH) in addition to cancer within the prostate.     

A comparative study of three cryopreservation protocols for effective storage of in vitro-grown mint (Mentha Spp.)

This study was designed to determine the response of diverse mint genotypes to three commonly used cryopreservation techniques. Four mints [Mentha x piperita nothosubsp. citrata (Ehrh.) Briq.; M. canadensis L.; M. australis R. Br, and M. cunninghamii Benth] were cryopreserved using three protocols: controlled rate cooling (CC), encapsulation dehydration (ED) and PVS2 vitrification (VIT). Regrowth of mint species following controlled rate cooling (93 percent) was significantly (P < 0.0001) better than encapsulation dehydration (71 percent) and vitrification (73 percent). All four genotypes responded well to the controlled rate cooling protocol but there was some variability with the other two protocols. Genotype specific response to the individual protocols showed that there were significant differences in the recovery of Mentha x piperita nothosubsp. citrata and M. australis with CC > VIT > ED. There were also significant differences in the recovery of M. cunninghamii and M. canadensis, with CC and ED significantly better than VIT. Regrowth of the shoot tips of these mints ranged from 60 percent to 95 percent for all but one treatment. The overall results of this study compare favorably to other techniques. These improved results may be due to a combination of favorable growth conditions, cold acclimation and recovery medium. Controlled rate cooling was the most successful technique for the storage of these diverse mint genotypes; however recovery of shoot tips from VIT and ED was high and these techniques could also be used for cryogenic storage of mint germplasm.     

Determination of genetic stability in surviving apple shoots following cryopreservation by vitrification

In vitro-grown apple shoot tips were successfully cryopreserved by vitrification, with an average survival and shoot formation of 80 percent and 76 percent respectively. Surviving shoots showed the same rate and regrowth patterns as those of non-treated controls. No significant differences (P < 0.05) were observed in morphological characteristics, including shoot length, leaf shape, leaf width/length ratio and root length, between the control and cryopreserved shoots. No different microsatellite alleles and ISSR fragments were detected between control and cryopreserved shoots using twelve pairs of microsatellites and eleven ISSR primers. These results show that cryopreservation using vitrification is a practical method for the long-term storage of apple germplasm.     

Geneticallly enginereed trehalose accumulation improves cryopreservation tolerance of chrysanthemum (Dendranthema grandiflorum kitam.) shoot-tips

Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48 percent and 67 percent for transgenic lines and between 33 percent and 36 percent for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci.     

The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance

The effects of fruit maturity, at the time of natural dispersal, on subsequent desiccation tolerance and sub-zero storage was investigated in three lots of Acer pseudoplatanus (sycamore) collected from northern to southern Europe. Fruits from the native plant distribution range in Italy had significantly higher desiccation tolerance (0.16 g water per g DW) than those from England (0.30) and Norway (0.50), confirming that the maximum potential desiccation tolerance in sycamore exceeds that of the recalcitrant type. In contrast, the unfrozen water content varied only slightly between seedlots, but systematically reduced with development (0.35 to 0.27 g water per g DW). Maximum survival (60 percent fruit germination) of seven days sub-zero temperature storage coincided with drying the Italian fruit lot to c. 0.2 g water per g DW followed by holding at -20 degree C, above the onset temperature for freezing, or at -196 degree C (liquid nitrogen). Fruit survival was much lower in the Italian fruits when held at this water content and -70 degree C, and in all other combinations of water content, temperature and fruit lot provenance. As the risk of nucleation in partially dried fruits held at -20 degree C is high, we recommend sycamore fruits are cryopreserved for long-term conservation.     

Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium sims. (Orchidaceae) by encapsulation-dehydration

Encapsulation-dehydration was employed for cryopreserving seeds and in vitro-cultured protocorms of Oncidium bifolium. Freshly harvested seeds, 120 days after pollination, were encapsulated in beads containing 1/2 MS medium with 3% sucrose and 3% calcium alginate and subsequently pretreated in agitated (80 rpm) liquid medium supplemented with 0.15 M sucrose (24 h) followed by 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h). The beads with seeds were dehydrated with silica gel for 5 h to 19.2% moisture content and immersed in liquid nitrogen for 1 h, thawed at 30 degrees C for 2 min, post-treated using the same series of liquid media [0.5 M sucrose (24 h), 0.25 M sucrose (48 h), 0.15 M sucrose (24 h)], and recultured on 1/2 MS medium with 0.1M sucrose and 0.7% percent agar. As much as 4.8% of the cryopreserved seeds produced complete plants. In-vitro cultured protocorms were successfully cryopreserved following the same procedure, allowing 11.3% of them to produce plants.     

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.     

Investigation of the influence of cell density of human fibroblasts cryopreserved inside collagen sponges at various cooling rates

The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10(5) to 10(7) cells/cm(3). Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50 degrees C/min in a solution of Dulbecco's Modified Eagle Medium, 20% fetal bovine serum and 10% dimethylsulfoxide, kept frozen below -185 degrees C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.     

Effect of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro

The effect of N6-benzyladenine (BA) on the recovery of cryopreserved shoot tips of the LN33 hybrid (Vitis L.) and Troyer citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis [L.] Osbeck.] cultured in vitro was examined. For the LN33 hybrid, the presence of BA in the recovery medium was essential for survival of control and cryopreserved shoot tips, although the BA concentration did not influence the survival percentage. BA at 5, 2, and 5 microM or higher induced callus formation in control, and shoot tips cryopreserved by vitrification, and by encapsulation-dehydration, respectively. While a BA concentration of 4 microM was found optimal for recovery of control shoot tips, 1 and 2-4 microM produced the best recovery of shoot tips cryopreserved by vitrification and encapsulation-dehydration, respectively. A similar pattern of effect of BA on recovery was found for 'Troyer' citrange. Low survival of control and cryopreserved shoot tips was observed with a BA-free recovery medium. The addition of BA to the recovery medium significantly increased survival. The BA concentration that induced callus formation in shoot tips cryopreserved by encapsulation-vitrification was higher than that which induced it in those cryopreserved by encapsulation-dehydration. Recovery of control shoot tips was best with an addition of 6-10 microM BA to the medium. Optimal recovery of shoot tips cryopreserved by encapsulation-vitrification and encapsulation-dehydration was achieved with 3-4 and 2 microM BA, respectively. Results from the present study suggest that an optimal BA concentration for recovery of control shoot tips may be different from that for cryopreserved shoot tips; furthermore, the optimal BA concentration for recovery of cryopreserved shoot tips may also differ among different cryogenic procedures.     

Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America

Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores     

Cryopreservation of peach palm zygotic embryos

Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.     

Rapid Consolidation by Spark Sintering of Rapidly Solidified 7075 Aluminum Alloy Powder

Compacts of 7075 aluminum have been produced from rapidly solidified powders by optimizing spark sintering parameters, such as pulse discharge time, fixed maximum temperature, holding time at this temperature, and method of cooling to room temperature after the sintering. High-grade compacts can be obtained by a short process (40-50 s) consisting of heating to 773 or 873 K at a heating rate of 9.6 K/s and holding this temperature for 10 s. The rapidly cooled compacts show the same supersaturated state at room temperature as the received as-atomized powder. Compacts quenched in water just after spark sintering at 873 K for 1.2 ks show the same age-hardening behavior as solution-heat treated compacts. Compacts that are quenched in water and aged after sintering at 873 K for 1.2 ks show the same elongation and flow stress as compacts aged after solution heat treatment. Elongation data suggest that compacts produced with longer holding time at a higher temperature and rapid cooling show a large amount of main alloying elements in solid solution and sufficient promotion of sintering.     

Cryopreservation of 'Nabali' olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification

Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30 degree C was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.     

Fabrication of fresnel lens by glass molding technique

In this paper, we describe the fabrication of a Fresnel lens with a diameter of 15mm by a glass molding technique. Tungsten carbide and K-CSK120 were used as the materials of the mold and lens, respectively. The optimal parameters determined for the molding process included a molding temperature of 560 °C, a molding force of 300 N, a temperature holding time of 180 s, and a cooling rate of 18.5 °C/min.     

Cryopreservation of shoot apices of in-vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols

Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.